Godoye al.
Bio echnology o Bio uels and Biop oduc s (2024) 17:51
h ps://doi.o g/10.1186/s13068-024-02498-1
RESEARCH Open Access
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Bio echnology o Bio uels
and Biop oduc s
Biosyn hesis o  ag ance 2-phenyle hanol
omsuga s byPseudomonas pu ida
Pa icia Godoy1, Zulema Udaondo2, Es ella Duque1 and Juan L. Ramos1*
Abs ac
Backg ound Pe ochemicals con ibu e o en i onmen al issues, wi h conce ns anging om ene gy consump ion
and ca bon emission o pollu ion. In con as , mic obial bio e ine ies o e eco- iendly al e na i es. The sol en - ole -
an Pseudomonas pu ida DOT-T1E se es as a sui able hos o p oducing a oma ic compounds, speci ically l-phenyla-
lanine and i s de i a i e, 2-phenyle hanol (2-PE), which ind widesp ead applica ions in a ious indus ies.
Resul s This s udy ocuses on enhancing 2-PE p oduc ion in wo l-phenylalanine o e p oducing s ains o DOT-T1E,
namely CM12-5 and CM12-5Δgcd (xylABE), which g ow wi h glucose and glucose-xylose, espec i ely. To syn hesize
2-PE om l-phenylalanine, hese s ains we e ans o med wi h plasmid pPE-1, bea ing he Eh lich pa hway genes,
and i was ound highe 2-PE p oduc ion wi h glucose (abou 50–60 ppm) han wi h xylose (< 3 ppm). To unde s and
he limi ing ac o s, we es ed he addi ion o phenylalanine and in e media es om he Eh lich and shikima e pa h-
ways. The esul s iden i ied in acellula l-phenylalanine as a key limi ing ac o o 2-PE p oduc ion. To o e came his
limi a ion, a cho isma e mu ase/p ephena e dehyd a ase a ian —insen i e o eedback inhibi ion by a oma ic amino
acids—was in oduced in he p oducing s ains. This led o inc eased l-phenylalanine p oduc ion and subsequen ly
p oduced mo e 2-PE (100 ppm). Random mu agenesis o he s ains also p oduced s ains wi h highe l-phenylala-
nine i e s and inc eased 2-PE p oduc ion (up o 120 ppm). The imp o emen s esul ed om p e en ing dead-end
p oduc accumula ion om shikima e and limi ing he ca abolism o po en ial pa hway in e media es in he Eh lich
pa hway. The s udy explo ed ag icul u al was e subs a es, such as co n s o e , suga cane s aw and co n-sy up
as po en ial C sou ces. The bes esul s we e ob ained using 2G subs a es a 3% (be ween 82 and 100 ppm 2-PE),
wi h glucose being he p e e ed suga o 2-PE p oduc ion among he monome ic suga s in hese subs a es.
Conclusions The indings o his s udy o e s a egies o enhance phenylalanine p oduc ion, a key subs a e
o he syn hesis o a oma ic compounds. The abili y o P. pu ida DOT-T1E o h i e wi h a ious C-sou ces and i s
ole ance o subs a es, p oduc s, and po en ial oxican s in indus ial was es, a e highligh ed. The s udy iden i ied
and o e come possible bo lenecks o 2-PE p oduc ion. Ul ima ely, he s ains ha e po en ial o become e icien
mic obial pla o ms o syn hesizing 2-PE om ag o-indus ial was e ma e ials.
Keywo ds 2-Phenyle hanol,
l-Phenylalanine, Pseudomonas pu ida, Mic obial p oduc ion, Biosyn he ic pa hways,
Chemical mu agenesis, 2G suga s
In oduc ion
Pe ochemicals a e one o he la ges g oups o com-
pounds used in he manu ac u e o housands o daily
used goods and a e one o he la ges con ibu o s o
ene gy consump ion and ca bon dioxide emissions.
