Reconstruction of the lantibiotic ruminococcin-A biosynthesis machinery in Escherichia coli and structural characterization [original]

Reconstruction of the lantibiotic
ruminococcin-A biosynth esis machinery i n
Escherichia coli and stru ctural
characterization
Elvis Legala Ongey - Dissertation

Rec ons tr uct i on of the l antibi otic ruminoc oc c i n - A b i osyn thes i s El vis Le ga l a Ong ey

Reconstruction of the lantibiotic ruminococc in- A
biosynthesis machinery in Escherichia col i and structural
characterization

vorgelegt von
M. Sc. Elvis Legala Ongey
geb. in Bali-Nyonga, Kamerun

von der Fakultät III-Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades

Doktor der Naturwissenschaften
- Dr. rer. nat. -
genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Prof. Dr. Jens Kurreck-TU Ber lin
Erstgutachter: Prof. Dr. Peter Neubauer, Institut für Biotechnologie - TU Berlin
Zweitgutachter: Prof. Dr. Nediljko Budisa, Institut für Chemie - TU Berlin
Drittgutachter: Prof. Dr. Oscar P. Kuipers, Groningen, The Netherlands

Tag der wissenschaftlichen Aussprache: 02 . August 2018

Berlin, 2018

Elvis Legala Ongey Rec onstruction of the lantibiotic ruminococcin-A biosynthesis

I
Abstract
Lanthipeptides are a group of therapeutically relevant proteinaceous compounds
predominantly produced by Gram-positive bacteria at the ribosomal level, contain ing thioether
cross-linked amino acids called lanthionine as a characteristic structura l element.
Lanthipeptides are attracting grow ing interests st emming from their w ide range of bioactivities
including antimicrobial, antiviral and anticancer. However, purifying them from native s ources
is challenging due low production yields and cultivation difficulties that accompany the natura l
producers. Ruminococcin -A (RumA) is an example which is naturally produced by the obligate
anaerobe Ruminococcus gnavus E1. Culturing R. gnavus E1 is obviously chal lenging and
would certainly negatively affe ct the developmen t of a stable high -quality productio n process,
limiting biotechnological development and therapeutic exploitatio n of RumA. In this study, we
amplified parts of the RumA bios ynthesis op eron from R. gnavu s E1, encoding the linear
precursor peptide (preRumA) and the lanthionine synthetase (RumM), and coexpressed them
in the heterologous bacteria l host E. coli .
Our results show that RumM catalyzed the introduction of dehydroamino acids into the
core peptide of preRumA and subsequently conjugated the dehydrated residues with cysteine
to generate thioether bridges. This was achieved with preRumA expressed as a chimeric fus ion
protein togeth er with GF P. Results here demonst rate that fusing a la rger protein carrier to the
N-terminus of the le ader peptide does not necessarily obstruct in vivo processivity of the
lanthionine -generating enzyme in modifying its substrate. A strong interactio n was observed
between the chimeric fusion product and RumM, supplying some interesting insights into the
catalytic mechanisms of class II lanthionine -generating enzymes. Characterizi ng the structure
of preRumA further revealed the pre sence of three thioether rings, contrad icting previous
report which concluded that the formation of a third thioet her bridge was not possible. Modified
preRumA was activated in vi tro by removing the l eader peptid e using trypsin to yield the active
product and biologica l activity was achieved. A production yield of 6 mg of pure modified
preRumA p er li tre of E. co li culture was attain ed. Cons idering the size ratio of the leader - to -
core segments of preRumA, this amount woul d produc e a final yield of approximate ly 1 -2 mg
of RumA wh en the leader peptide is removed. This yield exceeds the amount of RumA purified
from the native host in the o rder of 10 4 .
This study supplies a system that may be applied as a useful tool for studying peptide
engineering to generate analogues of RumA or completely new peptides with superior anti -
infective properties. The current system also provides an alternative st rategy to der ive
mechanistic insights on the co mplex modification machinery of lanthipeptides.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Zusammenfassung
Lanthipeptide sind eine Gruppe von therapeutisch bedeutsamen proteinogenen Naturstoffen,
die hauptsächlich von Gram-po sitiven Bakterien ribosomal produziert werde n. Als
kennzeichnendes Strukturelement ent halten sie über Thioether verknüpfte Aminos äuren,
sogenannte Lanthi onine. Auf Grund i hres breiten Wirksp ektrums, welches Bioak tivität sowohl
gegen Bakterien, Viren als auc h Krebszellen einschlie ßt, rücken Lanthipeptide zunehmend in
den Fokus der Forschung. Allerd ings ist die Gewinnung von Lanthipeptid en aus der en
natürlichen Produzenten äußers t schwierig, da diese nur in geringem Maße produzier t werden
und die Kul tivierung der entsprechenden Bakterien auf Grun d ihrer Leb ensweise sc hwierig ist.
Ein Beispiel hierfür ist Ruminococin-A (RumA), das vom Bakterium Ru minococcus gnavus E1
produziert wird. Eine Kultivierung von R. gnavus ist auf Grund seiner obl igat anaeroben
Lebensweise schwierig, wodurch die Entwicklung eines stabilen, hoc h effizi enten
Produktionsprozesse s erschwert und die biotechnologi sche Entwicklung bzw. die
therapeutische Erforschung limitie rt wird. Im Rahmen dieser Arbeit wurden Te ile des RumA
Biosyntheseoperons aus R. gnavus E1 amplifiziert und i m heterologen Wirtsorganismus
Escherichia coli exprimiert. Hierzu zähl en die Gene für das li neare „Precour sor“ -Peptid
(preRumA) sowie die Lanthioni ne -Synthetase (RumM).
Das Enzym RumM katal ysiert die Dehydratisierung einzelner Aminosäuren im
Kernpeptide von pre RumA und anschlie ßend die Bildung der typisch en Thioether Brücke n
durch Konjugation der dehydratisierten Reste mit einem Cystein. Hierfür wurde ein
Fusionsprotein aus preRumA und dem g rünfluoreszieren den Protein (GFP) produziert. Die
Resultate zeigen, dass die Fusion eines größeren Trägerproteins an den N -Term inus des
RumA „Le aderpeptides “ des sen Prozessierung in vi vo durch RumM nicht neg ativ beeinflusst.
Die beobachtete starke Interakt ion zwischen dem chimären Fusionsprotein und RumM
ermöglicht einen interess anten Einblick in den katalytischen Mechanismus der Klasse II
Lanthionin -Synthetasen. Im Gegensatz zur früheren Annahme, dass die Bil dung eines dritten
Thioetherrings ni cht möglich ist, konnte durch di e Aufklärung der Struktur von preRumA da s
Vorhandensein von drei Thioetherringe n nachgewiesen werden. Die Aktivierung des
modifizierten preRumA erfolgte in vi tro . Das Leader peptid wurde hi erfür durch Sp altung mit
Trypsin entfernt und so das biologisch aktive Produkt gewo nnen. Aus einem Liter E. coli Kultur
konnten hierbei bis zu 6 mg modifiz iertes preRumA gereinigt werden. Bedenkt man das
Größenverhältnis zwi schen Kernp eptid und Leaderpeptid entspricht diese Menge einer finalen
Ausbeute von ca. 1-2 mg aktivem RumA nac hdem das Leaderpep tid entfernt wurde . Dies
übersteigt die Menge an RumA, die aus dem natürlic hen Produzenten gewonnen werden kann,
um das 10.000 fache.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

III
Das im Ra hmen dieser Arbeit entwickelte Expressionssystem stellt ei n nütz liches
Werkzeug für die Untersuchung von Peptiden dar. Es könnte al s Grundlage für die Exp ression
von RumA Analoga oder anderer neu er bi oaktiver Peptid e mit unter Umständen besseren
antiinfektiven Eigenschaften dienen. Darüber hinaus bietet unser System ei ne alternati ve
Möglichkeit zur Untersu chung der komplexen Modifikationssysteme von Lanthipeptiden.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

The present work was performed fr om January 2015 – March 2018 in the research
group of Prof. Dr. Pete r Neubauer (Chair of Bioprocess Engineering ) at the Department
of Biotechnology, Technische Universität Berlin.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

V
List of Publications Contributing to this Work

I. Ongey, E. L.; Giessmann, R.; Fons, M.; Rappsilber, J.; Adri an, L. and Neubauer, P.
( 2018) Heterologous biosynthes is, modi fications and structural charac terization of
ruminococcin-A, a lanthipeptide from a gut bac terium, in E. coli. Front Microbiol
9:1688. https://doi.org/10.3389/fmicb.2 018.01688
II. Ongey E. L., Pflugmach er S., Neubauer P. ( 2018 ) Bioinspired designs , molecular
premise and tools for evaluating ecological importance of antimicrobial peptides .
Pharmaceuticals , 11(3), 68; https://doi.org/10.3390/ph11030068
III. Ongey, E. L.; Yassi , H.; Pflugmacher, S.; Neubauer, P. ( 2017) Pharmacologi cal and
pharmacokinetic pro perties of lanthipeptides undergoin g clinical studies. Biotechno l Lett
39: 473. https://doi.org/10.1007/s 10529- 016 - 2279 -9

IV. Ongey, E. L.; Neubaue r, P. ( 2016 ) Lanthipeptides: chemical synthesis versus in vivo
biosynthesis as tools for pharmaceutical prod uction. Microb Cell Fac t 15 : 97.
https://doi .org/10.1186/s12934- 016 - 0502 -y

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

List Abbreviations

[ppm]

Parts per million

ABG

Active beam guide

Abu

2-aminobutyric ac id

AMP

Antimicrobial peptide

CAI

codon adaptation inde x

CAP

catabolite activator protei n

CID

Collision-in duced dissociation

Direct current

Dha

Dehydroalanine or d ehydrated serine

Dhb

Dehydrobutyrine or dehydra ted threonine

DOT

Dissolved oxygen tension

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraac etic acid

ESI

Electrospray ioniza tion

ETD

Electron transfer di ssociation

FGA

Fibrinogen alpha chain

FPP

Farnesyl diphosphat e

GRAS

Generally regarded as safe

GST

Glutathione S-tran sferase

HCD

Higher-energy collis ional dissociation

HPLC

High-performance liq uid chromatography

IMAC

Immobilized metal ion affi nity chro matography

inducer peptide

IPTG

Isopropyl β -D-1-thioga lactopyranoside

LAB

Lactic acid bacteria

Lan

Lanthionine ring/product of lan gene

lan

Notation for genes c onstituting the lanthipeptid e biosynthesis
cluster

LanB

General notation for c lass I lanthipeptide deh ydratase

LanC

General notation for c lass I lanthipeptide cyclase

LanKC

General notation for c lass III lanthipeptide synthetase

LanL

General notation for c lass IV lanthipeptide syntheta se

LanM

General notation for c lass II lanthip eptide synthetase

LanP

General notation for l anthipeptide activation prote ase

LanT

General notation for l anthipeptide transport protein

LAP

Linear azol(in)e -containing peptide

LTQ

Linear trap quadrupole

MALDI

Matrix-assisted lase r desorption/ionizati on

ManPTS

Mannose phosphotran sferase system

MBP

Maltose binding protein

MeLan

Methyllanthionine ring

MRSA

Methicillin -resistant Staphylococcus aure us

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

VII

Mass spectrometry

MS 2

Tandem MS

MTP

Microtiter plate

MUB

Membrane-anchored ubiquitin-fol d

N AG

N-acetylglucosamine

nLC

Nano-liquid chromatograp hy

ORF

Open reading frames

ori

Origin of replicati on

PDB

Protein data bank

preRumA

Ruminococcin -A precursor peptide

PTM

Posttranslational modificati ons

PVDF

Polyvinylidene fluori de

Quorum sensing

RBS

Ribosomal binding si te

Radio frequency

RFU

Relative fluorescent u nit

RiPPs

Ribosomally synthesiz ed and post -translationally modified
peptides

Room temperature

RumA

Ruminococcin - A

RumM

Ruminococcin -A lanthionine synthetase

SAM

S-adenosylmethionine

SDS -PAGE

Sodium dodecyl sulfate polyac rylamide gel el ectrophoresis

SRM

Selected Reaction Moni toring

SUMO

Small ubiquitin -like modifier

TEV

Tobacco etch virus

Trx

Thioredoxin

UniProt

Universal protein reso urce

WHO

World Health Organi zation

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

VIII

Table of Contents
Abstract .................................................................................................... I
Zusammenfass ung ................................................................................. II
List of Publications Contributing t o this Work .................................... V
List Abbreviations ................................................................................. VI
Table of Contents ................................................................................ VIII
1. Introduction ....................................................................................... 1
2. Literature Review .............................................................................. 4
2.1 The Bacteriocins ...................................................................................................................... 4
2.2 Classification of bacteriocins ................................................................................................... 5
2.3 The classification of lanthipepti des ......................................................................................... 7
2.3.1 Class I lanthipeptides ....................................................................................................... 9
2.3.2 Class II lanthipeptides .................................................................................................... 12
2.3.3 Class III lantipeptides ..................................................................................................... 15
2.3.4 Class IV lantipeptides ..................................................................................................... 17
2.4 Biotechnological production versus che mical synthesis of lanthipe ptides ........................... 19
2.5 Pharmacological properties and therapeutic use of lanthip eptides ..................................... 19
2.6 Recent advances in engineering an d heterologous production of lanthipeptides ............... 19
2.7 Native biosynthesis and regulation of lantibiotic ruminococcin -A ....................................... 21
2.8 Isolation of ruminococcin -A from R. gnavus E1 .................................................................... 23
2.9 Carriers for heterologous expressio n of AMPs ...................................................................... 24
2.10 Objective of this work ............................................................................................................ 24
3. Materials and Methods ................................................................... 27
3.1 Materials ................................................................................................................................ 27
3.2 Software ................................................................................................................................ 27
3.3 Bacterial strains, growth and cultivation conditions ............................................................. 27
3.3.1 Cultivation media ........................................................................................................... 27
3.3.2 Monitoring bacterial growth with a spe ctrophotometer .............................................. 28
3.3.3 Automated fluorescence measureme nts ...................................................................... 28
3.4 Vectors ................................................................................................................................... 28
3.5 Molecular Biology techniques ............................................................................................... 28
3.5.1 Isolation of R. gnavus E1 Genomic DNA isolation ......................................................... 29

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

IX
3.5.2 Preparations of plasmid DNA ........................................................................................ 29
3.5.3 Oligonucleotides design ................................................................................................ 29
3.5.4 PCR techniques .............................................................................................................. 29
3.5.4.1 Analytical PCR ............................................................................................................ 29
3.5.4.2 Preparative PCR ................................................................ ......................................... 29
3.5.4.3 Colony PCR ................................................................................................................. 30
3.5.4.4 Splicing by overlap extension (SOE) PCR ................................................................... 30
3.5.5 Purification of DNA fragments and spectrophotometri c quantitation ......................... 30
3.5.6 Agarose gel electrophoresis .......................................................................................... 31
3.5.7 Conventional cloning using restrictio n enzyme digestion and ligatio n ......................... 31
3.5.8 Modification of expression vectors ............................................................................... 32
3.5.9 Site ‐ directed mutagenesis ............................................................................................. 33
3.5.10 Construction of expression vectors ............................................................................... 33
3.5.10.1 Amplification of gene fragment fro m R. gnavus E1 ch romos ome ........................ 33
3.5.10.2 Construction of His6 -preRumA expression vectors ............................................... 33
3.5.10.3 His6-preRumA vectors wit h alternative cleavage sit e at position -1 ..................... 33
3.5.10.4 Construction of His6 -GFP-TEV-preRumA expression vectors ................................ 34
3.5.10.5 Construction of His6 -SUMO-preRumA expres sion vectors ................................... 34
3.5.10.6 Construction of His6 -RumM expression vectors ................................................... 34
3.5.10.7 Construction of bicistroni c expression vectors ..................................................... 34
3.5.10.8 Construction of RumT125 expression vectors ....................................................... 35
3.5.11 Transformation of competent E. coli cells ..................................................................... 35
3.6 Protein production and an alysis ............................................................................................ 35
3.6.1 Expression optimization i n EnPresso B growth system ................................................. 35
3.6.2 Protein Expression in TB m edium .................................................................................. 36
3.6.3 Strain screening using GFP fluorescence ....................................................................... 36
3.6.4 Cell disruption ................................................................................................................ 37
3.6.5 SDS-PAGE analysis ......................................................................................................... 38
3.6.6 Native PAGE analysis ................................................................ ..................................... 38
3.6.7 Western blot .................................................................................................................. 38
3.7 Protein purification and peptide ex traction .......................................................................... 39
3.7.1 IMAC .............................................................................................................................. 39
3.7.2 Size exclusion chromatography ..................................................................................... 40
3.7.3 TEV Cleavage ................................................................................................................. 40
3.7.4 Extraction of preRumA fro m TEV-digest ed product ...................................................... 40
3.8 Measurement of protein concentra tions using Bradfo rd assay ............................................ 41

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

3.9 Iodoacetamide derivatization and trypsin digestion ............................................................. 41
3.10 Mass spectrometric analyses ................................................................................................ 42
3.10.1 MS sample preparation wit h ZipTiP .............................................................................. 42
3.10.2 LC -ESI-MS analyses of preRumA .................................................................................... 42
3.10.3 Tandem mass spectromet ric analyses of preRumA ...................................................... 42
3.11 Biological Assay ..................................................................................................................... 43
3.12 Identification of optimal production conditions using online DO T & pH .............................. 43
3.13 Computational analyses ........................................................................................................ 44
4. Results and Discussion ................................................................ . 46
4.1 Sequence analysis of ruminoco ccin-A p rocessing enzymes .................................................. 46
4.1.1 Sequence similarity between Rum M and other lanthionine syn thetases .................... 46
4.1.2 Sequence and structural similarities bet ween RumT and other A MS proteins ............ 47
4.1.3 Codon adaptability calculations and analysis ................................................................ 48
4.2 Vector construction and E. coli expression ........................................................................... 51
4.2.1 Features of expression vectors ...................................................................................... 51
4.2.2 Separate expression of His6 -preRumA and His6- RumM ............................................... 53
4.2.3 Two-plasmid coexpression system for His6-preRumA and His6-RumM ....................... 54
4.2.4 MS identification of produ cts from the two -plasmid system ....................................... 56
4.2.5 PreRumA Fused to GFP .................................................................................................. 59
4.2.6 LC -ESI-MS analyses of preRumA from WLEO grA and WLEO grA/ M .............................. 62
4.3 Quality enhancement, characterization & activation of preRumA ....................................... 65
4.3.1 Optimized vector for His6- RumM express ion ............................................................... 65
4.3.2 Expression of RumT peptida se domain ......................................................................... 66
4.3.3 Plasmid-encoded bicistronic operon ............................................................................. 68
4.3.4 Purification of His6-GFP-TE V-preRumA* and T EV digestion ......................................... 69
4.3.5 Interactions between His6 -RumM and His6 -GFP-TEV-preRumA* ................................ 71
4.3.6 Extraction and nLC- ESI-MS analyses of p reRumA* ....................................................... 73
4.3.7 Mass Spectrometric Fragm entation and MS 2 Analysis of PreRumA* ........................... 77
4.3.8 Alkyl derivatization and trypsin digestio n of preRumA* ............................................... 83
4.4 Bioassay analysis of trypsin activate d RumA ......................................................................... 85
4.5 Microtiter plate cultivations .................................................................................................. 87
4.5.1 Colony screening in microtiter plate usi ng GFP as a reporter ....................................... 88
4.5.2 Cultivation of WLEO grA* M1 in 24-well plate using TB medium ................................... 89
4.6 Strain optimization using multicultiva tion & screening strategies ....................................... 90
4.6.1 Characteristics of the bicistro nic and two-vector syst ems ............................................ 90

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

XI
4.6.2 Optimization of WLEO grA *M1 using process -relevant parameters ............................. 93
4.7 Overall discussion of results .................................................................................................. 97
5. Conclusions and outlook ............................................................. 102
5.1 Conclusions .......................................................................................................................... 102
5.2 Outlook ................................................................................................ ................................ 103
6. Appendix ....................................................................................... 105
6.1 Structure-function analys es, description of the biosynthesis opero ns, classification and
physicochemical characteristics of rando mly selected bacteriocins .............................................. 105
6.1.1 Class I: Posttranslationally modified peptides (<10 kDa) ............................................ 110
6.1.1.1 Ia — The lanthipep tides ............................................................................................. 110
6.1.1.2 Ib — Circular b acteriocins .......................................................................................... 112
6.1.1.3 Ic — Linear az ol(in)e-containing pep tides (LAPs) ...................................................... 114
6.1.1.4 Id — Sactipept ides ..................................................................................................... 115
6.1.1.5 Ie — Glycocins ........................................................................................................... 117
6.1.1.6 If — Lasso peptides ................................................................ .................................... 118
6.1.2 Class II: Unmodified ba cteriocins (<10 kDa) ................................................................ 119
6.1.2.1 IIa — pediocin -like ..................................................................................................... 119
6.1.2.2 IIb — Two-p eptide bacteriocins ................................ ................................................. 121
6.1.2.3 IIc — Leaderless b acteriocins ..................................................................................... 122
6.1.2.4 IId — Non -p ediocin-like single-peptide bacteriocins ................................................. 124
6.1.3 Class III: Unmodified high molecular weight bacteriocins .......................................... 125
6.2 Vector descriptions and vector ma ps .................................................................................. 126
6.3 Structure prediction and sequen ce similarity ..................................................................... 129
6.4 Codon-optimized gen e sequence of the peptidase -encoding domain of R umT (UniProt:
Q93JP5) ............................................................................................................................................ 132
6.5 MS 2 assignments of His6 -preRumA fragments .................................................................... 132
6.6 MS 2 characterization of preRumA* ..................................................................................... 134
6.7 Multi-parallel cultivations .................................................................................................... 137
6.8 The concept of mass spectro metry (MS) ............................................................................ 139
6.8.1 Mass spectrometry and peptide sequ encing .............................................................. 139
6.8.2 Electrospray ionization ................................................................................................ 140
6.8.3 The Orbitrap Fusion Tribrid mass spectrometer ......................................................... 141
6. 8.4 The quadrupole mass filter ................................................................ .......................... 143
6.8.5 Ion trap mass analyzer ................................................................................................ . 143
6.8.6 Tandem mass spectromet ry (MS 2 ) .............................................................................. 144
6.9 Tables and Figures for mat erials and methods section ....................................................... 146

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

XII

6.9.1 Instrumentation used .................................................................................................. 147
6.9.2 Expendables ................................................................................................................. 149
6.9.3 Software ...................................................................................................................... 149
6.10 Purification of TEV ............................................................................................................... 156
References .......................................................................................... 157
Acknowledgements ............................................................................ 179
Short Resume ..................................................................................... 182

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

1
1. Introduction
Economic sensi bilities and healthcare awarenes s are two major concerns in the world tod ay.
Clinical problems that range from minor nosocomial infections to substantially critical diseases
such as sepsis, in vasive endoc arditis , bone and so ft tissue intra -surgical infections are
examples of complica tions caused by staphylococci and enteroco cci forms of bacteria in
hospital settings. Methicillin-resistant Staphylococ cus aureus (MRSA) and vancomycin-
resistant enterococci are amongst the most disreputable forms of infec tious microorganisms .
According to World Health Organi zation’s report in 2014 , without potent anti -infec tive therapy,
a host of standard med ical procedures will fail or become co mpromised, resultin g in very
complex and high risk situations (WHO, 2014). The development of novel potent antimicrobial
compounds therefo re remains an ensuing challenge for humankind . The situations are
becoming more unp leasant as many of the available anti-in fective agents are persistently
losing their effect iveness against these pathogenic microbes. Some scientists have briefly
described the present condi tion as a “new pre - antibiotic era” (Rios et al., 2016), whic h is
perceived to be largely facil itated by the excessive use of conventional antibiotics and the huge
growth of b acterial resistances. This has consequently stimulated considera ble interests in
developing and applying natural compounds especially antimicrobial peptides (AMPs) as the
next generation d rug molecules (Zhang and Gallo, 2016 ).
The hist orical background of antib iotic research and the evolution of bacteri al resistanc es
highlight the nec essity to evolve with novel strategies to control pathogenic bacteria . Key
events record ed dur ing the past years to bring us to the econ omic reali ties we face tod ay and
the need for new antibiotics in medic ine have been analyzed elsewhere (Kirst, 201 3).
Interestingly, Kirs t hol ds the opinion that using non -lethal means to control bac teria may be the
ultimate antidote to the antibiotics dilemma. Zhang and Gallo on the other hand bel ieve tha t
controlling endogenous expressi on of AMPs may be an ideal approach for treating a wide
range of dise ases in humans and ani mals (Zhang and Gallo, 201 6). Either way, bo th
approaches require huge efforts to come to fruiti on. Attempt s to explore unculture d mic robes,
screening diverse resources and targets have proven that the bacteriocins family of A MPs may
be used as alternatives to conventional antibiotics bas ed on their remarkabl e potencies against
drug-resistant bacteri a (Cotter et al., 2013 ).
Nevertheless, a cr itical unders tanding of how target macro molecular structures in the cell are
recognized by a pep tide and how they govern the microbial killing process is nec essary to
facilitate the design of peptides with superior propertie s that can hamper the development of
resistances which perhaps may grossly restrict their use in medicine (Cotter et al., 2013). We
recently summarized molecular targets of some AMPs, identifying their evolutional

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

characteristics and indicating how the macromolec ules can be useful in des igning target -
specific peptides (Ongey et al., 2018 ) . T he desired application of a compound may pro vide
basis for rational design and production of AMP s with higher tenacity strong enough to esc ape
specific resistance machineries. In this regard, gene -encoded peptide eng ineering is one
advantage that nat ural peptides have over classical antibiotics. Although theoretic al
investigation of the pha rmacodynam ics of AMPs and conventional antibiotic with respect to
their abili ty to cause res istance evolutio n indicates less likelihood for AMPs (Yu et al ., 2017 ) ,
extensive investi gations are de sired t o obtain more in sights into the mechanisms of interaction
between complex fu nctional components involved in biosynthe sis, translocation and se lf-
immunization in order to prevent these si tuations from occurring . Furthermore, understan ding
the concept of biosynthesis , and macromolecular targets and their responses to AMPs’
interaction may create awareness of how to app ly bioengineering to rational ly tune bioactive
peptides to enhance propertie s like bi ostability , potency and sp ecificity as reported in a few
examples of class I bacteriocins, also ca lled lanthipeptides (Field et al., 2015 , Field et al.,
2015 ) .
Pharmacological studies including but not limited to mode of administratio n, dosing regimen,
half -life, distribution and cl earance rates, minimal inhibi tory conce ntration (MIC) and potentia l
drug -serum protein interactions are important to asse ss therapeutic relevance of b ioactive
peptides. This is partly because in many ca ses their intrinsic pro perties do not favour direct
use in medic ine as we recently discussed in a review of selected examples of lanthipeptides
(Ongey et al., 2017) . However, these studies require a good amount of the substance under
investigation which appear to be challenging to say the least. The absenc e of well -
characterized strate gies for produc ing these pep tides hampers the implementation of diverse
experimental protoc ols to evalu ate the m. We recently concluded from studying the literatu re
that ch emical synthesis is not a viable tool to achi eve econo mically fea sible production of
ribosomal ly synthes ized peptides with complex modifications, but emphasiz ed on biological
system as a realistic source to construct a production process (Ongey a nd Neubauer, 2016 ).
Nevertheless, yields obtained from natural isolates are low, and most native producers req uire
prolonged p eri ods of cultivation, cou pl ed with the high cost of cultivation strategies and growth
media supplements . The i nability to resolve these challenges have en couraged the use of
heterologous productio n hosts as the only realistic alternative .
A good example of a lanhthipeptide tha t poses signi ficant expression challenges is
ruminococcin-A naturally produced by the gut bacterium Ruminococcus gnavus E1. The
organism, besid es being an obligate anaerobe, large-scale produc tion and peptid e engineering
possibilities of the peptide may be limited by the relatively large size (12.8 kb) of the

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

3
rum inococcin -A biosynthesis gen e cluster (Gomez et al., 2002) . As reported for the cl ass II
lanthipeptides nuk acin ISK - 1 (Nagao et al., 2005) and lichenicidi n (Caetano et al., 2011b), and
many other lanthipeptides (Shi et al ., 2011), ac tive expres sion in E. coli does not require all the
genes pre sent in the biosynthes is clus ter. In the prese nt study, we utili zed dual -vector and
single-vector approaches to engineer the pat hway for the bios ynthesis and modifi cations of
ruminococcin-A in E. co li. The genes encoding the lanth ionine synthetase (RumM) and the
structural peptide (preRumA) were amplified from the genome of R. gnavus E1 and
coexpressed simultaneously in E. coli . We show that biosynthesis and modification of the
peptide can be successfully achieved by expressing the structural peptide as a fusion partner
to GFP. Further investigations in conju nction with the formati on of complexes between the
modifying enzyme and t he chimeric fusion product su pplied some interestin g insights into the
catalytic mechanisms of class II lanthionine -generating enzymes. We also ident ified a si mple
strategy to isolate the product and the yield overwhelmingly excee ded that attainable in the
natural producer by se veral thousand -fold. The structure of modified preRumA was fully
characterized, showing interesting new features . The systems developed here provide useful
tools for optimizing productio n of the peptide, as well as performing in vivo peptide engineering
to improve its physicoc hemical and pharmacolo gical features.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

2. Literature Review
2.1 The Bacteriocins
Bacteriocin s are ribos omally synthesiz ed proteinaceou s toxins se creted by bac teria and
archaea to prevent the growth of other microorganisms competing with them in their ecological
niches, and they have pote ntial clinical, aquaculture, livestock and food app licatio ns (Bali et
al., 2016 ) . They are produc ed predominantly by Gram-pos itive bacteri a including gut bacteria
(Hegarty et al., 2016) and especially lactic a ci d bacteria (LAB) (Cascales et al., 2007, Alvarez-
Sieiro et al ., 2016 ). Several conventional methods like the ammonium sulf ate precipi tation, pH -
mediated cell adsorption/desorption , me mbrane fil tration and a se ries of different
chromatographic techniques are currently appli ed to purify bacteriocins from complex cultu re
broth (Jamalud din et al ., 2017 ). However, app lying these technique s on the n atural host to
supply a sustainable production process is not trivial since m ost natura l produc er organisms
secrete the compounds in very minute quantities believed to play a role in signalin g and
repelling other microbes in the ir ecological environment (Chi kindas et al., 2018 ).
Although data on expressi on levels in their ecological niches are rare in the literature , studies
reveal that when bacterioc in s are applied at conce ntrations higher than those in the natura l
habitat of the host microorganisms, several different functions are observed amongs t which
the microbial inhibitory activities of the compound have been well characterized (Drider et al.,
2016 ). Bact eriocins usually inac ti vate their targets via me mbrane disruption or by forming
pores (Etayas h et al., 2016), or even terminate cell division by targeting and dissociating lipid
II (Hasper et al., 200 6) which serves as spec ific anchor molecul e for a host o f class I
bacteriocins including nisin, epidermin and gallidermin . Bacteriocins have hi gh potencies
against multi – drug-resis tant pathog ens, pos sess rel atively neg ligible toxi city to host cell s and
demonstrate significant stabilities under physicochemical conditions (de Oliveira Junior et al.,
2015 ). These characteristi cs position them on hi gh demand as alternative anti -infec tive age nts
in food processin g and as therap eutic drugs, which may be applied as narrow or bro ad spectra
anti microbials. Furthermore, the y are se creted nat urally by gut microbes and LAB frequently
found in many comme rcially useful products, and generally reg arded as safe (GRAS) for
hu man consumption (Nes et al., 2007 ). Additio nally, the fun ctions of bacteri ocins exceed
beyond the margin of ant imicrobial agents. For example , subtilosin A produced by Bac illus
subtilis show lethal activities against viruses (Quintana et al., 201 4) an d sperms (Sutyak et al.,
2008 ) , and others may be used in the tre atment of cancers (Kaur and Kaur, 2015 ) .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

5
2.2 Classification of bacte riocins
The growing number of bacteriocins isolated from different bac teria sources makes it a
challenging tas k to perfectly divide them into separate cl asses whic h explains why a pleth ora
of controversies exist about their classification. For example, carnobacterioc in X was recently
studied and from its structural information, it was rec lassified from class IId to class IIb (Tulini
et al., 2014 , Acedo et al., 2017) . Nevertheless, gen ome mining tools with a database like
Bagel3 (van Heel et al ., 201 3) and BACTIBASE ( Hammami e t al ., 201 0); and genome mining
tools like antiSMASH 2.0 (Blin et al., 2013) have been created to enable information collection
and to automatically screen for gene clusters that encode ribosomall y synth esized and post -
translationally modif ied peptide s (RiPPs). Other genome mining software that are commonl y
used to identify unique bios ynthetic machineries include BLAST, Clus terFinder and
RiPPPRISM (Hetrick and van der Donk, 20 17) . An empirical c omparison of these to ols show s
that Bagel3 is han dier and provides more insi ghtful i nformation (Gabere and Noble, 2017). For
example, an ana lysis of complete genome dat a extracted from public databases of 238 LAB
resulted in 785 gene clusters putatively belonging to bacteriocins (Alvarez-Sieiro et al., 2016 ).
Analyses of this nature ha ve greatl y facilitated the identification of valuable peptides with
enhanced functio ns and features that may ease t heir groupings into d ifferent classes .
Klaenhammer propos ed the fir st classificatio n of bacterioc ins which grouped them i nto four
distinct classes based on biochemical and genetic information exis ting at the time that made it
possible to predict structural features and mechanisms of action (Klaenhammer, 1993 ). Man y
other classific ations ensued taking into considerations criteria such as molecular sizes,
physical properties and chemical s tructures (Nes et al., 19 96 , Kemperman et al., 2003 , Cotter
et al., 2005 ). Cotter and colleagues revised previous classific ations (tha t es sentially
distinguished bac teriocins into five clas ses) and postulated a classi fication scheme that divided
them into two main categor ies namely; clas s I (cons titutin g the lanthionine -containing
lantibiotics) and cl ass II (the non -lanthioni ne-containing bacteriocin s), while the large murein
hydrolases were separately groupe d under bacteriolysins (Cotter et al., 2005). In an atte mpt
to solve the probl em of contra dictory classific ation that allows one bacteriocin to belong to
multiple classes, Zouhir and colleagues uti lized a host of bioin formatics tools to develop a
sequence -based fin gerprints tha t ena bled more tha n 70% known bacteriocin s to be divided
into 12 groups (Zouhir et al., 2010). However, bacteri ocin sequences with large evolutionary
distances would be difficult to fit in this classification because the s equence divergence wou ld
be hug e. This therefore pose s a significant limitation to the sequence homology -based
classification and as such, it may be di fficult to directly assign uncharacterized bacteriocins
under a particular gro up.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 1.1 Classification of bacteriocins. Examples depicted in light-green are from non -LAB sources.
Non-lytic class III members are yet to be structurally characterized. LAPs stands for Linea r azol(in)e -
containing peptides

Due to increasin g complexity of newl y identified co mpounds, man y oth er classi fications have
subsequently bee n propos ed bas ed on biosynthesis route and ac tivities of the pep tide (Arni son
et al., 2013 , Cotter et al., 2013). In 2013 Cotter and coworkers sugge sted a clas sification
scheme tha t depend ed on whether the peptide is modified (class I) or unmodified (class II),
and emphasize d the necessity to eliminate ribosomally synth esized antimicrobial proteins from
the list of bacteriocins. Thi s proposal was slightly m odified to include the bacteriolysin by

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

7
designating a third cl ass (class III) to ac commodate unmodified bacterioc ins (molecular
weights >10 kDa) that elicit a bacteriolytic or non -lytic action (Alvarez-Sieiro et al ., 2016).
The classification scheme displayed in Figure 1 .1 was suggested as an effective classification
model for LAB bac teriocins, but it may al so be expanded to include bacteriocins from other
organisms as indicated by the exa mples of sactibiotics and lasso peptides which are produced
by the non-LAB Bacillus su btilis 168 and Strepto myces cattleya , respectively. It was also
suggested that a more general definitio n for bacteri ocins may be considered as follo ws;
“proteinaceous substan ces produced at ribosomal le vel, having multifunc tional properties
whose antimicrobia l activities are concent ration- dependent” (Chikindas et al., 2018 ). This
largely el iminate the general consideration that they are AMPs, amphip athic in nature,
possessing an overall positive ch arge and since ani onic bacteri ocins such as subtilosin A do
exist ( Mathur et al., 2015). Structural description of the biosynthesis operons of the v arious
classes of bacteri ocins, the physicochemical characteristics of randomly selected examples,
and brief summaries of the main f eatures (including biosynthesis and kill ing mechanisms) are
described in Appendix 6 .1.
2.3 The classification of la nthipeptides
Lanthipeptides are RiPPs comprising of non -proteinog enic amino ac ids and thi oether cross -
linkages formed between dehydrated side cha ins of threonine/serine and the sulfhydryl group
of cysteine. The maturation of lanthipeptides involve a distinct group of enzymes that catalyze
three uni que reactio ns namely; deh ydration, cyclis ation and proteolysis. Other customized
enzymes may catal yze the introduction of additional PTMs which are necessary to ensure their
activities and/or stability (Repka et al ., 2017). The dehydrati on reaction usually occurs via
gl utamyl-tRNA- dependent glutamylation , fol lowed by eliminati on (Garg et al., 201 3 , Ortega et
al., 201 5 , Repka et al., 201 7). Michael addition cyclization reactions involving cysteine and
dehydroamino acids may in troduce a lanth ionine (Lan) ring in the case of deh ydrated serine or
dehydroalanine (Dha), and methyll ant hionine (MeLan) rin g when dehydrated threonine or
dehydrobutyrine (Dhb) is involved (Schnell et al., 1988 , Arnison et al., 2013 , Yang and van der
Donk, 2015 , Repka et al., 2017). Their polycyclic structure is made up of the MeLan /Lan rings
from which t hey derive their name. Addi tional features such as Dhb, Dha , N-termin al pyruvate ,
S-aminovinyl -cysteine and D-amino acid s may a lso be pre sent in the molecul e (Freund et al.,
1991 , Knerr and van der Donk, 2012 , Lohans et al., 2014 , Ortega et al., 2014 ). For decades,
they were globally refe rred to as lantibiotics because most m embers pos sessed antibacterial
activities (Schnell et al., 1988 ), but thi s has ch anged over the years as many new members

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

show diverse functions by produc ing different categories of biological effects (Knerr and van
der Donk, 2012 ) .

