De no o p oduc ion o p enylna ingenin compounds by a me abolically
enginee ed Esche ichia coli
Daniela Gomes
a
, Joana L. Rod igues
a,b,*
, Nigel S. Sc u on
c
, Ligia R. Rod igues
a,b
a
CEB-Cen e o Biological Enginee ing, Uni e sidade do Minho, Campus de Gual a , B aga 4710-057, Po ugal
b
LABBELS, Associa e Labo a o y, B aga/Guima ˜
aes, Po ugal
c
Manches e Ins i u e o Bio echnology, The Uni e si y o Manches e , 131 P incess S ee , Manches e M1 7DN, UK
ARTICLE INFO
Keywo ds:
P enylna ingenin
Esche ichia coli
Me abolic enginee ing
Syn he ic biology
He e ologous p oduc ion
CRISPR-Cas12a
ABSTRACT
P enylna ingenin (PN) compounds, namely 8-p enylna ingenin (8-PN), 3’-p enylna ingenin (3’-PN), and 6-p e-
nylna ingenin (6-PN), a e epo ed o ha e se e al in e es ing bioac i i ies. This s udy aimed o alida e a
biosyn he ic pa hway o de no o p oduc ion o PN in Esche ichia coli. A p e iously op imized E. coli chassis
capable o e icien ly de no o p oducing na ingenin was used o e alua e ele en p enyl ans e ases (PTs) o he
p oduc ion o PN compounds. As PT eac ion equi es dime hylallyl py ophospha e (DMAPP) as ex ended sub-
s a e ha has limi ed a ailabili y inside he cells, clus e ed egula ly in e spaced sho palind omic epea s
(CRISPR) and CRISPR-associa ed p o ein 12a (Cas12a) (CRISPR-Cas12a) was used o cons uc en boos ed
DMAPP-E. coli s ains. All he PTs, in combina ion wi h he na ingenin biosyn he ic pa hway, we e es ed in hese
s ains. Expe imen s in 96-well deep well pla es iden i ied wel e s ains capable o p oducing PN. E. coli M-PAR-
121 wi h he in eg a ion o he 1-deoxy-D-xylulose-5-phospha e syn hase (DXS) gene om E. coli (EcDXS) in o
he lacZ locus o he genome (E. coli M-PAR-121:EcDXS) exp essing he soluble a oma ic PT om S ep omyces
oseoch omogenes (CloQ) and he na ingenin biosyn he ic pa hway was selec ed as he bes p oduce s ain. A e
op imizing he p oduc ion media in shake lasks, 160.57 µM o 3’-PN, 4.4 µM o 6-PN, and 2.66 µM o 8-PN we e
ob ained. The p oduc ion was hen e alua ed a he bio eac o scale and 397.57 µM o 3’-PN (135.33 mg/L) and
25.61 µM o 6-PN (8.72 mg/L) we e ob ained. To he bes o ou knowledge, his wo k ep esen s he i s epo
o de no o p oduc ion o PN compounds using E. coli as a chassis.
1. In oduc ion
P enyl la onoids a e cha ac e ized by he p esence o a lipophilic
p enyl side-chain in he la onoid skele on, which con e s highe lip-
ophilici y and solubili y. This cha ac e is ic leads o imp o ed bioac-
i i y due o enhanced in e ac ion wi h a ge p o eins (Mukai, 2018;
Wen e al., 2021). P enylna ingenin (PN) compounds, namely 8-p enyl-
na ingenin (8-PN), 6-p enylna ingenin (6-PN), and 3’-p enylna ingenin
(3’-PN), a e epo ed o ha e se e al in e es ing biological ac i i ies,
such as an icance , an i i al, an i-in lamma o y and es ogenic
(F a a uolo e al., 2024, 2019; Hi zman e al., 2020; ˇ
S ulíko ´
a e al.,
2018). PN compounds a e na u ally p oduced in some plan species in
ace amoun s, which makes hei ex ac ion and u he inco po a ion
in o pha maceu ical and nu aceu ical compounds di icul (ˇ
S ulíko ´
a
e al., 2018). Using mic oo ganisms as mic obial cell ac o ies can p o-
ide an in e es ing al e na i e o hei p oduc ion, as i is a mo e
e icien , en i onmen ally iendly, and is po en ially a mo e
cos -e ec i e me hod. The mic obial p oduc ion o PN compounds om
a simple ca bon sou ce such as glucose depends on he e icien
exp ession o se e al enzymes (Fig. 1).
In a i s s ep, he na ingenin biosyn he ic pa hway should be
assembled o p oduce he accep o molecule in ele an amoun s om
glucose (Gomes e al., 2024). A e wa ds, he p enyla ion s ep is ca a-
lysed by a p enyl ans e ase (PT), ha uses dime hyllallyl diphospha e
(DMAPP) as ex ended subs a e and ans e s he p enyla ed chain o he
accep o molecule (na ingenin). DMAPP is na u ally p oduced in mi-
c oo ganisms h ough he me alona e (MVA) o me hyle y h i ol
phospha e (MEP) pa hways (Cha zi asileiou e al., 2019). The PT s ep
can be conside ed he c i ical s ep o he p oduc ion o p enyl la onoids
since i depends on he limi ed DMAPP a ailabili y inside he cells and
on he e icien exp ession o he PT enzymes (Peng e al., 2024).
Se e al a oma ic PTs de i ed om plan s o om mic oo ganisms
* Co esponding au ho a : CEB-Cen e o Biological Enginee ing, Uni e sidade do Minho, Campus de Gual a , B aga 4710-057, Po ugal.
E-mail add ess: [email p o ec ed] (J.L. Rod igues).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Bio echnology
jou nal homepage: www.else ie .com/loca e/jbio ec
h ps://doi.o g/10.1016/j.jbio ec.2025.05.017
Recei ed 2 Ap il 2025; Recei ed in e ised o m 20 May 2025; Accep ed 23 May 2025
Jou nal o Bio echnology 405 (2025) 215–228
A ailable online 29 May 2025
0168-1656/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
Fig. 1. Biosyn he ic pa hway esponsible o p enylna ingenin (PN) compounds p oduc ion. This pa hway can be di ided in o wo pa s: na ingenin biosyn he ic
pa hway and p enyl ans e ase (PT) eac ion. Malonyl-CoA and dime hylallyl diphospha e (DMAPP) a e used as ex ende subs a es in he i s pa and in he
p enyla ion eac ion, espec i ely. The pa hway is composed by y osine ammonia-lyase (TAL), 4-couma a e:CoA ligase (4CL), chalcone syn hase (CHS), chalcone
isome ase (CHI), and PT.
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
216
ha e been epo ed o pe o m p enyla ion in la onoids (An e al.,
2023; de B uijn e al., 2020; Mo i, 2020; Peng e al., 2024). Al hough
PTs om plan s a e he only ones in ol ed in he in i o p oduc ion o
p enyl la onoids, hese enzymes a e memb ane-bound enzymes, con-
aining signal pep ides ha hinde hei e icien exp ession in mic o-
o ganisms. In con as , mic obial PTs a e soluble enzymes making hem
easie o exp ess and ha e been ound o pe o m he p enyla ion o
se e al la onoid skele ons, including na ingenin (de B uijn e al., 2020;
Peng e al., 2024).
In ecen yea s, se e al e o s ha e been made o achie e he mi-
c obial p oduc ion o p enyl la onoids. Saccha omyces ce e isiae was
p ima ily exploi ed as chassis. De no o p oduc ion o PN compounds in
S. ce e isiae was i s epo ed by Le isson e al. (2019). Howe e , only
0.12 mg/L o 8-PN we e p oduced. La e , Guo e al. (2022) epo ed he
p oduc ion o 49.35 mg/L and 101.40 mg/L o 8-PN om glucose in
shake lask and 5 L bio eac o expe imen s, espec i ely (Guo e al.,
2022). As an al e na i e o S. ce e isiae, Esche ichia coli can also be
explo ed as a chassis o he mic obial p oduc ion o PN compounds. The
biocon e sion o na ingenin o PN compounds has been es ed in E. coli
using mic obial PTs (Qiu e al., 2021; Liu e al., 2023; Zhang e al.,
2024). Mo e ecen ly, E. coli was also enginee ed o he biocon e sion
o o he la onoids (silybin, daidzein, and baicalein) in o hei p eny-
la ed o ms using ungal PTs (Fan e al., 2025). Howe e , as a as we
know, his mic oo ganism has ne e been enginee ed o de no o p oduce
p enylna ingenin o o he p enyl la onoids.