In ac , i has been es ima ed ha he pe ochemical
indus y consumes abou 14% o he oil and gas used
*Co espondence:
Juan L. Ramos
[email p o ec ed]
1 Depa men o En i onmen al P o ec ion, Es ación Expe imen al del
Zaidín, CSIC, c/ P o eso Alba eda 1, 1808 G anada, Spain
2 Depa men o Biomedical In o ma ics, Uni e si y o A kansas
o Medical Science, Li le Rock, AR 72205, USA
Page 2 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
in he wo ld. This is because he p oduc ion o pe -
ochemicals equi es ex emely high p essu es and
empe a u es (400–900°C) [1]. Concomi an ly, he pe -
ochemical indus y p oduces a wide ange o was e
p oduc s ha pollu e he ai , wa e and soil.
One p omising way o educe he nega i e impac
o he chemical indus y is he de elopmen o mic o-
bial pla o ms ha , h ough e men a i e p ocesses,
can p oduce added- alue chemicals unde oom em-
pe a u e and ambien p essu e condi ions, and a e
en i onmen ally iendly [2–5]. Howe e , a numbe o
indus ially ele an chemicals a e oxic o many bu
no all mic oo ganisms, and adequa e p oduc ion pla -
o ms need o be in place [2–6]. The e o e, choosing
he igh chassis o he implemen a ion o syn he ic
ou es is o u mos impo ance o he success o he
indus y, as p oduc i i y is key o he economics o he
p ojec [7].
In he case o he bio-p oduc ion o a oma ic com-
pounds, such as l-phenylalanine, l- y osine and hei
de i a i es, a use ul pla o m is Pseudomonas pu ida,
because ce ain s ains o his species a e na u ally
equipped wi h nume ous ai s ha allow hem o h i e
in he p esence o high concen a ions o a wide ange o
a oma ic compounds [8, 9]. l-Phenylalanine is a ele an
in e media e o he syn hesis o a oma ic compounds o
indus ial alue such as s y ene, cinnamic acid, and a o-
ma ic alcohols, such as 2-phenyle hanol (2-PE). 2-PE is a
aluable ing edien in he cosme ic and pe ume indus-
ies [10] and i is also used as he subs a e o he syn-
hesis o addi i es and p ese a i es in he ood indus y
and he pha maceu ical sec o [11, 12]. Al hough 2-PE
can be ex ac ed om ce ain lowe s, he majo i y o
2-PE is chemically syn hesized [13, 14] in p ocesses ha
use oxic sol en s and ca alys s, agg essi e eac ion con-
di ions, which yield high amoun s o undesi able side-
p oduc s which dec ease he quali y o he esul ing
2-PE.
On he con a y, he biological p oduc ion o 2-PE is
an al e na i e eco- iendly p ocess ha can be achie ed
h ough di e en pa hways, including he phenylace al-
dehyde syn hase pa hway, he phenyle hylamine pa hway,
and he Eh lich pa hway, h ough which 2-PE is na u ally
p oduced by di e en yeas s, such as Saccha omyces ce -
e isiae, Kluy e omyces ma xianus, Pichia e men as and
Ya owia lipoli ica [15–22]. De no o p oduc ion o 2-PE
om glucose by enginee ed bac e ia has been achie ed
wi h Esche ichia coli, Bacillus licheni o mis, En e obac e
sp. and Pseudomonas pu ida [20, 23–26]; howe e , he
i e s a e o en low and indus ial suga sou ces ha e no
been used. In he Eh lich pa hway (Fig.1), s oichiome -
ic con e sion o l-phenylalanine in o 2-PE akes place in
h ee s eps [27]: Fi s ly, deamina ion o l-phenylalanine
o phenylpy u a e by a ansaminase; secondly, deca -
boxyla ion o phenylpy u a e o phenylace aldehyde by a
phenylpy u a e deca boxylase; and hi dly, educ ion o
phenylace aldehyde o 2-PE by an alcohol dehyd ogenase
(Fig.1).