R u m C l u s t e r 2
1 2 8 0 2 b p

La b y ri n t h o p e p t i n C l u s t e r
6 4 0 0 b p

V e n e z u e l i n C l u s te r
5 3 3 9 b p

Figure 2.1 Sum mary of the major features in the lanthipe ptides biosynthesis pathway. (a) Gene
clusters (drawn to scale) representing ex amples of each class, showing highly conserved motifs
and/or residues (indicated by co lour and ver tical lines on designated genes) in the lanthionine
synthetase encoding genes. The precursor peptides encoding genes is lanA is cloured red. (b )
Putative and characterized domains of lanthionine-generating enzym es : Class I (LanB, LanC), I I
(LanM), III (LanKC) and IV (LanL) . (c) Ca talytic core structure of NisC edited using X-ray
crystallographic data for NisC (PDB: 2G02) and Protein Workshop. The Zn 2+ cofactor is sh own to
coordinates residues Cys284, Cys330 a nd His33 1 in the acti ve site. The resid ues His 212 and Tyr285
which are much closer t o the Zn 2+ catalytic center are also shown.

The posttranslati onal modifications (PTMs) in lantipeptid es make them more res istant to
protease degradation (Rink et al., 2010 ) and accord them li mited conformational freedom
which confers target specificity (Goto et al., 2011). On the basis of their structure, they disp lay

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

9
rigid conformational flexibiliti es that render high bi ological ac tivities with very low minimal
inhibitory concentrati ons (MICs) against wide vari ety of infections ranging from antimicrobial to
antiallodynic effects (Repka et al., 2017). For these reasons they are seriousl y considered as
future alternative clinical agents against bacterial virulence (Dischinger et al., 2014 ).
The biosynthes is mach inery of lanth ipeptides i s regulated by a large cluster of genes pre sent
in the lan operon (“ lan ” is a genera l notation given to genes that are host ed in the lanthipeptide
biosynthesis cluster and their corresponding pro ducts are referred to as “Lan”). Note that “Lan”
may be used contextuall y to refer to the lanthionine ring (as mentioned above) or to name
proteins expressed by the lan operon (e.g. Lan M is a lanthionine synthetase encode d by lanM ).
Based on the structural arra ngement of the maturatio n enzymes on the lan operon, this class
of bacteriocins may be further grouped into four distin ct classes namely; class I where a
dehydratase (LanB) and a cycl ase (LanC) are involved in the ir maturat ion; class II modified by
a si ngle bifunctional enzyme Lan M (which performs both the dehy dra tase and the cycl ase
roles); cl ass III process ed by the LanKC enz yme; and class IV process ed by LanL (Knerr and
van der Donk, 2012 , Repka et al., 2017) . The arrangement of genes encoding the lanthionine -
generating enz ymes and oth er accessory proteins in the biosynthetic operon is unique for
different groups of these bioactive molecules (Figure 2.1a). The various domains of the
modifying e nzymes that dist inguish the four classes of lanthipeptid es are displayed in Fi gure
2.1b.
2.3.1 Class I lanthipeptides
The prototype member of lanthipeptides nisin bel ongs to class I where two enz ymes (LanB
dehydratase and LanC cyclase) are involved in their Lan/MeLan rings formation. LanB protei ns
usually have sizes around 1,000 amino aci d residues and altho ugh they catalyze the same
reaction, they sh ow on ly limited (≈ 30 %) seq uence identity across the family and share
negligible homology with other known proteins (Knerr and van der Donk, 2012) . However, the y
particularly share highl y cons erved struc tures in most of their biosynthes is genes ( Götz et al.,
2014 ) . Prev ious report s indicate that reactio ns catal yzed by LanB enzymes may involve
phosphorylation of se rine/threonine res idues (Chatterjee et al., 2005 , Repka et al., 201 7) .
Previous attempts to rec onsti tute LanB enzyme in vitro failed due to lack o f evidence that
supported dehydration of the peptide su bstrate (Xie et al., 2002 , Mavaro et al., 201 1) .
Nevertheless, very recent ly Garg and co workers repor te d that some bacterial cellular
components might be involved in the dehydration process since incubating the peptide
substrate with, ATP, Mg 2+ and E. coli ce ll extract supplemented with glutamate and spermidine
resulted in the desired bioac tivity of nisin (Garg e t al., 2013) .

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 2.2 Maturation of nisin and other class I lanthipeptides. (a) Suggested mechanism for NisC -
catalyzed reactions, illustrating the formation of the first N -terminal thioether cross-bridge of nisin. The
scheme begins with a water molecule being displaced from Zn 2+ ion by the thiol group of Cys7 residue
targeted for conjugation with Dha3. Furth er deprotonation of this group is believed to be achieved by
an active site base or water (Li et al., 2006) . The β -carbon of Dha then launches an attack on the
nucleophilic thiolate to produce an enolate intermediate which undergoes further protonation to
generate a D- configuration at the α -carbon. (b) Representative examples of class I lanthipeptide
structures, highlighting the canonical nisin-lipid II – binding motif and additional PTMs. (c) Proposed
model for nisin biosynthesis and secretion indicating how the ribosomally synthesized precursor
peptide is channeled to the lanthionine synthetase Nis B/NisT/NisC co mplex where the leader
sequence is recognized and bound. Nis in precursor peptide alternate (indicated by a mark) between
the active site of Nis B and NisC until all the five rings are formed. Fully modified nisin is then exported
via NisT and subsequent act ivation by the dedi ca ted mem brane -anchored NisP.

Nisin cyclase NisC of the LanC protein family shares about 2 0 – 30 % overall sequence identity
across the family (Knerr and van der Donk, 2012). The in vitro reconstitution of NisC and its X-
ray crystal structure ha ve been reported (Li et al., 2006 ). The se achievements uncovered
interesting insights on lanthi oni ne cyclase catal ytic mechani sm. Fi gure 2. 1c clearly show s how
the catalytic core of NisC coordinates Zn 2+ ion via a Cys284-Cys330-Hi s331 catalytic tr iad ( Li
et al., 2 006 ). The proposed catalytic mech anism for NisC (Figure 2.2a) il lustrates that the triad
activates the thiol group of cysteine t o enable in tramolecular nucle ophilic atta ck by Dha or Dhb
resulting from the dehydratase reactions (Li et al., 2006 ). The C-termin al residues Ala404,

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

11
Leu405, and Leu406 are most likely involved in stabilizing the catalytic core (Helfrich et al.,
2007 ). Rec ent st udies on NisC and the cl ass II lanthipeptide synth etase HalM2 showed that
the Michael -type addition reaction that produces the Lan/MeLan rings is reversible and that
these enzymes can actuall y ope n up all thioether rings in their produc ts (Yang and van der
Donk, 2015) . LanC cyclases are usually found in Bacteroidetes, Proteobacteria , Actinobacteria
and Firmicutes ; mean while se quences of LanB dehydratases from Bactero idetes and
Proteobacteria show anc estral links to Firmicutes (Zhang et al., 201 2). The re is no direct
correlation in the phylo genetic distribution of Lan Bs and LanCs, al though enzymes present in
the sa me bios ynthesis operon generally have si milar evolutionary origin (Repk a et al., 2017 ).
Examples of clas s I lanthipeptides other th an nisin ar e shown in Figure 2.2b.
Other protei ns in the class I clust er like LanT serves to export the modif ied peptid e while the
membrane-anchor ed leader peptide cleavage protein LanP remo ves the leade r peptid e and
consequently ac tivates the peptide (Chatt erjee et al., 2005 , Xu et al ., 2014 , Repk a et al., 2017 ) .
In the case of nisi n, the functions of NisT and NisP do not depend on one another as in other
classes (Kuipers et al., 2004 ). There exists evidence of steric hindrance in the active site of
NisP (Repka et al., 2017) indicati ng possible reasons why the protea se prefers modified NisA
(Kuipers et al., 2004 , Lagedroste et al., 2017). Nevertheless, a soluble variant of NisP
displayed subst rate promiscuity and add itional fin dings indica ted that the presenc e of
Lan/MeLan ring in the subs trate as well as some spec ifi c residues nearer to the cleavage site
are necessary for specific c leavage to occur (Montalbán -López et al., 2018 ).
NisA, NisB, NisC and NisT do interact to f orm a complex during biosynthes is of nisi n as
illustrated in Fi gure 2.2c, altho ugh their indi vidual activities are conserved even in the absence
of eac h other (Bakke s et al., 2008 , Lubel ski et al., 2008). Addditionally, dysfunctional Lan C
may also greatly affe cts active LanA synthesis as observed for nisin where the absence of
NisC resulted in increased levels of dehydrated NisA ( Lubelski et al ., 2009) . Little is known
about the order in which the reactions proceed and how much input is achieved by each
enzyme.
Although the deh ydratase reactions are indep endent of each oth er, mutagenesis studies on
NisA have r evealed that conjugation of the cysteinyl thiol to Dha/Dhb in the cyclization step
can prevent further dehydrati on of serine and threonine res idues (Kuipers et al., 200 8 , Lubelski
et al., 2009 ). This may explain why partially modified pro ducts have been observed in some
recombinant la nthipeptides expressi on systems . Biophysical characterization of purified NisA
and NisB indicated that the leader peptide creates a docki ng motif that allows NisB to bind and
interact with NisA ( Mavaro et al., 201 1). Furthermore, t he interactio n between dehydrated Nis A
and NisB is more strong er than either the unmodi fied or mature peptide (Mavaro et al., 2011 ) .
These results corroborated findings of Khus ainov and coworke rs where they showed that NisB

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

and Nis C were co-purified from L. lactis tog ether with C-terminal hexahistidine -tagged NisA
(Khusainov et al ., 201 1) . Since deleting the co re peptid e resulted in no co -purifica tion of Nis B
and NisC, thi s implies that coope rative interactions involving bot h the core peptide and the
leader seque nce exist between the modifying enzymes and Nis A . Thus, a model descr ibing a
unidirectional N→C terminal modification of NisA has been proposed (Lubelski et al., 2009 ) ,
wherein the cooperative a ction of NisB, NisC and Nis T prot eins are h ighlighted (Figure 2.2c ).
2.3.2 Class II lanthipeptides
Lacticin 481 and nukacin ISK -1 are typical examples of class II la nthipeptides whose PTMs
are installed by LanM protein which is a bifunctional lanthionine synthetase. The size range of
LanM is usually between 900 and 1,200 amino acid residues, possessing an N-terminal
dehydratase domain an d a C-terminal cyclase domain (K nerr and van der Donk, 2 012 ) .
Although the dehydratase domain catalyzes the same reaction as does LanB enzymes, bot h
proteins share no sequence homology. However, their C-terminal regions contains the
conserved zi nc- binding residues and share > 25 % homology to LanC proteins bu t negligible
similarities with each other (Knerr and van der Donk, 2012 ). Removal of the conserved zinc-
binding residues proportionately eliminated t he cross-linki ng abili ty of lacticin 481 synthetase
(LctM) (Paul et al., 2007 ).
In vitro and/or in vivo characteriza tion of LctM (Xie et al., 2004), bovic in HJ50 synth etase BovM
(Ma et al., 2014) and nukacin ISK-1 synthetase NukM (Shimafu ji et al., 2015 ) reveale d exciting
insights about how LanM enz ymes genera te the Lan/MeLa n rings. Whereas they essenti ally
lack the characteri stic ATP - binding motifs, their catalysis requires the hydrolysis of ATP to
supply phosphate for phosp horylation (Bierba um and Sahl, 2009 ). They also use ADP and
Mg 2+ as cofactor in the elimination step involving the pho sphate esters of serin e/threonine
residues in the core peptide to yield Dha/Dhb (Nagao et al., 2006). Furthermore, res idues that
are critical for ca talyzing phosphorylation are more inclined to those of typical se rine/threonine
kinase s (You and Van Der Donk , 2007). Tandem mass spectrometr y with a mutant whose
elimination properties i s removed re vealed that phosphor ylation occurs in a unidi rectional N -
to C-termina l ca talytic mode ( Le e et al., 2009). The order of modification is greatly influenced
by the leader peptide which add itionally improves catal ytic efficiency and product quality
(Thibodeaux et al., 2016 ) .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

Figure 2 .3 Representative ex amples of class II lanthipeptide structures, highlighting the canonical
mersacidin-lipid II – binding motifs, additional PTMs and the Dhx-Dhx-Xxx-Xxx-Cys motif (where Dhx
= Dha or Dhb).

The N-terminal A-ring of class II lanthipeptid es like geobacillin II, cytolysin, haloduracin, and
carnolysin share a Dhx -Dhx-Xxx- Xxx-Cys motif as illustrated for haloduracin β in Figure 2.3
(Garg et al., 2012 ). Geobacillin II was isolated from the thermophilic gram -positive bacterium
Geobacillus thermodenitrific ans NG80- 2 (Garg et al., 2012 ). Geobacillin synthetase GeoM is
thermostable and able to ca talyze PTMs formati on in geobacil lin II precursor peptide GeoAII
at temperatures ranging between 3 7 and 80 °C (Garg et al., 2 016).
The LanM enzymes are also known to exhibit wide ca talytic promiscuity as observed in the
case of ProcM and its ProcA pep tide substrat es (Li et al., 2010 , Tang and van der Don k, 2012 ,
Zhang et al., 2014 ) . However, this may al so cons titute a cost on the catalytic effi ciencies of
the enz ymes as a result of the burden exerted by the heterogeneou s Lan/MeLan ring
architectures required in the different sub strates as report indicates (Thibodeaux et al., 2014 ).
Although the mechanisms involved in th is promiscuou s catalysis remain to be estab lished, it is
suggested th at the peptide se quence, and not the modifying enz yme, determines both the
directionality of modif ication of nascent peptide and the final conformatio nal arc hitecture of the
product (Yang and van der Donk, 2 013 ). This may explain why it is possible for the enzyme to
catalyze the modif ications of several unrelated peptide s . Nevertheless, recent findings indicate
that the peptide sequence alone does not determine the stereochemical outcome s of
Lan/MeLan rin gs since it was revealed that the N-terminal A-rin g in geobacillin II has LL-
configuration while the rest of the rings possess the normal DL -configuratio ns (Garg et al.,
2016 ). Thi s was the very first report of a si ngle-compone nt la nthipeptide posse ssing the LL-
stereochemistry.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Class II lanthipeptides are exported from the cell by integral membrane proteins that belong to
the ATP- binding cassette (ABC) -transporter maturation and secretion (A MS) famil y of proteins
generally referred to as class II LanT s. This class of Lan T enzymes perform a dual functi on in
the biosynthesis of class II lanthipeptides ; (i) they cleave off the N -terminal le ader sequence s
of the lanthipepide precursor and (ii) transport the active peptide to the extracellular space
where they are required. The N -terminal domain of class II Lan T proteins possess residues
that are typical for papain-like cysteine proteases (Nishie et al., 2011 ). This domain acts as a
peptidase to cleave off the leader peptide at a conserved Gly -Gly mot if located at the C-
terminus of the leader s equence preceding the c ore structure (Chatterj ee et al., 2005 ) .
The protease domain s of other bifunctional transporters for non -lanthipeptid e bacterioc ins
have been characterized (Havarstein et al., 1995 , Wu and Tai, 2004 , Ishii et al ., 2006 ). N-
terminal 1- 15 0 amino a cid residues of lacticin 481 transporter LctT was the first class II
protease to be characterized. It was shown that this domain indiscriminately cleaves modif ied
and unmodified LctA (Furgerson Ihnken et al., 2008), indicating that the ABC transporter
domain does not affect the protease activity. However, nukacin ISK -1 transporter NukT
requires the cooperative action of both pep tidase and ABC transporter domains (Nishie et al.,
2011 ), and its protease d omain is located in the cytoplasmic side of the li pid bilayer membrane
(Nishie et al., 2009 ). Contrary to the protease domain of NukT, oth er lanthipeptides like the
class I prototype nisin is proce ssed pos t -excretory b y a me mbrane anc hored protea se NisP
(Lubelski et al ., 200 8). Interes tingly, the gene cluster of the class II two -co mponent lantibiotic
lichenicidin produc ed by Bacillus licheniformis cons titutes an add itional pro tease LicP that
further activates the β - component Bliβ’, produced by lich enicidin transpor ter LicT in a
processing event that initially cleaves off the leader peptide of Bliβ at the Gly -Gly site (Caetano
et al., 2011 ).
The non -lanthipeptide bacteri ocin-associated ABC transporter of Streptococcus Co mA
peptidase domain in duce the formation of an amphiphilic α -helic al structure in its su bstrate
ComC upo n binding to it. Moreover, mutagenesis studies revealed conserved hydroph obic
residues in the leade r peptide of ComC that faci litate this interactio n (Kotake et al., 2008 ) .
Existing theories su ggest that substrate recogn ition by LanT proteins may also occu r via the
amphipathic α -helic al conformation in the precursor pep tide (Furg erson Ihnken et al., 200 8 ,
Nagao et al., 2009 ).

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

15
2.3.3 Class III lantipeptides
Class III lantipeptides are synthesized by the RamC/LabKC family of lanth ionine/labionin
synthetases co nsisting of three ca talytic domains — an N-ter minal phosphoserine/
phosphothreonine lyase domain which has sequence homology with members of the outer
surface protein F (OspF) famil y of ef fec tor proteins [OspF catal yzes the remo val of the
phosphate gro up from phosp hothreonine (Li et al., 2007 )], a central Ser/Thr kinase domain,
and a C-terminal lanthionine cyclase domain (Goto et al., 2011) . The first member of this class ,
SapB, was isolate d slight ly over a decade ago from Strept omyces co elicolor and shown t o
possess structural characteristics of regular lantibiotics . It exhibite d no antimicrobial activities ,
but instead enhance d sporulation in stre ptomycete (Kodani et al., 2004 ) .
The C-terminal domain of the SpaB syntheta se (RamC) believed to be involved in the synthesis
of the Lan /MeLan rings showed very littl e sequence homol og y to the cyclase domai n of LanM
and lacks the conserved res idues that coordinate the Zn 2+ ion in the catal ytic core of a
lanthionine cyclase (Willey and van der Donk , 2007 ). Mutagenetic investigations of the N -
terminal lyas e domai n displ ayed structure -functional similarities to the OspF proteins (Goto et
al., 2010 ). The catalytic pathway that employs the kinas e -like domai n to produc e Dha/Dhb
follows a two- step pro cess involving phosphorylation and β -elimination (Goto et al ., 2011) .
Other lantipeptid es with simila r characteristics as SapB are SapT isolated from S. tendae
(Willey and Gaskell, 2010 ), labyrint hopeptins from Actino madura na mibiensis (Meindl et al.,
2010 ), catenulipept in from Catenul ispora acidi phila (Wang and Van Der Donk , 201 2) ,
erythreapeptin, avermipeptin and griseopeptin isolated from Saccharopolys pora erythraea ,
Streptomyces avermitilis and Strepto myces grise us respectively (Völler et al., 2012) .
Structures of selected examples are shown in Figure 2.4 .
The disc overy of labyrintho peptins enabled mechanist ic studies on the synthetic pathway of
class III lantipeptid es (Meindl et al., 2010). The labyrinthopeptins synthetase (LabKC)
responsible for processing thre e similar precursor peptides demonstrate d significant sequence
homology to RamC (Knerr and van der Donk, 2012 ). The bios ynthetic gene cluster for
labyrinthopeptins does not enc ode a thiol-disulfide oxidoreductas e to account for the di sulfide
bridge in the molecule (Figure 2.4 ) an d no dedic ated protease to remove the leader se quence.
T he lack of conserved Zn 2+ -binding residues in LabKC and the presence of two LanT-li ke
transporters also raise series of unanswered questions. In vi tro reconstitution studi es on
LabKC indicated GTP is involved in the kinase reaction and not ATP (Müller et al., 2010 ) .
However, a different scenario is observed for AciKC which is able to catalyze 5-fold dehydration
of AciA in the presenc e of all nucle oside triphosphates (Wang an d Van Der Donk, 2012 ).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

The directionality of LabKC ca talysis was establish ed to fol low a C→ N-termina l mod e when
processing labyrinthopep tin (Krawczyk et al. , 2012). Subsequent in vestigation with curvopeptin
using deuterium - label led CurA also indicated a C→N -ter minal modific ation order . The data
obtained from this study enabled the construction of a comprehensive biosy nth esis model that
included phosphorylat ion, elimination, and cyclization (Jungmann et al., 2014 ). The latter
findings supply an in sightful understanding of the post- translation al modificati on machinery
involved in the bi osynthesis of RiPPs.

Figure 2.4 Representative examples of class III lanthipeptide structures, showing the characteristic
carbocyclic rings, the Ser(Xxx) 2 Ser(Xxx) 3 Cys motif and additional PTMs like the N-glycosylation of
NAI-112

The two products of LabKC labyrinthopept in A1 and labyrinthopeptin A2 have been s tudied in
details (Meindl et al., 2010 , Müller et al., 2010 , Sambeth and Süssmuth, 2011 , Férir et al .,
2013 , Krawczyk et al., 2013) . The precurs or peptides of most class III lanthipeptides share a
Ser(Xxx) 2 Ser(Xxx) 3 Cys motif as shown in Figure 2.4 (Meindl et al., 201 0 , Wang and Van Der
Donk, 201 2) . The X-ray c rystallographic structure of l abyrinthopeptin A2 revealed the presence
of a disu lfide bond, as well as an unprec edented carbocycl ic labionin residue (Meindl et al.,
2010 ) , that have also been identified in other new members of the class. Other structural motifs
have been observed in the cours e of ch aracterizing oth er members su ch as erythreapeptin,
flavipeptin, and curvopeptin (Kraw czyk et al., 2012 , Völler et al., 201 2 , V  ller et al., 201 3) ,
indicating structural heterogeneity within class III peptides. Furthermore, the lanthipeptide NAI-

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

17
112 from Ac tinoplanes sp. DSM24059 has an N- glycosylated moiety (Iorio et al., 201 4), which
presents di verse pharmaceutical possibilities for this molec ule.
No dedicated pro tease has been id entified in the gene cluster for class III peptides. Preliminary
studies on erythreapeptin, avermip e ptin and griseopeptin suggested tha t a putative
aminopeptidase -like protease(s) may be involv ed in a stepwise leader peptid e removal,
produc ing a mixtures of peptides whose N -terminals are proce ssed differently (Völle r et al.,
2012 ). Neverthele ss, four gene clusters res ponsible for class III peptides biosynthes is were
found to encode prolyl oligopeptidase and a constituent member of this family of peptidases
FlaP was shown to remove the leader se quence of the class III lanthip eptide flavipeptin (V  ller
et al., 2013) . Unlike the redund ant subs trate specificity demonstrated by Lc tT (Furg erson
Ihnken et al ., 200 8) and NisP preference for modified Nis A (Kuipers et al., 2004 , Lagedroste
et al., 201 7) , FlaP is specific to modified flavipeptin and ca pable of di stinguishing between N-
and C-terminal rings (V  ller et al., 2013), indicating that other fac tors besides size and amino
acid sequence may prompt sequenc e recognition by this activat or pro tein.
2.3.4 Class IV lantipeptides
Class IV lanthipeptides are m odified by the lantipeptide synthetase LanL. It is a trifunctional
enzyme co mprising of three distin ct ca talytic domains namel y; lyase, kinase, and cyclase
domains that install PTMs in the lanthipeptide venez uelin from Strepto myces venezuelae ,
(Goto et al., 201 0). LanL enz ymes dehy drate thre onine/serine residues by employing the
kinase domain to phosphorylate side ch ain hydroxyl groups which are then su bsequently
elim inated b y the lyase domai n in a manner that was proposed to follow the scheme di splayed
in Fi gure 2.5 a (Knerr and van der Donk, 2012) . Unlike the class III LanKC, the cyclase domai n
of LanL possess the conse rved Zn 2+ - binding residues but share simi lar structural features in
the kinase and lyase domains (Goto et al., 2010 ). They also have one LanT -like transporter
that possess no protea se domain, and no evidence of a dedic ated protease was found in the
biosynthesis gene c luster (Knerr an d van der Donk, 20 12).
The simila rities between their synthetas es suggest that both cl ass III and IV lanth ipeptides
share a common ancestral lineage, completely di fferent from those of class I and II where the
synthetases lack apparent serine/threon ine kinases and phosphos erine/threonine lyases.
Phylogenetic comparison shows a broad range of phyla including fir micutes, actinomycetes,
Bacteroidetes, Cyanobacteria and Proteobacteri a, harboring putative LanB, LanM and LanL
(Goto et al., 2010 ) .

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 2.5 Features of class IV lantipeptide (a) Suggested mechanism of se rine/threonine
dehydration by LanL phosphothreonine/serine kinase and lyase domains. Structures of related class
IV lantipeptides venezuelin (b) and streptocollin (c).

So far two members of thi s class have been produc ed and charact erized; venezuelin ( see
above) and streptocollin from Strepto myces collin us Tü 365 (Ifti me et al., 2015). The structures
of venezuelin and streptocolli n (Figures 2.5b & 2.5c , respec tively) were elu cidated based on
mutagenesis and ESI tandem mass sp ectrometry (Goto et al., 201 0 , Iftime et al., 2015 ) . Both
have globular structures similar to duramycin and cinnamycin but do not inhibit human
phospholipase A2. Rather, streptocollin was shown to in hibit protein tyro si ne phosphatase 1B
by 33% at a concentration of 50 µM (Iftime et al., 2015 ), demonstrating that the mol ecule may
have therapeutic rel evance in related applications.
Four genes are implicated in class IV lanthipeptides biosynthesis, LanL, LanA, LanT, and
LanH, which are organize d in a single operon. Analyzing sequences adjacent to lanLATH
operon di d not reveal any evidence of genes associated with the biosynthesis and/or regulation
of lanthipeptide, nor an immunity factor (Iftime et al., 2015). Hypothetical ly, the putative LanT -
like ABC transporter may form a heterodimeric translocation complex with LanH (Méndez and
Salas, 2001) to actively ensure efflux of the peptide and prevent the product from harming the
producer hos t cells (Davidson and Chen, 2004 ) . Like class III, neithe r a dedicated N-terminal
leader peptide processing protein nor an enzyme containing a protease domain has been
detected in the gene cluster. Due to the inadequac y of empiricall y verifi ed sequences, it is
challenging to predict the cl eavage si tes for the leader peptid e of cl ass III and IV lanthipeptides

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

19
in silico , something which can be routinely done for class I and II lanthipeptides (Blin et al.,
2014 ).
2.4 B io technological production versus chemical s ynthesis of
lanthipeptides
These topics were extensively reviewed in publ ication I where the s uitability of biological
systems and ch emical approa ches were evaluated, and signi ficant evidences p ointed to
bio technological procedures as the only realist ic alternative for lanthipeptide production at the
moment (Ongey and Ne ubauer, 2016 ).
2.5 Pharmacological properties and therapeutic use of
lanthipeptides
This subject was sum marized in publicati on II where the therapeuti c relevant properties of
lanthipeptides under consideratio n for clinic al us e were ana lyzed while trying to gain
understanding of how extrinsic modifications affect the overall behaviour of the peptides and
how such information may be used t o engineer other peptides to improve their cl inical values
(Ongey et al., 2017 ).
2.6 Recent advances in engineering and heterologous production of
lanthipeptides
To fully exploit and understand how lanthipeptides can be engineere d to overcome challen ges
like in stability, in solubility and protease degradation , whic h rem ain the rapeutically
unfavourable under phy siological conditio ns, several tools have been developed (Fiel d et al.,
2015 ) and many others are still in the pipeline . Production levels of lanthipeptides from their
natural sources are very low and efforts are also being made to improve this. In add ition to
what has already been discussed in publication I on these subjects, some of the most recent
findings are reviewed he rein.
The in trinsic modulari ty of the Lan/MeLan rin gs and the promiscuous nature of lanthipep tide
synthetases ca n be exploited to generate a wide range of different pep tide archite ctures
(Montalbán-López et al., 2016). These perspectives can actually constitute a new genera tional
approach to identifying and characterizing novel anti -infective agents with enhanced
therapeutic potential. For instance t he promiscuity of ni sin biosynthetic machinery (NisBC) was

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

exploited to het erologous ly express and inst all dehydrations in 27 different peptides, with 5 o f
them demonstratin g good antimicrob ial activity, whil e 2 others were ful ly modifie d and
possessed enhanced antimic robial properties (van Heel et al., 201 6) . This study demo nstrated
an efficient approach to pro duce lanthipeptides and to easily identify m olecules that may b e
interesting to investigate furthe r.
Tailoring modific ations in RiPPs that usually oc cur on the side chain of amino acid s, and
sometimes within the peptide backbone (e.g. epimerization), or at the N - or C-terminus , can
inc rease stability of the peptides. Their activiti es may also be improved by the di verse chemic al
functionalities su pplied by these modificati ons, whic h a re necessary for opt imal interactions
with biological targets (Funk and Van Der Donk, 2017 ). Setti ng the platforms t hat may fac ilitate
these modifications is also important. One of such platforms was reported recently where t he
precursor peptide of Prochlorosin coupled to the C-terminus of bacteriophage M13 minor coat
protein pIII was heterologous expressed and subsequ e ntly modified by modifying enzyme
ProcM in the cytoplasm of E. coli , and then di splayed on the phage su rface (Urban et al., 2017) .
This technique may take advantage of the promisc uity of lanthionine synth eta ses to genera te
additional PTM s on a variety of peptides such as the N -glycosylation of NAI- 112 (Iorio et al.,
2014 ) or the D -amino acids of bicereucin from Bacillus cereus SJ1 (Huo and van der Donk,
2016 ). For example, by co-expres sing the bicereucin dehydratase BsjM and the zinc -
dependent dehydrogenas e NpnJ A from Nostoc punctifo rme PCC 73102 (Zhang et al., 2014) ,
it was possible to produce an analog of the D -amino acid-co ntaining opioid dermorphin in E.
coli (Huo and van der Donk , 2016) . In vivo and in vitro introdu ction of D- amino ac id into the
peptide are also possible using NpnJ A that reduces dehydroalanine to D-Ala in the presence
of NAPDH (Yang and van der Donk , 2015).
Furthermore, b y engineering the genetic code, it was possi ble to reassign the natural codon
that enable the subs titution of a tryptophan res idue in lichenicidin with an unu sual amino acid
L- β -(thieno[3,2 -b]pyrrolyl)alanine, and heterologously expres sed this bioactive congener of the
compound in an evolutionar ily adapted E. coli st rain (Kuthni ng et al., 2016 ). This st udy
supported the conc ept of bioco ntainment by showing that althoug h the non -canonical amino
acid was toxic to the heterologous host, evolutionary adaptatio n may help to alleviate the
situation. Moreover, co -expressing a dedicated suppressor aminoac yl -tRNA synthetase s and
the PTM enzymes f or ni sin NisBC in E. coli , nisin varian ts with α -chloroacetamide -containing
unnatural amino acid s were generated (Zambaldo et al., 2017 ). These peptide engineerin g
approaches allow for pos sible expansion of the chemical reactivity space of the peptides,
thereby broadening their activity spectrum.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

21
The outer membrane of Gram-negati ve bacteria prevents lantibiotics from gaining acce ss to
the inner phosph olipid membrane where li pid II is found and hen ce the ir inactivity against thi s
group of bacteria. Efforts to improve the efficiency at which the peptides can overcome this
barrier have recorded some progress very recently. For exampl e, part of the anti-Gram-
negative peptid e apidaecin 1b was fus ed to the C -terminu s of nisin, or truncated version s
thereof, producing a two -fol d increase in nisin activity again st E. co li CECT101 (Zhou et al.,
2016 ). Additional ly, a nano - engineering approach involving t he coupl ing of nisin molec ules to
gold surfac es was used to bro aden the ac tivity spec trum o f nisin to inclu de Gram negative
bacteria like E. col i and Pseudomonas aeruginosa ( Vukomanović e t al., 2017 ).
The bi osynthetic gene cluster of mathermycin from Marinactinos pora thermotolerans SCSIO
00652 was isolated and reconstituted in Streptomyces lividans and E. coli to produce the
cinnamycin-like lantibiotic that demonstrate d antimicrobial activity against a Bac illus strain
(Chen et al ., 201 7) . Althoug h purification from S. livi dans was not successful due to lo w
expression, se veral attempts to produce in the native strai n proved futile. Nevertheless,
overexpression was achieved in E. coli (Chen et al., 2017). The two-component lanth ipeptide
Flv System from Rumino coccus flavefaciens FD -1 was also rec ombinantly expres sed in E. coli
and found to have one highly conserved α -peptide and a set of eight struc turally diverse β -
peptides (Zhao and va n der Donk, 201 6). This work revealed new features in lanthipeptides
which have not been reporte d previously.
2.7 Native biosynthesis and regulat ion of lantibiotic ruminoc occin-A
The ability of bacteria to adapt and survive in an e colo gical co mmunity gre atly depend s on
their potential to sense local environmental changes and devis e means to sustain these
external influenc es usually by upre gulating or downregulating the expre ssion of specific genes
(Dunny and Leonard, 1997 ). The two-compone nt QS system is implicated in the bi osynthesis
of most bacteriocins includi ng the prototype lanthipeptide nisin from L. lactis (Kuipers et al.,
1995 , Lubelski et al., 2008) where nis in acts as an induc er in its biosynthetic pathway. Other
bacteriocins like plantaricin A from Lactobacillus plantarum C11 (Diep et al., 2001 ) is induced
by a bacteriocin -like peptide whic h is co -transcriptionally synthesiz ed si multaneously in a
three -component system. Similar to the latter QS system, a three-compone nt regulatory
mechanism is involv ed in the bi osynthesis of the cl ass II la ntibiotic ruminococ cin -A originally
isolated from Ruminococ cus gnavus E1, a constitu ent member of the human intes tinal
microbio ta (Gomez et al ., 2002).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 2.6 A hypothetical model describing the trypsin-dependent regulation of ruminococcin- A
biosynthesis in R. gnavus E1. (a) At lower cell density in the gut, R. gnavus E1 synthesizes an inactive
inducer peptide (pre-IP). The pre-IP is secreted to the extracellular space. Then at higher cell density,
trypsin a ctivates pre-IP to active IP. The histidine kina se RumK monitors environmental changes and
senses the accumulation of IP as it reach es a threshold concent ration. This leads to the activation of
the regulatory protein RumR which in turn triggers differential gene synthesis which also enables
RumA to be expressed and secreted concomitantly (Gomez et al., 2002). (b) The peptide sequence
of preRumA contains a leader peptide (position -23 to -1), the core peptide (position 1 to 24) and the
Gly -Gly motif (position - 2/ -1) where proteolytic cleavage occurs. Threonine, serine and cysteine side
chains involved in t hioether crosslinks formation are also indi ca ted.