In his wo k, we aimed o cons uc and alida e a biosyn he ic
pa hway o de no o p oduce PTs in E. coli o he i s ime. Using a
p e iously op imized na ingenin-p oducing E. coli s ain, se e al PTs
om plan s and mic obial sou ces we e es ed. Addi ionally, clus e ed
egula ly in e spaced sho palind omic epea s (CRISPR) and CRISPR-
associa ed p o ein 12a (Cas12a) (CRISPR-Cas12a) was used o
cons uc boos ed DMAPP-E. coli s ains o o e come his compound
limi a ion. A e sc eening all combina ions o PTs/s ains, he bes
p oducing s ain was selec ed o p oduc ion media op imiza ion and
bio eac o expe imen s. E. coli M-PAR-121 s ain wi h he in eg a ion o
he 1-deoxy-D-xylulose-5-phospha e syn hase (DXS) gene om E. coli
(EcDXS) in o he lacZ locus o he genome (E. coli M-PAR-121:EcDXS)
exp essing he soluble a oma ic PT om S ep omyces oseoch omogenes
(CloQ) and he na ingenin biosyn he ic pa hway was able o p oduce
397.57 µM o 3’-PN (135.33 mg/L) and 25.61 µM o 6-PN (8.72 mg/L)
a a bio eac o scale when wo addi ional glucose pulses we e p o ided,
co esponding o he i s epo o de no o p oduc ion in E. coli and he
highes de no o p oduc ion o PN epo ed in any hos .
2. Ma e ials and me hods
2.1. S ains, plasmids, chemicals and media composi ion
E. coli NZY5
α
(NZYTech - MB00401) and E. coli NEB5
α
(New England
Biolabs - C2987H) we e used o cloning and o he p opaga ion o
plasmids. E. coli M-PAR-121 was used as he pla o m s ain o he
cons uc ion o DMAPP-modi ied s ains (Koma e al., 2020). The he -
e ologous biosyn he ic pa hways o p oduce PN we e exp essed in E. coli
M-PAR-121 (wild- ype s ain) and in he E. coli M-PAR-121
DMAPP-modi ied s ains. The ea u es o all s ains cons uc ed and
used in his wo k a e p esen ed in Table S1. The plasmids used in his
s udy a e p esen ed in Table S2. Plasmids pWY16 and pWY24 we e
kindly p o ided by D . Shu-Ming Li (Yin e al., 2009, 2010). The p e-
nyl ans e ases (PTs) ca ying plasmids we e al eady a ailable in
Sc u on labo a o y. Plasmids pSIMcp 1, pTF-lacZ- p, and
pBbS8c-ddcp 1-Δ, ha we e used in he clus e ed egula ly in e spaced
sho palind omic epea s (CRISPR)-Cas12 s a egies, we e designed and
kindly p o ided by Je is e al. (2021).
Isop opyl β-D-1- hiogalac opy anoside (IPTG), 5-b omo-4-chlo o-3-
indolyl-β-D-galac opy anoside (X-Gal) and lysogeny b o h (LB) Mille
medium we e pu chased om NZYTech. LB aga , used o colonies
selec ion, was composed o 20 g/L LB Lennox (LabKem) and 20 g/L aga
(LabKem). M9 minimal medium was composed by 3 g/L KH
2
PO
4
(Riel-
deHa¨
en), 6 g/L Na
2
HPO
4
(Chem-Lab), 0.5 g/L NaCl (NZYTech), 1 g/L
NH
4
Cl (Pan eac), 110 mg/L MgSO
4
(Labkem), 15 mg/L CaCl
2
(Pan-
eac), 340 mg/L hiamine (The mo Fishe Scien i ic), 5 g/L CaCO
3
(Pan eac), and i amins (12.2 mg/L nico inic acid (Ac os o ganics),
10.8 mg/L pan o henic acid (Sigma Ald ich), 2.8 mg/L py idoxine
(Fishe BioReagen s), 0.84 mg/L ibo la in (Pan eac), 0.12 mg/L bio in
(Me ck), and 0.084 mg/L olic acid (Pan eac)). Te i ic b o h (TB) was
composed by 12 g/L yp one (Fishe Scien i ic), 24 g/L yeas ex ac
(LabKem), 9.4 g/L KH
2
PO
4,
and 2.2 g/L K
2
HPO
4
(Pan eac). M9 modi-
ied medium was composed by 5 g/L yeas ex ac (LabKem), 3 g/L
KH
2
PO
4
, 6 g/L Na
2
HPO
4
(Chem-Lab), 0.5 g/L NaCl, 1 g/L NH
4
Cl,
110 mg/L MgSO
4
, 15 mg/L CaCl
2
, 0.27 g/L FeCl
3
⋅6H
2
O (Pan eac) and
ace elemen s (0.2 g/L ZnCl
2
⋅4H
2
O (LabKem), 0.2 g/L CoCl
2
⋅6H
2
O
(Sigma Ald ich), 0.2 g/L Na
2
MoO
4
⋅2H
2
O (Ac os), 13 mg/L CuCl
2
⋅6H
2
O
(Sigma Ald ich), 5 mg/L H
3
BO
3
(Fishe Scien i ic), and 0.1 mL/L HCl
(Fishe Scien i ic). M9 minimal medium, M9 modi ied medium, and TB
medium we e supplemen ed wi h glucose (Ac os) a a inal concen a-
ion o 30 g/L. The ollowing an ibio ics we e used o s ain selec ion:
50 µg/mL kanamycin (NZYTech), 25 µg/mL chlo amphenicol (NZY-
Tech), 100 µg/mL o 50 µg/mL spec inomycin (Al a Aesa ), 150
μ
g/mL
hyg omycin (Fische Scien i ic).
2.2. Cons uc ion o he pa hway plasmids
To cons uc a single plasmid ca ying he comple e na ingenin
biosyn he ic pa hway (composed by y osine-ammonia lyase (TAL) om
Fla obac e ium johnsoniae (FjTAL), 4-couma a e:CoA ligase (4CL) om
A abidopsis haliana (A 4CL), chalcone syn hase (CHS) om Cucu bi a
maxima (CmCHS), and chalcone isome ase (CHI) om Medicago sa i a
(MsCHI)), he casse e con aining he A 4CL and MsCHI and espec i e
p omo e s and e mina o s was ampli ied by polyme ase chain eac ion
(PCR) using as empla e he p e iously cons uc ed ec o pACYCDue-
_A 4CL_MsCHI (Gomes e al., 2024). The p ime s used o his PCR hold
15 bp homology o e hangs o he pRSFDue _FjTAL_CmCHS ec o .
pRSFDue _FjTAL_CmCHS ec o was also linea ized by PCR. The con-
s uc ion o pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI was pe o med by
In-Fusion cloning using he In-Fusion® Snap Assembly ki om Taka a
Bio Eu ope.
Ele en PTs we e selec ed o be es ed in his s udy (Table S3). PT
om Humulus lupulus (HlPT) and PT om Sopho a la escens (S N8DT-1)
we e syn hesized wi h codon-op imiza ion o E. coli by Twis Bioscience
and hen ampli ied by PCR. AnaPT and CdpC3PT om Neosa o ya
ische i (wi hou codon-op imiza ion) we e ampli ied by PCR om
pWY16 and pWY24, espec i ely. These ou PTs we e cloned in o he
pCDFDue -1 ec o by es ic ion cloning. Codon-op imized e sions o
PT3 om Cannabis sa i a (CsPT3), coAnaPT, CloQ om S ep omyces
oseoch omogenes, PT om E. coli (EcPT), NphB om S ep omyces sp., PT
om S ep omyces sp. Ac 143 (SpPT), and UbiA om E. coli we e
ampli ied by PCR om he espec i e ca ying plasmids using p ime s
wi h 15 bp homology o e hangs o he pCDFDue -1 ec o . The
pCDFDue -1 ec o was linea ized by PCR and he cloning was pe -
o med by In-Fusion using he In-Fusion® Snap Assembly ki . The co -
ec cons uc ion o hese ec o s was con i med by colony PCR and
sequencing. The p ime s (Me abion / Eu o ins) used o ec o linea i-
za ion, genes ampli ica ion, colony PCR, and sequencing a e displayed
in Table S4.