Pseudomonas pu ida CM12-5,—a sol en - ole an
l-phenylalanine p oduce —is a P. pu ida DOT-T1E
de i a i e gene a ed by using a combina ion o gene ic
s a egies, namely, (1) chemical mu agenesis and he
selec ion o clones esis an o oxic analogues o l-phe-
nylalanine and (2) si e-di ec ed inse ional inac i a ion o
genes in ol ed in l-phenylalanine ca abolism [24]. When
P. pu ida DOT-T1E CM12-5 was ans o med wi h he
plasmid pPE-1, bea ing a his idinol phospha e ans-
e ase (PP_0967 om P. pu ida KT2440), a phenylpy u-
a e deca boxylase (kdc om Rhodospi illum ub um)
and a na i e alcohol dehyd ogenase (T1E_5478, adh om
P. pu ida DOT-T1E), he esul ing s ain was able o p o-
duce 2-PE om glucose [24]. In addi ion o glucose, Pseu-
domonas pu ida also uses uc ose as he sole C-sou ce
[28, 29], ano he majo suga used in indus y; howe e ,
i canno use xylose, he second mos abundan mono-
me ic suga a e glucose in enzyma ic hyd olysa es o
Fig. 1 2-Phenyle hanol biosyn hesis om glucose h ough he Eh lich pa hway. Glucose me abolized h ough he E ne -Doudo o and pen ose
phospha e pa hway (PPP) yield; phosphoenolpy u a e (PEP) and e y h ose-4-phospha e E4P; which a e channeled h ough he shikima e/
cho isma e o phenylalanine (PE). Then, he Eh lich pa hway enzyme p oduces 2-PE. AT, ansaminase; PDC, phenylpy u a e deca boxylase; ADH,
alcohol dehyd ogenase. (Adap ed om Qian e al. [20])
Page 3 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
lignocellulosic esidues (2G suga s) [30]. Se e al esea ch
g oups ha e cons uc ed a ian s o P. pu ida ha use
xylose as he sole C-sou ce, his equi es he inac i a ion
o he gcd gene encoding a glucose/xylose dehyd oge-
nase ha con e xylose in dead-end xylona e and inco -
po a ion o xylose anspo (xylE) and ca abolic genes
(xylAB) ha con e xylose in o xylose-5-phospha e ha
en e s in o he pen ose phospha e cycle [31–33].
The aims o his s udy we e: (i) o s udy he syn hesis o
2-phenyle hanol by sol en - ole an Pseudomonas pu ida
DOT-T1E de i a i es wi h a ious ca bon sou ces; (ii) o
iden i y he limi ing s ep in he biosyn hesis o phenyla-
lanine and i s con e sion o 2-PE in his P. pu ida chas-
sis; (iii) o gene a e mu an de i a i es ha a e mo e
e icien in he biosyn hesis o 2-PE, and (i ) o es di -
e en indus ial ca bon s ocks o he syn hesis o 2-PE.
This s udy iden i ies he syn hesis o l-phenylalanine as
he main bo leneck in he p oduc ion o 2-PE and ha
p e en ion o he p oduc ion o dead-end p oduc s om
shikima e inc eased he in acellula le els o l-pheny-
lalanine and i s subsequen con e sion o 2-PE. Glucose
has been iden i ied as he p e e ed suga in indus ial
sou ces o he biosyn hesis o he a oma ic alcohol.
Resul s
Syn hesis o 2‑PE omglucose andxylose byP. pu ida
CM12‑5 de i a i es
The wo L-phenylalanine p oducing s ains CM12-5
and CM12-5Δgcd (xylABE) ha me abolize glucose and
glucose and xylose, espec i ely, we e ans o med o
no wi h plasmid pPE-1 and syn hesis o 2-PE by he
ou s ains es ed wi h glucose, xylose and mix u es o
glucose:xylose (3:1) in assays ha las ed 24h. Ou esul s
a e shown in Fig.2. The wo s ains wi hou he pPE-1
plasmid did no p oduce 2-PE as expec ed (no shown).