RiPP-genes are ubiquitously present in anaerobic ba cteria where more than 25% host
biosynthetic pathways for the natural products (Letzel et al., 2014 ) . R. gnavus E1 is a strictly
anaerobic Gram-positive bac terium whic h was first id entified in human feces ( Ramare et al .,

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

23
1993 ). Ruminococcin C was also reported in R. gnavus E1 , pos sessing similar biological
activity as ruminococcin-A but differ ent structural and biochemical characteristics (Crost et al.,
2011 ) . The biosynthesis machinery of rumino coccin - A consists of 14 ORFs, two, whose
functions are yet to be det ermined. In the sc hematic drawing of the genet ic cluster presente d
in Figure 2.6a (b ottom right), following the ORF1 is a s eries of five genes rumFEGHR2 , which
have characteristics homologous to the ABC -transporte r and other accessory components
believed to particip ate in keeping the produc er R. gna vus E1 safe from its own toxic product.
Multiple co pies of the structural peptide-encodi ng gene appears to be a special feature in the
cluster of lanthipeptides from the genus Ruminococcus as observed with the rumA1A2A3 of
ruminococcin-A (Go mez et al., 2002 ) and the two-component la ntibiotic Flv System from R.
flavefaciens FD - 1 (Zhao and van der Donk, 2016). This physiological arrangement may be
necessary to ensure production efficienc y and strengthen a ntimicrobial activity.
Essential components of the gene cluster that are directly implicated in the bios ynthesis of
RumA are the genes that enc ode the pre cursor peptide pre RumA, the dual -functio nal
(dehydratase/cyclase) lanthionine synthetase RumM and the dua l -functional
(protease/transporter) AMS prot ein RumT (G omez et al., 2002). Native Rum A pro duction is
regulated by the abundant trypsin in the gut which activates an induc er peptide (IP) to trigger
a casc ade of events. The biosynthesis model propos ed in Figure 2.6a is an exp ansion of
previous studies (Dabar d et a l., 2001 , Gomez et al., 200 2 , G omez et al ., 2002 ), with additional
information curle d from extended litera ture. Although the amino acid sequence encoded by the
last ORF rumX displayed neg ligible se quence homology with known proteins, its N -terminal
130 residues showed similarity with DNA -associated pro teins (Gomez et al., 2002 ). Studies on
bacteriocin -negative R. gnavus mutant strain revealed the presence of rumA 1 A 2 A 3 , rumTX
genes and 3’ -truncated rumM, whereas the hypotheti cal regulatory ope ron ( rumRK ) and the
immunity operon ( rumFEGHR2 ) were both abs ent (Gomez et al., 2002). Although these
findings supported the predicted functions of these genes, the characterization of the variou s
products remained open for investigation. Figure 2.6b displays the schematic diagram of the
primary struc ture of preRumA, showing the Gly -Gly motif w hich is eng aged by the N -terminal
domain of RumT to activate the peptide, as well as threoni ne, serine an d cysteine side chains
supposedly targe ted for PTMs formation by RumM.
2.8 Isolation of ruminococcin-A from R . gnavus E1
Previous investigation in the early 90’s on gnotobiotic rats harboring R. gnavus E1 in its
digestive tract demonstrated the presence of a trypsin- dependent anti - Clostridium perfri ngens
(Ramare et al., 1993). Although the strai n at that time was characterized as belonging to the

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

genus Peptostreptococcus , subsequent studi es verified that it was actually a member of the
genus Ruminococcus (Dabard et al., 2001 ) . In vitro culturing identified RumA (Dabard et al.,
2001 ) and ruminococcin -C (Cros t et al., 2011 ) as the components respon sible for the observed
anti - Clostridium perfringens activities.
The strict anaerobic nature of R. gnavus E1 only allowed succes sful growth in an anaerobic
cabinet, applying brain heart infusion broth as the cu ltivation medium (Dabard et al., 200 1) .
The culture medium was further suppl emented with hemin and yeast extract , as well as trypsin
to trigger ruminococcin -A production. Upon all these efforts, Dabard and colleagues succ eeded
to achieve a product yield of 0.665 microgram RumA per litre of R. gnavus E1 culture (Dabard
et al., 2001). Suc h outcome und erscores the need to investigate alternative produc tion
systems since the curren t procedure is obviously not optimal for obtai ning quantities that would
allow the peptide to be evaluated for possible applica tions in therapeutic treatments, food
preservation and livesto ck farming.
2.9 Carriers for heterologous expres sion of AMPs
Heterologous expressi on challenges like agg regation, protease cleavage and toxic ity to host
cells, are prevent ed by fus ing AMPs to tags like glutathione S-transferase (GST) , small
ubiquitin -like modifier ( SUMO) , malt ose binding protein (MBP ) and thioredoxin ( Trx) (Li et al. ,
2009 , Li , 2011 , Bell et al., 2013 , Pane et al., 2016). These tags are then subsequent ly cleaved
to generate the functio nal peptides. Recombinant production of AMPs in E. coli that employ
these tags as fusion partners sometimes generate mixed out comes (Li, 2011 ). For instance,
the performance of GST in various attempts have been inconsistent since some of the syst em
even fai led to produce the desired results (Skosyrev et al ., 200 3 , Si et al., 2007 , Chen et al .,
2008 ). MBP whic h is even large r than GST was fus ed to Human b -defensins and expressed
in E. coli as aggregates which were then refolded to obtain the functional peptides ( Li and
Leong, 2011 , Tay et al., 2011 ). Other tags that h ave also been used successf ully in pro ducing
AMPs are thioredoxin and SUMO, w hich perhap s due to their smal ler sizes (11.8 kDa and 11.2
kDa respec tively) ha ve yielded higher titres of target precursor peptides expressed in the
cytoplasm (Gibbs et al., 2004 , Li et al ., 2009 , Bommarius et al., 2010 , Cao et al., 2010 , Li et
al., 2010 ).
2.10 Objective of t his work

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

25
Observations in se veral examples indicate that the natural produc ers of RiPPs are usual ly
challenging to cu lture and even when they are s uccess fully grown, the production lev el s of the
desired products are usually very low. Bioinformatics and recombinant technolog y have
allowed the charact erization of new sources of natural co mpounds via metabo lic sampling and
genome min ing approaches. These tec hnologies have ena bled the us e of e cologically close
relations to the native producer strains to produce lanth ipeptides in si tuations where optimiz ing
the natural host failed to produce the desired outcomes . Neverthele ss, characterist ics like short
generation time, high ce ll density growth, high produc t yield, and ease of manipulati on, which
are typical of a surrogate hos t like E. coli may have pos itive influenc es on produc tion titres and
facilitate certain as pects of peptide bioengineering thus fostering potential therapeutic ,
agricultural and food processing applicatio ns .
Since the production pathways of the se peptides incl ude complex modificati ons machineries
which are usually not present in the new expression hos ts, the y require the transfer of the
minimal essenti al components of the pathway i nto their own system to be able to produc e the
active biomolecule. The aim of thi s work was to recombinantly reconstruc t the Rum A
biosynthesis pathway in an autonomous hos t and test its amenability to genetic manipulation
and to improve the production level of the peptide. Specific biosynthes is genes from the Ru mA
biosynthesis cluster, hosted by the obligate anaerobic bacterium R. gnavus E1 , had to be
isolated and recons tituted in E. coli which was the host of choice.
It was desired to first achieve soluble express ion of the different genes involved by s eparately
expressing them in E. coli . Most importantly, the ruminococcin -A lanthionine syntheta se RumM
had to be active in order to catalyze formation of PTMs in the precursor peptide preRumA. To
address solubility and stability challenges of RumA, a highl y expressi ble and soluble fusion
partner was des ired. Different cloning and expression strategies were to be applied to des ign
the strains, meanwhile parallelized microtiter -plate screenings of cu ltivation and productio n-
relevant par ameters were also necessary. Active in vivo expression of Rum M capable of
install ing all the desire d modif ications in preRumA w as necessary for E. co li production to be
successful . Product identification and ch aracterization sh ould co nstitute an important phase of
the study. Furthermore, s electing parameters to optimize in both upstream and downstream
procedures shoul d be done rationa lly.
Secondly, the act ivation of modified preRumA which is th e final step in the maturation process
performed by the AMS protein RumT was to be tackled. Envis aging the challenges o f
expressing membrane protei ns and the fact that RumT does not engage directly in any PTM
formation, alternative activation methods were also to be exploited. This app roach involved
engineering the precursor peptide to contain alternative cleavage pos sibilities at position -1 in
the leader sequence of preRumA . T hese included the tobacco etch virus (TEV) protease, GluC,

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Trypsin and Fac tor Xa sites . Moreover, attempt s to express, purify and chara cterize the N -
termin al peptidase domain of RumT w as also anticip ated.
Finally , with a fully functional system in hand, engineering the peptide to expand and/or
increasing its ant imicrobial ac tivity by incorporati ng non -canonical amino acids into the peptid e
would be possible. The system woul d also offer irrefutable advantages over the native producer
in that scal e-up studies to achieve eco nomic feasibility would be attainable sinc e complic ated
and expensive experi mental set -ups would not be n ecessary.

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27
3. Materials and Methods
3.1 Materials
The variou s categories of materials used in this study are descr ibed in Appendix 6.9 ( tab les
A5.1-A5.3)
3.2 Software
Software used in this work are listed in Appendix 6.9 (Table A5 .4 )
3.3 Bacterial strains, growth and cultiva tion conditions
An overview of bac terial strains used in this work are listed in Appendi x 6.9 (Tabl e A5.5) .
Various E. coli strains were used for different purposes including cloning, plasmid maintena nce
and protein expression. Ruminococc in -A native biosynthesis strain R. gnavus E1 was us ed to
isolate the genome from whic h specific genes in the biosynthesis machinery of RumA were
amplified. For stora ge purposes, E. coli strains were grown overnight at 37 °C on an LB agar
plate supplemented with the appropriate antibiotic concentrations . Fresh LB medium
containing 2 0 % glycero l was added to the plates co ntaining colon ies and then washed off and
aliquoted into Roti -Store cryo vials (Carl Roth GmbH, Germany) and stored at -80 °C. R.
gnavus E1 was cultivated in Brain Heart Infusion (BHI) broth as described elsewher e (Daba rd
et al., 2001) . B. subtili s ATCC 6633 cultivations were performed at 30°C with an agitation speed
of 200 rpm.
3.3.1 Cultivation media
All media recipes (composed as listed in Table A5.6 , Appendix 6.9 ) were dissolved in ddH2 O
and pH was adj usted when necessary. After di ssolution, the media were the n autoclaved at
121 °C for 20 min. For solid media cultivation, 1.5 % agar was direc tly added to the mixture
prior to autoclavatio n, and appropriate amounts of required sterile -filtered antibiotics (as
described in Table A5.7 , Appe ndix 6.9 ) were added to the broth aga r medium precooled to
around 50 °C. The antibiotic -su pplemented brot h agar was then poured into st erile petri dish es
and allo wed to solidify at room temperature (RT). Liquid medi a were stored at RT while the
agar plates were stored at 4 °C. For cloning purposes, SOC medium was used to gro w freshly
transformed ce lls. Optimizati on and expressi on screening were performed in TB medium and

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

the EnPresso B growth system (Enpresso GmbH, B erl in, Germany) acco rding to the
manufacturer’s protoc ol.
3.3.2 Monitoring bacterial growth with a spectrophotometer
E. coli growth was monitored by estimating the concentration of cells using an Ultrospec 3300
spectrophotometer (Amersham). Opti cal densit ies (ODs) at 600nm were measured while
considering the ac curacy limit of the spectrophotometer (OD< 0.5 ) . Sample s were diluted in 0.9
% NaCl solution for measurements . For online OD s, measurements wer e performed every 10
min us ing the Synergy Mx plate reader (Biot ek). In this work, onl ine ODs only pro vided a
monitoring tool to asc ertain cell growth during fluorescence measureme nts.
3.3.3 Automated fluorescence measurements
Qualitatively, target product expres sions in strains expressing GFP-fus ed cons tructs were
estimated by measuring their fluorescenc e. The strains were cu ltivated in 96-well or 24-well
flat-bottom microtiter plates and the relative fluoresce nce emission of the cells were measured
using a Synergy™ Mx monoc hromator -based multimode microplate reader (Biotek ) . Ti me-
course GFP flu orescence signa l intensities were recorded as relative fluoresce nt unit (RFU)
every 10 min at 528 nm for 15-30 h. As a control, the auto-fluores cence of a strain which doe s
not express GFP was al so measured .
3.4 Vectors
An overview of clo ning and expression vectors modified or used in this study are describ ed in
Appendix 6 .2 (Table A1).
3.5 Molecular Biology techniques
Molecular biology procedures were performed following established protocols. T4 DNA ligas e,
restriction enzymes, nucleotide t riphosphates , as well as other DNA- modifying enz ymes were
purchased from Fermentas (Vilnius, Lithuania), Thermo Fish er Scientific (Walth am) and New
England BioLabs (NEB) (Frankfurt am Main, Germany), and used acco rding to the
manufacturers’ recomme nded procedures . Onl y sl ight modifications were made when it was
desired to improve reprodu cibility.

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3.5.1 Isolation of R. gnavus E1 Genomic DNA isolation
R. gnavus E1 culture was grown at 37°C overnight in a pre -reduced BHI (Difco Laboratories,
Detroit, MI) supplemente d with 5 g of yeast extract and 5 mg of hemin (Sigma-Aldric h) per litre.
The cultivation was performed in an anaerobi c cabinet. Cells were harvested by centrifuging 2
ml of the overnight culture for 2 min at 12,000 ×g at 4 °C. Genomic DNA was extracted usi ng
GenElute Bacterial Genomic DNA Kit (Sigma - Aldrich), following the manufacturer’s
instructions. The resulting chromosomal DNA was analyzed with 0.5 % agarose Gel, pre -
stained with GelRed ( Biotium).
3.5.2 Preparations of plasmid DNA
E. coli cells hos ting the desi red plasmids were cu ltivated overnight at 37 °C in LB medium. 2-
4 ml of the cu lture w as harvested by centrifugation for 1 min at 16,000 ×g. Plasmids were
purified from the cell pellets using the Invis orb Spi n Plasmid Mini Two Kit (Stratec ) according
to t he manufact urer’s protocol. The concent rations of isolated plasmid were estimated using
the Nanodrop ND-100 0 spectrophotometer (Pe Qlab) and stored a t 4 °C until required for use.
3.5.3 Oligonucleotides design
Vector NTI Advanc e 11 .5 (Invitrogen) was employed in designing oligonucleotide primers for
polymerase chain reactions (PCRs) and DNA sequencing. Chemically synthesized primers
used for PCRs were purchased from TIB M OLBIOL (Berlin, German y) . A n overview of the
primers is presented in Appendix 6.9 ( Table A5 .8 ).
3.5.4 PCR techniques
3.5.4.1 Analytical PCR
For ana lytical purposes, PCR were performed u sing the DreamTaq D NA polym era se (Thermo
Scientific) which has no pro of -reading proper ties. The reaction compos itions and conditions
were set up following to the manufacturer’s rec ommendatio n.
3.5.4.2 Preparative PCR
High-quality PCR pro ducts were desired for subsequent cl oning purposes. To achieve thi s, we
utilized the Phusion and Q5 high fidelity DNA po lymerases (NEB), both posse ssing the 3´→5´
exonuclease activity. All reactio n mixtures contain ing template D NA, deoxynuc leotide

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

triphosphate (dNTP) mix, primer pair and DNA polymerases were asse mbled and operated in
a gradient thermocycler under recommended conditions . Gradient PCR was used to optimize
the annealing temperature of each set of primers. Once the optimal amplific ation condition was
determined, it was the n used to run a preparati ve PCR.
3.5.4.3 Colony PCR
In thi s tec hnique, the DreamTaq Green DNA P olymerase ( Thermo Scientifi c) was used in the
reaction mix constituted accordi ng to the manufacture r’s instructio ns. Two Eppend orf tubes
were prepared for each colony, one contain ing 30 µl ddH 2 O and the other containing 100 µl LB
medium suppl emented with appropriate antibiotics concentration. Colonies were randoml y
selected from agar pla tes us ing sterile toothpi cks. Each colony was fi rst washed in the ddH 2 O
and then rinsed in the medium. The inoculated cultures were stored at +4 °C. The E ppendorf
tubes containing cells in ddH 2 O were incubat ed for 5 min at 95 °C to ens ure complete disrupti on
of the cell and su bsequently placed on ic e. 5 µl was obtai ned from eac h tube and used as
template for the DreamTaq Green reaction mix. Once positi ve clones were identified, they were
matched with the in oculated LB cultures , the plasmids were purified an d sent for sequencing.
3.5.4.4 Splicing by overlap extension (SOE) PCR
This te ch nique was applied to fu se gfp mut2 to the gene that encodes RumA precursor peptide
( rumA ). To do this, two sp ecial primers rumA s _f and gfp s _ r (Table A5 .8 , Appendix 6.9) were
designed su ch that their 5' overhangs had partially co mplementary regions . In a fir st PCR step ,
gfpmut2 and rumA wer e separately amplifie d from their respective templates using the set of
overlap pri mers respec tively designed for each of the target genes. This result ed in two double-
stranded DNA fragmen ts with overlapping regions in each of the sequences. In the second
stage, quasi-equimolar amounts of the two fragments were mixed together in a mega -primer
PCR where the two fragments preliminary se rved as primer s and as template at the same time .
They annealed to eac h other and were joined by amplific a tion using the external primers (see
Figure A5.4a and A5.4b, Appendi x 6.9, for illustrations ). The resulting chimeri c product
contained an NheI restriction site at the 5 ’ -end and a PstI si te at the 3’ -end.
3.5.5 Purification of DNA fragments and spectrophotometric quantitation
The quality and quantity of preparative PCR amplicons were preli minarily evaluated via
aga rose gel elec trophoresis. Gene fragments that were succes sfully amplified were purified

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31
using the HiYield PCR Clean -up/Gel Extrac tion Kit (Süd-Lab orbedarf) according to the
manufacturer´s protoc ol. This was don e in two ways: either a piece of the aga rose gel
containing a clear ban d at the des ired product length was ex cise d or the PCR products were
applied directly. The purified products were quantified using NanoDrop and directly used for
subsequent cloning steps or stored at -20 °C for further use.
3.5.6 Agarose gel electrophoresis
With regards to the size range of gene fr agments used in this study, 0.5 % or 1 % g els were
prepared by diss olving electrophoresis grade agarose in 1X TAE buffer (40 mM Tri s-HCl pH
8.5, 20 mM Acetic acid and 1 mM EDTA). The solute was completely dissolved by heatin g in
a microwave. Ethi dium bromi de (Carl Roth) or GelRed ( Biotium) was added (in the ratio of
1:10,000) to the molten gel soluti on precooled to about 50 °C and then poured into a
preassembled cast ing plate. Analysis of DNA w as performed by dilutin g samples in requi red
amount of ddH2O and mixing with 6x DNA loadi ng buf fer (Fermentas ) in the ratio of 1:6 . The
sample mixtures were loaded into precasted wells in the gel submerged in 1X TAE buffer in a
DNA electrophore sis unit (Biozym) , and an elec tric field was applied . DNA bands were
vis uali zed by UV light excitation.
3.5.7 Conventional cloning using restriction enzyme digestion and ligation
For analytical purposes, 10 µl of 400-500 ng conce ntrated DNA was assembled by addin g
target DNA, steri le ddH 2 O, FastDigest restriction enzymes and FastDigest buffer (Thermo
Scientific) according to the manufacturer’s manua l. The sample mixtures were incubated for
20 -30 min at 37 °C. For preparative sa mples, DNA up to 4 µg was digested for 1-2 h. In cases
where the pair of restriction enzymes us ed were both sens itive to higher temperatures, the
digested product was inactivated by heating for 15 -20 min at 80 °C. Otherwise the digested
DNA was analyzed by agarose gel electrophoresis and correspondi ng fragments were excise d
and purified as described earlier (sectio n 3.5 .5). Ligation reactions were performed with T4
DNA ligase (NEB) by asse mbling a 10 µl reaction according to the manufac turer’s pro tocol.
The amount of insert added to the mixture was calculated according to the following equation:

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Ligation reactions were performed for 10 -30 min at RT or overnight at 16 °C. T4 DNA ligase
w as inactivated by incubating for 20 min at 65 °C. The resulting products were us ed to
transformed competent E. coli cells.
3.5.8 Modification of expression vectors
The main vector backbone used in this study was the pCTUT7 which pos sessed a pBR322
origin of replic ation ( ori ) and an Isopropyl β -D-1 -thiogalact opyranoside (IPTG)- inducible CTU
promoter (Kraft et al., 2007 ), as well as the pJ L10 which possess ed an RSF ori and an IPTG-
inducible CU promoter (Li et al., 2015). pCTUT7 was der ived from pKA100 (Krebber et al .,
1996 ) and pDest15 , and pos sessed mutation s in the lacUV5 sequence of the origi nal pAK100,
−35 region from tac promoter, ribosomal binding site ( RBS ) of gen e 10 of bacteri ophage T7
(T7 RBS) and a recombination cassette amplified from pDes t15 (Kraft et al., 2007). T he
plasmid that hosted the CU pro moter (pJL10) was a modification of pRSF-1b where the strong
T7 promoter was substituted for the CU promoter (Li et al., 2015 ). The difference between the
CTU (stronger) and CU (relatively weaker) pro moters were sp eci fic mutations at their
respective -35 reg ions (Kraft et al., 2007). It was des ired to express the target genes under
control of different promoters . To achieve this, some features of the pCTUT 7 and pJL07 ( Li,
2013 ), as well as pSEVA1810 nee ded to be modif ied. pSEVA1810 is a vector that was
designed according to the Standard European Vector Architecture (SEVA) (Martínez-Garcí a
et al., 2014 ). It habours a pUC ori and a pBAD promoter.
To change the pUC ori of pSEVA1810 to p15A ori , FseI/AscI-fla nked p15A ori amplified from
pJL02 (Li, 2013 ) was digested and inserted into FseI/AscI -digested pSEVA181 0. The resulting
vector was named pSEVA15A. The Chloramphe nicol resistance marker in pJ L02 was replaced
with ampicil lin res istance by in serting HindIII/Bsu36I -flanked fragment amplifie d from pJL07
into HindIII/Bsu36I -digested pJL02 to yield pLEO p15A .
T he CTU pr omoter in pCTUT7 was replace d with CU promoter while mai ntaining its T7 RBS ,
by using SpeI and NdeI to remove an entire cassette comprising of the l ac I gene and the CTU
promoter from pCTUT7. SpeI / NdeI -flanked fragment containing the lacI gene together with the
CU promote r was amplifie d from pJL10 and subse quently inserted in to the backbone of the
SpeI / NdeI -digested pCTUT7 to yield pCUT7. Vec tor description and plasmid maps are found
in Appendix 6.2. Plasmids were purified and se quenced via LGC Genomi c (Berlin, Germany) .

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33
3.5.9 Site ‐ directed mutagenesis
Q5 Site-Direct ed Mutagenes is Kit (NEB) was us ed to generate mutants of preRumA that
allowed alternative mea ns to remove its leade r peptide and activate the compound . Trypsi n ,
GluC, TEV and factor Xa cleavag e sites wer e introduced between the leader and core
segments of preRumA. Q5 high-fidelity DNA polymerase produced linear chimeric products
which were di rectly used in the subsequent steps. After degrading methylated DNA followed
by ligation, the reaction mix was use d to transform chemical ly competent E. coli cells.
3.5.10 Construction of expression vectors
The general characteristics of expression vector s a re described in Appendix 6.2 (Table A1 ).
3.5.10.1 Amplification of gene fragment from R. gnavus E1 chromosome
R. gnavus E1 genome was used as a template together with primer pai r RumClus_f/ru mClus_r
(Table A5.8 , Appendi x 6.9) to amplify a segment of the rumA g ene cluster (GenBank access ion
no. AF32032 7), co ntaining the three genes encoding preRumA and the gene enc oding the
lanthionine synthetase Rum M. The blunt -ended amplico ns were directly inserted into a SmaI -
restricted pCTUT7 vector to yiel d pLEO rC2.
3.5.10.2 Construction of His6-preRumA expression vectors
For preRumA expres sion, NheI / PstI -flank ed rumA1 PCR pro duct was amplified from pLEO rC2 .
The resulting amplicons were di gested and ligated into NheI / PstI -di gested pCTUT7 vector
yielding pLEO rA .
3.5.10.3 His6-preRumA vectors with alternative cleavage site at position -1
To introduce Trypsin, GluC, Factor Xa and TEV cleavage sites at position -1 of preRumA, site
directed pri mers (Table A5.8 ) were designed for the respective purpos es. PCR, using pLEO rA
as template together with the Q 5 Site-Direct ed Mutagenesis Kit and the respective primer pairs
generated pLEO rA* , pLEO rA GluC , pLEO rA Xa , pLEO rA TEV , eac h pos sessing trypsi n, GluC, factor
Xa and TEV cleavage sites , respectively, at position -1 in the final preRumA e xpression
product.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

3.5.10.4 Construction of His6-GFP-TEV-preRumA expression vectors
The final NheI / PstI -flanked chimeric fragment from SOE PCR was digested and li gated into
corresponding NheI / PstI -digested pCTUT7, p JL07 and pLEO p15A to yield pLEO grA ,
pLEO p15A_grA and pLEO t0grA res pectively. To eliminate multiple products expression from
a single gene construc t, add itional features like a λt0 terminato r downstream and a tand em
stop codon at the 3’ -end of the chimeric gene fragment were included in the pLEO p15A_grA
and pLEO t0grA vectors. To subs titute the Gly-1 i n pre RumA fused to GFP with Arg, pLEO rA*
was used as template in the SOE PCR and the resulting pro ducts were digested and inserted
into pCTUT7 to yield pLEO grA*.
3.5.10.5 Construction of His6-SUMO-preRumA expression vectors
To fuse preRumA to SUMO, NheI / PstI -flanked gene fragments encoding the preRumA wer e
amplified from pLEO rA*, pLEO rA Xa and pLEO rA TEV respec tively. The resulting products were
digested and in serted into NheI / PstI -di gested pCTUT7- SUMO plasmid (Kraft et al., 2007 ) to
yield corresponding pLEO srA* , pLEOs rA Xa and pLEOs rA T EV .
3.5.10.6 Construction of His6-RumM expression vectors
For the expressio n of the ruminococci n -A lanthionine synth etase RumM, PCRs with
appropriate pri mer pa irs (Table A5.8) using pLEO rC2 as template were performed, and the
resulting amplicons were digested with either NheI / PstI, KpnI / PstI and Ba mHI / PstI, and
correspondingly ligated into the pCTUT7, pRSF-1b and pJL10, generating pLEO rM’ , pRSF- rM
and pLEO rM respectively. Similarly, NheI / PstI -digested rumM ampli cons were inserted into
NheI / PstI -restrict ed pCUT7 vector to yield pLEO rM1.
3.5.10.7 Construction of bicistronic expression vectors
Appropriate pri mer pairs (Tab le A5.8) were used to amplify a PstI/HindIII -flanked gene
fragment from either pLEO rM ’ or pLEO rM1 containing the CTU or the CU promoter
respectively, together with a T7 R BS and rumM. The resulting amplicons were digested with
PstI & Hind III and subsequently inserted in to PstI/HindIII -digested p LEO rA TEV , pLEO grA,
pLEO grA* , pLEO srA* , pLEOs rA Xa and pLEOs rA TEV to generate pLEO rA TEV M1 , pLEO grA M ,
pLEO grA*M1 , pL EO srA*M , pLEO srA Xa M and pL EO srA TEV M respectivel y.

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35
3.5.10.8 Construction of RumT125 expression vectors
The nuc leotide sequence encoding the first 125 N -terminal amino acid residues of the
bifunctional AMS protei n RumT was amplifie d from a cDNA optimized for E. coli expression
and purch ased from Thermo Fisher Scientific (Waltham). The resulting NheI / PstI -flanked PCR
amplicons were di gested and inserted into NheI / PstI - dig ested pCTUT7 and pCTUT7-SUMO to
yield pLEO rT125 and pLEO srT125 respectively.
3.5.11 Transformation of competent E. coli cells
Chemically competent and electrocompetent E. coli cells were prepared according to a
standard protocols (Sambrook and Russe ll, 2001) and stored at -80 °C. For chemical
transformation, 100 µl of competent cells were thawed on ice and 1 µl o f the des ired plas mid
or ligation product was added and mixed gently. The mixture was incubated on ice for further
30 min and subsequently plac ed at 42 °C for 45 s. The heat-shocked samples were inc ubated
on ice for 5 min. 900 µl of SOC medium was transferred into the transfected cell culture and
grown at 37 °C while shaking for 1 h. The tran sformed cells were spread on LB agar plates
prepared with app ropriate antibiotic conc entrations and cu ltivat ed overnight at 37 °C. Single
colonies were obtained from the plate and screened for positi ve clones via colony PCR. Major
strains used in thi s study are described in Appendix 6.9 ( Table A5 .5 ) .
3.6 Protein production and analysis
3.6.1 Expression optimization in EnPresso B growth system
Expression optimizati on were performed in 24 -well deep-well plates using the read - to -use
enzyme-based automated gluc ose -delivery EnPres so B growth system (Enpr esso Gmb H,
Berlin, Germany), follo wing the manufacture r’s recommende d pro cedure. E. coli W3110
transformed with the desired expres sion vectors were cultivated in an orbita l shaker (Infors HT,
Switzerland) at 30 °C, 250 rpm overnight. Prio r to induction (af ter about 12 -15 h of cultivation),
3 ml of the main culture was distribut ed into the 24-well pl ate and induced with different
concentrations of IPTG. Variable amounts of reagent A (the glucose -releasing biocatalyst that
slowly degrades a complex polymer compone nt of the EnPresso B medium tablet to deliver
glucose to the growing cells) were also su pplied to the different well s. The pl ate was seal ed by
Breathable Film (Starl ab, Hamburg) and placed back into the orbita l sh aker under the same
conditions as before. The optical densities of the cultures at 600 nm ( OD 600 ) were measured
at different time points durin g a period of culti vation ranging from 18 to 24 h post in duction . For

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

GFP fusion construct, protein expressi on was followed by measuring GFP fluorescence signa ls
online (section 3.3.3) . Cultivation was stopped by obt aining 2 ml of th e cultures into 2 -ml
Eppendorf tubes and harvesti ng the cell pellets b y ce ntrifuging in a Himac /CT15RE centrif uge
(VWR, Leuven) for 1 min at 16, 000 ×g at 4 °C. The cell pellets were stored at - 20 °C for fur ther
preparations and analysis.
3.6.2 Protein Expression in TB medium
Precultures of E. coli W3110 transformed with the des ired expression vectors were pre pared
by using glycerol st ocks from -80 °C to inoculate 20 ml of LB mediu m suppl emented with
appropriate antibiotics. The preculture was grown overnight at 37 °C, 200 rpm. For e xpres sion
screening purpos es, 100 ml of fresh TB medium containing adequa te antibiotic concentrations
in a 500-ml baf fled Ultra Yield Flask (Thomson Instrument Company, USA) was inoc ulated with
the overni ght cu lture to an in it ial OD 600 of 0.1. Larger preparative cultures were inoculated in
a 2-li tre Ultra Yield Fl ask containing 400 ml of fresh TB medium suppl ied with suitable
antibiotics. The flask w as sealed with air -permeable AirOtop Enhanced Seals (Thomson
Instrument Compan y, USA) and incubated in the orbita l shaker at 30 °C, 200 rpm. At induction
OD 600 between 0.8 and 1, 3 ml each from the 1 00 ml cu lture were distributed into a 24 -well
microtiter plate and induc ed with differ ent concentratio ns of IPTG, whil e the preparative culture
was induced with 100 µM IPT G (optimized from micro -scale cu ltivations) and further culti vated
for 18-30 h. The OD 600 of the cu ltures were measured at different time points while o nl ine GFP
fluorescence signals was used to monitor the expres sion of GFP fusion constructs. Small scale
cultures were harvested by collectin g 2 ml and ce ntrifuging for 1 min at 16,000 ×g at 4 °C. Cell s
from the preparative cu ltures were coll ected by centrifugation us ing the Eppendorf ce ntrifuge
5810R (Eppendorf, Hamburg) for 10 min at 6000 ×g at 4 °C. Cell pellets were store d at - 20 °C
for further preparations and analysis.
3.6.3 Strain screening using GFP fluorescence
The effi ciency and reprodu cibility of strains producing GFP fusion products were evaluate d
based on their GFP f luorescenc e over a longer culti vation period. These experi ments were
performed using the Hamilton Microlab STAR Li quid -han dling station (Hamilton, Martinsried)
coupled to the Synergy Mx plate rea der (Figure A5.5, Appendix 6.9). At the beginning, 96
colonies were sele cted manuall y from an LB ag ar plate and inoculated 150 µl of LB or TB
medium suppl ied with appropria te antibiotics in each of the wells of a 96 -well flat -bottom
microtiter plate. The cultures were induced automatically with 100 µM IPTG 8 h after the