2.3. E alua ion o PTs exp ession
E. coli M-PAR-121 ca ying he PTs plasmids we e g own a 37 ◦C in
LB Mille , un il eaching an op ical densi y a 600 nm (OD
600nm
) o 0.6.
The p o ein exp ession, he p epa a ion o samples o sonica ion, he
se ings used o sonica ion, he p epa a ion o p o ein samples and
u he quan i ica ion we e p e iously desc ibed in Gomes e al. (2024).
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
217
Soluble and insoluble ac ions we e subjec ed o sodium dodecyl sul a e
(SDS) polyac ylamide gel elec opho esis (SDS-PAGE) gel (4 % s acking
gel and 10 % unning gel). Colo P es ained P o ein S anda d, B oad
Range (10–250 kDa) (NEB), Blue P es ained P o ein S anda d, B oad
Range (11–250 kDa) (NEB), and NZYColou P o ein Ma ke II (NZY-
Tech) we e used as e e ence p o ein ladde s.
2.4. Cons uc ion o DMAPP-modi ied E. coli s ains
DMAPP-modi ied E. coli s ains we e cons uc ed so ing o he
CRISPR-Cas12a sys em designed by Je is e al. (2021).
2.4.1. Genome in eg a ion o he e ologous and na i e DXS and IDI genes
2.4.1.1. Cons uc ion o a ge -speci ic in eg a ion ec o s. To pe o m he
genome in eg a ion o DXS and IDI genes, he pTF-lacZ- p ec o p e-
iously cons uc ed by Je is e al. (2021) was used. This ec o holds a
CRISPR a ay and 500 bp ups eam and downs eam homology a ms o
he lacZ locus o he E. coli genome o e icien deli e y and in eg a ion
o ca go DNA. The ed luo escence p o ein coding gene ( p), ha was
used as ca go DNA o in eg a ion in he s udy pe o med by Je is e al.
(2021), was emo ed by PCR using p ime s ha anneal in o he ex-
emi ies o he lacZ a ms. Linea ized pTF-lacZ was used as backbone o
ecei e he casse es o genome in eg a ion.
He e ologous DXS gene om Bacillus sub ilis (BsDXS) and IDI genes
om S. ce e isiae (ScIDI) and Bacillus licheni o mis (BlIDI) we e syn he-
sized wi h codon-op imiza ion o E. coli by Twis Bioscience. Na i e
DXS and IDI om E. coli we e ampli ied om E. coli M-PAR-121 genomic
DNA ex ac ed using Mona ch Genomic DNA Pu i ica ion Ki (NEB). The
sequences o he na i e and he e ologous codon op imized genes a e
lis ed in Table S5. Single and double in eg a ion o DXS and IDI genes
wi h imp o ed ac i i ies (BsDXS, ScIDI, and BlIDI) we e designed.
Al e na i ely, single and double in eg a ion o he na i e DXS and IDI
genes om E. coli we e also designed. Fo in eg a ion o single genes, he
genes we e ampli ied by PCR wi h speci ic p ime s holding 15 bp ho-
mology a ms o he linea ized pTF-lacZ ec o . In he case o double
gene in eg a ion, he IDI gene was ampli ied wi h a o wa d p ime wi h
15 bp homology o he co esponding DXS gene and a e e se p ime
wi h 15 bp homology o linea ized pTF-lacZ ec o and he DXS gene
was ampli ied wi h a o wa d p ime wi h 15 bp homology o linea ized
pTF-lacZ ec o and a e e se p ime ha anneals in he end o he gene.
The agmen s we e assembled by In-Fusion cloning using he In-
Fusion® Snap Assembly ki . The ollowing a ge -speci ic in eg a ion
ec o s we e cons uc ed: pTF-lacZ-BsDXS, pTF-lacZ-ScIDI, pTF-lacZ-
BlDI, pTF-lacZ-BsDXS-ScIDI, pTF- lacZ-BsDXS-BlIDI, pTF-lacZ-EcDXS,
pTF-lacZ-EcIDI, and pTF-lacZ-EcDXS-EcIDI. The co ec cons uc ion o
hese ec o s was con i med by colony PCR and sequencing. The p ime s
(Me abion / Eu o ins) used o ec o linea iza ion, genes ampli ica ion,
colony PCR, and sequencing a e displayed in Table S6.
2.4.1.2. Clus e ed egula ly in e spaced sho palind omic epea s
(CRISPR) edi ing. CRISPR edi ing o he in eg a ion o he a ge -
speci ic casse es was pe o med as desc ibed by Je is e al. (2021).
E. coli M-PAR-121 chemically compe en cells we e ans o med wi h
pSIMcp 1. Then, elec ocompe en cells o E. coli M-PAR-121 ca ying
pSIMcp 1 we e p epa ed and ans o med wi h he cons uc ed
a ge -speci ic in eg a ion pTF-lacZ ec o s. Recombinan colonies we e
selec ed a 30 ◦C in LB aga wi h 150
μ
g/mL o hyg omycin and
50
μ
g/mL o spec inomycin. Since he casse e in eg a ion dis up ed he
lacZ gene, he blue-whi e sc eening es was pe o med by epla ing
single colonies in LB aga con aining he equi ed an ibio ics (hyg om-
ycin and spec inomycin), 0.1 mg/mL o X-GAL and 1 mM o IPTG. Whi e
colonies (posi i e in eg a ion ecombinan s) we e selec ed and g own
o e nigh a 30 ◦C in LB Mille wi h he equi ed an ibio ics. A e wa ds,
he genomic DNA was ex ac ed using Mona ch Genomic DNA
Pu i ica ion Ki (NEB). The co ec in eg a ion in o he lacZ locus was
con i med by PCR using he genomic DNA o each selec ed colony as
empla e and by sequencing.
A e con i ma ion, CRISPR plasmids we e emo ed om he con-
s uc ed s ains as desc ibed by Je is e al. (2021). Eigh
DMAPP-modi ied E. coli M-PAR-121 s ains we e cons uc ed: E. coli
M-PAR-121:BsDXS, E. coli M-PAR-121:ScIDI, E. coli M-PAR-121:BlDI,
E. coli M-PAR-121:BsDXS-ScIDI, and E. coli M-PAR-121:BsDXS-BlIDI,
E. coli M-PAR-121:EcDXS, E. coli M-PAR-121:EcIDI, and E. coli
M-PAR-121:EcDXS-EcIDI. A schema ic ep esen a ion o he s a egies o
single and double in eg a ions can be obse ed in Fig. S1.
2.4.2. Gene egula ion using CRISPR in e e ence (CRISPRi) sys em
2.4.2.1. Cons uc ion o he CRISPRi ec o s. Two s a egies using he
CRISPR in e e ence (CRISPRi) sys em de eloped by Je is e al. (2021)
we e designed o down egula e he ge anyl diphospha e/ a nesyl
diphospha e syn hase (ispA) gene. The p o ospace adjacen mo i s
(PAM) TTTV, ha is ecognized by Cas12a o clea age, we e iden i ied.
Since a ge ing di e en PAM sequences o he gene can lead o di e -
ences in he down egula ion le els, wo di e en 23 bp a ay sequences
we e designed (Je is e al., 2021). The i s a ay was designed o he
i s PAM sequence (6 bp ups eam o he ispA s a codon) on he 5
′
end
o he gene coding sequence and he second a ay was designed o he
PAM sequence 154 bp ups eam o he ispA s a codon. ispA gene wi h
he PAM sequences and a ays highligh ed a e ep esen ed in Table S7.
These a ays we e in oduced in o he pBbS8c-ddcp 1-Δ by PCR using
speci ic p ime s ha we e designed including he 23 bp a ay sequences
and 15 bp homology a ms o he ec o o allow he ec o ci cula i-
za ion using he In-Fusion® Snap Assembly ki . The co ec cons uc ion
o pBbS8c-ddcp 1-ispA1 and pBbS8c-ddcp 1-ispA2 was con i med by
sequencing. The p ime s (Eu o ins) used o ec o linea iza ion and
inse ion o he a ays and o sequencing a e displayed in Table S8.