We ound ha he P. pu ida CM12-5 (pPE-1) and P.
pu ida CM12-5Δgcd (xylABE) (pPE-1) p oduced abou
50 ppm 2-PE when glucose was he g ow h subs a e
(Fig.2). Howe e , wi h xylose, negligible 2-PE accumu-
la ed, i.e., less han 3ppm was de ec ed in he cul u e
medium (no shown). When glucose and xylose we e
p esen simul aneously, a p oduc ion le el o 2-PE simila
o ha ob ained wi h glucose alone was achie ed (Fig.2).
These s ains accumula ed low amoun s o phenylalanine
in he cul u e medium (< 3ppm) unde all g ow h condi-
ions (no shown), which sugges s ha almos all pheny-
lalanine p oduced in acellula ly was channeled owa ds
2-PE p oduc ion.
Since xylose is me abolized h ough he pen ose phos-
pha e pa hway (PPP), i is likely ha phosphoenolpy u-
a e (PEP) le els in CM12-5Δgcd (xylABE) a e limi ing
he ope a ion o he shikima e pa hway (Fig.1), which is
c i ical o p oduc ion o l-phenylalanine and i s subse-
quen channeling owa ds 2-PE.
E ec o  headdi ion o  heEh lich pa hway andshikima e
pa hway in e media es on2‑PE p oduc ion
To de e mine i he limi ing ac o in he p ocess was
ela ed o he p oduc ion o he p ima y subs a e, l-phe-
nylalanine (l-Phe), o pa hway in e media es, i.e., phenyl
ace aldehyde (PA) and phenylpy u a e (PP), we ca ied
ou es ing cells assays in which 1mM l-Phe (165ppm),
1 mM PA (120 ppm) o 1 mM PP (186 ppm) we e
added o cul u es o CM12-5 (pPE-1) and CM12-5Δgcd
(xylABE) (pPE-1), in a medium wi h glucose o wi h
xylose as he C sou ce. Figu e3 shows ha , ega dless o
he C sou ce used (glucose o xylose), 2-PE p oduc ion
eached concen a ions o abou 100–120ppm 2-PE in
bo h s ains, inc eases ha ep esen p oduc ion abou
200% highe han hose ob ained in he absence o Eh -
lich pa hway in e media es. The e o e, he ans o ma-
ion o l-Phe in o 2-PE h ough he Eh lich pa hway was
no comp omised. This sugges s ha he ini ial amoun
o l-Phe in he cells is he main limi ing ac o in he p o-
duc ion o 2-PE.
On he o he hand, l-phenylalanine in P. pu ida is
made h ough he shikima e pa hway, in which shiki-
ma e and cho isma e a e in e media es (Fig. 1), and
hence we es ed whe he he addi ion o 1mM shiki-
ma e (174ppm) o 1mM cho isma e (226ppm) would
Fig. 2 P oduc ion o 2-PE in he p esence o glucose (open ba s)
o mix u e o glucose:xylose (closed ba s) as he C sou ce. P. pu ida
CM12-5 (pPE-1) and CM12-5Δgcd (xylABE) (pPE-1) cells we e
g own o e nigh wi h glucose as he C sou ce. The cul u es we e
hen washed and dilu ed o DO660 o 0.1 dilu ed in esh medium
wi h glucose (0.5% w/ ) o glucose:xylose (0.375% w/ :0.125%
w/ ) as he C sou ce, and 2-PE p oduc ion was measu ed a e 24 h
incuba ion. The igu e shows he a e age o h ee independen
assays
Page 4 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
also lead o an inc ease in 2-PE p oduc ion. Supple-
men a ion wi h cho isma e o CM12-5 (pPE-1) and
CM12-5Δgcd (xylABE) (pPE-1) g owing wi h glucose o
xylose (Table1) led o p oduc ion le els o abou 1 ppm
105 ppm 2-PE (Table 1), a alue simila o ha eached
when Eh lich pa hway in e media es we e added. How-
e e , his was no he case wi h shikima e, as i was only
pa ially consumed by he cells, and also because i was
bio ans o med in side dead-end p oduc s (3-hyd oxy-
shikimic acid, quina e and ace a e) bo h wi h glucose o
xylose as he C sou ce (da a no shown). This implies ha
an app op ia e channeling o shikima e o cho isma e is
a c i ical issue in he syn hesis o 2-PE by his hos pla -
o m. Hence, ou da a suppo he hypo hesis ha he
in acellula le els o l-Phe maybe he limi ing s ep in
he biosyn hesis o 2-PE. Mo eo e , his was pa icula ly
no iceable, when xylose was used as he sole C sou ce,
since wi h his C-sou ce a clea limi a ion o he a ailable
in e media es o he syn hesis o l-Phe was iden i ied.