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

37
cultivation start ed . Prior to induction, the rob ot was programmed to sa mple 10 µl each from all
96 wells and transfer into a new plate (containing 10 µl of 30 % glycerol prepared in LB medium)
placed in one of the 96 -well microtit er plate (MTP) posit ions on the deck (Figur e A5.5, Appe ndix
6.9 ). The latt er plate was collected and stored at -80 °C. IPTG solution used for induction was
also stationed in one of the 96 -well MTP positi ons. Measurements were performed e very 20
mins. The robot transported th e culture plate from the inc ubator to the plate read er and
transferred it back into the incubator once measurements were fin ished. Online OD 600 were
also measured to deter mine if the cells were growing.
3.6.4 Cell disruption
Prior to purification, E. coli cells were lyse d by a co mbined enzymatic and ultrasonication
procedure. Frozen cells from -20 °C were thawed a t RT and resolubilized in th e IMAC binding
buffer (50 mM NaH 2 PO 4 ·H 2 O, pH 8, 300 mM, 10 m M Imidazole ). Fresh lysozyme and
benzonase were added to the cell suspension to a final concentration of 1 mg ml -1 and 1 U ml -
1 respectively. Ultra sonic ation was performed on ice for 5-12 min usin g 30 sec on/off intervals
using the UP200S sonicator (Hie lscher Ultrasonics, Teltow). Dependi ng on the sample size,
different probes were used. Samples from screening experiments were proce ssed using the
detergent-bas ed cell disruption technique, BugBuster (Merck, Darmstadt, Germany) protocol,
following the manufacturer’s rec ommendations. Smaller lysates were clarified by centr ifugation
for 15 min at 16,000 ×g, 4 °C using a in a Himac centri fuge, while larger sample volumes were
clarified by centrifuging for 20 -30 min at 10,000 ×g, 4 °C using an Eppendorf ce ntrifuge 581 0R .
The samples were filtered thro ugh a 0.4 5-µm pore-sized syringe fil ter (C arl Roth) and us ed for
subsequent purifica tion steps. When whole cell extracts were desired for SDS -PGAE
comparative analyses, BugBuster was added to the collected cell pellets according to the
following equation:

where original culture volume is the volume of culture collected; OD 600 is the final optical density
of the cultu re; 2 is the dilution factor include d because of the 2X lo ading buffer used for SDS
sample preparation; 67 is the estimation factor; and 1000 converts volume units from milliliter
to microliter.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

3.6.5 SDS -PAG E analysis
Insoluble fractions from the BugBuster experiments were resuspen ded in resolubilization buffer
(100 mM Tris -HCl pH 8. 0, 8 M Urea , 100 mM DTT, 1 mM EDTA) with the same volume as
used in resuspending the cell pellet. The suspension was vortexed and centri fuged for 5 min
at 16,000 ×g at 4 °C. The su pernatant, as well as solub le protein sa mples were mixed in a one -
to -one ratio with 2X SDS sample buffer (100 mM Tris-HCl pH 6.8, 200 mM DTT, 4 % SDS, 20
% Glycerol 0.20 % bromphenol bl ue). The samples were boiled for 5 min at 95 °C and then
cooled to RT. 10 µl of eac h sample were loade d into the wells of the SDS gel. Roti -Mark
TRICOLOR (Carl Roth) was us ed as molecular weight s tandard.
Larger proteins were pr ocessed using 10 % res olving, with 4 % st acking polyacrylamide gel
prepared following the protocol des cribed by Sambrook and Russel (2001). The SDS running
buffer (25 mM Tris-HCl pH 8.3, 192 mM Glycine, 500 mM Urea, 0.10 % SDS) was us ed in the
electrophoresis chamber. A voltage of 70 V was app lied across the two electrodes
(anode/cathode) for ~20 min and subsequently increased to 120 V until the bromophenol blue
dye fron t was almost out of the gel. Smaller pepti des (<10 kDa) were analy zed us ing 16 %
Tricine PAGE as descr ibed else where (Schägger, 2006) . Gels were rinsed with ddH 2 O and
then washed twic e for 2 min with warm ddH2 O. All gels were visualized by staining with
colloidal blue silver Coomas sie G-250 as reported elsewhere (Candiano et al., 2004) .
Background staining were removed by rinsi ng the gels severally with distilled and when the
bands were suffici ently clear, the gels were documented usin g a sca nner.
3.6.6 Native PAGE analysis
Sample preparations and running of the gel were performed in the same manner as described
for SDS -PAGE except for the fact that det ergents (SDS and urea) and DTT were not included
in the native PAGE as sembling, sample and running buffers. Also, the sample heating step (95
°C, 5 min) was not performed. Additionally, 8 % resol ving, with 4 % stacking polyacrylamide
gels were used for the a nalysis.
3.6.7 Western blot
Semi-dry electrob lotting was appli ed usin g a horiz ontal apparatus (Cleaver Scientifi c). Gels
from SDS-P AGE was rinsed with ddH 2 O and incu bated in the transf er buffer (25 mM Tri s -HCl
pH 8.3, 192 mM Glycine, 10 % MeOh, 0.1 % SDS) for 15 min. Six pieces of equally sized (the

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

39
same size as the gel containing proteins to be transferred ) Whatman Filter pap er (Whatman,
UK) previously soaked in the transfer buffer were placed on the anode plate of the
el ectroblotting unit. The blot sandwich was completed by plac ing a same -si zed nitrocellulose
membrane, followed by the gel and ano ther set of si x wet fil ter papers. The upper plate
(cathode) was fitted onto the blot sandwic h and a voltage of 20 V was appli ed across the
electrodes for 1 h at RT. To detect the poly -histidine tag on the protein of intere st, the bl otted
membrane was first incubated in Blocking buffer (5 % skimmed milk po wder in TBST) while
shaking for 1 h at RT. The membra ne was subsequ ently rinsed with TBST buffer (25 mM Tris-
HCl pH 7.4, 150 mM NaCl, 0.1 % Tween 20) an d in cubated in 1:20,000 dilution of anti - penta -
His mouse IgG1 (Quiagen) in blocking buffer (primary antibo dy). The membrane was incubated
for 1 h at RT , washed and further incubated in 1:10,000 di lution of alkaline phosphatase ( AP )-
conjugated anti -Mouse IgG antibody (Sig ma) in blocki ng buffer. The membrane was rinsed
with AP buffer (100 mM Tris -HCl pH 9.5, 100 mM Na Cl, 5 mM MgCl2) and visualization was
achieved via the reaction on th e ch romogenic BCIP/NBT substrates catalyzed by alkaline
phosphatase [80 μl BCIP (20 mg ml -1 BCIP in 100 % DMF) plus 60 μl NBT (50 mg ml -1 NBT in
70 % DMF) in 10 ml AP Buffer].
3.7 Protein purification and peptide extrac tion
3.7.1 IMAC
For analytic al purifi cation of His 6-preRumA, His6-GFP-TEV-pre RumA a nd His 6-RumM, the
clarified lysates obt ained after cell disruption were applied to His SpinTra p columns (GE
Healthcare) and processed according to the manufact urer’s in structions. For comparative
analysis, all samples were pro cessed following the same procedure and eluted in the same
amount of elution buffer. Equa l volumes were then used for SDS -PA GE anal ysis. Preparative
purifications were per formed with the Äkta Avant 25 instrument by loading the clarified bacterial
lysates onto a 1-ml HisTrap FF Crude column (all from GE Healthcare) at a flow rate of 1 ml
min -1 . UV signals at 280 nm, 260 nm and 484 nm (GFP flu orescence) were used to monitor
purification. The column was initi ally flushed with five column volumes of bi nding buf fer (50 mM
NaH 2 PO 4 ·H 2 O, pH 8, 300 mM, 10 mM Imidazole ) and eluted by applying a gradient range of
0 – 100 % of eluti on buffer (50 mM NaH 2 PO 4 ·H 2 O pH 8, 300 mM NaCl, 500 mM imidaz ole). The
purified constructs were co ncentrated using Amicon Ultra centrifugat ion tubes (Merk Millipore,
Darmstadt) with the appropriate molecular weight cu t -off limits (10 kDa for SUMO fused
proteins, 30 kDa for GFP fus ed constructs and 1 00 kDa for His6-RumM). Protein
concentrations were me asured using the Bradfor d assay.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

3.7.2 Si ze exclusion chromatography
The IMAC conc entrated sample was injected onto a HiLo ad 16/60 Superdex pg 200 column
(GE Healthcare) and eluted with the gel fil tration buffer (20 mM NaH 2 PO 4 ·H 2 O pH 8, 150 mM
NaCl, 10 % glycerol) at a constant flow rate of 1.5 ml min -1 . Samples were obtained fr om each
of the 2 ml eluti on fraction and analyzed via SDS-PAGE. Fractions were pooled and
concentrated to app rox. 4 -5 ml. Concentrations were measured using Bradford assay and
aliquot s were stored at -20 °C.
3.7.3 TEV Cleavage
TEV was applied to cleave off fusion partners from the des ired pro duct by addin g it to the target
sample according to the following equation:

where V represents the volume; m, mass ( mg) of targ et protein to be digested; an d C,
concentration (mg ml -1 ) o f TEV. All reactions were performed overnight at 4 °C. The procedure
followed one of three ways: (i) by add ing fresh homemade TEV (see Appendi x 6.10) to the
IMAC concentrate d sa mples and directly loade d into an appropri ate dia lysis membr ane and
then dial yzed in 1 -2 ×10 3 volumes of dialysis buffer (20 mM NaH 2 P O4·H2O, pH 8, 150 mM
NaCl, 1 mM DTT, 0.5 mM EDTA, 1 mM PMSF); (ii) by buffer -excha nging the protein sa mples
in di alysis buffer and TEV was subsequently added; and (iii) by thawing gel filtration sa mples
from -20 °C on ice, and 1 mM DTT, 0 .5 mM E DTA and TEV were added.
3.7.4 Extraction of preRumA from TEV-digested product
To extract the desired preRumA p eptide from TEV -digested samples , the sa mples were run
through a Ni 2+ -NTA column and the flow through containin g the desired product was colle cted,
dialyzed in water and dried using Concentrator Plus vacuum concentrator (Eppendorf,
Hamburg, Germany). The dried sa mples were stored at -20 for furt her use. Alte rnatively, the
TEV- diges ted sa mples were extracted with 1- butanol by adding 1 volume of the solvent and
stirring the mixture at R T for 1-2 h. Ex trac ts were separated by ce ntrifugation for 5 min at 3000
×g. The upper organic layer (containing the peptide) was obtained and the conc entrations were

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

41
measured using the Nano Drop technique. Recovered pep tide extracts w ere dried and stored
at -20 for subsequent applica tions.
3.8 Measurement of protein conce ntrations using Bradf ord assay
Bradford Coomassi e Brilliant Blu e method (Bradford, 1976) was used to measure protein
concentrations . Samples were assembled by adding 10 μl protein solution to 90 μl of Roti -
Quant Bradford solu tion (Carl Roth). The resul ting solution was mixed and incubated in 96 -
microwell plate for 5 min at RT. Meas urement wa s performed using the Infinite 200 plate rea der
(Tecan). The absorption value at 595 nm was co mpared with a standard curve prepared using
known concentrations of BSA.
3.9 Iodoacetamide derivatization an d trypsin digestion
Iodoacetamide derivatization was used to determine the availabili ty of side chain cysteinyl
thiols in the core of preRumA following presumed lanthionine modific ation s. Meanw hile trypsin
was app lied to cleave the leade r pep tide from preRumA and consequentl y ac tivating the
compound. Dried extracts from -20 °C were dissolved in 100 mM amb ic (pre pared by addin g
0.79g ammonium bica rbonate to 100 ml ddH 2 O) to obtai n ~1 mg ml - 1 of soluble pep tide. 30 µl
(~30 µg of peptide) wa s obtained and 2µl of 16 0 mM DTT (final c onc. = 10 mM) was add ed to
the peptid e solution and incubated at 55°C for 1 h. 400 mM fresh stock solution of
iodoacetamide (Sigma) was pre pared by weighing 36 mg of the solute crystals and di ssolving
in 100 mM ambic . 2 µl of the alkylating agent was added to the reduced sample (final conc. =
25 mM) and the n incubated at room t empera ture for 45 min in the dark. Af ter the deriv atization
was completed, 6.3 µl of 0.1 µg µl -1 mass spectrometry grade trypsi n (Promega, Lyon, France)
stock solution was added and incub ate d on a shaking pl atform at 37 °C overni ght. For peptide
identification purposes , gel pieces were excise d from SDS -PAGE and in-gel tryptic di gestion
was performed as descr ibed elsewhere (Shevch enko et al., 2006 ) . Prior to mass sp ectrometric
analyses, the tryptic dig ests were purified usi ng ZipTip.
For modified and non -modified His6-SUMO-Rum A*, the derivatization step was not performed.
The IMAC-purified sa mples in elution buffer 2 (20 mM TRIS-HCl, pH 8, 250 mM NaCl, 300 mM
imidazole) were dialyzed in water and the n in 100 m M a mbic. 15 µl of trypsin stock solution
(1.5 µg) was add ed to 1 00 µl of protei n samples obt ained from eac h of t he modif ied and non -
modified His 6-SUMO-R umA* and then incu bated on a sha king platform at 37 °C overnight .
SDS -PAGE samples were obtained from the tryptic diges ts and the res t w as us ed for bioassa y
analyses.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

3.10 Mass s pectrometric anal yses
3.10.1 MS sample preparation with ZipTiP
ZipTip (Merck) is a 10 µl pipette tip possessing a non- polar (C18) chromatographic matrix
immobilized inside the end of the tip. This technique is ideal for pur ifying and concentrating
peptides for MS analyses. To do this, two tubes were prepared with 10 µl each of 30 or 80 %
acetonitrile (ACN) in HPLC-grade H 2 O, containing 0.1 % formic acid (FA). Dried sa mples from
-20 °C were diss olved in 0.1 % FA a nd plac ed in ul trasonic bath for 30 s to ens ure complete
dissolution. The dissolve sample was aspirated and dispensed severally through the column
matrix of a pre- equilibrated ZipTip. The loaded ZipTip column was washed thrice with 0.1 %
FA by aspirati ng and dispensing the solvent to remove contaminants, and then eluted in the
tube containing 10 µl of 30 % ACN tube, followed by the 80 % ACN tube. The column w a s
equilibrated again, and the ZipTip procedure was performed three times. The two elution
fractions were combined and dried using a vacuum concentrator. Furthermore, w hen desired,
the ZipTi p pro cedure was also applied directly to the purifi ed TEV -digested samples or tryptic
digests.
3.10.2 LC - ESI -MS analyses of preRumA
For the liquid ch romatography-elec trospray ionizatio n-mass spectrometry (LC - ESI -MS)
analyses, dried ZipTip extracted samples were suspended in 15 -20 µl of 0.1 % FA and
dissolved by placing in ultraso nic bath for 30 s. 5 µl was injected in to an Agilent 1290 Infinity
HPLC syst em (Agilent Technologies, Waldbronn, Germany), followed by an ESI -Triple-
Quadrupole LC-MS 6460 electron spray ionization mass spectrometry analysis us ing multiple
reaction monitorin g. The material used for the column and pre -co lumn was Poroshell 120 EC-
C8 (2.1 × 50 mm, 2.1 μm). Solven ts allocation was designated as: H 2 O for A and ACN as
solvent B. The introd uced sample was eluted with an ACN g radient from 5 % to 20 % solvent
B within 0.5 min, follo wed by 70 % B in 4 min and then to 100 % B in 0.2 min. An isoc ratic
elution with 100 % B was finally appl ied for 1.3 min. The flow rate was set at 0.7 ml min − 1 .
3.10.3 Tandem mass spectrometric analyses of preRumA
Whereas LC-MS analyze s only the precursor io n, LC- MS 2 filters out the precurs or ion, fragment
it and analyze the product ions with a se cond mass analyzer ( see Appendix 6 .8.5 ). Nano- LC -
ESI - MS 2 is a widely used method to identify peptides in a complex mixture. Dried sa mples from

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

43
-20 °C we re resolubilized as described in the previous sectio n . For LC- MS 2 anal ysis, the
samples were directly injected into an HPLC - c oupled LTQ Orbitrap XL™ Hybrid Ion Trap -
Orbitrap mass spectrometer with a nanoelectrospray ion source. Highly intense ions identifi ed
in the full MS were directed to the orbitrap analyzer where they were fragmented and
processed in the linea r ion trap (see Appendix 6 .8.5 ). For the n LC - ESI - MS 2 , the dried samples
were red issolved in 50 µl of 0.1 % FA. The pro cessed samples were direct ly inje cted in to the
Orbitrap Fusion mass spectrometer (Thermo Scientifi c). Spec ific ions were selected and
fragmented with HCD, CID or ETD ( Appe ndix 6 .8.3 ). Different fragmenta tion energies (in volt)
were applied.
3.11 Biological Assay
B. subti lis ATCC 6633 was reporte d by Dabard et al. (2001) as one of the indicator strains for
RumA bi ological activity. The strain was cu ltivated in M + medium (Table A5.6, Appendi x 6.9 )
as described earlier and then spread on M + -agar plate s containing punche d wells. The plates
were air-dried and 50 µl of each sample was pi petted into separate wells an d incubate d at
30 °C overnight.
3.12 Identification of optimal production conditions using online DOT & pH
To determine certain parameters tha t may influence a pro duction process , 24 diffe rent
representative experi ments were designed by altering the conce ntrations of IPTG and reagent
A at the point of induction as sh own in Figure 5.6. The se experiments were aimed at optimizing
the expres sion of His6-GFP-TEV-pr eRumA and His6-Rum M during cult ivation in EnPresso B
growth system. Three different plates were set up in parallel us ing the same conditions. One
of the plates was a normal 24 -well shallow plate which was us ed to measure online OD 600 and
GFP fluoresc ence. The other two plates were; a 24 -well deep-well plate possessing a pH
sensor at the bottom of each well (HydroDis h HD24) and a 24 - well shal low plate with oxygen
sensors at the bottom of each well (OxoDish OD24), all from PreSens. The culture volume in
the wells of the shallow plates was maintained at 1 ml per well whil e the deep -well plate was
set at 2.5 ml per well . To prevent unpr ecedented evaporation of li quid between the wells, Duetz
System san dwich covers and cover clamps (Enzyscreen, Heemstede, Netherlands ) were used
to close the micr otiter plates (Duetz et al., 2000).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure A5.6 24 -well plate experimental design for screening optimal growth and production condition s
in EnPresso B m edium. The concentrations of reagent A and IP TG are indicated for each well.

The cultivations were performed for 20 h after induction at 30 °C and 200 rpm while measuri ng
dissolved oxygen tension (DOT) and pH online using the SensorDi sh Reader (PreSens). GFP
fluorescence and OD 600 were measured onl ine at 10 min intervals with the Synergy Mx plate
reader (Biotek ). At line OD measurements of cultures in the first row (A1 -6) of the deep -well
plate (the pH measurement pl ate) were performed at differ ent time points by dilu ting 10 µl of
the culture in 990 µl of 0.9 % NaCl solution in 1.5 ml Eppendorf tubes and measure d using the
Ultrospec 3300 spectrophotomete r (Amersham). Expressi on of His6 -GFP-TEV-preR umA was
determined by the GFP fluorescence while both His6 -GFP-TEV-preRumA and His6-Ru mM
were determined by SDS -PAGE anal ysis of samples obtai ned at the end of the cultivation.
3.13 Computat ional analyses
For illustration purposes, protein structure data were obtai ned from the protein data bank (PDB)
and edi ted using protein work shop (Moreland et al., 2005). Nucleotide sequence data were
obtained from the National Center for Biotec hnology Info rmation (NCBI) GenBank databa se
(Benson et al ., 2014 ) and the European Molecular Bio logy Laboratory (EMBL) nucleotide
sequence database (Kulikova et al., 200 6). Info rmation about lanthip eptides and other AMPs
were ac quired from Bag el3 ( van He el et al., 2013 ), BACTIBASE (Hammami et al., 2010) and
the AMP dat abase, APD (Wang et al ., 2016). Polypeptide sequence information was acquired
from the UniProt database (Consortium, 201 6) .
The NCBI’s Basic Local Alignmen t Search Tool (BLAST) (Alt schul et al., 1990 ) was used to
investigate similarities between nu cleot ide or protein sequence s. Vector NTI Ad vance 11. 5
(Invitrogen) was used for in silico molecular biology experi mental evaluation including primer

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

45
design, cloning, analysis of sequencing data and manipulation of DNA and protein sequences .
The Vec tor NTI was also used to graphicall y view DNA sequence s and to generate plasmid
maps and li near graphi cal prese ntation of DN A sequ ences. The codon us age between E. co li
and R. gna vus E1 was compared using the Graphical Codo n Usage Analyzer 2.0
( http://gcua.schoedl.de ) and the Rare Codon An alysis Tool (GenScript) was us ed to analyze
rare codons and calculate the Codo n Adaptation Index (CAI). Rapt or X server (Käl lberg et al.,
2012 ) was us ed to predic t pro tein struc tures and the prediction data was processed using
PyMOL 2.1 (Schrödinger , New York, US).
Peptide masses and iso topic distribution were calculated using mMass 5.5 .0 (Strohal m et al.,
2010 ). Mass spectra data acqui sit ion and analyses were performed with either the Anal yst
1.4.2 software (AB SCIEX) or the MassHunter Qualitative Analysis Software (Agilent) or
Xcalibur 4.1 (Thermo Scient ific ). MaxQuant (Cox and Man n, 2008 ) and Xi Spectr um Viewer
( http://spectrumviewer. or g/ ) were also used for MS data an alys es. MASCOT search eng ine
(Matrix Sci ence, UK) was used for sequence id entification by searching the MS and MS 2 data.
Chromatographic data obtained during pro tein purification wi th the Äkta avant 25 system were
acquired using the UNICORN 6.1 software (GE He althc are). Chemical structures and
schematic representation of biomolecular structures were created with ChemBioDraw Ultra
14.0 (PerkinElmer). ChemBioDraw Ultra 14.0 was also used to d ete rmine the chemical
formulae of pep tides using their ami no acid seque n ces. MicrolabSTAR VENUS one software
(Hamilton) was used to contro l the Micro labSTAR liquid handling station. Graphs were
generated with SigmaPlot (Systat Software).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

4. Results and Discussion
4.1 Sequence analysis of ruminococcin -A proces sing enzymes
4.1.1 Se quence similarity between RumM and other lanthionine synthetases
The amino acid sequences of thre e dehydratase s (La nB enzymes) of class I lanthipeptides
and their correspondi ng cyclases (LanC enz ymes) were aligned together with three cl ass II
lanthionine synthetases (LanM enzymes ), including RumM. Although the dehydratase domains
of the LanM enzymes show ed negligible sequence homol ogy with eac h other and none with
their class I equivalents, their C-terminal region was found to contain the conserved zi nc-
bin ding residues present in lanthionine cyclases (Li et al ., 2006 , Helfrich et al., 2007 ). This
domain also share d significant homology with each other as well as o the r LanC enzymes. In
fact, the C- term inus of RumM di splayed >30 % identity and >60% similarity with nisin cycl ase
(NisC), indicating possible structure -function similaritie s. This type of structure-function
similarity has been described in a variety of the lanthionine -generatin g enzymes (Knerr and
van der D onk, 201 2 , Repka et al., 2017) . The conserved active site residues are indicated in
Figure 4. 1. The reaction ca talyzed by cyclases in volves dep rotonation of cystein yl thiol and
protonation of the resulting enolate intermediates , formed vi a a conjugate addition reaction
between the α -carbon of Dha/Dhb sub strates as described earlie r for nisin (se e Figure 2.2a,
section 2. 3.1) .

Figure 4.1 The highly conserved domains of selected class I cyclases including subtilin [SpaC, Swiss-
Prot accession number P33115 (Klein et al., 1992)], nisin [NisC, CAA48383 (Engelke et al., 1992 ) ]
and epidermin [EpiC, CAA44254 (Schnell et al., 1992)]; as well as class II LanM cyclase domains of
ruminococcin-A [RumM, Q9L3F1(Gomez et al., 2002) ], lacticin 481 [LctM: P37609 (Rince et al., 1994) ]
and actagardine [GarM, C4NFI1 (Boakes et al., 2009)]. The underlined residues are responsible for
coordinating the central Zn 2+ ion as shown in the crystal structure of NisC (Li et al., 2006). The
residue s indicated by arrows ar e essential for an active modification of subtilin, whereas those
indicated by single orbitals appear to have no effect on SpaC ca talytic activity (Helfrich et al., 2007) .
The conserved Gly residue denoted by an open arrow was shown to be highly implicated in the
maturation of epiderm in (Agustin et al., 1992) . [see section 3.13 for analysi s methods]

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

47
The conserved tyrosine residue in cl ass I cyclases ( Figure 4.1) h as been shown to be
obligatorily implicated in subtilin biosynthesis, serv ing as an acid/base catal yst (Helfrich et al.,
2007 ). Interestingly, histidine seems to play this rol e in class II cyclases. Addi tionally, the
co nserved non-esse ntial Ser residue in clas s I cyclases seems to be subs tituted for conserved
Asn in the cyclase domains of LanM enzymes. The mere fac t that these substitutions do not
alter the putative functions at these positions (i.e. proton donor/accepto r), reflect more on the
similarities between the enzymes.
4.1.2 Sequence and structural similarities between RumT and other AMS
proteins
The structure of a related member of a pro tein family may provide vital in formation with re gards
to its function and how it m ay be expressed . This was the ca se with RumT wh ere the crystal
structure of rel ated family members like PCAT1 from C. the rmocellum (L in et al ., 2015) and
the peptidase domain of ComA from Streptoc occus pneumoniae (Ishii et al., 2010 ) have been
determined. Although the peptidas e domain of the class II la nthipeptide AMS protein LctT has
been characterized in vitro (Furg erson Ihnken et al., 2008), no structures exist for this class so
far. To enable a comparability assessment, RaptorX protein structure model ing tool was used
to predict the monomeric struc tures of five cl ass II lanthipeptide transporters namely; LctT,
NukT, MrsT, RumT and BovT.
Although these proteins displayed very diss imilar amino acid sequences, the final predicted
structures all si x pro teins showed strong structural homology . They possess features si milar to
the crystal structure of the AMS protei n PCAT1 (Figure A2, Appendix 6.3). They contai n a
transmembrane domain with of six membrane - spanning α -helic es, an ATP- binding domain a nd
a peptidase domain ( Figures 4 .2a & 4.2b). The crystal st ructures of the peptidase domains of
ComA (PDB: 3K8U) and PCAT1 (PDB: 4RY2) show that the peptidase domain has two
subdomains namely: N -terminal s ubdomain and the C -terminal s ubdomain.
These subdomains are made up of a serie s of α - helices and antiparallel β -strands arranged
such that some of the stran ds are connec ted via a short α - helix (Ishii et al., 2010 , Li n et al.,
2015 ) , as well as a short β -strand. All these features were also visible in the predicted LanT
peptidase dom ains ( Figures 4.2c & 4.2d). The active site of t he predict ed peptidase domain of
RumT was ana lyzed in co mparison with the crystal st ructure data of the peptidase domain of
ComA (P DB: 3K8U). These results indicate a tight correlation between hydrogen bon d
distances measured for putative active si te res idues in Rum T and those performed with the
actual residues in ComA (Figure A3, Appe ndix 6 .3).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 4.2 Structure prediction of two LanT proteins: RumT and LctT using RaptorX. The predicted
structures of LctT (a) and RumT (b) show strong h omology to the crystal structure of the polypeptide
processing and secretion transporter PCAT1 (Lin et al., 2015), po ssessing a trans membrane domain
(TM),, an ATP-binding domain (ABD) and a peptida se domain (PEP). The peptidase dom ains of LctT
(c) and RumT (d) are also homologous to the crystal structure of the peptidase domain of ComA (Ishii
et al., 2010). They have two subdomains: SD1 and SD2 . SD1 consists of three alpha -helices (α1 -
α3) while SD2 is co nsists of 6 beta- strands (β1 - β6) and an alpha helix (α4 ) connecting β1 and β5 .
β2 and β3 are linked by another short β -strand . The catalytic site is formed b y residue s in α1 and β3.
[see section 3.13 for com putational analysis methods used]

4.1.3 Codon adaptability calculations and analysis
Organisms differ in the way the y sele ct and apply the triplet codon during the synthesi s of a
nascent polypeptid e chain. How this codon usage bias come about is a subj ect under strong
debate within the scientific community. Howe ver, some suggestions with regards to their role

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

49
in temporal protein regulation have been proposed (Shin et al., 2015). Codo n bias may have a
multiplicative effect on the effi ciency of protei n synthes is as well as misi ncorporation whic h
may occur durin g heterologous protein exp res sion (Kurland and Gallant, 19 96) . Codon us age
analysis comparing E. coli and R. gnavus was performed us ing the gra phical co don usage
analyzer (Fuhrmann e t al., 2004 ). A graphical presentatio n is shown in Figure 4. 3.

Figure 4. 3 Comparison of codon usage between E. coli and R. gnavus. Green bars re present the
relative adaptiveness or the usage of each codon in R. gnavus against the black bars which represent
codon usage in E. coli. For each amino acid, the codon that is used most frequently by the designated
organism has the highest relative adaptiveness (100 %) and used as a reference point to scale the
other codons for the sam e amino acid.

The mean difference in codon usage between R. gna vus with tha t of E. co li is 25 %. Al l codons
used by R. gnavu s (green bars) with high frequenci es have moderate frequenc ies in E. coli
(black b ars), and so genes from R. gnav us source can be heterologous expressed in E. co li .

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

However, it would be extremely challengi ng to express genes from E. coli source in R. gnavu s ,
as many co dons like ACC (Thr), ACG (Thr), CGC (Arg) and GCC (Ala) that are freq uently
utilized by E. coli are rare in R. gnavus .
Using E. coli as a surrogate host may pose some problems if codons like AGA (Arg), AGG
(Arg), CGA (Arg), ATA (Ile), CTA (Leu), GGA (Gly), CGG (Arg) and CCC (Pro) are pre sent in
the target gene (Nakamura et al., 2000). However, exce pt for AGA (Arg) which has low
frequency of usage in E. coli compared to R. gnavus (100 % → 43 %), the usage pre ferences
for all the other codons whic h are rarely used in E. coli do not vary very much in R. gna vus ,
indicating that expression of thos e genes may be relativel y tolerable in E. coli.

Table 3 .1 Properties of the RumM and RumT encoding gene seq uences

Frequency of Occu rrence

Rare codon

Amino acid

rumM

rumT

AGG

Arg

AGA

Arg

CGG

Arg

CGA

Arg

GGA

Gly

AUA

Ile

CUA

Leu

CCC

Pro

Total #codons

—

915

689

CAI

—

0.66

0.61

GC content

—

29.9

32.29

We ana lyzed RumM and RumT coding sequences using GenSc ript rare codon analysis tool
and found out that there are a variety of these rare codons which may cause expression
problems in E. coli (Table 3.1). We also calculated the codon adaptation in dex (CAI) of rumM
and rumT. C AI is the rel ative adaptive ness of a fore ign gene to the biosynthes is machinery of
the host organis m usually reporte d as a value between 0 and 1. Higher values within this range
indicate that there is similar codon usage patter n in the gene of interest as there are in the
reference gene (Sharp and Li, 1987). The lower the CAI, the higher the ch ances for poor
expression (Wu et al., 2005 ) . Although the CAIs ca lculated for rumM and ru mT (Tab le 3 .1)
were low er than the expected value to guarantee good expres sion in E. coli (>0.8), studies
in dicate that the CAI is not a strong det erminant of heterologous gene expres sion (Welch et

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

51
al., 2009 ). Furthermore, few tandem rare co dons were found in rumM (Figure A4, Appe ndix
6 .3) that may neg atively influence heterologous e xpression of the gene (Kim and Lee, 2006).
Additionally, several tandem and eve n a quadr uple rare codon cluster s were found in rumT
that can significantly slow down tra nslation and caus e expressi on problems (Clarke IV and
Clark, 2008) . For this reason, a codon-optimized gene sequence encoding the first 125 amino
acid residues of RumT (Appendix 6.4 ) was purchased for E. coli expression.

4.2 Vector construction and E. coli expression
4.2.1 Features of expression vectors
The biosynthes is gene cluster of RumA is huge (12.8 kb ) and thi s may limit overexpressi on if
the whole cluster is isolated and introduc ed into the heterol ogous hos t . In order t o fac ilitate
product optimization, we constructed single -vector and two-vector systems habouring genes
that enc ode the structural peptide and the PTM enzyme, as well as regulatory features that are
required for expres sion in E. coli. Studies on lichenicidin which is also a class II lant hipeptide
identified genes in its biosynthetic cluster which are not directly involved in the biosynthesis
and modification of the compone nt peptides (Caeta no et al., 2011). Followi ng this example,
and considering data reported by Gomez et al. (2002), we proposed in this study that the
hypothetical genes for immunit y ( rumFEGHR2 ), tran sport/process ing ( rumTX ) and regula tory
operon ( rumRK ) are access ory components and not directly involved in the biosynthesis and
PTMs forma tion in preRumA, conc luding that rumA and ru mM genes are the two essential
elements required.
To maintain two plasmids in the same expression host, the co -transformants were construc ted
to possess compatible origins of replication and distinct antibiotic res istance genes .
Conventional re striction and ligatio n cloning was employed to facilitate insert ion of the gene
encoding preRumA and the lanth ionine-generating enzyme RumM und er contro l of variab le
promoters. Two of the most prominen t promoters used here were the Isopropyl β -D-1-
thiogalactopyranoside (IPTG)- inducible CTU and CU promoters (Kraft et al., 2007) . The
promoters are described in detail in section 3.5.8 . Others like the pBAD and the T7 -RNA
po lymerase promoter were also applied. Compatible pair of ori s used for the two -plasmid
systems were ei ther pBR322/RSF or pBR322/ p15A . At fir st, our strategy involved placing
rumM under control of the relatively weak CU promoter and the rumA c onstruct under the
relatively stronger promoter CTU . The plasmid maps of all resultant expression vectors are

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

displayed in Appe ndix 6 .2 and det ails on the co nstruction procedures can be fou nd in section
3.8.10 .
The upstream reg ion of the backbone vectors hosting the CU a nd CTU promoters contain ed
the transcriptional terminator tHP and mutations in the catabolite activator protein (CAP) si te
that weaken CAP binding and reduce transc riptional activation prior to induction (Krebber et
al., 199 6 , Šiurkus et al., 2010 ). Eve n with this tight arrangement, leaky expres sion was still
visible when traces of lactos e were present in the culti vation medium , as will be se en later for
complex media which have yeast extract as one of their major co nstituents.

Figure 4.4 Isolation of rA1A2A3M fragment from RumA biosynthe sis gene cluster. (a) RumA
gene cluster illustrating gradient PCR amplification of RumA and RumM encoding gene s from purified
genomic DNA of R. gnavus E1. E1 genomic DNA was analyzed on a 0.5 % agarose gel (b) wh ile
gradient PCR amplified rA1A2A3M fragment was analyzed on a 1 % agarose gel (c) . [see method
section 3.5 for m olec ular biology tech niques]

Genomic DNA purified from R. gnavus E1 was us ed as a template to amplify a segment of the
rumA gen e cluster via gradient PCR u sing the ann ealing temperature settings of 55 °C ± 7 °C.
Results show ed that the chromosomal DNA pur ification from R. gnavus E1 was successfu l
(Figure 4.4b). The target rA1A2A3M fragment (3370 bp) was also su ccessfully amplified and
the correc t size was id entified on agarose gel (Figure 4.4c). The resulting PCR amplicon was
inserted into pCTUT7 to yield pLEO r C2 (section 3.5.10.1 ).