2.4.2.2. Quan i ica ion o down egula ion le els by e e se ansc ip ion-
quan i a i e polyme ase chain eac ion (RT-qPCR). E. coli M-PAR-121
was ans o med wi h pBbS8c-ddcp 1-Δ (con ol), pBbS8c-ddcp 1-ispA1
and pBbS8c-ddcp 1-ispA2. Th ee colonies o each ans o med s ain
we e g own o e nigh in LB Mille medium supplemen ed wi h 25 µg/
mL o chlo amphenicol. These cul u es we e eshly cul i a ed (1:50)
and g own a 37 ◦C un il eaching an OD
600 nm
be ween 0.2 and 0.4. A
his poin , 10 mM L-a abinose and 0.1 mM IPTG we e added o in-
duc ion. The cells we e cul i a ed a 37 ◦C and 200 pm un il eaching
an OD
600 nm
be ween 1 and 1.4 and we e hen cen i uged o eco e he
pelle . The pelle was ozen in liquid ni ogen. A e wa ds, he pelle s
we e used o RNA ex ac ion using he Di ec -zol RNA Minip ep Ki
(ZymoResea ch) ollowing he manu ac u e ’s ins uc ions. RNA sam-
ples we e un in 1 % (w/ ) aga ose gel o check i s in eg i y. The sam-
ples we e quan i ied using Nanod op One (The mo). A e wa ds,
1000 ng o RNA o each sample was used o cDNA syn hesis. cDNA
syn hesis was pe o med using he iSc ip ™ gDNA Clea cDNA Syn hesis
Ki (Bio-Rad) ollowing he manu ac u e ’s ins uc ions. P ime s o
e e se ansc ip ion-quan i a i e polyme ase chain eac ion (RT-qPCR)
we e designed using IDT P ime Ques Tool (Table S9). The house-
keeping genes pu a i e 3-phenylp opiona e anspo e coding gene
(hcaT) and glucona e anspo e coding gene (idnT) we e chosen as
e e ence genes o no maliza ion (Je is e al., 2021; Zhou e al., 2011).
RT-qPCR eac ion was pe o med in echnical iplica es o each sample
using iTaq™ Uni e sal SYBR G een Supe mix (Bio-Rad) in Ligh Cycle ®
480 Sys em (Roche), ollowing he manu ac u e ’s ins uc ions.
2.5. P oduc ion expe imen s
Chemically compe en cells o E. coli M-PAR-121 (wild- ype (WT)
s ain) and DMAPP-modi ied E. coli M-PAR-121 s ains we e eshly
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
218
p epa ed and ans o med wi h he pa hway plasmids using he hea -
shock me hod. The ans o man s we e selec ed in LB aga wi h he
equi ed an ibio ics.
2.5.1. 96-well deep well pla es expe imen s
O e nigh g own cul u es o E. coli M-PAR-121 (WT s ain) and
DMAPP-modi ied E. coli M-PAR-121 s ains ans o med wi h he na -
ingenin pa hway plasmid (pRSFDue _FjTAL_A 4CL_CmCHS_MsCHI) and
he cons uc ed PT plasmids we e eshly cul i a ed (1:100) in 1 mL o
TB medium supplemen ed wi h 4 g/L glucose and 100 µg/mL spec i-
nomycin in 96-well deep well pla es sealed wi h b ea hable sealing ilm.
The 96-well deep well pla e was incuba ed a 37 ◦C and 1000 pm in a
pla e incuba o shake un il eaching an OD
600 nm
be ween 1.5 and 2.0.
A his poin , IPTG was added a a inal concen a ion o 0.1 mM and he
empe a u e o incuba ion was changed o 30 ◦C. A e 2 h, glucose was
supplemen ed a a inal concen a ion o 30 g/L o be used as subs a e.
The cul u es we e also main ained a 30 ◦C and 1000 pm o 120 h. All
he expe imen s we e conduc ed in iplica e and samples we e collec ed
a he inal ime poin o me aboli es analysis.
2.5.2. Shake lasks expe imen s
O e nigh g own cul u es o he p oducing s ains ca ying he
na ingenin pa hway plasmid (pACYCDue _FjTAL_A 4CL_CmCHS_MsCHI)
and he PT plasmids selec ed in 96-well deep well pla es expe imen s
we e used o inocula e 50 mL o LB Mille medium con aining he
equi ed an ibio ics in 250 mL shake lasks, a an ini ial OD
600 nm
o 0.1.
The cul u es we e main ained a 37 ◦C and 200 pm. IPTG (0.1 mM) was
added when he cul u e a ained an OD
600 nm
o 0.9 and he cul u e was
hen main ained a 26 ◦C and 200 pm o 5 h. A e wa ds, a cen i u-
ga ion was pe o med o pelle he cells (5000 pm, 10 min) and he
pelle was esuspended in M9 minimal medium supplemen ed wi h
30 g/L glucose and he ele an an ibio ics. The expe imen was main-
ained a 26 ◦C and 200 pm o 120h (Gomes e al., 2024).
The bes p oduce was also es ed in single M9 modi ied and TB
p oduc ion media. O e nigh g own cul u es we e used o inocula e
50 mL o M9 modi ied o TB media supplemen ed wi h 4 g/L glucose
and he equi ed an ibio ics, a an ini ial OD
600 nm
o 0.1. The cul u es
we e main ained a 37 ◦C and 200 pm un il a aining an OD
600 nm
o
0.9. A e wa ds, 0.1 mM IPTG was supplemen ed, and he cul u e was
main ained a 26 ◦C and 200 pm o 5 h. A his ime, glucose a a inal
concen a ion o 30 g/L was supplemen ed o be used as subs a e. The
expe imen was main ained a 26 ◦C and 200 pm o 120 h. G ow h was
moni o ed o e ime by measu ing OD
600 nm
. All he expe imen s we e
conduc ed in iplica e and samples we e collec ed o e ime o me-
aboli es analysis.
2.5.3. Bio eac o expe imen s
O e nigh g own cul u e o he bes p oducing s ain was used o
inocula e 100 mL TB medium in 500 mL shake lasks a an ini ial
OD
600 nm
o 0.1. This p e-cul u e was hen incuba ed a 37 ◦C and
200 pm o 12 h. A e wa ds, he OD
600 nm
was e alua ed and he
olume o cul u e equi ed o ini ia e he bio eac o expe imen s a an
ini ial OD
600 nm
o 0.1 was cen i uged o pelle he cells a 5000 pm o
10 min. The pelle was hen esuspended in TB medium and used o
inocula e he bio eac o s. These expe imen s we e pe o med in he 2 L
DASGIP® Pa allel Bio eac o Sys em (Eppendo ). All he bio eac o
expe imen s we e pe o med in duplica e and 2 mL samples we e
collec ed o e ime o measu e OD
600 nm
and o me aboli es analysis.
Ba ch expe imen s we e pe o med in 400 mL o TB medium. Re-
ac o s we e au ocla ed a 121 ◦C o 20 min con aining 350 mL o TB
medium. The o he 50 mL o TB medium we e au ocla ed sepa a ely in a
shake lask and used o dissol e he cells o inocula ion. An ini ial
glucose concen a ion o 4 g/L and he equi ed an ibio ics we e sup-
plemen ed a he beginning o he expe imen . The expe imen s we e
ca ied ou a an ini ial empe a u e o 37 ◦C and ini ial agi a ion o
300 pm, wi h a cons an oxygen eeding o 0.5 m (12 L/h). The
dissol ed oxygen pe cen age (%DO) was main ained abo e 30 % by
adjus ing s i ing speed up o 1000 pm. pH se ing was 6.5 and he pH
con ol was pe o med by au oma ic eeding a 2 M NaOH (LabSol e)
solu ion. The cul u es we e main ained a 37 ◦C un il he cul u e
a ained an OD
600 nm
o 0.9. A his OD
600 nm
, IPTG a a inal concen-
a ion o 0.1 mM was added o he eac o and he empe a u e se ing
was changed o 26 ◦C. A e 5 h o induc ion, glucose was supplemen ed
o he bio eac o s a a inal concen a ion o 30 g/L. The expe imen was
hen main ained a 26 ◦C o 120 h.
Ano he ba ch expe imen was pe o med in he same condi ions.
Howe e , wo addi ional pulses o glucose we e supplemen ed du ing
he expe imen . Glucose concen a ions we e moni o ed o e ime using
high-pe o mance liquid ch oma og aphy (HPLC). Two pulses o glucose
a a inal concen a ion o 10 g/L we e added o he bio eac o s when
he emaining glucose concen a ion on he medium was be ween 5 and
10 g/L (a 24 h and 63 h ime poin s). The expe imen was main ained
a 26 ◦C o 144 h. The p oduc ion o p-couma ic acid, na ingenin, and
PN compounds we e also moni o ed h oughou he expe imen ime
using ul a-high pe o mance liquid ch oma og aphy (UHPLC).