Inc easing phenylalanine p oduc ion and2‑PE le els inP.
pu ida
I has been desc ibed ha l-Phe exe s eedback inhibi-
ion o i s own syn hesis h ough in e ac ion o he amino
acid wi h he R-domain o cho isma e mu ase/p ephen-
a e dehyd a ase, PheA, a bi unc ional enzyme in he shi-
kima e pa hway o he syn hesis o a oma ic amino acids
[34]. Molina-San iago e  al. [24] cons uc ed PheA b ,
a PheA mu an a ian in which he R-domain o PheA
was dele ed and he allos e ic sensi i i y o phenylala-
nine was elimina ed. This PheA b mu an a ian was
exp essed in plasmid pPHE1. This s udy con i med ha
CM12-5 (pPHE1) accumula ed highe i e s o L-phe-
nylalanine in he cul u e medium i.e., up o 350 ± 10mg
L−1 e sus a ound 300 ± 5mg L−1 o CM12-5 (Fig.4A).
Then, CM12-5 (pPHE1) was ans o med wi h pPE-1 and
2-PE measu ed in he medium. I was ound ha 2-PE
inc eased om abou 50–60 ppm o nea 95 ± 5 ppm
(Fig. 4B), p oduc ion le els simila o hose achie ed
wi h exogenous supplemen a ion o l-Phe in es ing
cell assays. This con i med ha he limi a ion in he
Fig. 3 P oduc ion o 2-PE by es ing cells in he p esence o Eh lich pa hway in e media es: l-phenylalanine (l-Phe), phenylace aldehyde (PA)
o phenylpy u a e (PP). CM12-5 (pPE-1) (blue ba s) and CM12-5Δgcd (xylABE) (pPE-1) ( ed ba s) cells we e g own in he p esence o glucose as he C
sou ce un il eaching a DO660 o 1, he cul u es we e hen washed and concen a ed in 1xM9 + 0.5% (w/ ) glucose o xylose o each a DO660
o abou 10, and he e ec o 1 mM l-Phe PA o PP supplemen s on 2-PE p oduc ion was measu ed a e 24 h o incuba ion. The igu e shows
he a e age alues o h ee independen assays
Table 1 E ec o shikima e pa hway in e media es cho ismic
acid (CA) and shikimic acid (SA) on he syn hesis o 2-PE in P.
pu ida CM12-5 (pPE-1) and CM12-5Δgcd (xylABE) (pPE-1) s ains
The assay was ca ied as desc ibed in he legend o Fig.3 excep ha 1mM
cho ismic acid (CA) o 1mM shikimic acid (SA) we e added. Values and s anda d
de ia ion a e he a e age o h ee independen assays
C sou ce Supplemen [2‑PE] (mg L−1)
CM12‑5 (pPE‑1) CM12‑5Δgcd
(xylABE) (pPE‑1)
M9 Glucose None 39.3 ± 0.5 27.3 ± 0.1
+ 1 mM CA 104.3 ± 1.8 120.5 ± 0.9
+ 1 mM SA 72.9 ± 3.8 83.5 ± 2.2
M9 Xylose None 16.0 ± 0.3 30.1 ± 0.4
+ 1 mM CA 105.4 ± 1.7 124.8 ± 1.6
+ 1 mM SA 51.4 ± 1.0 88.6 ± 3.7
Page 5 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
p oduc ion o l-Phe is an ob ious s ep in he p oduc ion
o 2-PE.