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

Figure 4. 5 Growth and protein exp ression. The quasi- linear growth profile s of WLEOrA (a) WLEOrM’
(b) in EnPresso B cultivation medium. (c) SDS-PAGE analysis of HisTrap spin column-purified
extracts from WLEOrM’ strains induced with different IPTG concentrations (indicated at the top of
each well). (d) Comparing His6-RumM expression in extracts from E. coli W3110 (W), Rosetta 2 ( R)
and Rosetta DE3 (R (DE3) ). IMAC (IM ) and gel fil tration (GF) purificat ion of His6- RumM from WLEOrM’.
(e) Soluble (S) and ins oluble (I) fractions of His6-preRumA extracted from WLEOrA. His 6-RumM was
analyzed using 10 % standard SDS-PAGE while His6-preRumA was analyzed with 16 % tricine SDS-
PAGE gel. Protein bands were visualized using the colloidal blue silver Coomassie G-250 as reported
elsewhere (Candiano et al., 2004) [see section3.6 for protein analysis m ethods].

4.2.2 Separate expression of His6-preRumA and His6-RumM
Following p rocedures described in secti on 3.5.11, E. coli W3110 was separately transformed
with pLEO rA (e xpressing His 6-preRumA under control of CTU pro moter) and pLEO r M’
(expressing His6 -RumM under CTU promoter) , yielding strains WLEO rA and WLEO rM’. In a
similar procedure , E. coli Rosetta 2 (DE3) was separately transf ormed with pLEO rM
(expressing His6-RumM under CU promoter) and pRSF - rM (e xpressing His6-RumM under T7
promoter), respectively . The strains were cult ivated in 24 deep -well plates, using the
automated in si tu glucose delivery EnPresso B growth mediu m system (sectio n 3.6 .1) . Varyin g

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

concentrations of IPTG rangi ng from 10 to 500 µM were suppl ied to the cultures. Cells were
further cul tivated for 18 to 24 h . OD 600 of WLEO rA and WLEO rM’ strains were measured and
plotted as shown in Figures 4.5a and 4 .5 b res pectively. Endpoint samples were obtained and
processed using immobilized metal ion affinity chromatography (IMAC) as desc ribed in the
experimental part (section 3.7 .1 ) .
Although the cu ltures all followed a quasi -linear growth profile as expec ted, IPTG concentrati on
only sl ightly decreased the gro wth rate but did not produc e any signific ant effect on the
expression levels of His6-Rum M (Fi gure 4.5c). We investigated if producing RumM in a system
that supplies the tRNAs that encode the rare codons (e,g Rosetta) could improve its yield .
Results in Figure 4. 5d demonstrates an even poorer expressi on in the Rosetta (DE3) system.
However, in Rosetta 2, the express ion level was comparable to W3110 that was original ly
used. We therefore decided to continue with the wild -type E. c oli W3110 strain throughout the
study and only swi tched when the need was critical to establish some fac ts. We further
cultivated the WLEO r M’ strain and purified His6-RumM via IMAC and size excl usion
chromatography (se ction 3.7.2 ). Approximately 0.75 mg l -1 of purified protein was obt ained
after gel filtration ( Figure 4.5d). His6-preRumA expres sed in WLEO rA was large ly present in
the insoluble fraction ( Figure 4. 5e) , suggesting that the his-tagged precu rsor peptide must have
been expresse d in incl usion bodies as reported for other lantipept ide precursors expressed in
the absence of the PTMs enz yme ( Li et al., 2009 , Li et al., 20 10 ).
4.2.3 Two-plasmid coexpression system for His6-preRumA and His6-RumM
The RumM encoding gene was cloned in a vector with a we aker promoter compared to the
vector that hosted the preRumA encoding gene (see secti ons 3.5.10.2 and 3. 5.10.6 for det ails
on how individual His6 -preRumA and His6 -RumM vectors were constructed) . Results fro m
colony PCR and control digests (Figure 4.6a), together with se quencing indi cated that the
genes of interes t were succ essfully clone d in -frame into the ir respective vectors. The
arrangement of various expressi on-relevant features on a plasmid are shown in Figure 4.6 b.
The conc epts that guided the expres sion vectors desig n was adopted to limit physiological and
metabolic stress on the hos t organi sm and enab le high-quality expressi on yield. On the one
hand, RumM is relatively la rge (914 aa) and thus require more cellular resources for its
biosynthesis and a slower process from transcription to translation for the protein to be properl y
folded and soluble. Strong overexpr ession of RumM may result in truncated pro ducts and/or
aggregation which woul d be wasteful . On the other hand, strong overexpression of the
structural peptide may have littl e physiological effect on the host since it is relatively small (47
aa). As such, this strategy may help to conceal the effects of cellular proteases on the small

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

55
peptide th ere by in creasing cellular availability of the unmodified substrate and the chances for
RumM to install PTMs on preRumA.

p L E O r M 2
6 3 5 1 b p
r u m M
L a c O
T 7 p r o m
l a c I
K a n R
6 H i s
S - t a g
R S F o r i
A m p p r o m
T 7 t er m / p r i m e r
T 7 t er m i n a t o r
P s t I ( 2 8 5 4 )
K p n I ( 1 0 3 )

p L E O r A 1
3 9 5 7 b p
CA T s i g n a l
6 x H i s
L i n k e r
Cm R
I n i _ A T G
l a c I
r u m A
p l a c C U
S D T 7
p B R3 2 2 o r i g i n
- 1 0
- 3 5
P s t
I ( 1 5 8 6 )
N h e
I ( 1 4 3 2 )
K p n
I ( 1 1 7 3 )

Figure 4. 6 Cloning of pr eRumA and RumM for coexpression in E. coli. (a ) Colony PCR and control
digest analyses of pLEOrA (1) and PLEOrM (2). (b) A sketch of the plasmid m aps indicating th e main
features.

Figure 4.7 Growth and protein expression test. Varying concentrations IPTG (indicated in the graph
and at the top of each well of the SDS-P AGE) were u sed to in duce protein expression. The graphs in
(a) illustrates the growth of WLEOrA/M strain in EnPresso B m edium system under th e diff erent IPTG
concentrations. (b) Soluble WLEOrA/M lysate extracted from cultures grown under varied IPTG
concentrations, showing expression of His6 -preRumA. The letter P, IMAC-purified His6-preRumA
from WLEOrA/M. (b) Soluble His6-RumM purified from the same c ultures. His6-Rum M was analyze d
using 10 % standard SDS-PAGE wh ile His6 -preRumA was analyzed with 16 % tricine SDS -PAGE
gel. Protein bands were visualized using the blue silver staining protocol. [see section 3.6 for protein
analysis methods]

E. coli W3110 was cotransformed with the expression vectors pLEO rA and pLEO rM , resultin g
in the expre ssion strain named WLEO rA /M . To optimize the per formance of strain WLEO rA/M
in producing both His6-preRumA and His6-RumM, the strain was cultivated in EnPres so B and
processed as described in the previous section. Additional ly, prior to induction, the booster

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

tablet (a complementa ry component of the gro wth syst em) was not added to one hal f of the
overnight EnPresso culture. The other half was boosted, and the cultivation was continued as
described in the experimental part ( section 3.6 .1 ). Growth of c ultures proceeded as expected.
Due to limited growth resources, the non -boos ted cultu res grew very slow and produced end-
point ODs that were approximately 1.5 times less than their boosted co unterparts (Figure 4.7a).
Consequently, the booster table ts were us ed for all subsequent st udies performed with the
EnPresso B growth system.
IPTG influenced growth and His6-preRumA e xpression (Figure 4. 7b) but did not show any
significant effect on the expres sion levels of His6 -Rum M (Figure 4.7c) . Results indicate that
PTMs improve the solubi lity of the precursor peptides (Shi et al., 2011 ). This may explain why
soluble His6-preRumA was present in extracts from WLEO rA/M and not in WLEO rA. Although
we were able to purify His6-preR umA from WLEO rA/M via IMAC (Figure 4.7b), further
purification steps proved to be challenging as the IMAC samples precipitated shortly after
elution . Several different buf fer conditions were tested in an effo rt to prevent the formation of
the preci pitate or to resolubiliz e the m without success. Gels in Fi gures 4 .7b and 4.7c show that
soluble His6 -RumM and His 6-preRumA were indeed expres sed in E. co li W3110. Furthermore,
the SDS-PAGE bands of IMAC-purified His6 -preRumA (lane P, Figur e 4. 7b) and gel filtration -
purified His 6-RumM (lan e GF, Figure 4.5d, section 4 .2.2) were excised and subjected to in -gel
tryptic digestion preceded by an iodoac etic acid derivatizatio n step for identification via MS .
Note shoul d be taken here that the set-up procedures and react ion co nditions of the iodoace tic
acid reaction were the same as those of the iodoacetamide (section 3.9) since both chemicals
share similar physicoc hemical prop erties.
4.2.4 MS identification of products from the two-plasmid system
The extracted gel samples from the previous section were desalted and co ncentrated usin g
ZipTip (section 3.10 .1 ). 5 µl was ap plied to an Orbitrap-coupl ed HPLC system for LC - MS /MS 2
analysis (secti on 3.10.3) . Figure 4.8 shows MS peaks of the four tryptic peptides that were
produced f ollowing co mplete digestion of the His6 -preRumA. Modi fications like S-
carboxymethyl-c ysteine and oxidation of methionine were apparent. The measured masses of
all the tryptic peptides were co nsistent with the calc ulated exact masses . Worthy to note is the
fact that if preRumA from WLEO rA/M was modified by RumM, carboxymet hylation of cysteine s
would be blocked due to the presence of the th ioether cross-bridges formed between cysteine
and Dha/Dhb residues in the core peptide . Additionally, dehydrati on of threonine and serine
residues are acc omplished as a prelude to t he cyclization reaction (Arnison et al., 2013 , Yang
and van der Donk, 2015 , Repka et al., 2017 ). We did not find any evidenc e of dehydration in

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

57
the tryptic pep tides, especiall y in the core fragment, compellin g us to conclude that there was
no modification in the isolated preRumA.

Figure 4.8 Sequence verification of His6-preRumA . Mass spectra and corresponding retention
times (indicated by arrows) of trypsin-digested His6-preRumA obtained from LC - MS analysis. The
mass peaks repr esent t he masses of tryptic His6-preRumA fragments. Modifications such as
oxidation, methylation (CH 2 ) and carbamidom ethyl (cm)-cysteine, corresponding to mass shifts of
+16, +14 and +57 Da respectively are also indicat ed . The calculated exact masses of each peptide
sequences are labelle d in their respective spectra. [se e section 3.10 for mass spectrometri c analysis
methods used]

Although our results in the previous section suggest ed that RumM ma y have facilitated
solubility of His6- preRumA in the WLEO rA/ M strain, MS 2 data obtained by fragment ing the
precursor ions (Fi gure 4.8 ) did not proffer any evidence to suggest tha t the precursor peptid e
was modif ied in the two -vector system, even tho ugh expression of RumM was apparent.
Assignments of product ions produced as a result of fragmenting the tryptic fragments of His6-
preRumA are presente d in Appendix 6.5 . The fragmentation pattern was characteristic of a
linear peptide. The peptide sequence exhibited b- and y-type ions with high intensities which
nicely fitted to all amino acids from N - to C-terminus (Figure 4. 9) . Thi s is unlike lanthi onine -
modified peptide s whose rin g structures cause them to resist fragmentatio n (Müller et al.,
2010 ) . Consequently, there was no ring structure in the preRumA measu red. Th ese resul ts
also supported the outco me of LC -MS scans . Furthermore, subsequent MASC OT searc h of
the MS and MS 2 data from the orb itrap analyzer identified the pre RumA pep tide sequen ce
(UniProt ID: P83674) and Rum M sequence (UniProt I D: Q9L3F1).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

It is challenging to say exactly why preRumA was not modified when coexpressed
simultaneously with RumM although the coexpression fostere d solubi lity of the precursor
peptide . We may just b e tempted to su ggest that there was a simple bi nding interaction
between His 6-preRumA and His6-RumM that facilitated t he solubility of the former. Our
suggestion is su pported by the fact that interactions between nonmodified peptide precursor
and its modifying enzyme have b een reporte d in previous studi es (Lubelski et al., 2009 ,
Khusainov et al., 2011 , Mavaro et al., 2011 , Repka et al., 2017) . Meanwhile Nagao et al. (20 05)
had demonstrated that applying a two -vector co expression system to produce modified His -
tagged lantibiotic in E. coli is a rather laborious venture, Basi-Chipalu and co workers applied
the same procedure to modify pseudomycoic idin in E. co li (Basi-Chipalu et al., 2015). Their
data indicate that functional expression of lanthip eptides in E. coli may vary from p eptide to
peptide, whi ch obviously also involv e several diff ere nt factors in-between. Nevertheless, other
methods have been used to successfully expres s lanth ipeptides in E. coli (Shi et al., 2011 , Shi
et al., 2012 , Tang and van der Donk, 2012 , Kuthning et al., 2015).

Figure 4. 9 Annotated M S 2 spectrum of His6-preRumA . MS 2 - ESI -Orbitrap spectra of gel -extracted
His-tagged RumA precursor peptide, following iodoacetic acid derivatization and tryptic digestion. B-
and y-type ions produced by the c omplete fragmentation of preRumA tryptic digest. Data was
analyzed using xiSpe c V2 [ http://spectrumviewer.org ]

Perhaps protei n misfolding which is common in heterologous E. coli expression and other
unknown factors may be the reason f or the inactivity of the modifying enzyme in the His6 -
preRumA/His6-RumM c ombinatorial system as earlier proposed (Nagao et al., 200 5) .
However, we concluded tha t beside unfavoura ble conditio ns in E. coli which may impair proper
folding of the modifying enzyme leading to loss of activity, the stability of the precursor peptide
may also be crucial in determining the fate of the final pro duct. This is becaus e, by its si ze, the

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

59
expressed precursor peptide may face intracellular bi ochemical challenges that may lead to
aggregation and/or digestio n by host proteases .
4.2.5 PreRumA Fused to GFP
To overcome regular expres sion challe nges, fusion partners like se lf-cleavable carrie rs,
aggregation-promoting and solubility-enh ancing conjugates are us ually employed to pre vent
proteolytic inactivation, conceal lethal effects towards heterologous hosts and promote
production of AMPs in E. coli (Li, 2011 , Bell et al., 2013 , Pane et al., 2016). Our res ults from
the pre vious section, supported by earli er studies, compelled us to posit that the timing
between production of the precursor peptide preRumA and subs equent modification by Rum M
may be cruc ial in determining the fate of the small peptide , s ince it may encounter di verse
physical and bioc hemical challenges before the PTMs are installed . We therefore decided to
provide a physical support to preRumA by fusing GFP to its N -termin us as describ ed earlier
(section 3.5.4.4) . Transformation of E. coli W3110 with the si ngle plasmid pLEO grA, and
cotransformation with pLEO grA an d pLEO rM (see section 3.5.10 for vectors constructio n)
resulted in st rain WLEO grA (expr essing His6-GFP-TEV-preRumA) and strain WLEO grA/M
(expressing both His6-GFP-TEV-preRumA and His6-RumM on separate plas mids) .
Expression of the chimeric fusio n protein was monit ored onl ine in a 24-well flat-bottom plate
via GFP flu orescenc e signal intensities (see section 3.3.3 ). Diffe rent concentrations of IPTG
were tested and results showed an inducer concentra tion - dependent expre ssion (Figur es
4.10a & 4 .10b ).
Cultures of the strai ns WLEO grA and WLEO grA/M were in duced with 100 µM IPTG and time -
course flu orescence signal intensities were measured ( Figure 4. 10c). In all cases, the relative
fluorescent unit (RFU) of the c ontrol strain remained constant throughout the enti re cultivation
period. A dr amatic drop in fluorescence signals of strain WLEO grA/M was obs erved. This was
also reflected in the amount of purified His6 -GFP-TEV-preR umA obtained from this strain in
comparison with WLEO g rA (data not shown). A p ossible reason for thi s may be the metabolic
burden imposed by the presence of two distinct plasmids. Th e large modifying enzyme RumM
may also utilize a su bstantial portion of the availabl e resources in the combina torial system
and hence reduced the expression levels of His6 -GFP-TEV-pre RumA.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 4. 10 Expression of GFP-RumA fus ion constructs. (a) GFP fluorescence signals of WLEOgrA
cultures induced with varying IPT G concentratio ns (b) SDS-PAGE analyses of His6-GFP-TEV-
preRumA purified from respective WLEOgrA cultures. (c) Comparison between the GFP fluorescence
signals of WLEOgrA and WLEOgrA/M strains, cultivated under the same growth conditions . WLEOrM’
was used as a control. (d) UV signals showing Gel filtration purif ication of His6- GFP -TEV-preRum A
from WLEOgrA/M lys ate. (e ) SDS-PAGE analyses of gel filtration-purified His6- GFP - TEV-preRumA
from WLEOgrA lysate (lane Q) and WLEOgrA/M lysate (lane R). (e’) Western blot analyses of gel
filtration-purified His6-GFP-TEV-preRumA from WLEOgrA (lane Q) and WLEOgrA/M (lane R) us ing
anti-Histag antibody and biochemical chromogenic detection method . (f) SDS-PAGE analyses of
IMAC-purified GFP, His6- GFP -TEV-preRumA from WLEOgrA lysate (lane F1), WLEOgrA/M lysate
(lane F2), TEV-digested His6- GFP -TEV-preRumA from WLEOgrA (lane F1’) & TEV -digested His6-
GFP -TEV-preRumA from WLEOgrA/M (lane F2’) [see section 3.6 for protein analysis methods].

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

For larger expression experiments, WLEO grA and WLEO grA/M were cu ltivated in TB medium
as des cribed in the exp erimental part (section 3.6.2) and purified using IMAC and ge l fil tration.
Figure 4.10d sh ows a gel filtration chromatogram represent ing the elution of His6 -GFP-TEV-
preRumA purified from WLEO grA/M lysa te. We obtai ned approx. 100 mg of total protein pe r
litre of WLEO grA culture and app rox. 15 mg l -1 from WLE O grA/M after I MAC and gel filtration
purifications. Results demonstrated dissimilar migrat ion properties of products from the two
systems on SDS-PAGE (Fi gure 4. 10e), clearly indicating that His6 -RumM has an effect on the
His6-GFP-TEV-preRu mA.
Purified His6-GFP-TEV- RumA was d ialyzed in TEV digestion buffer and fresh homemade TEV
from -20 °C (see Appendix 6 .10 for purification results) was added to the sample as described
in section 3.7.3. The digested products tog ether with the non -digested sam ples were ana lyzed
on SDS-PAGE (Figure 4.10f). Interestingly, the expected cleaved pre RumA product ban d was
not obs erved in digested His6 -GFP-TEV-preRu mA purified from WLE O grA as opposed to that
obtained from WLE O grA/M (Figure 4.10f ). One reason for this may be degradation by host
proteases carried over from the IM AC purifi cation sinc e cleavage ma y have exposed the
unmodified peptide to such challenging environment. If the peptid e is modified, protea se
degradation would be unlikely sinc e the presence of PTMs would confer stability onto it (Rink
et al., 2010). However, additional experiments with protease in hibitors did not show any
noticeable improvements (data not shown).
The unequal mobility of His 6-GFP-TEV-preRumA obtai ned from the two syst ems on
SDS_PAGE and the ab sence of a preRumA band in the TEV -digested sample purified from
the system expressing His6-GFP-TEV-pre RumA alone supplie d the first evidence for the
activity of His6-RumM on the GFP fus ion construct. It was h owever not pos sible at th at time to
determine if this was just a simple bindi ng activity or if RumM was actuall y introd ucing the
expected PTMs on preRumA. Additional ly, multi ple bands were visibl e in the SDS-PA GE which
both were f urther co nfirmed via Western blot analysi s ( section 3.6.7 ) to belong to the His6 -
G FP -TEV-preRumA (Figure 4. 10e’) . This may have been produced via host protease
degradation or incomplet e translation during expres sion.
To evaluate the real nature of the issue, a ll four bands in Figure 4. 10e (lanes Q and R) were
excised and processed for i dentification via MS. The y all showed completely di fferent amino
a cid sequence structures with both bands of lane Q and the lower band of la ne R showing
evidences of truncated His6 -GFP-TEV-preRumA. Only the upper band in lane R conta ined full-
length His6 -G FP -TEV-preRumA (data not sh own). We inse rted tandem st op codons in to the
preRumA enc oding gene and a downstrea m λt0 terminator (section 3.5 .10.4 ) to ens ure that

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

these two bands were not the result of read -through translation (Ryoji et al., 198 3) . Since
neither of these pro cedures resolved the issu e, we conc luded that it was not a read - through
translation problem, narrowi ng down the possibilities to degradation only ; where the C-terminal
protease-prone precu rsor peptide were readi ly digested by host pr oteases.
4.2.6 LC - ESI - MS analyses of preRumA from WLEO grA and WLEO grA/M
TEV- dige sted His6 -GFP-TEV-preRumA purified from WL EO grA and WLEO grA/ M were
extracted with butanol ( section 3.7.4 ). The extracts were resolved by SDS -PAGE and results
showed no band for extrac ts from WLEO grA and again, a si ngle band for extract s from
WLEO grA/M (Figure 4. 11 a, lanes E1 and E2 re spectively). The E1 and E2 extrac ts were dried
and re-dissolved in ACN/H 2 O/formic acid (50:50:0.2%). LC- ESI -M S analysis of E1 di d not
produce any charg ed ion correspon ding to unmodifie d preRumA (schematical ly represented
in Figure 4.11b), which further co rroborated results obtained from SDS -PAGE. Major pea ks
resolved in the reversed -phase ch romatogram (Fi gure 4.11c) was analyzed by ESI -MS and
found to contain charged ion correspon ding to truncated preRumA fragments ( Figure 4.11d),
indicating that the peptide may have undergone host protease degradation as suggested in
the previous section.
Interestingly, analysis of extract E2 yielded a mixture of fourfold dehydrated, triple- dehydrated
and non-dehydrated preRumA (Figu re 4.11e). The latter observation suggests that RumM may
have conferred some form of stabilizing role on the precursor peptide sinc e we could not
identify non -dehydrated preRumA in extracts pu rifie d from the strain expressing His6- GFP-
TEV-preRumA alone. This results further supported our suggestion that biophysical
interactions between His 6-pre RumA and His 6-RumM may have fac ilitated the so lubility of the
former without necessarily in troducing t he desired modific atio ns in the peptide (see se ction
4.2.4). Nevertheles s, mixtures of partially dehydrated products are pos sible since
investigations reveal ed that the coupling of cysteinyl thiol to Dha/Dhb via Michael addition
cyclization can prevent further modification of serine and/or threonine residues (Kuipers et al.,
2008 , Lubelski et al., 2009), although the format ion of Dha and Dhb are indep endent of one
another. The peak correspon ding to fourfold dehydrated p reRumA was further zoomed to
reveal its isotop ic distribution (Figure 4.11f). The meas ured exact mass es sh owed very tight
consistencies with t he calc ulated exact masses with an error margin of ± 0.05 Da (s ee Table
3.3 for the accurac y of measurement s expressed in parts per million [ppm]). These results
supplied enough evidence to demonstrate that biosynthesis and modification of RumA is
achievable in E. c oli .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

El vi s_ G F PrAM_ Bu t # 265 R T : 5 . 6 1 AV: 1 NL:
T: F T MS {1 , 1 } + p ESI F u l l ms [ 5 0 0 . 0 0 -2 5 0 0 . 0 0 ]
1060 1080 1100
m/ z
0
20
40
60
80
100
Relat iv e A b undanc e
1069.91
z=5
1073.51
z=5 1083.72
z=5

El vi s_ G F PrAM_ Bu t # 267 R T : 5 . 6 6 AV: 1 NL:
T: F T MS {1 , 1 } + p ESI F u l l ms [ 5 0 0 . 0 0 -2 5 0 0 . 0 0 ]
1069 1070 1071 1072
m/ z
20
40
60
80
100
Relat iv e A b undanc e
1069.91
z=5
1070.31
z=5
1069.71
z=5
1070.51
z=5
1069.51
z=5 1070.71
z=5
1071.11
z=5
1069.31
z=5

El vi s_ G F PrA_ Me C N # 236 R T : 4 . 5 7 AV: 1 NL:
T: F T MS {1 , 1 } + p ESI F u l l ms [ 5 0 0 . 0 0 -2 5 0 0 . 0 0 ]
800 820 840 860 880
m/ z
20
40
60
80
100
Relat iv e A b undanc e
GMRNDVLTLTNPME [Mr=1589.75 Da ]
GMRNDVLTLTNPMEE [Mr=1718.80 Da ]
795.88
z=2
860.40
z=2

R T : 3 . 2 2 - 7 . 8 5
4 5 6 7
T i me (mi n )
0
20
40
60
80
100
Relat iv e A b undanc e
4.57
5.07
5.18 7.68
6.90
4.43 6.37
5.98
3.72
N L : 1 . 2 5 E8
T I C F : F T MS {1 , 1 }
+ p ESI F u l l ms
[ 500. 00- 2500. 00]
MS
El vi s_ G F PrA_ Me C
N

Figure 4. 11 Butanol extraction and LC- ESI -MS. (a) Butanol extracts of TEV-digested His6-GFP-TEV-
preRumA purified from WLEOgrA (lane E1) and WLEOgrA/M (lane E2). (b) The primary structure of
preRumA showing threonine, serine and cysteine side chains that are targeted for modifications by
RumM, as well as Gly-24 which is added to the N-terminus of the peptide following TEV cleava ge. (c)
Reverse-phase chromatog ram indicating a major elution peak (peak M). (d) Mass spectrum showing
charged ions mass peaks obtained from MS scan of peak M. (e) ESI mass spectrum indicating a
mixture of mass peaks associated with the loss of four H 2 O molecules (5344.55 Da), three H 2 O
molecules (5362.55 Da) and non-dehydrated preRumA (5413.60 Da). The calculated exact masses
of the fourfold, threefold and non-dehydrated preRumA were 5344.54, 5362.50 and 5413.55
respectively. (f) Isotopi c distribution of fourfold dehydrated p reRumA peak.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

In this section, we sh ow ed that fus ing preRumA to GFP linked via a TEV cl eavage si te and co-
expressing the chimeric construct simultaneously with the RumA lanthioni ne synthetase RumM
on separ ate plasmids resulted in in vivo modific ations of the preRumA core peptide . Non-PTMs
bacteriocins like enteroc in P and enterocin A have been produced in E. coli using in tein chitin ‐
binding domain as fusion partner (Ingham et al., 2005 , Klo cke et al., 2005 ). Other fusion tag s
like GST, SUMO, MBP and TRX ha ve been su ccessfully us ed to express AMPs in E. coli (Li
et al., 2009 , Li, 2011 , Bell et al., 2013 , Pane et al., 2016 ). Although GST falls within the sa me
size range as GFP, applicati on of the latter as a fusion partner in hetero logous AMP produc tion
is rath er unpopular. Even MBP which has a larger molecular weight compared to both GST
and GFP, has been applied for peptide production in E. coli (Li and Leong, 2011 , Tay et al.,
2011 ) . However, rep orts where GFP has been utilized as a f usion par tner for AMP product ion
are rare in the literature. Additional ly, no study has reported the fusio n of larger protein carriers
to the N-terminus of a lanthipep tide precursor to achieve active E. co li production. This st udy
therefore constitutes the first report of its ki nd for lanthipeptide. Sin ce the modifying enzyme
requires the leader peptide as a docking si te to direct its activity on the core structure, resul ts
here also demonstrate that a larger atta chment to the N -terminus of the leader peptide does
not obstruct in vivo processivity of LanM enzymes in intera cting and modifying the core pep tide.
To the best of our knowledge, this is the fir st time that a lanthipeptide from a strictly anaerobic
Gram-positive micro be is heterologously expre ssed in a Gram - neg ati ve host. Our approach
here differed from earlier studies where either the whole biosy nthes is cluster was isolated and
inserted into a pl asmid and su bsequently expressed in E. coli (Caetano et al., 2011 ), or where
the genes for the struc tural peptide and the modif ying enzymes were constructed in si ngle
polycistronic plas mids (Shi et al ., 2011 , Shi et al., 2012 , Tang and van der Donk, 2012 ,
Kuthning et al., 2015 ) .
M odific ation was not achieved in the combinatorial system expressi ng both His6 -preRumA and
His6-RumM. In co ntrast, a similar system exp ressing His6-GFP-TEV-preRumA and His6-
RumM, modificati on was successful. Since the fusion partner used here exhibits high
expression and solu bility in E. coli , we deduced that GFP offered supportive role to the
precursor pep tide allowing enough time for Ru mM to act. It w as also interesting to observe that
non -dehydrated preRumA was i dentified in extracts obtai ned from strain WLEOgr A/ M , but not
in extrac ts obt ained from WLEOgrA . This obs ervation prompted us to further suggest tha t the
presence of RumM in vi vo foster ed a cooperative molecular interactions with both the leade r
sequence and the core peptide or preRumA, preventing it fro m degradation by host protease.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

65
Another reason for also noticing highe r degradati on of His6 -GFP-TEV-preRu mA in the
combinatorial system may be linked to the a mount of His6 -RumM enzyme produce d by the
weakly expressing vector. The ratio of His 6-RumM to His6-GFP-TEV-pre RumA e xpression
was too low whic h may have not been optimal for complete modif ication of the precursor, thus,
the reaso n why we al so observed a mixture of fully deh ydrated and intermediate products.
However, such drawback may be averted by reen gineering the ho st vector to increase Rum M
expression.
4.3 Quality enhancement, characterization & ac tivation of preRumA
4.3.1 Optimized vector for His6-RumM expression
The chimeric His6 -GFP-TEV-preRumA enc ountered some expres sion challenges as reporte d
in the previous sect ion. We decided to enhanc e rumM expres sion by optimizing the host vector.
To do thi s, we designe d an expression vector named pC UT7 tha t hosted the CU p romoter and
the ribosomal binding site ( RBS ) of gene 10 of bacteriophage T7 (sect ion 3.5 .8 ). Th is vector
was designed to replace the CTU pro moter in pCTUT7 vecto r with CU pro moter because rumM
expression und er the CTU promoter which is consi dered the stron gest in that se ries (Kraft et
al. , 2007 ) was inefficient (section 4.2.2). rumM PRC amplicons were digested and inserted into
pCUT7 vector to yield p LEO rM1 (se e section 3.5 .10.6 ) .
Transformation of E. coli W31 10 with pLEO rM1 res ulted in strai n WLEO rM1 . WLEO r M1
[expressing His6-Rum M under control of the CU _(+T7 RBS) promoter] and WLEO rM ’
[expressing His6 -RumM under control of the CTU _(+T7 RB S) promoter] were grown in TB
medium as described in the method s part ( section 3.6.2 ) exce pt for the fact that the 100 -ml
culture was distributed into 24 -well deep-well pl ate at the point of induction. Varying IPTG
concentrations were applied to one row of the wells and then replicated for the remaining three
rows. ODs of the cultures were measured at different time poi nts. Results in Figures 4.12a and
4.12b show that both strains shared similar growth ch aracteristics. His6 -RumM production was
drastically improved in the WLEO rM1 system (Figur e 4. 12b). Since there was no signi ficant
influence on growth for both plasmids, we can only conclu de that the CU promoter and T7 RBS
combination favoured expressi on of His6 -RumM. There was no evidence of aggregati on in
both systems expressing His 6-Rum M but minor signs of degrada tion were apparent after the
IMAC purification.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 4. 12 Growth and product optimization. Growth curves of WLEOrM1 (a) and WLEOrM’ (b)
cultures induced with varying IPTG concentrations in TB. (b) Expression of His6-RumM in pCTUT7
(WLEOrM’ strain) versus pCUT7 (WLEOrM1 strain). The concentrations of IPTG are indicated in the
plots and at the top of each lane on the gel. The optim al IPTG concentration range is 100-200 µ M.

4.3.2 Expression of RumT peptidase domain
As a general ch aracteristic for class II la nthipeptides, the ABC -type transporter/activator
protein RumT cleaves off the leader peptid e for preRumA after PTMs have been installed and
export the activated RumA out of the cell (Gomez et al., 2002 , Chatterjee et al ., 200 5 ,
Furgerson Ihnken et al ., 200 8 , Bierbaum and Sahl , 2009 , Nishie et al., 201 1) . Here, the
nucleotide sequence encoding the first 125 amino acid residues of the N- terminus of RumT
was codon-optimiz ed for E. coli expressi on and cloned in pCTUT7 and pCTUT7 -SUM O vectors
to yield pLE O rT125 and pLEO srT125 (see sect ion 3.5.10.8 for details ). T he pl asmids were
subsequently us ed for tran sformation of different E. coli strains and tested for expression. His6-
SUMO-TEV-RumT125 was solubly produced while the His6-TEV-RumT125 went into incl usion
bodies (Figure 4. 13a). We resolu bilized and refolded the His6-TEV-RumT 125 incl usion bodies.
All attempts to cleave off the SUMO tag from His6 -SUMO-TEV -RumT125, or the Histag from
resolubilized His6 -TEV-RumT125, usi ng TEV protease were un successful.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

Figure 4.1 3 Expression RumT and activity evaluation. (a) SDS-PAGE showing whole cell soluble
extracts of E. coli W3110 (lane 1) and BL21 (lane 2) expressing pLEOrA, compared with E. coli W3110
(lane i) and BL21 (lane ii) expressing pLEOsrT; as we ll as purified His6-TEV-RumT125 inclusion
bodies (lane P1 ) and purified soluble His6-SUMO-TE V-Ru mT125 (lane P2). The bands repres enting
His6-TEV-RumT125 (~17.13 kDa) and His6 -SUMO-TEV - RumT125 (~29.12 kDa) are indicated by
bold arrows. (b) SDS-PAGE showing purified His6- GFP -TEV- preRumA (lane 1), activity of TEV on
His6-GFP-TEV-preRum A (lan e 2), no activity for purified His6- SUMO-TEV-Rum T125 (lane 3) and
resolubilized/refolded Hi s6- TEV-RumT1 25 (lane 4) on His6-GFP-TEV-preRumA.

We tested both constructs for their ab ility to cleave His6 -GFP-TEV-preRumA at the Gly-Gl y
motif at the C-terminus of the leader peptide of preRumA and there was no evidence of any
activity (Fi gure 4. 13b). Such inactivity as well as non -specificity were also reporte d when a
GST tag was fused t o the N-terminus of Lc tT peptidase domain (Furgerson Ihnke n et al., 2008) .
Here, the activity of the proteas e was evaluated only based on an expected shift in the mobility
of the upper band of His 6-GFP-TEV-pre RumA on SDS-PAGE as demonstrated for TEV in lane
2 (Figure 4.13b). Furt her investigatio ns on this as pect and/or optimization of the co nstructs
were put on hold since the rationale for such experiments did not reflec t much on the focus of
the current study.
The bas is for requesting furt her experiments arise from the fact tha t leader peptide cleavage
by LanT proteins does not have a g eneral mechanism. For instance, lacticin 481 transporter
LctT cleaves both modified and unmodified preLctA indiscriminately (Furgerson Ihnken et al.,
2008 ) while nukacin ISK -1 transp orter NukT is only ac tive in the presence of membrane
vesicles and requi re s modified preNukA to serve as its substrate (Nishie et al., 2009 ). Whereas
a cooperative ac tion between peptidase and ATP bindi ng domains of NukT (section 4 .1.2) are
intricately involved in preNukA leade r peptide processi ng (Nishie et al., 2011 ), the N-terminal
peptid ase domains of LagD, ComA, CvaB and Lc tT (all AMS proteins) do not req uire ATP
binding domain for activity (Havarstei n et al ., 1995 , Wu and Tai, 2004 , Ishii et al., 200 6 ,
Furgerson Ihnken et al., 2008 ) .

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

4.3.3 Plasmid-encoded bicistronic operon
Introducing altern ative leader peptide cleavage sites in preRumA was necessary to act ivate
the peptide in vi tro since expres sion of the peptidas e domain of Ru mT was un successful
(section 4.3.2). TEV, factor Xa, Trypsin and GluC cleavage sites were incorporated into
preRumA via site-directed mutagenesis (section 3.5.9 ). The sequence description s are
illustrated in Figur e 4. 14a.