2.6. Ex ac ion o me aboli es
2.6.1. Me hanol ex ac ion
The me aboli es p oduced in 96-well deep well pla es expe imen s
we e ex ac ed using 100 % ( / ) me hanol. An aliquo o he cul u e
(100 µL) was ans e ed o a 96-well mic opla e and an equal olume o
100 % ( / ) me hanol was added. The 96-well mic opla e was o exed
o 2 min and hen cen i uged o 10 min a 4000 pm. The supe na an
was eco e ed and added o a new 96-well mic opla e o ul a-
pe o mance liquid ch oma og aphy (UPLC) analysis.
2.6.2. E hyl ace a e ex ac ion
The me aboli es p oduced in 50 mL shake lasks and bio eac o ex-
pe imen s we e ex ac ed using 100 % ( / ) e hyl ace a e. The ex ac-
ion was pe o med om 1 mL whole b o h cul u e in a 1:1 a io.
Samples ex ac ion and p epa a ion we e pe o med as p e iously
desc ibed in Gomes e al. (2024). A e wa ds, he samples we e analyzed
by UHPLC.
2.6.3. Analy ical me hods
The me aboli es p oduced in 96-well deep well pla es expe imen s
we e e alua ed by UPLC using he 1290 In ini y III Agilen LC sys em
(Agilen , San a Cla a, Uni ed S a es) accoupled wi h a Kine ex® 5µM
XB-C18 100 Å LC column (50 ×2.1 mm). The diode a ay de ec o
measu ed he abso bance a 290 nm. A bina y mobile phase composed
by 0.1 % ( / ) o mic acid in wa e (A) and ace oni ile (B) was used.
The sepa a ion was achie ed using a cons an low a e o 0.5 mL/min.
The ollowing elu ion g adien was used: 5 % o B om 0 o 1 min, 5 %-
95 % o B om 1 o 5 min, and 95–5 % om 5 o 6 min. p-Couma ic acid,
na ingenin, 8-PN, 3’-PN, and 6-PN we e quan i ied based on he peak
a eas a 1.0 min, 1.9 min, 2.8 min, 3.0 min, and 3.3 min, espec i ely.
The me aboli es p oduced in 50 mL shake lasks and bio eac o ex-
pe imen s we e e alua ed by UHPLC using he Shimadzu Nexe a-X2
sys em (Shimadzu Co po a ion, Kyo o, Japan) accoupled wi h a
Kine ex® 2.6
μ
m Pola C18 100 Å LC column (150 ×4.6 mm). SPD-
M20A de ec o measu ed he abso bance a 290 nm. The bina y mo-
bile phase was also composed o 0.1 % ( / ) o mic acid in wa e (A)
and ace oni ile (B), and he g adien was also main ained cons an a a
low a e o 0.5 mL/min. The ollowing g adien was used: 5 %-95 % o
B om 1 o 10 min, 95 %-5 % o B om 10 o 12 min, and linea ly 5 % o
B om 12 o 15 min. p-Couma ic acid, na ingenin, 8-PN, 3’-PN, and 6-
PN we e quan i ied based on he peak a eas a 7.5 min, 9.2 min,
10.6 min, 10.8 min, and 11.2 min, espec i ely.
Glucose consump ion was e alua ed in shake lasks and bio eac o
expe imen s by HPLC so ing o a JASCO sys em connec ed wi h he RI-
203 de ec o and a Aminex HPX-87H column (Bio-Rad). The column was
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
219
main ained a 60 ◦C. A cons an low a e o 0.5 mL/min o 5 mM H
2
SO
4
was used, and glucose was de ec ed and quan i ied based on he peak
a ea a 10.9 min.
2.7. S a is ical analysis
G aphPad P ism So wa e. Inc., e sion 8.0.1. was used o pe o m
he s a is ical analysis o he esul s. All he esul s p esen ed co espond
o he mean alue o h ee independen es s ±s anda d de ia ion (96-
well deep well pla e and shake lask expe imen s) o wo independen
es s ±s anda d de ia ion (bio eac o expe imen s). S a is ical signi i-
cance was e alua ed using O dina y one-way ANOVA es s. When p-
alue was <0.05, he di e ences we e conside ed signi ican .
3. Resul s
3.1. Valida ion o PTs ac i i y and hei abili y o p oduce PN compounds
de no o
Func ional PTs a e equi ed o p oduce PN compounds. In his s udy,
ele en PTs om di e en sou ces we e selec ed due o hei epo ed
ac i i y o use la onoids as accep o subs a es o he p enyla ion
(Mo i, 2020; Ozaki e al., 2009; Rea e al., 2019; Sasaki e al., 2011,
2009; Tello e al., 2008; Tsu uma u e al., 2012, 2010; Winkelblech
e al., 2015; Zhou e al., 2015). Th ee PTs om plan s we e selec ed: PT
om H. lupulus (HlPT), N8DT-1 om S. la escens (S N8DT-1), and PT3
om C. sa i a (CsPT3). These plan -de i ed a oma ic PTs we e selec ed
because hey ha e been epo ed as being able o p oduce se e al p e-
nyl la onoids (including PN compounds) in i o in hei na i e plan
sys ems (Chen e al., 2013; Rea e al., 2019; Sasaki e al., 2011, 2008;
Tsu uma u e al., 2012, 2010). Codon-op imized e sions o he genes
o E. coli we e syn hesized o a oid possible ansla ion e o s. How-
e e , plan PTs a e memb ane-bound enzymes being mo e di icul o be
e icien ly exp essed in E. coli since his mic obial chassis does no
possess in acellula compa men s and an endomemb ane sys em
simila o he ones p esen in plan cells. As an al e na i e, eigh PTs
om mic obial sou ces we e selec ed. These mic obial a oma ic PTs a e
soluble enzymes being mo e easily exp essed in E. coli. In wo cases he
o iginal sequence wi hou codon op imiza ion was es ed: CdpC3PT and
AnaPT om N. ische i. Mo eo e , codon-op imized e sions o he
ollowing PTs we e also es ed: AnaPT om N. ische i (coAnaPT), CloQ
om S. oseoch omogenes, PT om E. coli (EcPT), NphB om S ep o-
myces sp., PT om S ep omyces sp. Ac 143 (SpPT), and UbiA om E. coli.
Al hough hese PTs do no pe o m p enyla ion eac ions in he mic o-
bial hos s, hey we e selec ed because hey ha e been epo ed o ca-
alyse such eac ions using la onoid molecules, including na ingenin,
as accep o subs a es (Mo i, 2020; Ozaki e al., 2009; Rea e al., 2019;
Sasaki e al., 2011, 2009; Tello e al., 2008; Tsu uma u e al., 2012,
2010; Winkelblech e al., 2015; Zhou e al., 2015). All PTs we e cloned
in o he pCDFDue -1 backbone ha is widely used as exp ession ec o
o E. coli (Tolia e al., 2006). The cons uc ed pCDFDue _PT ec o s
we e exp essed in E. coli M-PAR-121, a y osine-o e p oducing s ain
cons uc ed by Koma e al. (2020). E. coli M-PAR-121, E. coli K-12
MG1655 (DE3) and E. coli BL21 (DE3) we e p e iously es ed owa ds
he p oduc ion o na ingenin. Highe p oduc ions o na ingenin using
glucose as subs a e we e ob ained when E. coli M-PAR-121 was used as
chassis (Gomes e al., 2024). Conside ing his, his s ain was used as
chassis o e alua e PN p oduc ion. The exp ession o he ele en PT genes
in E. coli M-PAR-121 was es ed and e alua ed by SDS-PAGE gel (Figs. S2
and S3). As expec ed, he SDS-PAGE gels o plan -de i ed PTs did no
e eal he desi ed band o he p o eins, indica ing ha hese h ee PTs
a e no being e icien ly exp essed in E. coli o he amoun o exp essed
p o ein is no enough o be isible on he gel. In con as , he SDS-PAGE
gels o a oma ic PTs om mic obial sou ces p esen ed he desi ed
p o ein bands indica ing ha hese soluble enzymes a e mo e easily
exp essed in E. coli.