The ea e , i was decided o sea ch o clones ha
can p oduce mo e l-Phe han he CM12-5 and CM12-5
(pPHE1) s ains. To his end, he P. pu ida CM12-5 and
CM12-5 (pPHE1) s ains we e subjec ed o andom
mu agenesis wi h EMS and selec ion o clones esis -
an o high concen a ions (> 3mg mL−1) o he l-phe-
nylalanine analogue p- luo o-phenylalanine (FPA), he
mu agenesis was ca ied ou as desc ibed in Ma e ials
and Me hods. One clone o each s ain (named PG1E
and PG2E), which we e able o accumula e abou 300
and 450ppm L-Phe, espec i ely, when g own wi h 1.5%
(w/ ) glucose (Fig.4A) we e kep . These o e p oduce
s ains we e subsequen ly ans o med wi h pPE-1 plas-
mid and 2-PE accumula ion de e mined. Wi h P. pu ida
PG1E, a ound 100ppm 2-PE accumula ed and he high-
es le els o 2-PE we e eached wi h PG2E ha accumu-
la ed a close o 120ppm (Fig.4B).
As he highes l-Phe and 2-PE p oduc ions we e
achie ed wi h PG2E s ain, i was decided o sequence
i s genome o de e mine which genomic changes we e
esponsible o l-Phe o e p oduc ion. When compa -
ing he genome o his l-Phe o e p oduce de i a i e
PG2E wi h DOT-T1E genome, we ound ha , apa om
he i e mu a ions in ol ed in l-Phe deg ada ion pa h-
ways gene a ed by di ec mu agenesis and desc ibed in
Molina-San iago e al. [24], up o 400 SNP (see Addi-
ional ile2: TableS1) we e ound. To e ine he s udy, he
SNP in he genes ha encode p o eins ela ed o l-Phe
me abolism (Addi ional ile4: TableS3) we e analyzed. A
H431P change in shikima e dehyd ogenase (QuiA) ha
may p e en he mis ou ing o shikima e o 3-hyd oxyski-
ma e was iden i ied; oge he wi h a se o mu a ions in
genes encoding enzymes o he phenylace ic acid (PAA)
deg ada ion pa hway. As sugges ed by L324P in PaaH_2,
Y154C in PaaK, V154A and F284L in PaaA (Addi ional
ile3: TableS2). Indeed, due o he mu a ions in he paa
genes, he PG2E s ain los he abili y o me abolize
PAA (see Addi ional ile1: Fig. S1). The e o e, he highe
accumula ion o l-Phe in he s ain p oducing he high-
es l-Phe le el, and he subsequen 2-PE ob ained, may
be ela ed o a mo e e icien channeling o shikima e o
l-Phe.
The use o ag icul u al was e p oduc s as eeds ocks
o 2‑PE p oduc ion
To explo e he po en ial o Pseudomonas pu ida as a
chassis o p oduce 2-PE om 2G subs a es, he capa-
bili y o p oducing 2-PE om co n s o e (PCS) and
suga cane s aw (PSCS) hyd olysa es, p epa ed as
desc ibed in Ma e ials and Me hods was assayed. To
his end, P. pu ida CM12-5 (pPE-1), CM12-5(pPHE1)
(pPE-1), CM12-5(pPHE1)-FPA (pPE-1) and CM12-
5Δgcd (xylABE) (pPE-1) s ains we e g own in M8 mini-
mal medium wi h KNO3 as he N sou ce, and 3% (w/ )
PCS o PSCS. 2-PE p oduc ion le els we e de e mined
a e 24h and he esul s a e shown in Table2. All he
s ains p oduced be ween 82 and 110mg L−1 2-PE, ei he
om 3% PCS o 3% PSCS hyd olysa es. The concen a-
ion o suga s was also conside ed in mola e ms o es i-
ma e mola yields, which we e in he ange o 8.6–11.1%
ega ding glucose u iliza ion (Table2).