Figure 4. 14 Site-directed mutagenesis and bicistronic vector construction. (a) preRumA peptide
sequences, showing the various cleavage sites engineered in the peptide via site -directed
mutagenesis and residues targeted for PTMs. Position -1 is indicated by the yellow shading. The
symbols *, represents the Gly-1/Arg pr eRumA mutant construct that will be wide ly used for
subsequent investigations in this work. (b) The arrangement of genes encoding the His6-GFP-TEV-
preRumA* an d His6-RumM on pCUT7 vector. (c) Analysis of His6- GFP -TEV-preRumA* and His6-
RumM extracted from E. co li W3110 and BL21 expressing the bicistronic vector pLEOgrA*M1. The
letter C, co ntrol ex tract from WLEOrA strain (section 4 .2.2). The symbols i, 20 µM; ii, 50 µM; iii, 100
µ M IPTG. (d ) Analysis of purified GFP (lane 1); His6 - GFP -TEV-preRumA encoded by the bici stronic
vector pLEOgrAM in W3 110 (lane 2) and BL21 (lane 3).

To furt her red uce the metaboli c burden impos ed by the presenc e of two distinct plas mids in
the same expressio n host, we decided to cl one the two genes encodi ng the chimeric preRumA

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

69
construct and the modifying enzyme RumM on a single -pl asmid bicistronic expres sion vector.
The plasmids were constructed as described in the experimental part ( se ction 3.5. 10.7). All
plasmids were used to tran sform E. co li W3110 or BL21 and tested for expres sion. All the si x
constructs showed positive production of the desire d product via SDS -PAGE analy sis (data
not shown). One of the E. coli W3110 strains (na med W LEO grA*M1 ) hostin g the bicistronic
pLEO grA*M1 vector, and expressing both His 6-GFP-TE V-preRumA* and His6-RumM,
exhibited impressive protein yields. Note here that the symb ol (*) den otes the variant containing
trypsin cleavage site at position -1 of preRumA which was gen erated by replacing Gly -1 with
Arg. For clarity, this mutant is referred to as preRumA* while the wil d -type remains preRumA
throughout this report.
The bicistronic operon control led by the CTU and CU promoters are arranged as schematically
represented in Fi gure 4.14b. Expres sion test s demonstrated IPTG depe ndent expression of
both targ ets, with very good yields es timated by th e thi ckness of bands on SDS-PAGE (Figure
4. 14c). It is important to not e that the single plasmid -encode d bicistronic system obtained by
cloning the RumM encoding gen e under control of the CTU promoter (i.e. pro moter/rumM
fragment amplified from pLEO r M’ ) di d not resolve the degradation issues associated with His6 -
GFP-TEV-preRumA (Fig ure 4. 14d). However, e xpression results from the pLEO grA*M1 vecto r
system (where rumM is under contro l of the CU promoter) indicated almost complete
eradication of this pro blem. Thus, reports in the following sections will foc us more on the
product of this system.
4.3.4 Purification of His6-GFP-TEV-preRumA* and TEV digestion
Bacterial lysates obtained from 500 - ml WLEOg rA*M 1 or WLEOg rAM cultu re were pur ified via
IMAC. Figur e 4. 15a represents the chromatogram of a single IMAC ru n, indicating a sh arp
elution peak. Elution fractions representing this pea k were subseque ntly colle cted and pooled
together and further purified via gel filtration. The elution chromatograms show ed UV signa ls
at 260 and 280 nm (represent ing protein absorption) and at 484 nm (repr esenting fluoresce nce
absorption ).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

0
2
4
6
0 20 40 60
280 n m
260 n m
484 n m

0
100
200
300
60 70 80 90
2 8 0 nm
2 6 0 nm
4 8 4 nm

0
100
200
60 80 100
2 8 0 n m
2 6 0 n m
4 8 4 n m

Figure 4. 15 Expression and purification of GFP-preRumA fusion constructs. Bacterial lysates were
purified by IMAC followed by size exclusion chrom atography. (a) Chromatogram of IMAC purification
of lysate extracted from WLEOgrA*M1. (b ) Size exclusion chromatogram showing elution of His6 -
RumM (peak A) and His6- GFP -TEV- pr eRumA (peak B) both purified from WLEOgrAM strain. (c) Size
exclusion chromatogram showing elution of His6 -RumM/His6-GF P- TEV-preRum A* complex (peak
A’) and His6 - GFP -TEV- preRum A* (peak B’). (d ) SDS -PAGE analyses of the elution peaks, IMAC
purifications of lysate from WLEOgrA* (1) and WLEOgrAM (2), as well as gel filtration purification of
IMAC extract from WLEOgrA* (lane 3). Letters atop the gel; A, B, A’, B ’ and E are pooled fractions
from peak labels in (a), (b) and (c). (e) SDS-PAGE analysis of individual fractions that constitute the
chromatographic pea k A ’.

The peaks marked “ A ” represented elution of His6 -RumM and peak B represented elution of
His6-GFP-TEV-Ru mA (Figure 4. 15 b). Furthermore, peak A’ represented elution of a complex
mixture of His 6-RumM and His6-GFP -TEV- preRumA* while peak B’ belonged to His6 -GFP-
TEV-preRumA* (Figure 4.15 c). The pooled fractio n co llected for each of these elution peaks
were analyze d vi a SDS- PAGE (section 3.6.5 ) and results cl early indica ted that the system with
enhanced RumM production resolved the degrad ation probl em since product purified from
strain WLEO grA*M1 di d not show multi ple bands corresponding to His6 -GFP-TEV-preRumA*
as did constructs purifi ed from WLE O grA* or W LEO grAM (Figur e 4. 15 d). Overexpressi on of
His6-RumM was also visibly apparent in samples purified from WLEO grA*M1 stra in. The
concentrations of the ch imeric co nstruct purified from various syst ems are di splayed in Tabl e
3.2.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

71
His6-GFP-TEV-preRu mA* was deg raded when produced without or in the presence of lo w
amount of RumM, but not when high amount of RumM w as present. These res ults are very
important si nce they clearly show tha t the presence of sufficient amount of the m odifying
enzyme in vivo st abilizes preRumA fused to the C-terminus of GFP. It shoul d be noted that we
have earlier shown that the deg radation occurs within the preRumA sequence (s ection 4 .2.6).
Results from SDS-PA GE analyses of elu ted fractions collected f rom ch romatographic peak A’
also supported the fact that His6 -GFP-TEV-preRumA* and His6 -RumM were coeluted as a
single co mplex (Figure 4. 15e). It is worthy to note that the amount of His 6-GFP-TEV-preRumA *
stays fairly constant from lane 4 to around lane 9 while the amount of His6 -RumM decrease s.
This observation was further i nvestigat ed by native PAGE (s ee section 4 .3.5).

Table 3 .2 Purification yield s of total protein per litre

Purification
step

His6 -GFP-
TEV-preRum A
(mg)

His6 -GFP-TEV-
preRumA* (mg)

‡ His6 -GFP-
TEV-preRum A*
(mg)

preRumA
(mg)

preRumA*
(mg)

‡ preRumA*
(mg)

IMAC

84.53

97.61

Size exclusion

65.32

39.01

79.40

TEV-digested
IMAC FT

3.23

4.45

6.14

TEV-digested
butanol extract

1.34

2.50

4.75

The symbol ‡ , po oled fracti ons from peaks A’ and B’. This specifically applies to His6 -GFP-TEV-preRumA* which appeared in
two separate chromatograp hic peaks during purifi cation. FT; flow th rough

4.3.5 Interactions between His6-RumM and His6-GFP-TEV-preRumA*
His6-RumM and His6 -GFP-TEV-preRumA * were coeluted together and appeared as a single
peak during size exclusion chromatographic purific ation as shown in the previous sectio n
(section 4.3.4) des pite their enormous size difference (~73 kD a). Samples were obtained from
the co mbined elute d fractions that represente d the chromatographi c peaks and digested with
TEV. Results in Figure 4.16a show that His6- GFP-TEV-preRumA* complexed with His6 -RumM
was only pa rtially susceptible to TEV cleavage while the same construct not complexed with
His6-RumM was readily diges ted during the overnight rea ction. These results sugges ted
possible molecular interactions between His6 -RumM an d the preRumA fused to the C -
terminus of GFP. Most highly, su ch interactions could pos sibly sh ield the cleavage site making
it inaccessible to TEV.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

The prese nce of both digested and undigested products in the TEV cl eavage reaction involving
the His6 -RumM/His6-GFP-TEV- preRumA* co mplex furt her su ggests that the in teractions
between His6 -GFP-TEV-preRum A* and His 6-Rum M may be either cooperative or
noncooperative. Cooperativity here would refer to interactions with both the core peptide and
the leader peptide at the same time. Assuming that RumM interacts noncooperatively with the
core peptide only, the leader peptide whic h is closest to the TEV si te would be free, creating
enough sp ace for TEV to engage. However, if RumM interact s coope ratively with both leader
and core segments, or non cooperatively with the leader pep tide alone , there is a chance that
the TEV site woul d be buried and rendered inacce ssible to the protease . The formation of
complexes bet ween precu rsor peptide and modifying enzymes is well -established in the
literature (Khus ainov et al., 2011 , Mavaro et al., 2011 , Repka et al., 2 017) .

Figure 4. 16 TEV digestion and Native PAGE analysis of His6 -RumM/His6- GFP -TEV -preRumA*
complexes. (a) SDS-PAGE analysis of His6-RumM/His6- GFP -TEV-preRum A* complex (lane 1),
noncomplexed (free) His6- GFP -TEV -preRumA* (lane 2), overnight TEV digest s of complexed His6-
GFP -TEV-preRumA* (lane 3) and free His6 - GFP -TEV- pr eRumA* (lane 4). (b) Native PAGE of purified
His6-GFP-TEV-preRum A from WLEOgrAM strain (lane N1) and complexed His6-GFP-TEV-
preRumA* (lane N2). The latter show s multiple bands above the expected molecular weight of His6-
RumM corresponding to 1 or 2 molecules of His6 -GFP-TEV-p re RumA* co mplexed to one molecule
His6-RumM

A sample from the His6-RumM/His6 -GFP-TEV-preRumA* complex was obtained and run o n
native PA GE emplo ying purified His6 -GFP-TEV- pr eRumA from WLEO grAM strain (with low
RumM expres sion) as a control. Result in Figure 4. 16b shows the usual two ban ds reported
for the latter, while four distinct bands were obtained for the former belonging to His6 -GFP-
TEV-preRumA*, His 6-Rum M and possible His6-RumM/His6 -GFP-TEV-preRumA complexes

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

73
as in dicated on the gel. The res ult further suggest s th at the complex formation may have
occurred between His6 -RumM and dehydrated preRumA* si nce Mavaro et al. (2011)
demonstrated that the interactions between modif ying enzyme and d ehydrated precursor
peptide is much stron g er . It woul d be interesting to determine if this is actually the case with
the His6-RumM/His6 -GFP-TEV-pre RumA* complex.
The aspect of in teractions between modifying enzymes and pre cursor pep tide ha s been
investigated in detail for the prototype lanthipeptide nisin. Biophysical ch aracterization of
purified NisA and NisB indica ted that the leader peptide creates a docking motif that al lows
NisB to bind and interact very strongl y with the unmodified, d ehydrated or mature NisA (Mavaro
et al., 2011) . Cooperative interactions in volving both the core and the leader segments of the
precursor peptide, and the modifying enzyme were previously observed (Khusainov et al.,
2011 ) .
4.3.6 Extraction and nLC- ESI - MS analyses of preRumA*
Having a si mple method to is olate the peptide may fac ilitate characterizat ion of se veral mutant
constructs. In this study, TEV-digested samples were processed via a Ni 2+ -NTA co lumn to
remove the His6-GFP-TEV component and/or extracted with but anol as described in the
methods part ( section 3.7.4 ). The concentrations of the respective extracts and purifi ed
products are presente d in Table 3.2. Dried samples from -20 °C were redissolved in water and
processed for SDS- PAGE analysis. Purified preRumA extr acts are shown in lanes 1 and 2 o f
Figure 4.17a.
Dried samples from -20 °C were e qually resuspended in 0.1 % formic acid and processed
using the C18 pipette tips ( se ction 3.10.1 ) and analyzed by mass sp ectrometry. The Orbitrap
Fusion nLC- ESI -MS analysis ( section 3.10.2 ) of both butanol and IMAC fl ow through extracts,
as well as direct ZipTi p purifi cation of the crude di gest and the aqueous phase of the but anol
extract pro duced the mass spec tra in Figures 4 .17b -e, indicating fourfold deh ydrated
preRumA* (st ructure is schem atically rep resented in Figure 4.17f ). The mass- to -charge (m/z)
ratios of the resulting five times ch arged ion [M - 4H 2 O+5H ] 5+ measured for preRumA* in all
cases were consistent with the calculated m/z of 1089.72 co rresponding to preRumA* including
the loss of four water molecules.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Rum A _Cr ude_S F 80 # 1 R T : 0 . 0 0 AV: 1 NL:
T: F T MS + p N SI si d = 8 0 . 0 0 F u l l ms [ 9 0 0 . 0 0 . . .
1080 1100
m/ z
0
50
100
Relat iv e A b undanc e
1089.72
z=5
1093.32
z=5

R u mA_ BT # 70- 131 R T : 1. 62- 1. 99 AV: 18
T: F T MS + p N SI F u l l ms [ 1 0 8 9 . 0 0 0 0 -1 0 9 1 . . . .
1080 10 90 1100
m/ z
0
50
100
Relat iv e A b undanc e
1089.72
z=5

Rum A _Cr ude_S F 80 # 1 R T : 0 . 0 0 AV: 1
T: F T MS + p N SI si d = 8 0 . 0 0 F u l l ms [ 9 0 0 . . .
1080 109 0 1100
m/ z
0
50
100
Relat iv e A b undanc e
1089.72
z=5
1093.32
z=5

R u mA_ F T _ BT # 147- 182 R T : 2. 41- 3. 02 AV:
T: F T MS + p N SI F u l l ms [ 9 0 0 . 0 0 0 0 -1 2 0 0 . 0 0 0 0 ]
1080 1 090 1100
m/ z
0
50
100
Relat iv e A b undanc e
1089.72
z=5
1092.92
z=5
1085. 75
z = ?

Figure 4. 17 TEV-digested His6- GFP -TEV - preRumA*, extraction of preRumA* and nLC-ESI-MS
analyses. (a) SDS-PAGE analysis of IMAC flow through extract (lane 1) and butanol extract (lane 2)
from purified His6-GFP-TEV-RumA* TEV digest. EIS-MS spec trum of preRumA* extracted from TEV-
digested His6- GFP -TEV-RumA* using butanol (b), ZipTip purification of the aqueous phase of butanol
extract (c), direct ZipTip purification of crude digest (d) and IMAC flow through (e). The calculated
molecular m ass of the unm odified peptide [M +5H] 5+ = 1104.1 2. (f) Schematic structure of preRum A*,
indicating the dehy droamino acids, an extra residue (Gly -24) that is inc luded in the peptide following
cleavage by TEV and A rg that replaced Gly-1 in wild-type p reRumA to enable trypsin cleavage .

We deduced from the mass spectra of samples purified via the different methods that butanol
extraction isolated only the four times dehydrated produc t since traces of three times
dehydrated preRumA* were present in all the other extracts but abs ent in the butanol extract.
Furthermore, applying the ZipTip procedure to pu rify the aqueous phase of the butanol extract
indicated that the organic so lvent extraction isolated only a fraction of the fully modified peptid e
as traces of fourfold and threefold dehydrated pro ducts were al so visibly apparent in the mass

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

75
spectrum when the aqueous phase extract was ana lyzed. The accuracies of measurements
are expressed in [ppm] and recorded in Table 3 .3. These res ults sugge st activity of RumM as
dehydration of Ser/Thr in the co re peptide of preRumA* are detectable . Comparing this dat a
with the litera ture pointed, that these losses occurred at Thr7, Thr16 and Thr22 to yield three
Dhb, and Ser9 to produce Dha as il lustrated in Figure 4 . 17f.

Figure 4. 18 Proposed reaction mechanism through wh ich ruminococcin-A synt hetase or other LanM
enzymes convert Ser/Thr residues in the core peptide into dehydroamino acids via phosphorylat ion
and subsequent phosphate elimination from pSer/pT hr

The scheme di splayed in Figure 4.18 illustrates a suggested mechanism for the react ion
catalyzed by the dehydratase domain of RumM. Accord ing to this scheme, RumM, like oth er
LanM enzymes incl uding LctM, BovM and NukM, requires ATP hydrol ysis to supply phosp hate
for phosphorylation using Mg 2+ as cofactor, followed by an elimination reaction whic h also
utilizes ADP as a cofactor to convert the phosphate esters of Ser/Thr (pSer/p Thr) to Dha/Dh b
(Xie et al., 2004 , Knerr and van der Donk, 2012 , Ma et al., 2014 , Shimafuji et al., 2015 , Repka

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

et al., 2017 ). Earlier investigations revealed that the dehydratas e introd uces pSer/ pThr in to the
core peptid e via a unidi rectional N- to C-terminal catalytic mode (Lee et al., 2009 ), while the
leader peptide plays a determinant role in ensuring catalytic efficiency (Thibodeaux et al.,
2016 ).
Our data sh ows that t he dehydratase domain of Rum M successful ly catalyzed the installatio n
of dehydroamino acids into the core pep tide of preRumA in E. coli . These PTMs were identif i ed
by multiple charged io ns in the mass spectra correspon ding to the molec ular mass of the
precursor peptide, with an equivalent loss of four water molecules. These data supplied
incontestable evidence that in vi vo biosynthesis and modification of the (Gly-1/Arg)p reRumA
mutant is also ac hievable in E. co li . The informati on pro vided herein may then be used to
design in vitro experi ments to characteri ze th e activity of RumM.

Table 3 .3 Predicted and empirical molecular masses measured in the LC - ESI -MS and
Orbitrap Fusion nLC- ESI -MS & MS 2 spectra of in vivo E. coli synthesized
preRumA

Molecular
mass [c]

Chemical
Formula

Exact
mass
calculated

Mass
found

Error
[ppm]

Charge

Wild-type preRum A

C232H369N63O74S6

1083.7143

1083.7201

5.35

M -3H2O

C232H363N63O71S6

1073.5085

1073.512

3.26

M -4H2O

C232H361N63O70S6

1069.9064

1069.9128

5.98

preRumA*

C236H378N66O74S6

1104.1208

M -3H2O

C236H372N66O71S6

1093.3244

1093.3249

0.45

M -4H2O

C236H370N66O70S6

1089.7223

1089.7211

1.10

preRumA* [b-ions]

b3 +

C13H25N6O3S

345.178

345.1775

1.44

b4 +

C17H30N8O5S

459.2131

459.2125

1.30

b5 +

C21H35N9O8S1

574.2401

574.2392

1.56

b6 +

C26H44N10O9S1

673.3085

673.3075

1.48

b7 +

C32H55N11O10S2

786.3925

786.3916

1.14

b8 +

C36H62N12O12S1

887.4402

887.4412

1.12

b9 +

C42H73N13O13S1

1000.5243

1000.5228

1.49

b10 +

C46H80N14O15S1

1101.5717

1101.5712

0.45

b11 +

C50H86N16O17S1

1215.6147

1215.6128

1.56

b12 2+

C55H93N17O18S1

656.8374

656.8366

1.21

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

preRumA* [y-ions]

y- 23 3+

C234H367N65O69S6

179 6.5264

1796.5266

0.11

y° - 22 3+

C229H357N64O67S5

1747.8464

1747.8414

2.86

y° - 21 3+

C223H343N59O67S5

1695.4767

1695.4703

3.77

y- 20 3+

C219H340N58O65S5

1662.7982

1662.7937

2.70

y° - 19 3+

C215H333N57O61S5

1619.12

1619.1201

0.06

y° - 18 3+

C210H324N56O60 S5

1586.0968

1586.0925

2.71

y- 17 3+

C204H315N55O60S5

1553.7383

1553.7401

1.15

y- 16 3+

C200H308N54O58S5

1520.0557

1520.0538

1.24

y- 15 3+

C194H297N53O57S5

1482.361

1482.3607

0.20

y- 14 3+

C190H290N52O55S5

1448.6784

1448.6804

1.38

y- 13 3+

C186H284N50O53S5

1410.664

1410.6622

1.27

y- 12 3+

C181H277N49O52S5

1378.313

1378.3112

1.30

y- 11 5+

C176H268N48O51S4

1334.6329

1334.6313

1.19

y- 10 5+

C171H261N47O48S4

1292.6181

1292.6145

2.78

y-9 5+

C166H254N46O45S4

1248.2709

1248.2687

1.76

y-8 5+

C160H242N44O44S4

1205.5726

1205.5714

0.99

y-7 5+

C155H235N43O41S4

1162.5584

1162.557

1.20

y-6 5+

C149H224N42O40S4

1124.5298

1124.5311

1.15

4.3.7 Mass Spectrometric Fragmentation and MS 2 Analysis of PreRumA*
The processing of the precursor peptide by some class III lanthipeptide biosynthesis enzymes
have been shown to proceed in a C→N -terminal mode (Krawcz yk et al., 2012 , Jungmann et
al., 2014), establishing an in clusive biosynthes is model involv ing pho sphorylation, elimin ation
and cyclization (Ju ngmann et al., 2014 ) . We have demonstrat ed in t he pre vious se ctions
(sections 3.2.6 and 3 .3.5) that the ruminococcin-A lanthionine synthetase RumM is able to
introduce dehydroamino acids into preRumA. However, this data di d not contain information
about cyclization of the pep ti de because bot h the linearly dehydrated pe ptide (with no thioether
rings) and the cycl ized version (with thioether rings) are identical with re spect to their molecular
mass (the y bot h have an average molecular mass of 544 4.27 Da) . Furthermore, if the C -
ter minus of RumM is dysfunctional, accu mulation of dehydrated preRumA would still be
possible as reported for nisin where nisi n cyclase was in activated (Lubelski et al., 2009) . Thus,
it would be pre mature to assume that dat a presenting evidences of dehydration directly indicate
the prese nce of thioethe r rin gs in the precu rsor peptid e. Here, we report the ch aracterization
of modified full -length preRumA* using tandem MS analysi s.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

A combination of CID, HCD and ETD at different frag mentation energies (in Volts) was used
to genera te different fragmentation pat terns (Appendix 6.6). In the CID and HCD spectra, high -
intensity b- and y-type produc t ions were ident ified, that fit ted per fectly to the fir st 19 amino
acid residues of the leader pep tide ( Figure 4.19). With res pect to the primary structure of the
pep tide, no high-intensity b- or y-type ions were easily identified at the C-terminus, associated
with fragmentation of the core peptide. This is reasonable since data from the literature reveal
that the prese nce of cyclic thioether cross-bri dges in the core peptide confer resistance to
fragmentation (Goto et al., 2010 , Meindl et al., 201 0 , Müller et al., 2010 , Sambeth and
Süssmuth, 2011 , Krawczyk et al., 201 2 , Völle r et al., 2012 , Férir et al ., 2013 , Krawczyk et al.,
2013 , V  ller et al., 2013). However, a serie s of intensive produc t ion s that fitted to succ essive
loss of residues from the core peptide were observed ( Fi gure 4.20a).

Rum A _c r ude_f r agm ent at ion_20180117122411 # 331- 1242 R T : 16. 05- 30. 23 AV: 834 NL: 6 . 4 6 E2
T: F T M S + p NS I s id= 80. 00 F ull m s 2 1089. 7200@ hc d18. 00 [ 150. 0000- 2000. 0000]
300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
m/z
0
10
20
30
40
50
60
70
80
90
100
Relat iv e A b undanc e
574.24
786.39
887.44
869.43
673.31
1000.52
358.14
542.28
491.76
444.22
323.16 972.53
843.41
362.20 758.40
298.18 599.30
389.22 945.45
726.31
533.28 656.84
421.22
923.81
803.42 687.32
568.23

Rum A _c r ude_f r agm ent at ion_20180117122411 # 331- 1242 R T : 16. 05- 30. 23 AV: 834 NL: 4 . 4 2 E2
T: F T M S + p NS I s id= 80. 00 F ull m s 2 1089. 7200@ hc d18. 00 [ 150. 0000- 2000. 0000]
1400 1450 1500 1550 1600 1650 1700 1750 1800
m/z
0
10
20
30
40
50
60
70
80
90
100
Relat iv e A b undanc e
1410.66
1810.53
1448.68 1482.36 1657.46
1404.65 1695.47
1443.00 1476.68 1796.53
1388.01
1553.74 1747.84
1520.05 1586.09
1427.66 1460.73 1702.80
1501.70 1787.19
1662.79 1619.12
1686.79
1637.10

Figure 4.19. Tande m MS 2 -experimen ts and product ions assignment for the leader peptide of
preRumA*. Orbitrap Fusion MS 2 spectra representing ion series with intensive peaks produced from
fragmentation of t he N- terminal leader peptide of preRumA*. [M’+5H] 5+ = 1089.7 2.

Note that in labelling the product ion peaks in Figures 4.19 and 4.20 the following notations
were co nsidered: C n denotes the core peptide residue (C) at specific position (n); while C n-n´
denotes core peptide residues from positi on n to positi o n n´ followi ng a C- to N-terminal
direction. No further high-inten sity product ions were identified within the core pep tide. The

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

79
current dat a describes the struc ture of preRumA* sc hematically rep resented as shown in
Figure 4. 20b. Figure 4. 21 describes the structure as sociated with product io ns generated as a
result of the loss of each su ccessive core peptide res idue. The measured molecular mass es
nicely fitted to the struc tures of the fragment presented in the first column.

Rum A _c r ude_f r agm ent at ion_20180117122411 # 516 R T : 19. 17 AV: 1 NL: 1 . 5 6 E3
T: F T M S + p NS I s id= 80. 00 F ull m s 2 1089. 7200@ hc d20. 00 [ 150. 0000- 2000. 0000]
940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080
m/z
0
10
20
30
40
50
60
70
80
90
100
Relat iv e A b undanc e
1037.89 1060.31
1008.28 1086.32
1067.31
1084.12
1034.48
1000.52
1082.91
992.96 1054.91 1080.92
977.07
1004.87
1031.09
945.45 1025.28
997.07
1073.91
1021.28
1012.08
986.27 1043.29
972.53
1049.10
965.80
982.86
959.70
951.46

Rum A _c r ude_f r agm ent at ion_20180117122411 # 460- 539 R T : 18. 41- 19. 51 AV: 70 NL: 9 . 2 7 E2
T: F T M S + p NS I s id= 80. 00 F ull m s 2 1089. 7200@ hc d18. 00 [ 150. 0000- 2000. 0000]
1100 1120 1140 1160 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360
m/z
0
10
20
30
40
50
60
70
80
90
100
Relat iv e A b undanc e
1215.61
1296.86
1260.09
1228.08
1181.56
1101.57 1325.38
1292.61
1255.84
1239.34 1334.63
1177.30
1217.62
1320.88 1361.64
1264.85 1285.61
1160.80 1207.32
1112.02 1246.60 1317.94
1187.62 1355.96
1124.53 1140.29
1304.12

Figure 4. 20 MS 2 -experiments and product ions assignment for preRumA* core peptide. (a) nLC - MS 2
spectra showing intensive ion series resulting from fragmentation of C- terminal core peptide of
preRumA*. [M’+5H] 5+ = 1089.72 RumA*. C n denotes the position of the co re peptide amino acid
residue (e.g. C 21 - 17 denote residues 21 to 17) (b ) Proposed schematic structure of modified preRumA*.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 4. 21 Analysis of fragmented pr oduct ions obtained from C-terminal core peptide of preRumA*.
This figure describes the possible cross- linked structu res that gave rise to the resulting ions id entified
in the mass spectrum. The ch arges and measurement precisions in parts per million [ppm] are also
indicated.

To further expatiate on th e structural description of the various frag ments that gave rise to the
intensive product ion peaks identified in the mass sp ectrum, different structures were proposed
of which those presented in Figure 4.21 fitted precise ly to the measured m/z. Notice that each
structure represent an internal double cleavage of the polypeptide backbone from the C -
terminus tha t resul ted in product ions asso ciated with the successive loss of amino acids in the

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

81
direction indicated by the arrow. Any loss of an intern al res idue sh ould aut omatically res ult in
b- and/or y-type ion(s) or other associated ion types li ke a, c, x and z. T his was not the case
observed with the fragmentation of C-termin al preRumA *. Not even the x- and z-type which
are observed during de novo peptid e seq uenci ng were identified. Instead, the dou ble cleavage
fragments hosted residues present upstream and downstream of the cleavage positi on. This
is only possible if the re is a bond linking the C -terminal fragment to the N-termin al segment.
The presence of cysteine or Dhb/Dha residue adjacent to these cleavage pos itions allowed us
to allocate thioether cross -linkages. Moreover, Figur e 4.21 shows details of how the various
structures were associated to their respective molecular masses , with very low mass error s (in
ppm).
Whereas the present data are consistent with those obtained by Dabard and coworkers
(Dabard et al., 2001 ), our findings further indicated the presence of a third MeLan ring formed
between Thr7 and C ys12. It is worth noting that Dabard et al. (2001) co ncluded in their st udy
that the formation of thioether cross -bridge between these res idues was not possible. Their
conclusion was however, bas ed on Edman degr adation assay which has some limitations in
studying the structure of lanth ipeptides since it is blocked by Lan /MeLan rings as well as
Dhb/Dha resi dues, and no seque nce information can be obtained thereafter (Lohans and
Vederas, 201 4). We confiden tly state tha t the final struc ture determined h erein represents an
improved struc ture of modified ruminococcin -A precurs or pep tide because the third MeLan ring
identified in this study also co nstitutes the mersaci din -lipid II- binding motif which appears to be
common in all class II lanthipept ides (Knerr and van der Don k, 2012 ).
The RumM-ca talyzed Michael addition cyclisation reactio ns may follo w the scheme proposed
in Figur e 4.22. The scheme is simila r to the mechanism propos ed elsewhere for NisC (Li et al.,
2006 ). Gene rally, the re is an overwhelmingl y low sequence identity between constituent family
members of lanthionine -generating enzymes. However, the C-termin us of Rum M po ssesses
conserved ac tive site residues which have been ident ified in other lanthionine cycl ases to be
critically impli cated in th e ca talytic process (section 4 .1.1 ). Hence, by inference , there exis t
possible structure-function similarities and closely similar mode of action as discussed
elsewhere in the litera ture (Knerr and van d er Donk, 2012 , Repka et al., 2017 ).
The scheme illu strates that dedic ated active si te residues of the cyclas e domain of RumM
coordinates a hydrated Zn 2+ cofac tor whose water molecul e is displaced by the cystei nyl thiol
(Cys12) which is targ eted for conjugation with Dhb 7 in the core peptide of pre RumA. As a
result, the thiol group by itsel f becomes activate d, creating a nucleophilic active center. The
displacement of the water molecule or the activation of the cysteinyl thiol may be facilitated b y
His778 ( Figure 4.1, section 4 .1.1) which probably plays the role of an acid/base catalyst like

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

the Tyr304 in SpaC ( Helfrich et al., 2007). The electrophilic carbon atom of the Dhb7 now
launches an attack on the activated Cys12, forming an enolate intermediate which is
subsequently protonated to g enera te the thioether cross bridge.

Rum A _Cr ude_S F 80 # 1 R T : 0 . 0 0 AV: 1 NL:
T: F T MS + p N SI si d = 8 0 . 0 0 F u l l ms [ 9 0 0 . 0 0 0 0 -1 . . .
1080 1100
m/ z
0
20
40
60
80
100
Relat iv e A b undanc e
1089.72
z=5
1093.32
z=5

Figure 4. 22 Proposed mechanism of Michael-type addition reaction catalyzed by RumM and catalytic
efficiency. (a) The C-terminus of this bifunctional enzyme pos sesses the conserved residues that have
been shown to coordinate Zn 2+ ion in the active site of some lanthionine cyclase. The scheme
illustrates the form ation of the first N-terminal lanthionine ring of RumA. (b ) Comparison between the
old and the new enhanced system with respect to pr oducts intermediates formed. Note that the
differences in masses arise from the fact the new old system expressed the wild -type while the
enhanced system expressed the Gly-1/Arg mutant.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

83
The WLEO grA*M1 strain did not only resol ve the degradation issues observed with the
chimeric GFP fusion co nstruct, but it also improved yields of the des ired product s drastically.
In addition, onl y traces of triple -dehydrated pre RumA* inter mediate w ere obs erved in the mass
spectrum, and no unmodifi ed product as was the case in the WLEO grA/M system (Figure
4.22b) . Although reports indicate that the formation of thioe ther cross -linkage may circ umvent
the dehydratas e from further co nverting Thr/Ser to Dhb/Dha (Kuipers et al., 2008 , Lubelski et
al., 2009), we cannot at this time report whether t he intermediate produc ts obs erved contained
at leas t one of the rings or not. Nevertheless , the data presented here show that low levels of
RumM may have been responsibl e for the inefficienc y of the WLEO grA/ M system.
Results from the tandem M S chara cterization show that RumM catal yzes the deh ydration of
Thr/Ser residues in the core pep tide of preRumA into dehyd roamino ac ids, and subsequently
conjugate the Dhb/Dh a resid ues with the su lfhydryl groups of specific cysteine side chains to
produce a modified version of preRumA containing thre e MeLan/Lan cross-linkages and an
additional D hb . The dehydratase reactions involve Thr7, Ser9, Thr16 and Thr2 2 while the
cyclase reactions in volve the co njugation of the following pair of residues; Dhb7 /Cys12,
Dhb22/Cys24 and Dha9/Cys23 via Mic hael-type addition cyclization reactions.
4.3.8 Alkyl derivatization and trypsin digestion of preRumA*
Naturally, trypsin does not cl eave at positi on 6 of the core pep tide due to modification on the
adjacent Thr7 (Dabard et al., 2001 , Gomez et al., 200 2 , Gomez et al., 2002 ) . The PTMs on
Thr7 render the lysi ne residue inaccessible to trypsin . It was on this basis that we selected
trypsin to serve as an alternative leader peptide pro cessing strategy. Prior to trypsin cleavage,
pr eRumA* was fir st derivatized with iodoacetamide (see secti on 3.9) . The alkylating agent
reacts irreversibly with the sulfhydryl group of the cysteine side ch ain ac cording to the scheme
illustrated in Figure 4. 23 a . Assuming one cysteine side chain were to be involved in this
reaction, t he mass spectrum of the derivatized sample would show an alkylated species with
an overall mass shift of +57 Da corresp onding to the alkyl der ivative. Therefo re, it was expected
that if preRumA* did not contain any Lan/MeLan rings, al l three cysteines in the core peptid e
would have bee n free to engage in the alkylation reaction. Interestingly, we were u nable to
identify charged ions asso ciated with either single, double or triple alkylation of fu ll -length
preRumA* and the vari ous tryptic peptid e fragments, including the matu re RumA labelle d in
Figure 4. 23b. Note that a ll the three cyst eine res idues in preRumA* p eptide sequence are
present in the core peptide. Thus, it was expected tha t the alkyl modifications would occur in
thi s segment of the peptide but results plainly displayed no ev idence to suggest that there were
any alkyl derivatives in the sample.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Figure 4. 23 Alkyl derivatization and trypsin digestion of preRumA*. (a) Scheme showing how
iodoacetamide react with thiol group of cysteine side chain. (b) Schematic re presentation of preRumA*
showing five different fragments expected from the tryptic digestion. (c) Charged ions observed in
nLC - ESI -MS spectrum, representing th e var ious trypti c fragments of preRumA*. The pred icted exact
masses for each of the fragments are labelled in their respective spectra. (d) Tandem MS ex periments
and assignment of trypsin-activated RumA fragment ion peaks. Major product ions in the spectrum
were identified t o fit successive loss of Gln18 , Trp17 and Dhb16. [ M’ +3H] 3+ = 892.40*.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

85
These outcomes su pplemented results from the previous section by supplying mor e data t o
corroborate the fact that all cysteine resid ues in the core peptide were involved in la nthionine
cross-bridge formation. This also further verifies the activity of RumM in catalyzing PTMs
installation in preRumA* sinc e one could accurately state that the cysteinyl thiols were
inaccessible to the io doacetamide derivatiza tion becaus e they were al ready blocke d by
thioether cross - lin kages gen erated dur ing the cyclization step of the bi osynthesis pathway .
Furthermore, when the same experiments were performed with un modifie d His 6-preRumA
(section 4 .2.4), all the cysteine residues were S -carboxymet hylated (Figur e 4.9).
Results from trypsin diges tion also supported what Dabard and colleagues already reporte d
with regard s to cleavage at Lys6. The mass sp ectrum of the trypsin -digested sample indicated
charged ion species correspondi ng to the various tryptic peptides (Figure 4. 23 c). We were
unable to identify fragments resulting from cleavage at Lys6, indicating that this position was
not acce ssible to cleavage by trypsi n as modif ications at Thr7 forbid this. Fragmentation of the
mature core peptide (RumA) precursor ion yielded a fragmentatio n spectrum with very little
information due to low concentration . Nevertheless , we also deduced that the MS 2 spectrum
contained characteristic peaks that were identical to th ose of preRumA* (Figur e 4.23d ).
So far, we h ave shown that ruminococc in-A lanthioni ne syntheta se RumM can ca talyze in vivo
installation of PTMs including 2 MeLan rings , one Lan ring and an α,β -didehydrobutyrin e into
preRumA when the two proteins are coproduced simultaneousl y in E. coli . The data here
demonstrat es that a larger fus ion partner to the N-terminus of the leader peptide does not
interfere with in vivo processi vity of RumM in catalyzing both the deh ydratase and cyclizati on
reactions. We also determined that the fully modified peptide host three thioether cross -bridges
and possessed a sp ecific lipid II -binding motif that is common to most class II peptides. This
study, to the best of our knowledge reports the first su ccessful het erologous production of fully
modified lanthipep tide originally isolated from a strictly anaerobic Gram-pos itive microbe, in a
Gram-negative host. With reg ards to product titre, we report here a total yield of appro ximately
6 mg of cycl ic-modified pr eRumA p er litre of E. coli culture. This may reduc e to about 1-2 mg
of pure active Ru mA per litre of cultu re when the leader peptide is removed si nce bot h the
leader peptide and the core pep tide have almost the same si ze with respect to the number o f
amino acid res idues in their sequence s. This yield is very important as it overwhelmingl y
exceeds that obtained earlier (Dabard et al., 2001) in the order of app roximately 10 4 .
4.4 Bioassay analysis of trypsin activated RumA
Biological assays were assembled as describ ed in the experimental part ( section 3.11 ). 50 µl
of the tryptic digest of preRumA* contain ing the active RumA (sectio n 4 .3.8) was obtained and

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

dispensed into a punched well initiall y created in an agar plate sp read with Bacil lus su btilis
ATCC 663 3. The set - up was su bsequently incubated overnight at 30 °C. Results in Figure
4.24 a shows a distinct zone of inhibition , suggest ing that the trypsin-digested sample contai ned
a growth inhibitory component with activity again st B. subtilis ATCC 663 3 . In this case RumA
was the only possibl e suspect .