Due o he ele ance o p oducing PN compounds om a simple
ca bon sou ce, he ini ial sc eening o he se e al PTs was pe o med
di ec ly om glucose by assembling he comple e biosyn he ic pa hway
in E. coli M-PAR-121. In o de o educe he possible me abolic bu den o
E. coli cells imposed by he exp ession o se e al plasmids, a single
plasmid holding he p e iously op imized combina ion o genes o he
na ingenin pa hway was cons uc ed (pRSFDue _FjTAL_CmCH-
S_A 4CL_MsCHI). The p oduc ion o na ingenin by E. coli M-PAR-121
exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI was alida ed and
492.98 mg/L o na ingenin we e p oduced (Fig. S4). This s ain was
u he ans o med wi h he pCDFDue _PT ec o s and he cons uc ed
s ains we e u he es ed in 96-well deep well pla e expe imen s using
30 g/L o glucose as subs a e. Howe e , he p oduc ion o PN was no
de ec ed in any combina ion. This sugges ed ha in acellula DMAPP
a ailabili y was a limi ing ac o o PTs e icien ac i i y posing a sig-
ni ican challenge o he success ul p oduc ion o p enyla ed com-
pounds. Conside ing hese esul s, he imp o emen o DMAPP
a ailabili y inside o E. coli M-PAR-121 cells is essen ial o a ain highe
p oduc ions o PN compounds.
3.2. Cons uc ion o DMAPP-modi ied Esche ichia coli s ains
DMAPP is na u ally syn hesized in E. coli h ough he me hyle y-
h i ol phospha e (MEP) pa hway. Howe e , his pa hway con ains a e-
limi ing s eps impai ing he p oduc ion o DMAPP which limi s i s
in acellula a ailabili y. Beyond his, DMAPP is also used in compe ing
pa hways o p oduce ge anyl diphospha e (GPP), a nesyl diphospha e
(FPP), and ge anylge anyl diphospha e (GGPP) and in he syn hesis o
e penoids and s e ols (Cha zi asileiou e al., 2019; Hen y e al., 2018).
Consequen ly, inding s a egies o imp o e he pool o DMAPP is
manda o y o cons uc an e icien s ain able o p oduce
p enylna ingenin.
DXS and IDI we e p e iously iden i ied as he majo a e-limi ing
s eps in he MEP pa hway impac ing he o e all pa hway lux
(Bane jee e al., 2013; Li e al., 2017; Rinaldi e al., 2022; Yuan e al.,
2006; Zhou e al., 2012). Rega ding DXS, i s ac i i y is igh ly egula ed
by eedback inhibi ion and a ansc ip ional and pos - ansc ip ional
le els, playing a cen al ole in he con ol o he pa hway lux (Di
e al., 2023). Rega ding IDI, i s ac i i y in ol es complex edox e-
ac ions and di ec ly impac s also he ac i i y o DXS co obo a ing he
ele ance o op imizing bo h s eps o he MEP pa hway (Li e al., 2017).
Modi ying hese nodes ins ead o o he s eps o he MEP pa hway can
esul in a la ge inc ease in o e all DMAPP p oduc ion compa ed o less
a e-limi ing s eps (Rinaldi e al., 2022). Conside ing ha , we a emp ed
o o e exp ess hese genes o imp o e hei lux. BsDXS was selec ed o
be in eg a ed and o e exp essed in he E. coli M-PAR-121 genome since
i has p e iously shown posi i e e ec s in he p oduc ion o e penoids,
lycopene and isop ene, ha also equi e DMAPP as ex ende subs a e,
in enginee ed E. coli (Chen e al., 2013; Rinaldi e al., 2022; Zhao e al.,
2011). Mo eo e , ScIDI and IDI om BlIDI we e selec ed since hese
genes showed highe ac i i y o he con e sion o IPP in o DMAPP (Gao
e al., 2016). The single in eg a ion o hese genes and he combina ion
o BsDXS wi h each IDI gene in o he lacZ locus o he genome was
pe o med and con i med by PCR and sequencing. The genes we e in-
eg a ed in o he lacZ locus since i is a well-cha ac e ized locus ha is
widely used in CRISPR s a egies. This locus is conside ed a neu al si e
allowing s able genomic in eg a ion wi hou dis up ing essen ial
cellula unc ions (Je is e al., 2021; Zhao e al., 2016). The s ains
E. coli M-PAR-121:BsDXS, E. coli M-PAR-121:ScIDI, E. coli M-PAR-121:
BlDI, E. coli M-PAR-121:BsDXS-ScIDI, and E. coli M-PAR-121:
BsDXS-BlIDI we e success ully cons uc ed using he CRISPR-Cas12a
in eg a ion sys em (Fig. S5). Na i e DXS and IDI om E. coli we e also
o e exp essed by in eg a ing ano he gene copy in o he lacZ locus o he
genome. Single EcDXS, single EcIDI and a casse e holding bo h genes
we e in eg a ed in o he E. coli M-PAR-121 and he success ul in eg a-
ion was con i med by PCR and sequencing. The s ains E. coli
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
220
M-PAR-121:EcDXS, E. coli M-PAR-121:EcIDI, and E. coli M-PAR-121:
EcDXS-EcIDI we e success ully cons uc ed using he CRISPR-Cas12a
in eg a ion sys em (Fig. S6).
Two s a egies o down egula ion o he ge anyl diphospha e/ a -
nesyl diphospha e syn hase (ispA) gene, esponsible o DMAPP con-
e sion in o FPP, we e also designed. Since ispA is conside ed an
essen ial gene o E. coli due o i s unc ion in he isop enoid pa hway
and p oduc ion o essen ial lipids o cell wall main enance, he com-
ple e gene knockou would p obably esul in cell dea h (Mendez-Pe ez
e al., 2017; Shiomi and Niki, 2011). Two di e en a ays we e designed
o a ge wo di e en PAM sequences o he gene using CRISPRi since i
was al eady epo ed ha a ge ing di e en PAM sequences can lead o
di e ences in he down egula ion le els. Mo eo e , i was epo ed ha
he gene ep ession le el is highe he close he PAM egion is o he
ini ia ion ansla ion codon (Je is e al., 2021; Tao e al., 2018). The
gene ep ession was e alua ed by RT-qPCR by compa ing he ispA
exp ession le els in E. coli M-PAR-121 wi h pBbS8c-ddcp 1-ispA1 and
E. coli M-PAR-121 wi h pBbS8c-ddcp 1-ispA2 wi h he exp ession le els
o he con ol s ain (E. coli M-PAR-121 wi h pBbS8c-ddcp 1-Δ) and wo
housekeeping genes (hcaT and idnT). The mel ing cu es o he p ime s
used o each a ge o ensu e p ime e iciency a e ep esen ed in
Fig. S7. A e calcula ing he exp ession old change, i was possible o
conclude ha E. coli M-PAR-121 wi h pBbS8c-ddcp 1-ispA1 (a ay a -
ge ing he i s PAM sequence a e he ispA s a codon) leads o an ispA
down egula ion o 73 %. As expec ed, E. coli M-PAR-121 wi h
pBbS8c-ddcp 1-ispA2 wi h he a ay a ge ing a PAM sequence 154 bp
ups eam o he ispA s a codon, leads o a lowe le el o ispA down-
egula ion (46 %). The g ow h o all he cons uc ed s ains was assessed
and g ow h cu es a e p esen ed in Figs. S8 and S9.
3.3. E alua ion o de no o PN p oduc ion in he cons uc ed DMAPP-
modi ied E. coli s ains
3.3.1. Sc eening in 96-well deep well pla es expe imen s
The en DMAPP-modi ied E. coli cons uc ed s ains we e ans-
o med wi h pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and he pCDFdue -
1 ec o s holding he di e en PTs. One hund ed and en (110) combi-
na ions o s ains/PTs we e ob ained o pe o m p oduc ion expe imen s
in 96-well deep well pla es. Since he comple e biosyn he ic pa hway o
p oduce PN om a simple ca bon sou ce was cons uc ed, only glucose
(a a inal concen a ion o 30 g/L) was supplemen ed o hese expe i-
men s o be used as subs a e. Ou o he 110 combina ions, only 12 we e
able o de no o p oduce PN om glucose (Table 1). 3’-PN was he mos
p oduced PN compound and 6-PN was also de ec ed in wo o he
combina ions.