Simila assays we e also done wi h all o hese 2-PE
p oducing s ains in M9 minimal medium, bu using 1G
subs a es as he C sou ce, such as co n sy up, which
was used a 1/80 ( / ). Ou esul s a e shown in Table3.
Su p isingly, he o al p oduc ion o 2-PE wi h 1G sub-
s a es was lowe han wi h 2G subs a es, since hese
s ains accumula ed be ween 10 and 84mg L−1 o 2-PE
and he mola yields we e below 4.8%. This is in acco d-
ance wi h low l-Phe accumula ion in he cul u e medium
when he CM12-5 s ain was g own wi h uc ose ins ead
o glucose as he sole C sou ce (no shown). This may be
due o he high phosphoenolpy u a e (PEP) and ene gy
Fig. 4 l-Phe p oduc ion and 2-PE p oduc ion om 1.5% (w/ )
glucose o Pseudomonas pu ida CM12-5 de i a i e s ains
wi hou and wi h he pPE-1 plasmid. Cells we e g own o e nigh
wi h glucose as he C sou ce, hen he cul u es we e dilu ed o DO660
o 0.1 in esh medium wi h 1.5% (w/ ) glucose, and l-Phe p oduc ion
(panel A) o 2-PE (panel B) was measu ed a e 24 h incuba ion. The
igu e shows he a e age alues o h ee independen assays

Page 6 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
equi emen s o he uc ose in ake h ough he PTS
ac i e anspo sys em [35, 36]. The lowe yield ag ees
wi h he ac ha glucose and uc ose we e no con-
sumed comple ely a e 48h; in ac , on a e age, only
30% o glucose was deple ed, while 5% o he uc ose was
consumed (no shown).
The e o e, indus ial subs a es such as co n sy up
(1G subs a e), and co n s o e and suga -cane s aw
hyd olysa es (2G subs a es) a e sui able o using in
2-PE p oduc ion in a mo e sus ainable and en i on-
men ally iendly way; howe e , amongs i s compo-
nen s, glucose is p e e ably consumed o e uc ose o
xylose, and he p esence uc ose may slow his p ocess
down.
Table 2 2-PE p oduc ion om 2G lignocellulosic hyd olysa es PCS and PSCS in Pseudomonas pu ida CM12-5 de i a i e s ains
The assays we e ca ied ou as desc ibed in M8 minimal medium wi h 10mM KNO3 and 3% (w/ ) o hyd olysa ed PCS o PSCS. Glucose and xylose we e de e mined
a he beginning o he assay and 24h la e . The concen a ion o 2-PE was de e mined a he end o he assay. The alues and s anda d de ia ion a e he a e age o
h ee independen assays
3% PCS 3% PSCS
(pPE‑1) (pPHE1)
(pPE‑1) PG2E (pPE‑
1) Δgcd(xylABE)
(pPE‑1) (pPE‑1) (pPHE1)
(pPE‑1) PG2E (pPE‑
1) Δgcd(xylABE)
(pPE‑1)
mg L−1 Ini ial glu-
cose 1630 ± 78 1753 ± 130 1638 ± 107 1562 ± 87 1533 ± 111 1576 ± 99 1576 ± 166 1576 ± 135