Figure 4. 24 In vitro a ctivation of preRumA* and bioassa y. (a) Mass spectrum showing prom inent ion
peak corresponding to active RumA purified from the t rypsin digest of cyclized preRumA* (section
4.3.8), as well as the activity of the resulting product (b) SDS-PAGE analysis of IMAC-purifie d modified
(lane 1) and non -modified (lan e3); as well as trypsin-digested modified (lane 2) and non-modified
(lane 4) His6-SUMO-preRum A*. (c) The bioactivity of modified and non- modified trypsin digests
against B. subtilis ATCC 6633 grown on a culture plate. (d ) The effect of active Ru mA on B. su btilis
ATCC 6633 liquid culture.

PreRumA* was also fused to a SUMO tag as d escribed in the experi mental part ( section
3.5 .10.5). The resulting plasmids (pLEO srA* and pLEO srA* M ) were used to transformed E.
coli W3110 and the resulting strains were cultivated in 100 ml TB medium. Purified products
from both systems were digested with trypsin to yield a mixture of peptide fragments including

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

87
the putative mature RumA. One half of the digested products was used for SDS -PAGE (Figure
4. 24b) while 50 µl of the other half was pipetted into punched wells in an agar pl ate spread
with B. subtilis ATCC 6633 . Following an overnight incubat ion at 30 °C, a distinct zone of
inhibition was obs erved for digested His6 -SUMO-preRumA* purified from the system
expressing pLEO srA*M but no activity was apparent for extracts purified from the pLEO srA*
expressing host (Figure 4.24c), suggesting that only the modi fied product has growth inhibitory
activity. These results show that preR umA fused to the C -termin us of SUMO was also modifie d
by RumM a s does the GFP fusion construc ts, indicating that SUMO tag may al so be used as
fusion partner inst ead of GFP. Alth ough we did not per form MS characteriza tion of products
derived from the SUMO-fused constructs, the growth in hibitory ac tivity demonstrated by the
modified construct and none for the non -modif ied one allowed us to draw a positive conclus ion
with regards to the funct ionality of the system.
The ability of the tryptic digest of His6 -SUMO-pr eRumA* to inhibit growth of B. subtilis ATCC
6633 in liquid cultures was also evaluated by monitoring the DOTs of di fferent cultures online
using the PreSens OxoDish OD24. B. subti lis ATCC 6633 was grown to OD 600 of ~0.5 and then
distributed into the 24 wells of the OxoDish. Ampicilli n, modified and non -modified tryptic
digests of His 6-SUMO-preRumA * w ere add ed to the wells in f our replicates and the DOT of
the respective wells were measured over time. Results show that the modified extract exerted
a si gnificant overall effect on growth while the non -modified construct fairly followed the DOT
profile of the cultu res without any supp lement ( Figure 4.24d). In t he positive co ntrol, where the
culture was su pplemented with ampici llin, the DOT fairly r emained constant throughout the
cultivation, indica ting no cell growth. Thi s data su ggests a simple method to screen for
biological activ ity of several active compounds or RumA mutants online. This strategy may
reduce the experimentati on time and enable eas y identification of interesting c andidates.
We ca nnot say much abo ut the mechanis m of action of RumA at this time, but b inding to lipid
II and inhibiting cell wall biosynthesis (Götz et al., 2014 ), inhibition of spore pro liferation (Gut
et al., 2011), formati on of por es in the phospholipid membrane s (Breukink et al ., 1999 , Has per
et al., 2004 , Hasper et a l., 2006) and membrane disr uption (Bakhtiary e t al., 2017) are some
of the general mechanisms of the antimicrobial activity of lanthipep tides. Furthermore, RumA
also possess a distinct lipid II -binding motif which has bee n shown in other class II lantibiotics
to interact with the cell wall precu rsor with out form ing pores in the membrane (Islam et al.,
2012 ) but r ather induce intens e cell wall stress responses in their targets (Sass et a l., 2008).

4.5 Microtiter plate cultivations

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

4.5.1 Colony screening in microtiter plate using GFP as a reporter
Besides co lony PCR te chnique (section 3.5 .4.3), online GFP fluorescence signals were also
used to identify pos itive cl ones. In this proce dure, single colonies were select ed from LB agar
plate containing E. coli Top10 transformed with pLEO grA*M1 and used for inoculat ing 96 -well
microtiter plate conta ining LB medium supplem ented with the appropria te antibiotics. These
experiments were performed using the Hamilton Microla b STAR Liqui d - handling station as
described in the method s par t (section 3.6.3) . The same amount of IPTG was automatica lly
supplied to all well s at the point of induction , approximately 9 h after cu ltivation start ed . Res ults
indicated that not all co lonies produced GFP fluoresc ence ( see Appe ndix 6 .7 ), meaning that
some of the strains growing on the agar plate did not hab our the correct vector or perhaps the
gene of inte rest was wrongly inserted in th e vector.

Figure 4. 26 Online GFP fluorescence and ODs of WLEOrA*M1 cultivated in 96-well microtiter plate.
(a) GFP fluorescence curves representi ng six of the culture s in the microtiter plat e (b) Corresp onding
online OD curves of the cu ltures.

Since dat a from reaction kinetics studies of lanthipeptide synthetases revealed that these
enzymes require enough time to catalyze PTMs formati on in their substrate (Thibodeaux et al.,
2014 ), having information about the stabili ty of the substrate over the culti vation time may help
to estimate the optimal duration of cultivation. We further selected the st rain tha t demonstrated
the most pr ominent ch aracteristics in terms of g rowth and GFP fluore scence (s ee Appendi x
6.7, Figure A8) and used it for further experime nts. The pLEO grA* M1 pl asmid was purified
from this strain, sequenced and further transferred into E. coli W31 10. In a similar proce dure
like the colony id entification experiment, we isolate d single colonies from LB agar plate
containing the WLEO grA*M1 culture and inoc ulated a 96 -well microtiter plate. Results showed

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

89
that all colonies exhibited similar growth and fluorescence emission characteristics (see
Appendix 6.7, Figur e A9). Six of the 96 cultures are pre sented in Figure 4.26. One of the
WLEO grA*M1 cultures was then randomly se lected and us ed for subsequent experiments
which have been descr ibed in the previous sections

Figure 4. 27 Performance of WLEOgrA*M1 in TB medium. (a) Growth of TB medium cultures of
WLEOgrA*M1 induced with vary ing concentrations of IPTG. (e) GFP fluorescence signals of
corresponding cu ltures. Concentratio ns of IPTG are indicated in the plots. (c) SDS-PAGE analysis o f
samples extracted from the cultures. IPTG concentrations are labelled at the top of eac h gel lane. For
the ODs and fluorescen ce measurem ents, the cultures were set up in three replicates.

4.5.2 Cultivation of WLEO grA*M1 in 24-w ell plate using T B medium
Microcultivations were per formed in 24 -well microtiter plate with varying IPTG concentrations
( 20 to 1000 µM). The gro wth of WLEO grA*M1 show ed sl ight but co nsistent responses to IPTG
concentrations (Figure 4. 27a) , rec ording a final OD 600 betwe en 13 and 15 for al l the cultu res .
The influence of IPTG on product formation (estimated by GFP fluorescenc e signal) was not
uniform (Figure 4.27b). For instance, higher production rates were observed in cultu res
induced with intermediate IPTG concentrations in the ra nge 200 - 400 µM, while cultures
induced with higher IPTG concentrations li ke 500-1000 µM generated production rates

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

comparable to those induced in the lower concentration range (20 -100 µM IPTG). Thus, there
wa s no direct correlation between growth and fluoresc ence intensities measured for the
different cultures .
His6-RumM and His6-GFP-TEV-preRumA* were highly produced (Figure 4. 27c). No
degradation of His6 -GFP-TEV-preRumA* was observed. We deduc ed from the multi -scale
parallel sc reening that the co ncentration of IPTG necessary f or optimal growth and expressi on
lies between 50 and 100 µM estimated by the thicknes s of bands on SDS -PAGE.
4.6 Strain optimization using multicultiv ation & scre ening strategies
4.6.1 Characteristics of the bicistronic and two-vector systems
The strains WLEO grA M (expressing the bicistronic vector pLEO grAM ) and WLEOg rA/ M
(habouring pLEO grA a nd pLEO rM ) were cultivated in LB and the enzyme-based fed -batch
EnPresso B medium. ODs and GFP fluore scence signals were measured online. Resul ts
indicated quite similar growth and production charac teristics for all the cultures gro wn in the
complex medium, with obvious and noticeable effects associated with variations in IPTG
concentrations (Fi gures 4 .28a and 4 .28b). Alth ough the WLEO grA/M grew si gnificantly, GFP
fluorescence measurem ent remained comparative ly low despite the fact that it was producing
both His6-GFP-TEV-pre RumA and His 6-RumM li ke the WLEO grA M system. The EnPresso
cultivation system produc ed a quasi-linear growth characteristic for all cultures . Whereas
intensive fluorescence signals were rec orded for the WLEO grAM strain, almost n o
fluorescence signal was noti ceable with the WLEO grA/ M until about 8 h after induction (Figures
4.28c and 4 .28d).
The resul ts reported here in dicate that the lo wer GFP fluorescenc e observed in the two -
plasmid system ma y be attributed to metabolic burden incurred by hosting the two pl asmids,
and not dependent on the cultivation medium. Furthermore, production seems to rea ch
maximum earlier in the complex medium cultures than in the defined EnPresso medium. This
is unders tandable becaus e co mplex media are deficient in divalent ca tions w hich are
necessary t o maintain membrane stability and cells grown in them are therefore susceptible to
lysis (Wee and Wilkinson, 1988 ). These cations especially Mg 2+ have very low conte nt in LB
broth: 30 to 40 μM (Papp-Wallace and Maguire, 2008) and we canno t rule out the possibility
that this may contribute to the early stationary phase observed with E. coli growth in LB
cultures. Furthermore, E. coli grown in complex media is associated with alterati ons in c arbon
nutrition in which the easi er- to -uti lize ones get depleted easily and the cul tured organism then

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

91
switches to the difficult - to -uti lize carbon sources (Sez onov et al., 2007 ). The se observations
and the fact that the medium composition vary from batch - to -batch make LB broth very
unreliable to obtain reproducible parameters in a m iniature cultivation set-up that are amenable
to large-scale pro duction.

Figure 4.28 Cultivation of WLEOgrAM and WLEOgrA/M in different media conditions. (a) Online GFP
fluorescence signals measured in LB cultures. (b) Online ODs measured during LB cultivation. (c)
Fluorescence signals measured in EnPresso B cu ltures. (d) Online ODs m easured during EnPresso
cultivation.

To investigate the reproducibility of the EnPresso cultures, we applied different glucos e
releasing rates by supplying varying amoun ts of reagent A to the cu ltures at the point of
induction, without boosting. Panels (a) to (d) of Figure 4.29 clear ly demon strate that supplying
an excess of gluc ose to the culture is detrimental to product formation. M ost es pecially, when
the amount of the reagent A was more than double the usual recommended amount of 0.8
units, production began to decline just about 12 h after induction and the fin al RFUs of the
cultures decreased to about half the maximum attai ned during the entire period of cultivation.
Moreover, the final pH of the cultures displayed a dec reasing trend with increasing amount of

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

reagent A add ed (Figure 4.29e); meanwhi le the final OD 600 increased as the amount of reagent
A added to the culture also increased ( Figure 4.29f).

Figure 4. 29 Effects of different glucose-releasing rates on the production of His6- GFP -TEV-preRumA
by WLEOgrAM and WLEOgrA/M. Two separate IPTG-induction concentrations were used for each
culture supplied with 0.8 unit (a), 1.2 units (b), 1.6 units (c) and 2 units (d) of reagent A, at the point
of induction (No boosting). The final pH and OD 600 of the cu ltures were measured and plotted in (e)
and (f) respectively. Value s in brackets represent µM IPTG concentrations.

We also noticed tha t although the fluorescence si gnals produced by WLEO grA M re mained
exceedingly higher in comparison to those produced by WLEO grA/ M , the final ODs of cultures
growing the latter strai n were more than 1.5 times higher than those growing the for mer. These
outcomes indicated that the fluorescence signals were not dependent on the optical density of
the culture as al ready suggested by res ults fr om TB mediu m cu ltures (section 3 .6.2) . Whereas
reagent A appeared to cause only slight changes in the overall growth, its influenc e on the fin al
pH of the cultures were rather si gnificant. These aspects are expla ined further in the next
section .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

93
4.6.2 Optimization of WLEO grA*M1 using process-relevant parameters
To evaluate the behavior of WLEO grA*M1 under process-rel evant cond itions, parallelized
microtiter plates were used to screen process-relevant parameters like di ssolved oxygen
tension (DOT) and pH . In this case, the EnPres so B culti vation system was applied to study
how DOT and pH may vary durin g a standard fermentati on process. The WLEO grA*M1 strain
was first cultivated in a PreSens shake flask containing an oxy gen and a pH sens or integrated
into the polycarbonate material at the bot tom of the flask. The first 14 h of cultivation indicated
a typical growth profile of E. coli (Fig ure 4. 30a) . During the fir st 5 h of cultivation, the cell s took
up free glucose in the EnPresso medium (free glucose is derived from nonspecific degradation
of the complex pol ymer component of EnPresso) and exhibit ed ch aracteristics that were typical
for a batch cultivation, demonstrated by the constant drop in DOT until about 40 %. Within 2 h
after this drop, the DOT rapidly increased to 75 % where it remained at a stable level. This
rapid transition indicat ed a switch to gluc ose-limited growth. The pH decreased slowly
through out the cultivation period.
After 14 h of cultivation, the cu lture was spli t into t hree 24 -well microtiter plate containing
varying conce ntrations of IPTG and reagent A. Measurements were performed as described
in the method part (see section 3.12 ). Plate 1 was used to monitor the DOT , plate 2 (24-well
deep -well pl ate) to monit or pH chang es and plate 3 to measur e GFP fluores cence signals over
time. The optical densities of cultures in the first row of plate 2 were also measured. At the st art
of plate measu rements, all 24 cu ltures were nearly in an ana erobic st ate of growth, with initial
DOT values around 0 %. This rapidly changed as DOT increased to about 25 -40 % dependi ng
on the amount of reagent A that each of the cultures received ( Fi gure 4.30a). This transie nt
change occurred bec ause there was no aeration when the culture was di stributed into the wells
of the pl ates, but aeration resumed once the plates were placed in to the shak er. It is worthy to
note that bet ween 14 and 20 h, growth was rapid due to ex cess gluc ose present, supplied by
the additional reagent A ( Figures 4 .30a and 4 .30b ). It seems that between 19.5 and 27 h mos t
of the cultures experienced transient alteratio ns between b atch and glucos e-limited growth
before attaining a steady D OT level. T he pH was contro lled by nitrogenous sources in th e
booster tablets added to the culture prior to splittin g in the plates. However, the pH profil es
shown in Figure 4. 30 c indicated slight decreases with increasing amount of reagent A but
maintaining the phys iological range more tightly than the non -boosted cultures reporte d earlier
(section 4 .7.1) .

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

0
50
100
6 . 6
6 . 7
6 . 8
6 . 9
0 10 20 30
C2
D2
A3
B3
C3
D3
C5
D5
A6
B6
C6
D6
pH
A1
B1
C1
D1
A2
B2
A4
B4
C4
D4
A5
B5

6.5
7
7.5
8
0 10 20

0
5
10
15
20
0 10 20 30 40
2.8 U Reag en t A
2.4 U
2.0 U
1.6 U
1.2 U
0.8 U

Figure 4. 30 Screening ferm entation-relevant parameters. (a) DOT of cultures before and after
inductions, as well as pH before inducti on (red line). (b) Growth of cultures in the first row of the 24 -
well plate induced with 20 µ M IPTG (see Figure A10, Appendix 6.7) (c) pH of cu ltures aft er
boosting/induction.

Whereas there appeared to be no significant difference in the behaviour of the different cultures
with respect to DOT, culture in well D6 whic h received maximum amoun t of rea gent A and the
highest concentration of IPTG app eared to di splay a pronounced deviation ( blue curve in
Figure 4.30a). We also notic ed that the pH of thi s par ticular cultu re was most of the time
relatively lo w compared to the oth ers during the cultivation (blue curve in Figure 4.30c) . It is
worth menti oning that increasin g the amount of reagent A consequently increases the rate of
glucose release into the m edium and excess of the rea gent also su pplies excess glucose to
the culture , causing acetate accumulatio n.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

2
3
4
5
0 10 20
A1
A2
A3
A4
A5
A6

Figure 4. 31 Production optimization. The left panel indi ca tes online GFP fluorescence
measurements of WLEOgrA*M1 stra in grown in the 24-well microtiter plate. Each graph represents
a row on the plate and the well numbers ar e indicated in the graphs. The concentration of reagent A
(in Units) added to eac h well of a row from left to right, are labelled in the top-left graph . The IPTG
concentrations added to the wells in each row are indicated by the double arrows in between the
graphs and the SDS gels. The right panel shows the SDS -PAGE analyses of corresponding His6-
RumM ( r1 ) and His6- GFP -TEV-preRum A ( r2 ) extracts obtained at the end of the cultivation. See
Figure A10 in t he Appendix for reagent A concentrations ad ded to each well.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

Furthermore, applying high concentrations of IPTG to induce a recombinant gene strongly
inhibits E. coli ’s glucose upt ake capaciti es and t heir abi lities to res pire optimally le ading to
formation and accumulation of acetate (Neubauer et al., 200 3). Accumulation of excess
acetate may in hibit gro wth of E. coli (Roe et al., 2002 , Neubauer et al., 200 3), even though the
organism may later oxi dize the organic acid as an alternative ca rbon so urce. Acetate
accumulation may explai n why the pH of the cultures decreased with increasing concentration
of reagent A (Figur e 4.30a). These res ults further corrobor ated those reported in se cti on 4 .7.1.
Examining the GFP fluorescenc e signals recorded for all the 24 cultivation conditions, we could
easily identify that product formation was greatly influ enced by the IPTG co ncentration. The
trend is visible as one navigate s vertically through the wells in eac h column from A to D as
depicted in the left panel of Figure 4.31. Conversely, increasing the concentra tion of reagent
A res ulted in a significant decrease in fluorescence signals starting 10 h after induction.
Extracts from all the wells were purifi ed with HisTrap spin colu mns and ana lyzed via SD S -
PAGE. Resul ts show that the productio n levels of targets increased with increasing IPTG
concentrations (right panel, Figure 4. 31). The inducer concentration increases vertically from
the gel at the t op to the bottom. Interestingly, we also observed the same trend on SDS -PAGE
as for the measured GFP fl uorescence signals.
The final pro duct extracted from t he wells demonstrated higher produc tion levels if more
reagent A was added but excess amount of th e reagent (like in colu mn 6 of the plate ) instead
negatively affec ted expressi on especiall y in cult ures where the co ncentration of the induc er
w as lo w (rows A and B: 1 st and 2 nd gels from top-right panel). However, we took notice of the
fact that some of t he end GFP flu orescence signals do not di rectly correlate to the final amount
of products (es timated by the thickness of bands on SDS -PAGE). Therefore, in vivo GFP
fluorescence may be used to qualitatively determine expressi on but may not be applied as a
q uantitative method to assess product formation. Nevertheless, it would be interesting to
determine produc t formation at the point where the fluorescence signals begin to dec line
(around 18 h) to asc ertain if the same conclusion can s till be drawn in tha t situation.
Results here demonst rate tha t supplying excess glucose to the EnPres so B culture increases
growth rate and cons equently produces higher biomas s yields, but the same procedure ha s
contrary effect s with regards to target protein expressi on levels . If the flu orescence resul ts
were to be taken as a standard, then culti vation should be stopped after 10 h in duction since
product for matio n begin s to decline. However, t his would imply produc ing a mixture of partially
modified product since the modifying enzyme may require long er time to catalyze formation of
PTMs (Thibodeaux et al., 2014 , Repka et al., 2017). Our res ults show that an IPT G

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

97
concentration of 100 µM in combination with up to 1.6 units of reagent A (i.e. double amount
of the recommended stand ard) add ed to the cultures at the point of induction/boo sting is safe
to ensure opt imal production of His6-GFP-TEV-Ru mA* and Hs6-RumM using st rain
WLEO grA*M1 . Our data also present growth and pro duction characteris tics which may be
easily adapted to larger scale culti vations.

4.7 Over all discussion of results
In this work, we isolated the biosynthetic machinery of the lanthipeptide ruminococcin -A from
the natura l pro ducer and heterologously reconstituted the pathway in E. coli. We successfully
expressed and modified the peptide as a ch imeric fusion product togeth er with GFP. F using
the structural gene for the precursor pep tide to the gene encoding GFP and co -expressing the
chimeric construct simultaneously with the dedicated lanthionine synthetase RumM did not
alter the formation of thioet her cross-bridges. This was surprising since installation of PTMs in
the core peptide by RumM requires the N-terminal leader sequence as a docking site to direct
its a ct ivity. One may expec t that emplo ying a larger fusion partner as such would influence the
activity of the enzyme bu t this app eare not to be the case. We obtained fully modified peptide
possessing thioether bridges and an additional dehydroamino acid residue. The results of this
study have been submitted and is currently under consideration for publication (Ongey et al.,
2018 ) . This study, to the best of our knowle dge, is the first reported case il lustrating that the
precursor peptide can still be correctly modi fied with su ch a large fusion partner to the leader
sequence. The data reported herei n supplies an al ternative experimental design to gain more
insights in to mechanistic events that drive the generation of MeLan/Lan rings as well as
dehydroamino acid s in l anthipeptides.
One of the main objec tives of thi s study was to improve the production strategy thereb y
enabling further biotechnological development and engineering possibilities. The current
system recorded imme nse performance in the regard. The amoun t of modified precu rsor
peptide obtained was 6 mg per litre of E. coli culture. Upon removing the leader peptide and
subsequent purifi cation of the active RumA, we e xpect to rec ord a yield ranging between 1 and
2 mg of pur e product, taking into consideration the size ratio of the leader- to -core segment
(~1:1). This product concentratio n surpas ses the amount of RumA initi ally purified from the
natural source by several thousand -fold. Daba rd et al. (20 01) only succeeded in achieving a
yield of 0.665 micro gram rum inococcin -A per litre of R. gnavus E1 cultu re (Dabard et al., 2001 ) .
Perhaps the nature of R. gnavus E1 with respect to growth and optimization contri buted largely
to this outcome. It is improbable that regul ating cultivation parameters and engineering

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

cultivation media would pro duce a meaningf ul improvement. Ass uming that would be the case,
the overall production cost would by skyrocketed by additional expenses incurred by
supplementing the c ultivation medium with trypsi n and hemin .
Be ing able to produce the pep tide now in E. coli which is simple, easy to manage and have a
fast generation time, we expect that transferri ng the current system to proce ss scale production
would require less time and efforts. It may be w orthwhile consid ering other approaches like
performing f urther optimizati on in cluding strain engineer ing or employi ng the use of smaller
fusion partners to achieve higher production yields like other systems reported in the literature
(Shi et al., 2011 , Shi et al., 2012 , Tang and van der Donk, 2012 , Kuthning et al., 2015).
Employing a single -plasmid trici stronic operon to si multaneously express preRumA, Rum M
and RumT, may enable biosynthes is and secretion of the peptide. Of co urse this would reduce
some steps in the purification proced ure that may co nsequently produce a positive impac t on
the yield. Our study did not include such approach because we were unab le to express RumT
separately. Neverthe less, it is possible that if there exist a biosynthesis complex (involving al l
three components) like the one proposed herein, it may mask the toxicity of RumT, allo wing
the peptide to be pro duced and exported. However, for the purpose of studying peptide
engineering and LanM catalysis, the current system represents a sufficient tool. Additional ly,
expanding and/or increasing the antimic robial activity of Ru mA or other related compound s
may be eas ier to apply with the present system. An exampl e of such approach includes the
use of amber stop codon su ppression to incorporate non -canonic al amino acid s into the
peptide (Steiner et al., 2008 , Chatterjee et al., 2012 , Oldach et al ., 2012).
With respect to structural ch aracterization of modified preRum A* , data prese nted herein are
consistent with those obtained by Dabard and coworkers, with the excep tion of one additional
methyllanthionine rin g between Thr7 and Cys12 whic h was never reported. In fact, Dabard et
al. (2001) concl uded in their study that the MeLan ring formation between these two res idues
was not possi ble. Neverthele ss, they arrived at this conclusion using i nformation derived fro m
Edman degr adation assay. However, the Edman sequenc ing method is not very accurate in
studying the structure of lanthipeptides because of limitations associated with the fact tha t any
modification at the N-terminus of the peptide sequence will block the experiment. Additio nally,
Edman degrad ation is bloc ked by Lan/MeLan rings as well as Dhb/Dha residues, and no
sequence information can be obtained therea fter (Lohans and Vederas, 201 4 ). We ma y
conclude with certainty that the structu re described herei n represents the actual structure of
modified ruminococc in-A precursor peptid e because the third ring identi fied herein also
constitutes the core active feature referred to as the mersacidin -lipid II- binding motif which is
common in all c lass II lanthipeptides (Knerr and van der Don k, 2012 ).

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

99
This implies that in E. coli , the dedicate d ruminoco ccin -A lanthio nine synthetase Rum M is
capable of catalytically installing three thioether cross - linkages and one add itional α, β -
unsaturated amino acid into the core structure of pre RumA. T he dehydratas e domain of RumM
catalyzes the dehydration of Thr7, Thr16 and Thr2 2 to three Dhb, and Ser 9 to Dha.
Subsequently, the cyclase domai n engages in a Michael -type addition -cyclisation reactions
involving Dhb7 and Dhb22, and activated thiol groups of Cys12 and Cys24 to produc e two
MeLan ring s, while the Dha9 and Cys 23 produc e a Lan ring. We have pr oposed mechani sms
for RumM- catalyzed reactions which may be studied further. For instance, uti lizing
m utagenesis studies in conjunction with m ass spectrometry and/or bioassay analyses may be
employed to characteri ze the two domain s of RumM. Additionally, the crystal st ructure of the
full-length enzyme may be more resourceful.
In this study, we initi ally considered using two plasmids to separately control biosynthesis of
the precursor peptide preRumA and the modifying enzyme RumM. We d esigned the plasmids
so that rumM expressi on was co ntrolled by a weaker CU promoter while r umA was under
control of the stronger CTU pro moter . This strategy was conceived to limit metaboli c burden
and to increase cellular availability of the unmodified preRumA substrate for RumM to catalyze
PTMs formation . However, this approac h failed to produce the desired modifications in
preRumA. Although just recently, Basi-Chipalu a nd coworkers emplo yed a similar approach to
modify pseudomycoicidin in E. coli (Basi -Chipalu et al., 2015 ), we cannot say exactly why
preRumA w as not modified when His6 -preRum A was co expressed simultaneously with His6 -
RumM. We ant icipate that the enhanced solubility (an d no modification) of preRumA in the
WLEO rA/M strai n was facilita ted by simple binding intera ctions between H is6 -preRumA and
His6-RumM since interactions between nonmodified peptide precursor and its modifying
enzyme hav e been reported (Lub elski et al., 2009 , Khusainov et al., 2011 , Mavaro et al., 2011 ,
Repka et al., 2017 ).
Furthermore, a mixture of modified a nd non-modified preRumA were identified in extract s
obtained from strain WLEO grA/M (expressi ng His 6-GFP-TEV-pre RumA and His6-RuM on
separate plasmids), and non-modified preRumA was not appare nt in extract from WLEO grA
(expressing His6-GFP-TEV- preRumA alone ), but rather truncated products of preRumA.
These outcomes also obl iged the reasoning that RumM may have f ostered some form of
molecular intera ctions that in turn conferred a stabili zing role on the precu rsor peptid e since it
is believed that the enz yme forms a complex with the prec ursor peptide (Lubelski et al., 2008 ,
Lubelski et al., 2009 ) . Exis ting data also sugge st a constant in teraction with both the non -
modified and the modified su bstrate (Yang and van der Donk, 2015 ) . Thi s Resul t further
support our su ggestion that biophysic al interactions between His6 -preRumA and His6-RumM
may have fac ilitated the solu bility of the former without necessarily introducing the des ired

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

100

modifications in the peptide. Neverthe less, mixtures of partially dehydrated products are
possible since investigations revealed that the coupling of cysteinyl thiol to D ha/Dhb via
Michael addition cyclization can prevent further modific ation of serine and/or threonine
residues (Kuipers et al., 2008 , Lubelski et al., 2009 ) . However, we canno t say whether the
intermediate products observed contained at least one of the rings or not. Nevertheless, the
data prese nted here sh ow that low levels of RumM may have been respon sible f or the
inefficiency of the WLEO grA/M system.
The amount of enzyme produc ed by the weak ly expressing vecto r may not be su fficient for
complete modificati on of the precursor pep tide, whic h may explain why unmodified preRumA
was identified in extract s from th e str ain WLEO gr A/M . This appeared to be the case as this
drawback was mitigate d by reengineering the host vector to favour adequa te pro duction of
RumM. It is important to note tha t applying the single -plasmid bici stronic expression vector
system did not improve the quali ty of His6 -GFP-TEV-preRu mA when RumM was expressed
under co ntrol of the CU promoter and RBS from the lactose operon. However, by replacing the
RBS from th e lactos e operon with that of gen e 10 of bacteriophage T7, RumM expres sion was
enhanced and the degradation problem was solved. In fact the WLEO grA*M1 strain produc ed
only minute traces of three-fol d dehydrated peptide and no unmodified peptide compared to
WLEO grA/M . Therefore, it is important to ensure adequate expression of the modifying
enzyme wh en trying to develop a combinatorial system for the production of lanthipeptides in
E. coli.
The strong interactions observed between His6 -GFP-TEV-preRumA * and His6 -RumM may be
further investigated to supply more insights into the nature of comple xing. I t ma y be wo rthwhile
to crystallize the complex to pro vide data required to ch aracterize these complexes, wh ich may
reveal some struc tural features in LanM enz ymes yet to be known. Our data suggests that
dehydrated preRumA* may be predominant in the His6-GFP-TEV-preRumA* / His 6-RumM
complex since anothe r report showed that the interact ions bet ween modifying enzyme and
dehydrated precursor peptide is much stronger (Mavaro et al., 2011 ). This may be true since
a LanM enzyme was shown to ca talyze reversible openi ng o f the thioether ring (Yang and van
der Donk, 201 5) and thus, RumM may constantly su pply the dehydrated precursor, and
subsequently eng ages the free substrat e to form complexes.
Lastly, our optimiz ation experiments pro duced results to demonstrate that supplying excess of
glucose to E. coli cultures expres sing the ch imeric GFP fus ion product simultaneously with the
modifying enzyme, may produce a positive re sponse on growth, but constitute counter effects
with regards to targ et protein expressi on levels. Furtherm ore , our data in dicated that strength

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

101
of GFP fluorescenc e signal may not be employed as a tool to determine when to end the
cultivation as the signals start decreasi ng at a point when it is not expected that full
modifications must have oc curred in the preR umA. Additionally, our results indicated an
optimal IPTG concentration in the range of 100 -200 µM for expression of His 6-GFP-TEV-
RumA* and Hs6-RumM using the strain WLEO grA*M1 . Thes e data may be easily adapted to
larger scale cultivation s.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

102

5. Conclusions and outlook
5.1 Conclusions
We successfully constructed the biosynthesis pathway of ruminococcin -A in E. coli and showed
for the first time that fusin g the precursor pep tide to a large r ca rrier like GFP and subs equent
coexpression with the lanthioni ne synthetase resulted in high -level in vivo production of the
active co mpound. Sin ce the modifying enz yme requires the leade r pep tide as a docki ng site to
direct its activity on the core structure, our results demon strate that a larger attachment to the
N-terminus of the leader sequenc e does not hamper the activity of the lanthioni ne -generating
enzymes. Interac tions between the modifying enzyme and the GFP fusion cons truct yielded
some insights into the mechanis m of RumM catalysis . No rep ort exist tha t des cribes the fus ion
of large protein carriers t o the N -terminus of the leader peptide and so the current system ma y
be hel pful in providing further mech anistic insights on the modific ation machinery. Our data
further indicated that the fully modi fied compound possess a total of three thioether cross -
bridges (two MeLan and one Lan) instead of two as previous ly reported. The third r ing identified
in this study actually constitutes the mersaci din -lipid II-binding motif whic h is common in all
class II lanthipeptides.
Sequence – structure – function relationship studies showed tha t lanth ionine -generating
enzymes of class II lanthipeptides share distinct domai n homologies within their respective
family members , with the C-terminal domain of RumM sharing >60 % similarity with the cl ass
I nisin cyclase. In the abs ence of RumM, the precu rsor peptide preRumA was largely
expressed as insol uble pr oduct but regai ned its solubility when it was coexpressed
simultaneously with RumM. However, modificati ons were observed only in preRumA
expressed as a fusi on partner to a larger protein tag and not to His-tag alone.
Microscale cultivations enabled us to determine the optimal growth and expressi on conditions
that were eas ily ada pted to larger scale cultivations. We attained a product yield of
approximately 6 mg of c yclized preRumA per litre of E. coli culture. This yield corres ponds to
the modifie d pre curs or pep tide and we estimate d that upo n removing th e leader peptid e and
subsequent purification of the active product, this amount may reduce to about 1 -2 mg of pure
and active RumA per litre of culture . This is because the leader peptide and the core peptid e
share a ratio of 1:1 with respect to their average molecular weights. The yield reported here
overwhelmingly excee ds values reported earlier by 10 4 order. Alterna tive strategies were used
to remove th e leader peptide and consequently activating preRumA. Gro wth inhibi tory activity

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

103
of trypsin- activated Ru mA against B. subtilis ATCC 6633 was achieved, suggesting tha t the
modified product poss ess biological activity.
The hetero logous production system developed here in, offers irrefutable advantages over R.
gn avus E1 (native host) since scale-up st udies and reaching econo mically feasible production
are practicabl e. Compl icated and expensive experimental se t -ups are not required .
Furthermore, this work opens up pro spective studies in the area of peptide engineer ing aimed
at expand ing and/or increasing the antimic robial activity of RumA or generating novel
compounds with enhan ced biological functions.
5.2 Outlook
The system herein may be exploited as a new generatio nal app roach to identify and
characterize new peptide variants with improved therapeutic effici ency via peptide eng ineering.
This is possible by c ombining sp ecific mutati ons in the peptide together with the pro miscuous
catalytic properties of the class II lanthionine -generating enzyme RumM. O ther tai loring
modifications li ke the N-gl ycosylation and non-canoni cal amino ac ids may also hel p to in crease
stability and enh ance biological functions by providin g a bro ad di stribution of chemical
functionalities that would strength en interactions with biological targ et components. For
example, specific residues in the core peptide preRumA ma y be replaced by unnatu ral amino
acids like those containing α -haloacetamide via genetic code engineering to allow expansio n
of the chemical reactivity space and broaden the activity spectrum of the peptide.
This study also opens avenue s for large- scale studies and to inve stigate the mode of action of
RumA since we now have the peptid e in an easy- to -cultivate host and preliminary asse ssment
of the production level seems very encouraging. Some vecto rs that were developed during this
work were not fully characterized e.g. pLE O rA TEV M1 and pLEO srA Xa M1 . It would be interesting
to further evaluate these constructs containing TEV or Factor Xa sit es at position -1 of
preRumA in order to evolve with a system tha t would facilitate purifi cation of the active peptide.
We have suggested a coupl e of mechanisms like those involving the dehydratase and cyclase
domains of RumM that require empirical evaluation by conduct ing the appropriate experiments.
Additionally, we postulate that the biosynthesis and export of RumA in R. gnavus E1 requir e
the synergi stic involvement of RumM and Rum T fun ctioning as a complex machinery durin g
the maturation process. Adeq uate information is suppl ied that may be used to design in vivo
and in vitro experiments to evaluate these concepts. Further optimization, expression and
characterization of the peptidase domain of RumT would be important to add to the growing
list of char acterized class II LanTs. Additiona lly, engineerin g a polycis tronic expression vector

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

104

that simultaneously exp resses preRumA, RumM and Ru mT may foster in vivo expression and
secretion of active RumA tha t may easily be purifi ed from the culture broth.