In he o he 98 combina ions, no peak was de ec ed o PN
compounds. Mo eo e , PN p oduc ion was no de ec ed when he
CRISPRi s ains (E. coli M-PAR-121 wi h pBbS8c-ddcp 1-ispA1 and E. coli
M-PAR-121 wi h pBbS8c-ddcp 1-ispA2) we e used as mic obial chassis.
The p oduc ion o PN was only de ec ed in he E. coli M-PAR-121:BsDXS,
E. coli M-PAR-121:EcDXS, E. coli M-PAR-121:BsDXS:ScIDI, and E. coli M-
PAR-121:EcDXS:EcIDI. Among he es ed PTs, PN compounds we e no
de ec ed when plan a oma ic PTs we e exp essed. The bes p oducing
s ain in hese expe imen s was E. coli M-PAR-121:EcDXS exp essing
pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and pCDFDue _CloQ. This s ain
was able o p oduce 88.34 ±15.22 µM o 3’-PN and 6.20 ±1.20 µM o
6-PN. In addi ion o PN p oduc ion, highe amoun s o p-couma ic acid
and na ingenin we e p oduced and accumula ed in his s ain compa ed
wi h he o he p oducing s ains.
3.3.2. E alua ion o he iden i ied p oducing s ains in shake lask
expe imen s
The abili y o he 12 iden i ied s ains o de no o p oduce PN was
u he e alua ed in shake lask expe imen s. The p oduc ion expe i-
men s we e pe o med in a wo-s ep app oach using he combina ion o
LB Mille and M9 minimal media supplemen ed wi h 30 g/L o glucose.
The i s s ep using LB Mille is pe o med o cell g ow h. Then, he cells
a e cen i uged, and he pelle is esuspended in 50 mL o M9 minimal
medium con aining he ca bon sou ce used as subs a e. This ype o
expe imen wi h one i s phase o g owing ollowed by one second
phase o p oduc ion in o he medium has been used o p oduce se e al
aluable compounds and i was also p e iously op imized o he p o-
duc ion o na ingenin (Rod igues e al., 2020; Gomes e al., 2024). The
p oduc ion o p-couma ic acid, na ingenin, 8-PN, 3’-PN, and 6-PN by he
12 p oducing s ains was e alua ed (Table 2).
PN compounds we e de ec ed o all he 12 combina ions in he
shake lask expe imen s using he combina ion o LB Mille and M9
minimal media. The p oduc ion o 3’-PN was lowe han he p oduc ion
achie ed in 96-well deep well pla es expe imen o some o he com-
bina ions. As occu ed in 96-well deep well pla es expe imen s, E. coli M-
PAR-121:EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and
pCDFDue _CloQ was he bes p oducing s ain being able o p oduce
71.71 ±7.18 µM o 3’-PN and 1.66 ±0.17 µM o 6-PN. Taking hese
esul s in o accoun , his s ain was chosen o p oceed wi h u he
op imiza ions.
Due o he ele ance o hese compounds and he impo ance o
implemen ing an indus ial p ocess, se e al op imiza ions mus be ca -
ied ou . One o he p oblems wi h using he LB+M9 combina ion in
hese shake lask expe imen s is he ac ha his is no a one-s ep p o-
cess and he e is a need o eco e he cells and esuspend hem in he
p oduc ion medium. This makes he expe imen s mo e p one o
con amina ion and is di icul o implemen on a la ge p oduc ion scale.
Table 1
De no o p oduc ion o p enylna ingenin (PN) compounds in 96-well deep well pla es expe imen s. P oduc ion expe imen s we e pe o med in iplica e o he 110
combina ions o s ains exp essing he pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI (NAR plasmid) and he pCDFDue ec o s holding p enyl ans e ases (PTs). Only he 12
combina ions able o de no o p oduce p enylna ingenin a e he ein ep esen ed. The p oduc ion o p-couma ic acid (CA), na ingenin (NAR), 3’-p enylna ingenin (3’-
PN), and 6-p enylna ingenin (6-PN) was e alua ed by ul a-high pe o mance liquid ch oma og aphy (UHPLC).
P oduced compound (µM)
S ain PT CA NAR 3’-PN 6-PN
E. coli M-PAR-121:BsDXS CdpC3PT 546.13 ±14.20 44.70 ±8.56 21.64 ±2.27 -
CoAnaPT 539.29 ±37.72 41.72 ±6.45 19.55 ±0.05 -
CloQ - 40.62 ±2.49 - 3.31 ±0.04
NphB 150.62 ±18.83 57.92 ±5.26 24.66 ±1.49 -
SpPT 463.29 ±10.34 57.55 ±3.67 37.01 ±2.34 -
UbiA 182.96 ±0.99 13.64 ±2.45 20.56 ±1.08 -
E. coli M-PAR-121:EcDXS CoAnaPT - 15.49 ±0.53 15.29 ±0.73 -
CloQ 1427.79 ±360.00 57.79 ±18.38 88.34 ±15.22 6.20 ±1.20
NphB 50.29 ±2.84 18.69 ±0.30 27.46 ±2.27 -
SpPT 268.07 ±9.44 33.50 ±3.65 19.64 ±0.48 -
E. coli M-PAR-121:BsDXS:ScIDI CloQ - 42.16 ±2.67 20.64 ±2.30 -
E. coli M-PAR-121:EcDXS:EcIDI EcPT 764.15 ±80.78 84.47 ±4.85 49.55 ±1.57 -
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
221
Mo eo e , he p oduc ion s ep in M9 minimal medium does no allow
he e alua ion o cell g ow h due o he p esence o CaCO
3
in suspen-
sion. Implemen ing a one-s ep p oduc ion s a egy would make he
p oduc ion p ocess mo e economically iable and easie o scale-up
(Cou o e al., 2017). Conside ing ha , shake lask p oduc ion expe i-
men s using he E. coli M-PAR-121:EcDXS s ain exp essing
pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and pCDFDue _CloQ we e pe -
o med in TB and M9 modi ied media (Fig. 2). M9 modi ied medium
di e s om he M9 minimal medium used in he LB+M9 expe imen s
due o he p esence o ace elemen s and yeas ex ac ins ead o i a-
mins. Yeas ex ac was supplemen ed o imp o e E. coli g ow h and o
inc ease he p o ein exp ession and p oduc ion (Chen e al., 2024, 2022;
Tachibana e al., 2021). TB medium was p epa ed as used in he 96-well
deep well pla es expe imen .
As can be obse ed in Fig. 2, he p oduc ion o PN compounds was
highe using TB medium. Using his p oduc ion medium, E. coli M-PAR-
121:EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and
pCDFDue _CloQ was able o p oduce 160.57 ±23.60 µM o 3’-PN, 4.40
±1.85 µM o 6-PN, and 2.70 ±0.15 µM o 8-PN. Mo eo e , 3’-PN is he
mos p oduced compound in 96-well deep well pla es expe imen s and
in shake lask expe imen s. Ne e heless, he p oduc ion o his com-
pound was signi ican ly imp o ed (2.2- old) using TB medium ins ead o
he combina ion o LB+M9. In con as , he p oduc ion le els using M9
modi ied medium we e sligh ly lowe han he ones ob ained using he
combina ion o LB+M9. Howe e , he di e ences we e no s a is ically
signi ican . Despi e his, om an indus ial and scale-up poin o iew, i
would be mo e ad an ageous o use he M9 modi ied medium han he
combina ion o LB+M9.
The p oduc ion p o ile o all he compounds and he p o ile o
glucose consump ion was e alua ed du ing all he p oduc ion expe i-
men s o all he p oduc ion media es ed (Fig. 3).
As can be obse ed in Fig. 3., he glucose consump ion was simila in
he es ed p oduc ion media. Only 16–17 g/L o glucose we e consumed
in hese expe imen s. Rega ding he in e media ies p-couma ic acid and
na ingenin, highe amoun s o hese compounds we e accumula ed in
he p oduc ion expe imen using LB+M9. By o he side, 3’-PN was only
de ec ed 72 h a e he beginning o his expe imen and 6-PN is only
de ec ed in he inal ime poin (120 h). In he expe imen using M9
modi ied medium, 3’-PN was de ec ed a 24 h and he p oduc ion o his
compound was exponen ially inc easing du ing he expe imen . As
occu ed in LB+M9, 6-PN was only de ec ed in he inal ime poin s o
he expe imen (96 h and 120 h). Rega ding TB expe imen , highe
amoun s o 3’-PN we e de ec ed since he 24 h o he expe imen and he
p oduc ion also inc eased o e ime. Con a ily o he o he expe i-
men s, 6-PN was also de ec ed since he 24 h o he expe imen . Mo e-
o e , 8-PN was also de ec ed in he inal ime poin s o he expe imen
(96 h and 120 h), being only de ec ed in his expe imen . G ow h cu es
in bo h media a e p esen ed in Fig. S10. Conside ing hese esul s, he
use o M9 modi ied o TB media can be conside ed ad an ageous
compa ed o he use o he combina ion o LB+M9 since PN compounds
a e p oduced and de ec ed ea lie in he expe imen .