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

105
6. Appendix
6.1 Structure-function analyses, description of the biosynthesis
operons, cla ssification and ph ysicochemical chara cteristics of
randomly selected bacteriocins

D y s g a l a c t i c i n
3 0 4 3 b p

A F 2 4 2 3 6 7
3 3 4 1 b p

D Q 1 9 8 0 8 8
7 3 1 2 b p

S S U 6 6 8 8 3
5 4 1 1 b p

F J 9 3 8 0 3 6
4 9 1 8 b p

Figure S1. 1 Schematic organization of biosynthesis gene clusters involved in production
bacteriocins (drawn to scale). Bacteriocins structural genes, red; modification genes, blue & gre en;
maturation and transporter genes, dark grey; light green, immunity genes, yellow; accessor y/s ecretory
or other immunity 0g enes, brown; genes with unknown f unctions, black.

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

106

Table S 1 Sources and physicochemical characteristics of selected examples of different classes of
bacteriocins

Class of
bacteriocin

Example

Native producer

AA
residues

MW (kDa)

Reference

Ia
(Lanthipeptides)

Elgicins

Paenibacillus elgii
B69

4.5 – 4.8

(Teng et al., 2012)

Nisin

Lactococcus lac tis

3.4

(Field et al., 2015)

BacCH91

Staphylococcus
aureus CH-91 DSM
26258

2.07

(Wladyka et al.,
2013 )

NAI- 107

Microbispora sp.

2.2

(Castiglione et al.,
2008 )

Haloduracin

Bacillus halodurans
C- 125

28; 30

2.3; 3.0

(McClerren et al.,
2006 )

Pseudomycoicidin

Bacillus
pseudomycoides
DSM 12442

2.7

(Basi-Chipalu et al .,
2015 )

Ruminococc in -A

Ruminococcus
gnavus E1

2.6

(Dabard et al., 2001)

Ib
Circular peptides

Amylocyclicin

Bacillus
amyloliquefaciens
FZB42

6.3

(Scholz et al., 2014)

Circularin A

C. beijerinckii ATCC
25752

7.6

(Kemperman et al.,
2003 )

Enterocin 4

E. faecalis

7.1

(Joosten et al., 1996 )

Butyrivibriocin AR10

Butyrivibrio
fibrisolvens

6.0

(Kalmokoff et al.,
2003 )

Subtilosin

B. subtilis

3.4

(Babasaki et al.,
1985 )

Uberolysin

S. uberis

7.0

(Wirawan et al.,
2007 )

Garvicin ML

L. garvieae

6.0

(Borrero et al., 2011)

Leucocyclicin Q

Leuconostoc
mesenteroides
TK41401

6.1

(Masuda et al.,
2011 )

Carnocyclin A

Carnobacterium
maltaromaticum
UAL307

5.8

(Martin-Visscher et
al., 2008)

Gassericin A

Lb. gasseri

5.6

(Kawai et al., 1998 ,
Kawai et al., 2001 )

AS - 48

E. faecalis

7.1

(Gálvez et al., 1986)

Ent53B

E. faecium

6.3

(Himeno et al., 2015)

Plantazolicin A

B. amyloliquefaciens
FZB42

1.3

(Scholz et al., 2011)

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

107

Ic
LAPs

Microcin B17

3.1

(Baquero and
Moreno, 1984)

Streptolysin S

Group A
streptococcus (GAS)

2.7

(Nizet et al., 2000)

azolemycins A – D

Streptomyces sp.
FXJ1.264

0.68

(Liu et al., 2015)

Goadsporin

Streptomyces sp.
TP -A0584

1.6

(Igarashi et al.,
2001 )

Id
Sactibiotics

Hyicin 4244

Staphylococcus
hyicus 4244

<10

(de Souza Duarte et
al., 2017)

Subtilosin A

B. subtilis 168

3.3

(Babasaki et al.,
1985 )

Thuricin CD

B. thuringiensis DPC
6431

30 & 31

2.7, 2.8

( Rea et al., 2010 )

Thurincin H

Bacillus thuringiensi s
SF361

3.1

( Lee et al., 2009 )

Propionicin F

P. freudenrechii

4.3

(Brede et al., 2004 )

Ie
Glyocins

Glycocin F

Lactobacillus
plantarum KW 30

5.2

(Stepper et al., 2011 )

Thurandacins A & B

Bacillus thuringiensi s
serovar
andalousiensis
BGSC 4AW1

~10.5 –
11.0

(Zwick et al., 2012 ,
Wang et al., 2013)

enterocin F4-9

Enterococcus
faecalis F4-9

5.5

(Maky et al., 2015 )

Sublancin 168

B. subtilis

3.8

(Paik et al., 1998 ,
Garcia De Gonzalo
et al., 2014)

If
Lasso peptides

capistruin

Burkholderia
thailandensis E264

2.04

(Knappe et al., 2008)

Sviceucin

Streptomyces
sviceus ATCC 20983

2.08

(Kersten et al., 2011 ,
Li et al., 2015)

Lassomycin

Lentzea
kentuckyensis sp

1.8

(Gavrish et al., 2014)

Microcin j 25

Escherichia coli

2.14

(Bayro et al., 2003)

cattlecin

Streptomyces
cattleya

2.68

(Sugai et al., 2017)

IIa
Pediocin-lik e
peptides

Pediocin PA-1

Pediococcus
acidilactici

4.6

(Chikindas et al.,
1993 )

Enterocin A

Enterococcus
faecium,

4.8

(Aymerich et al.,
1996 )

Bacteriocin T8

Enterococcus
faecium T8,

4.2

(De Kwaadsteniet et
al., 2006)

Bacteriocin 31

Enterococcus
faecalis

4.9

(Tomita et al., 1996)

Enterocin P

Enterococcus
faecium

4.6

(Cintas et al., 1997)

Leucocin A-UAL 187

Leuconostoc
gelidum.

3.9

(Hastings et al.,
1991 )

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

108

Maltaricin CPN

Carnobacterium
maltaromaticum CPN

4.4

(Hammi et al., 2016)

Mesentericin Y105

Le uconostoc
mesenteroides

2.5 – 3.0

(Héchard et al.,
1992 )

Enterocin HF

Enterococcus
faecium M3K31

4.3

(Arbulu et al., 2015)

Sakacin P

Lb sake Lb706

4.4

(Tichaczek et al.,
1992 )

Curvacin A

Lb curvatus
LTH1174

38 - 41

4.3

(Tichaczek et al.,
1992 )

IIb
Two-peptide
bacteriocins

Lactococcin G

Lc lactis

α;39
β;35

4.3
4.1

(Nissen-Meyer et al.,
1992 )

Plantaricin EF

Lb plantarum C11

PlnE;32
PlnF;34

3.5
3.7

(Diep et al., 1996 ,
Ekblad et al., 2016)

Thermophilin 13

Streptococcus
thermophilus

ThmA;62
ThmB;43

5.7
3.9

(Marciset et al.,
1997 )

Plantaricin JK

PlnJ;25
PlnK;32

2.9
3.5

(Diep et al., 1996)

Enterocin C

E. faecalis C901

α;39
β;35

4.2
3.8

(Maldonado-
Barragán et al.,
2009 )

lactococcin Q

Lc. lactis

α;39
β;35

4.2
4.0

(Zendo et al., 2006)

Enterocin X

E. faecium KU- B5

α;40
β;37

4.4
4.0

( Hu et al., 2010 )

Carnobacteriocin X Y

Carnobacteria

X; 33

3.5

(Acedo et al., 2017)

IIc
Leaderless
bacteriocins

Aureocin A53

Staphylococcus
aureus A53

6.0

(Netz et al., 2002)

Aureocin A70

Staph. aureus

AurA: 31
Aur B: 30
AurC: 31
AurD:31

2.92
2.7
2.95
3.0

(Netz et al., 2001)

Enterocin L50

E. faecium

L50A: 44
L50B: 43

5.19
5.17

(Cintas et al., 1998)

Enterocin Q

3.9

(Cintas et al., 2000)

Lacticin Q

Lc. lactis
Lc. lactis

5.9

(Fujita et al., 2007)

LsbB

3.4

(Gajic et al., 2003)

Garvieacin Q

L. garvieae BCC
43578

5.3

(Tosukhowong et al.,
2012 )

Enterocin EJ97

E. faecalis

5.3

(Gálvez et al., 1998)

Garvicin A

L. garvieae

4.6

(Maldonado-
Barragán et al.,
2013 )

Enterocin K1

E faecium
EnGen0026

(Ovchinnikov et al.,
2017 )

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

109

IId
Non -pediocin-lik e
single peptides

Laterosporulin

Brevibacillus sp.
strain GI- 9.

5.6

(Singh et al., 2012 ,
Singh et al., 2015 )

Lactococcin A

Lactococcus lac tis
subsp. cremoris LMG
213 0

5.7

(Holo et al., 1991)

Lactococcin 972

Lactococcus lac tis
IPLA 972

7.5

(Mart ı  nez et al.,
1999 )

Lactococcin B

Lactococcus lac tis
subsp. cremoris 9B4,

5.3

(van Belkum et al.,
1992 )

Enterocin B

Enterncoccus
faeciurn T I 36

5.4

(Casaus et al., 1997 )

Microcin V

E. coli

8.7

(Håvarstein et al.,
1994 )

Epidermicin NI01

Staphylococcus
epidermidis strain
224

6.0

(Sandiford and
Upton, 2012 )

Lacticin Q

Lactococcus lac tis
QU 5

5.9

(Fujita et al., 2007)

II I
Large heat-labile
bacteriocins

Lytic

Enterolysin A

E. faecalis LMG
2333

316

34.5

(Nilsen et al., 2003)

Zoocin A

Streptococcus equi
subsp.
zooepidemicus

262

27.9

(Simmonds et al.,
1997 )

Millericin B

Streptococcus mill eri
NMSCC 061

259

28.

(Beukes et al., 2000)

Lysostaphin

Staphylococcus sp.

246

~27

(Schindler and
Schuhardt, 1964 ,
Bastos et al., 2010)

Non-lytic

Dysgalacticin

S. dysgalactiae
subsp. equisimilis

192

21.5

( Heng et al., 2006 )

Caseicin

Lactobacillus c asei
strain 138

~42

(Müller and Radler,
1993 )

Helveticin-J

Lactobacillus
helveticus

255

37.5

(Joerger and
Klaenhammer, 1990)

Unsorted
bacteriocins

Closticin 574

C. tyrobutyricum

7.0

(Kemperman et al.,
2003 )

PAMP

P. jensenii

6.3

(Faye et al., 2002)

Plantaricin 163

Lb. plantarum 163

3.5

( Hu et al., 2013 )

Bactofencin A

Lb. salivarius
DPC6502

2.7

(O'Shea et al., 2013)

plantaricin LpU4

Lb. plantarum LpU4

4.8

(Milioni et al., 2015 )

Enterocin 7B

Enterococcus
faecalis

5.2

(Martín-Platero et al.,
2006 )

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6.1.1 Class I: Posttranslationally modified peptides (<10 kDa)
6.1.1.1 Ia — The lanthipeptides
The nisA biosynthesis cluster encodes an operon consiting of 11 genes (Figure S1.2a).
nisA operon is regulated by a two -component quorum sensing (Q S) system invol vi ng
nisRK that codes for a regulator protein and a histidine kinase which is sensitive to
nisin concentration (Lubelski et al., 2008). Nisin cluster encodes the gene for the
structural peptide, nisA ; a dehydratase, nisB ; a cyclase, ni sC ; a transporter for
secretion of the peptide, nisT ; an immunity fact or for self-immunization of the producer,
nisI ; and a protease that cleaves off the leader sequence to activate peptide, nisP . An
ATP-binding cassette (ABC) transporter system which is also responsible for immunity
is encoded by nisFEG ( Lubelski et al., 2008). In some cases, the activation protein may
not be located in the cluster (e.g., subtilin). Pep5 for example is acti vated intracellularly
while nisin is activated after secretion. The situation is different for type II which is
activated by the transporter prior to secretion (Knerr and van der Donk, 2012 , Repka
et al., 2017) .
Most class I lantibiotics exert their biological functions by binding to lipid II. For
example, gallidermin may i nteract with the bactoprenylpyrophosphate moiety of lipid II
to inhibit transglycosylation (Götz et al., 2014). Studies on the mode of action of nisin
began way back in the 80’ s (Ruhr and Sahl, 1985). Howe ver, actual data that
specifically demonstrate this has only been generated during the last decade. The A/B
ring str ucture (Figure S1.2b) appears to be a peculiar featur e obs erved in se veral other
members of class I lantipeptides such as epidermin, gallidermin, microbisporicin and
mutacin 1140. NMR studies of Nisin complexed to lipid II shows that the N -terminal A
and B rings provide the amide backbone (Fig ure S1.2c) that coordinates intramolecular
hydrogen bonding with the pyrophosphate moiety of the lipid intermediate (Hsu et al.,
2004 ). Nisin binding to lipid II triggers membrane insertion, creating pores via a
stoichiometric assembly of eight nisin and four lipi d II molecules as illustrated in Fi gur e
S1.2d (Breukink et al., 1999 , Hasper et al., 2004 , Hasper et al., 2006). Mechanistic
studies on self-immunity of L. lactis to nisin show that the C- terminus of NisI protects
Lipid II from nisin binding and hence prevent pore formation (AlKhatib et al., 2014) .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

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Figure S1.2 Production and structure-func tion relationship of prototype lanthipeptides nisin.
(a) Schematic drawings of nisin biosynthesis gene clusters (drawn to scale); structural genes, red;
modifications, blue & green; leader peptide cleavage and/or transport, dark grey; immunity gene,
yellow; accessory or other immunity genes, brown; (b) Schematic stru cture of nisin; (c) solution
structure of the nisin, showi ng the A, B & C rings, an d the lipid II- [C -terminal A/B rings] com plex (lipid
II is co loured grey). Abbreviations: Dh a, dehydroalanine; Abu, 2-aminobutyric acid; Dhb,
dehydrobutyrine; NAG, N-acetylglucosamine; FPP, farnesyl diphosphate; FGA, fibrinogen alpha
chain; MUB, membrane-anchored ubiquitin-fold. (d) Mechanism of action of nisin, illustrating binding
(I), membrane insertion (II) and pore formation (III). Nisin-lipid II complex was edited using solution
NMR data for nisin (P DB: 1WCO) and Protein Workshop toolkit (M oreland et al., 2005).

Nisin also inhibits outgrowth of spores and disrupts th e propagation of vegetative cells (Gut et
al., 2011). These events may be coordinated by covalent interactions between the lantibio tic
and the spores as suggested elsewhere (Morris et al., 1984). Other experimental data identify
the dehydroalanine a t position 5 of nisin to be responsible for its spore inhibitory role (Chan et
al., 1996 ). Same is true for subtilin (Liu and Hans en, 1993 ). Howev er, recent results from
fluorescently labeled ni sin analogues produced no evidenc e for a covalent mech anism of
inhibition (Gut et al ., 2011 ). Given t hat these effe cts are based on distinct structure -activity

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relationships, the biological effects of su ch compounds should the refore proce ed via two
distinct molecular mechanisms . Furthermore, molecular basis that supports the ability of the
two- peptide lanthipeptide lac ticin 3147 to rapidly disr upt target cell membran e in the absenc e
lipid II was recently characterized and res ults indicate that precise ring geometries in the two
peptides are essential for synergistic activity (Bakhtiary et al., 2017 ). Althoug h their pore -
forming potentials may be weakened by the thickness and composition of the lipid bilayer
membrane of the target strain (Wiedemann et al ., 2006), so me type II la ntibiotics interact via a
distinct lipi d II - binding motif but do not form pores (Islam et al., 2012). Rather, they induce
intense cell wall stress respon ses in their targets (Sass et al., 2008).
There is no record of types II I and IIV exhibiting antimicrobial activity, howe ver, they have
shown activities in other aspects such as the activity of labyrinthopeptin A1 again st HIV/AID S
and herpes si mplex virus (HSV) (Férir et al., 2013), and the efficacy of la byrinthopeptin A2
against a modelled mous e in flicted with neuropathic pai n (Meindl e t al., 2010) .
6.1.1.2 Ib — Circular bacteriocins
The main feature of this group of RiPP is the head - to -tail (N- and C-te rmini) peptid e bond
linkage which generate the cyclic nature of the molecules (Maqueda et al., 2008 ). All members
pos sess α -helices of similar sizes tha t fold into a tertia ry structure having a central por e
surrounded by a compact globular bundle comparable to the saposin folds (Montalbán-López
et al., 2012 , Acedo et al., 2015 , Himeno et al., 2015 ). Note that other circular peptide antibiotics
also exist like vancomycin a nd gramici din S, but they are non -ribosomall y produced.
Circularin A and enteroci n AS -48 are two prominent example s of cyclized bacteriocins. The
latter is produced by a clus ter of 10 genes arranged on a 68 kb pheromone -re sponsive
conjugative plasmid in a two-co mponent operon system (Ma rtínez ‐ Buen o et al ., 1998 , Diaz et
al., 2003) . These genes include as -48A , as - 48B , as - 48C , as -48C1D , as -48D1 and as -48EFGH
which res pectively cod es for the structural pep tide, a putative cyclas e, a DUF95 protein
implicated in immunit y and biosynthes is, a putative ABC tr ansporter, immunity prote in, and
additional ABC transporter responsi ble for self -immunization (Maqueda et al., 2008 , Mu et a l.,
2014 ). The equivalenc e of as -48ABCDD1 in circularin A was determined to be the cirABCD E
(Figure S1.3a), and constituted the mini mal s et of genes required for active biosynthes is
(Maqueda et al., 2008). The additional immunity gen es are n on -essenti al and play ju st a minor
role since they appear to be absent in some know gene clusters (Maqueda et al., 2008 ,
Gabrielsen et al., 2014 ) . In sili co anal ysis of a putative circ ular bacteriocin cluster in
Streptococcus pneumoniae also id entified a putative regulator (Bogaardt et al., 2015). Studies

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

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on how the various components of the biosynthesis operon cooperate to produce active AS -
48 have been reported extensively (Sanc hez -Hidalgo, 2011 , Cebrián et al., 2014), and other
data point to the fact that the biosynthes is mechanism is not necessarily the same for all
circular bacteriocins (Gabrielsen et al., 20 14).

Figure S 1. 3 Production machinery, structure and function of ci rcular bacteriocins . (a)
Schematic representation of circularin A biosynthesis gene cluster (drawn to scale): structural gene,
red; modifications, blue & green; immunity gene, yellow; accessory genes, brown. Ribbon
representation of the structures of (b) carnocyclin A and (c) enterocin AS-48, showing saposin folds.
The first (Leu1) and the last (Leu60) residues of carnocyclin A are shown to illustrate the C - to N-
termini linkage. (d) Structural model for enterocin AS-48 molecular mechanism, illustra ting how two
protomeric units interact to produce two dimeric forms. The free-state dimeric form (I) trans forms to
dimeric form (II) at the membrane ’s surface, burying the hy drophobic helices into the hydrophobic
lipid core while the polar helices interact with the polar heads. Structures (b) and (c) were edited us ing
the solution NMR data for carnocyclin A (PDB: 2KJF) and the X-ray crystallographic data for enterocin
AS -48 (PDB: 1O82).

ATPase activity may be nec essary to suppl y the energy requi red to form the h ead - to -tail
peptide bond during tr ansport since there is no C -terminal extension and an additional
processing step seems to be nec essary to remove the leader peptide (Montalbán-López et al.,
2012 , Gabrielsen et al., 2014 , Scholz et al ., 2014 , Al varez-Sieiro et al., 2016).

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Reports suggest that circular bacteriocins may have multiple mechanisms of action, however ,
a vast majority of the ir members pos sess a common struc tural motif that is similar to the fol d
of mammalia n sa posins (Figur e S1.3b & 1.3c ) and hence may have a di rect di sruptive effect
on the membrane s or activate lipid -deg rading enzymes (Martin-Visscher et al., 2009) .
Nevertheless, some deviation from this common principle exist. For example, enterocin AS -48
forms pores in lipid membranes and cause leakage of io ns and low -molecular-weig ht
compounds (Gálvez et al., 1991 ) whereas carnocyclin A forms voltage- dependent pores that
are selective to anions (Gong et al ., 200 9). The formation of multimeric structures in aqueou s
solution which subsequently undergoes conformational readjustment to bur y the non -polar
core in the lipid membra ne is characteris tic of AS -48 as illustrated in Figure S 1.3d ( Cruz et al.,
2013 , Cebrián et al., 2015 ) .
Whether or not cyclized AMPs in general require a docking molec ule to exert their inhibitory
effects on target organisms have been a subj ec t under considerable debates in recent times .
This was la id to rest when Gabrielse n and co lleagues demonstrated increase sensitivity of L.
lactis to garvicin ML re sulting from the expressi on of a maltose ABC transporter complex
(Gabrielsen et al., 2012 ). This study recorded the first putati ve target re ceptor for a circ ular
bacteriocin. Furthermore, receptor - independent bactericidal effects was observed with high
concentrations of enterocin AS -48 and ca rnocyclin A, indicating a concentrati on -dependent
mode of action and that nonsp ecificity occur at higher bacteriocin concentrations while receptor
on target cells maintain spec ific activity at the lower extremes (Gabrielse n et al., 2014 ).
6.1.1.3 Ic — Linear azol(in)e- co ntaining peptides (LAPs)
The biosynthesis pathway of some bacterioc ins involves ATP - dependent cyclodehydration of
threonine, cysteine and serine to produce flavin - dependent substrates which subsequently
undergo dehydrogenation to form a mixture heterocycl ic thiazole and (methyl)oxazol e within
the peptide chain (Melby et al ., 2011 ). These types of peptides are referred to as Linea r
azol(in)e-contai ning peptides (Figur e S 1.4a) .

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

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Figure S1.4 Structural features and production machinery of streptolysin S . (a) Schematic
structure of streptolysin S. (b) Organization of genes in streptolysin S biosyn thesis operon (drawn to
scale); structural gene, r ed; modifications, blue & green; immunity gene, yellow; accessory genes,
brown.

The biosynthesis machinery of Streptolysin S w hich happens to be the most prominent member
of this class composed of sagA, sagB, sagCD and sa gE (Fi gure S1.4b) whic h res pectively
codes for th e structural pep tide, a dehydrogenase, a cyclodehydratase and a protease (Cox et
al., 201 5). The ABC-type transporter SagGHI and SagF, may be responsible for immunity (Le e
et al., 2008 ). With respect to their mode of action, LAPs remain extensively uncharacterize d
(Alvarez-Sieiro et al., 2 016)
6.1.1.4 Id — Sactipeptides
Sactipeptides are bacteriocins that conta in sulphur - to - α -carbo n linkages (Arnison et al., 2013 ,
Mathur et al., 2015). Those that possess antimicrobial activities are also called sactibiotics .
Extensive studies performed with subtilosi n A show that the carbon -sulfur linkage s and hairpins
are common structural el ements (Kawulka et al., 2003 , Maqueda et al., 2008 , Murphy et al.,
2011 ). Subtil osin A has three sulfu r - to - α -carbon cros s-linkages (Figure S1.5 a and S1.5b) and
demonstrate wide activity spectrum against a variety of bacterial strains (Montalban -Lopez et
al., 201 1 , Mathur et al., 2015 ). There are also two -component sactibiotic like thuricin CD with
enhanced activity against Clostridium difficile (Rea et al., 2010). Thuric in CD and thu ricin H are
both produced by Bacillus thuringiensis , but the latter is a single peptide co ntaining f our sulfur-
to - α -carbon linkages (Mathur et al., 2015).
The biosynthesis cl uster of subtilosinA constitutes 8 open reading frames (ORF) including
sboA , ywiA and yRQPONM most of whose molecu lar functions are unknown (Figure S1.5c).

Reconstruction of the lantibiotic ruminococcin-A biosynthesis Elvis Legala Ongey

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sboA encode s the st ructural peptide, ywiA codes for modification protein, while yRQP ONM are
believed to fun ction in process ing, immunity and export of the peptide (Zhe ng et al., 199 9 ,
Stein et al ., 2004). YwiA is an S-ade nosylmethionine (SAM) enzymes containing the
CXXXCXXC conserved motif that forms a [4F e-4S] clusters necessary for the reductive
cleavage of SAM into meth ionine and a 5′ -deoxyadenosyl radic al ( Fl ü he et al., 2013 ).

Figure S1.5 Biosynthesis operon and structure of subtilosin A. (a) schem atic structure of
subtilosin A and (b) its so lution structure, showing the coordination of the sulfur- α -carbon bridges. (c)
Organization of genes in the biosynthesis cluster of subtilosin A (drawn to scale); structural gene, re d;
modifications, blue & green; trans port, dark grey; accessory genes, brown. Structure (b) was edited
using the solution NM R data for subtilosin A (PDB: 1PXQ).

Unlike thu rincin H which seems not to affect the permeability of the pho spholipid bilayer
membrane (Wang et al., 2014), the bacterici dal mechani sm of subtilosin A in volves partial
insertion of the peptide into the hydrop hobic core of target cell membran e. The buried pep tide
ultimately caus es a disarray within the area leading to the creation of transient pores (Noll e t
al., 2011 ).

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

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6.1.1.5 Ie — Glycocins
Glycocins are a group of RiPPs with one or more residues in the peptide chain linked to a
carbohydrate moeity (Arnison et al., 2013). Glyc ocin F produced by Lactobacillus plantarum is
a prototype example of this cl ass to be structura lly characterize d (Stepper et al., 2011 ). Other
examples li ke thurandacin A & B (W ang et al., 2013 ), Sublancin 168 (Garcia De Gonzalo et
al., 2014) and entero cin F4 - 9 (Maky et al., 201 5) have also been described. Sublancin 168 and
thurandacin A are β -S-link ed glycosylated bacteri ocins; enterocin F4 -9 contains β -O- linked
glucose moiety (Figur e S1.6), while glyco cin F and thurandacin B conta in both β -S-linke d and
β -O-linked glycos ylated moieties. Glucose, N -acetylgl ucosamine and N-acetylhe xosamine are
the carbohydrate compounds that have been identifi ed so far.

Figure S1.6 Structural features and biosynthesis of glycopeptide bacteriocins. (a) NMR
structure of sublancin 168 (PDB: 2MIJ), showing a glucose moiety linked to Cys22 and two
intramolecular dis ulfide bridges. (b) Organization of genes in the biosynthesi s cluster of enterocin F4 -
9 (drawn to scale); structural gene, red; modifications, blue & green; transport, dark grey; immunity
gene, yell ow . Structure in (a) was edited using the solution NMR data for sublancin 168 (PDB: 2MIJ).

Enterocin F4-9 is a bacteri ostatic peptid e (unlik e glycoc in F) and its biosynthes is pathway
comprises five gen es inc lu ding entT , ent A49 , enfB , enfC and enfI , which respectively encodes
a putati ve ABC -transporte r; the structural pep tide, the glycosyltran sferase, a thioldisulfide
isomerase and a s elf-protectin g protein (Maky et al., 20 15) .

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Although data on the mechanism of action are rare, resul ts with O -deglycosyla ted glycocin F
shows that the O-li nked N-acetylglucos amine may be involve d in a reversible interaction with
target cells (Stepper et al., 2011 ).
6.1.1.6 If — Lasso peptides
Lasso peptides are bacterioc ins that pos sess a ring formed via an amide bond between the
first res idue of the core pe pti de and a negatively charged core residue at positions +7, +8 or
+9; afte r which the ring then embod ies the linear C -terminus of the sequence (Arnison et al .,
2013 , Hegemann et al., 2015 , Alvarez -Sieiro et al., 2016 ). Additional modific ations such as
intramolecular disulfide bridges like in the ca se of sviceucin (Figure S1.7a) are also possi ble.
Such structural arrangement confers high stability to the peptides and hence they may be used
as peptide scaffolds (Alvarez-Sieiro et al., 2016) .

Figure S1.7 Structural features and biosynthesis of lasso peptides. (a) NMR structure of
sviceucin, showing two intramolecular disulfide bridges between Cys1 and Cys13, and Cys7 and
Cys19. (b) Organization of genes in the biosynthesis cluster of microcin J25 (drawn to scale);
structural gene, red; modifications, blue & green; immunity gene, yellow; transport, dark grey.
Structure in (a) wa s edited using the solution NMR da ta for sviceucin (PDB: 2LS1).

Microcin J25 , from Escheric hia coli was the first lass o peptide with antimicrobial activities to b e
characterized. Genome mining approach on 87 different proteobac terial strains recent ly

Elvis Legala Ongey Rec ons truction of the lantibiotic ruminococcin-A biosynthesis

119
identified 108 put ative biosynthetic gen e clusters for lasso peptides and heterologous
expression of 12 members from this list was successfull y per fomed in E. coli (Hegemann et
al., 2013) . The bios ynthesis clus ter of microci n J25 constitutes four genes namely ; mcjA , mcjB ,
mcjC and mcjD (Figure S1.7b) which respectively encodes the structural peptide, the
peptidase, the cyclase and the A BC transporter (Yan et al., 2012) .
With respect to the mode of actio n, it ha s earlier been es tablished that Microcin J25 enters
target bacterial cells via FhuA receptor in the outer membrane (Sal omón and Farías, 1993) as
well as the SbmA protei n in the inner membrane (Salomón and Farias , 1995) to in hibit RNA
polymerase (Mathavan et al., 2014 ), tri ggers the production of reactive oxygen sp ecies that
facilitates the inhibition process (Chalon et al., 200 9). Capi struin al so inhibits bacterial RNA
polymerase (Kuznedelov et al., 2011), unlike lassomycin whic h targets Mycobacteri um
tuberculosis by inhibi ting pro teases (Gavrish et al., 2014). Like other antimicrobial peptides,
biological activities of lasso peptides expand beyond antimicrobial s to incl ude antiviral or
anticancer (Maksimov et al., 2012 ).
6.1.2 Class II: Unmodified bacteriocins (<10 kDa)
6.1.2.1 IIa — pediocin-like
Pediocin-like bacteriocins constitute the largest bac teriocin group, heat -stable and are
produced by a variety of LAB (Cui et al., 2012). They are broad spectrum antimicrobials with
strong activity against li steria (Kjos, 2011 ). Clas s IIa peptides has two distinc t reg ions in their
structures separated by a flexible hinge (Haugen et al ., 2008). The N-terminus has an overall
positive charge, a disulfide bridge formed between two cysteine res idues and a conserved
YGNGVXC cons ensus motif (Figure S1.8a), which may be actively involved in target
recognitio n and the killing process (Cui et al., 2012 , Perez et al., 2014 ). The disulfide bridge
may play a st ability role and not necessa ry directly involved in the killing process since its
replacement by hydrophobi c interaction did not abolish the activity of leucocin A (Sit et al.,
2012 ).
Pediocin PA-1 is the prototype member of this class and has bee n extensively studied. Its
biosynthesis cluster is haboured on a plasmid (Ennahar et al., 1999). As shown in Figure
S1.8b, the ped operon is enc oded by four genes, namely; pedA and pedB , encoding the
structural peptide and the immunity protei n respectively, and pedC and pedD, enc oding the
accessory factor component tha t constitu tes part of the ABC transport systems and the ABC
transporter respec tively (Miller et al., 200 5). The leade r sequence in the structural peptide
serves the purpose of recognition and direc ts the unp rocessed molec ule to a dedicated ABC
transport systems where it is processed and secreted (Alvarez -Sieiro et al., 2016). Additionally,

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