3.4. E alua ion o de no o PN p oduc ion by Esche ichia coli M-PAR-
121:EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and
pCDFDue _CloQ a a bio eac o scale
Wi h he aim o inc easing p oduc ion le els, he scale-up o he
p oduc ion p ocess o 2-L lab-scale s i ing bio eac o was conside ed.
Since he p oduc ion o 3’-PN and 6-PN was signi ican ly highe when
TB was used in shake lask expe imen s, his p oduc ion medium was
selec ed o bio eac o expe imen s. The p oduc ion p o ile o all he
compounds and glucose consump ion p o ile we e e alua ed h oughou
he p oduc ion expe imen . Mo eo e , he cul u es g ow h was also
e alua ed du ing he expe imen (Fig. 4).
Compa ing wi h he shake lask expe imen s, signi ican ly highe
amoun s o he in e media es p-couma ic acid and na ingenin we e
p oduced and accumula ed du ing he expe imen . A he inal ime
poin , 462.06 ±74.61 µM o p-couma ic acid and 212.28 ±26.54 µM o
na ingenin we e p oduced, ep esen ing a 42.1- old and 4.2- old
Table 2
De no o p oduc ion o PN compounds in shake lask expe imen s using he combina ion o LB Mille and M9 minimal media. P oduc ion expe imen s we e pe o med in
iplica e o he 12 iden i ied s ains exp essing he pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI (NAR plasmid) and he pCDFDue ec o s holding p enyl ans e ases (PTs)
able o de no o p oduce PN in he 96-well deep well pla es expe imen s. The p oduc ion o p-couma ic acid (CA), na ingenin (NAR), 3’-p enylna ingenin (3’-PN), and
6-p enylna ingenin (6-PN) was e alua ed by ul a-high pe o mance liquid ch oma og aphy (UHPLC).
P oduced compound (µM)
S ain PT CA NAR 3’-PN 6-PN
E. coli M-PAR-121:BsDXS CdpC3PT 219.13 ±49.73 15.23 ±1.41 1.54 ±0.64 -
CoAnaPT 340.09 ±32.82 22.94 ±6.09 3.38 ±1.43 -
CloQ 165.93 ±23.26 6.29 ±4.72 0.56 ±0.12 -
NphB 1660.32 ±84.39 32.10 ±9.35 4.26 ±9.35 -
SpPT 124.44 ±13.98 25.27 ±3.57 29.85 ±4.92 -
UbiA 58.97 ±5.04 16.53 ±3.41 12.45 ±2.16 -
E. coli M-PAR-121:EcDXS CoAnaPT 384.38 ±42.00 68.90 ±5.98 39.10 ±4.35
CloQ 442.74 ±70.39 137.81 ±12.36 71.71 ±7.18 1.66 ±0.17
NphB 436.03 ±89.08 11.90 ±1.27 4.53 ±0.93 -
SpPT 219.18 ±16.90 40.39 ±9.15 21.90 ±3.79 -
E. coli M-PAR-121:BsDXS:ScIDI CloQ 106.48 ±2.59 1.10 ±0.48 2.68 ±0.58 -
E. coli M-PAR-121:EcDXS:EcIDI EcPT 183.19 ±32.67 23.88 ±5.42 9.96 ±1.32 -
Fig. 2. De no o p oduc ion o PN compounds by Esche ichia coli M-PAR-121:
EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and pCDFDue _CloQ
in shake lask expe imen s using di e en p oduc ion media. The combina ion
o LB+M9 and he use o only M9 modi ied medium and TB medium we e
es ed. The p oduc ion expe imen s we e ca ied ou using glucose as he sole
subs a e. Resul s co espond o he a e age o h ee independen expe imen s
±s anda d de ia ion.
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
222
inc ease compa ing wi h shake lask expe imen s, espec i ely. In
con as o shake lask expe imen s, glucose was comple ely consumed
wi hin he i s 48 h (Fig. 4B). Rega ding he p oduc ion o PN com-
pounds, he p oduc ion o bo h 3’-PN and 6-PN was inc easing du ing
he expe imen ime and 191.94 ±2.40 µM o 3’-PN (65.34 mg/L) and
9.9 ±0.24 µM o 6-PN (3.37 mg/L) we e de ec ed in he inal ime poin
o he expe imen . Mo eo e , 8-PN was de ec ed a 36 h and 48 h.
Howe e , i was no de ec ed in he nex ime poin s indica ing ha his
compound was p obably deg aded. Al hough he di e ences in he 3’-
PN p oduc ion we e no s a is ically signi ican compa ed o he shake-
lask expe imen s, highe amoun s o his compound we e de ec ed, wi h
a 1.2- old imp o emen in he p oduc ion le els. Rega ding 6-PN, he
p oduc ion achie ed in he bio eac o expe imen ep esen s a 2.3- old
imp o emen compa ed o he p oduc ion in shake lask expe imen s.
Howe e , he di e ences we e no conside ed s a is ically signi ican
since p- alue is abo e 0.05.
Since glucose (30 g/L) was ully consumed wi hin he i s 48 h o
his expe imen , wo addi ional glucose pulses (10 g/L in each pulse)
we e adminis e ed (Fig. 5). These pulses we e in oduced when he
glucose concen a ion d opped o a ound 5–10 g/L o ensu e a su icien
ca bon sou ce was a ailable and p e en a shi in he E. coli me abolism
ha could induce s a a ion, leading o an inc ease in he espi a o y
me abolism and o he ac i a ion o me abolic pa hways o me abolize
o he ca bon sou ces (Li e al., 2022). Mo eo e , main aining glucose
concen a ions in his ange minimizes he o ma ion o inhibi o y
by-p oduc s, such as ace a e, ha could comp omise cell iabili y and
he p oduc i i y o he he e ologous compound (Gecse e al., 2024;
Pinhal e al., 2019). Due o he addi ional glucose, he expe imen was
ex ended o 144 h ins ead o 120 h.
Despi e he wo addi ional glucose pulses (10 g/L), all he supplied
glucose was consumed in 72 h. Compa ed o he shake- lask expe imen s
and o he i s ba ch expe imen , signi ican ly highe amoun s o he
in e media es p-couma ic acid and na ingenin we e p oduced and
accumula ed du ing he expe imen . A he inal ime poin , 1879.18
±73.79 µM o p-couma ic acid and 932.84 ±357.07 µM o na ingenin
we e p oduced. In his expe imen , 8-PN was no de ec ed in any ime
poin . This esul indica es ha he compound may no be p oduced o
may be p oduced in such low amoun s ha i is no de ec ed o is being
deg aded. As obse ed in he i s expe imen , he p oduc ion o bo h 3’-
Fig. 3. P o ile o glucose consump ion and me aboli es p oduc ion by Esche -
ichia coli M-PAR-121:EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI
and pCDFDue _CloQ in shake lask expe imen s. A. P oduc ion expe imen
using he combina ion o LB+M9. B. P oduc ion expe imen using M9 modi ied
medium. C. P oduc ion expe imen using TB medium. Resul s co espond o he
a e age o h ee independen expe imen s ±s anda d de ia ion.
Fig. 4. E alua ion o me aboli es accumula ion, glucose consump ion and cell
g ow h in he ba ch expe imen in bio eac o by Esche ichia coli M-PAR-121:
EcDXS exp essing pRSFDue _FjTAL_CmCHS_A 4CL_MsCHI and pCDFDue _CloQ.
A. P o ile o me aboli es p oduc ion and accumula ion and glucose consump-
ion. B. Op ical densi y a 600 nm (OD
600nm
) o he s ain du ing he expe i-
men . Resul s co espond o he a e age o wo independen expe imen s
±s anda d de ia ion.
D. Gomes e al.
Jou nal o Bio echnology 405 (2025) 215–228
223