Consumed
Glucose 1334 ± 32 1682 ± 149 1456 ± 131 1425 ± 116 1441 ± 121 1548 ± 103 1358 ± 247 1443 ± 195
P oduced
2-PE 86 ± 19 109 ± 17 110 ± 25 85 ± 14 82 ± 15 97 ± 16 93 ± 15 84 ± 11
Ini ial xylose 2596 ± 178 2627 ± 59 2600 ± 37 2633 ± 115 2034 ± 203 2305 ± 18 1910 ± 394 2065 ± 205
Consumed
xylose 1990 ± 232 1690 ± 230 1302 ± 431 1978 ± 254 1698 ± 380 1788 ± 102 1367 ± 305 1574 ± 181
mM Ini ial glu-
cose 9.05 ± 0.43 9.20 ± 0.72 9.09 ± 0.59 8.67 ± 0.48 8.51 ± 0.61 8.75 ± 0.55 8.75 ± 0.92 8.75 ± 0.75
Consumed
Glucose 7.40 ± 0.18 8.68 ± 0.83 8.08 ± 0.73 7.91 ± 0.64 8.00 ± 0.67 8.59 ± 0.57 7.54 ± 1.37 8.01 ± 1.08
P oduced
2-PE 0.70 ± 0.15 0.82 ± 0.14 0.90 ± 0.21 0.69 ± 0.12 0.67 ± 0.12 0.80 ± 0.13 0.77 ± 0.12 0.69 ± 0.09
Ini ial xylose 17.29 ± 1.19 17.50 ± 0.40 17.32 ± 0.25 17.54 ± 0.77 13.55 ± 1.35 15.36 ± 0.12 12.73 ± 2.62 13.76 ± 1.36
Consumed
xylose 13.26 ± 1.55 11.26 ± 1.53 8.67 ± 2.87 13.18 ± 1.69 11.31 ± 2.53 11.91 ± 0.68 9.11 ± 2.03 10.49 ± 1.21
(G) Mola yield (%) 8.85 ± 1.68 9.51 ± 1.82 11.14 ± 1.76 8.80 ± 1.66 8.68 ± 1.28 9.24 ± 0.89 10.17 ± 0.26 8.64 ± 1.06
Table 3 2-PE p oduc ion om co n sy up (CS) in Pseudomonas pu ida CM12-5 de i a i e s ains
The assays we e ca ied as desc ibed in he oo no e o Table3 excep ha co n sy up was used. Glucose and uc ose we e de e mined a = 0 and 24h, and 2-PE
was measu ed a he end o he assays. The alues and s anda d de ia ion a e he a e age o h ee independen assays
CS 1/80 ( / )
(pPE‑1) (pPHE1) (pPE‑1) PG2E (pPE‑1) Δgcd (xylABE) (pPE‑1)
mg L−1 Ini ial glucose 4725 ± 94 4737 ± 95 4722 ± 89 4729 ± 100
Consumed glucose 2375 ± 48 2117 ± 42 2579 ± 52 2204 ± 44
Ini ial uc ose 6351 ± 127 6323 ± 162 6318 ± 120 6349 ± 127
Consumed uc ose 429 ± 9 357 ± 7 273 ± 5 474 ± 9
P oduced 2-PE 28.7 ± 0.57 43.8 ± 0.87 84.1 ± 1.68 10.5 ± 0.21
mM Ini ial glucose 26 ± 0.52 26 ± 0.53 26 ± 0.52 26 ± 0.52
Consumed glucose 13 ± 0.26 12 ± 0.24 14 ± 0.28 12 ± 0.24
Ini ial uc ose 35.3 ± 0.71 35.3 ± 0.70 35.3 ± 0.70 35.3 ± 0.71
Consumed uc ose 2.4 ± 0.05 2.0 ± 0.04 1.5 ± 0.03 2.6 ± 0.05
P oduced 2-PE 0.23 ± 0.01 0.36 ± 0.02 0.69 ± 0.03 0.09 ± 0.01
(G) Mola yield (%) 1.78 ± 0.03 3.05 ± 0.05 4.81 ± 0.09 0.70 ± 0.01
Page 7 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
Discussion
The in e es in he de elopmen o echnology o g een
chemis y inc eased a e a se ies o s udies e ealed ha
nea ly wo hi ds o all chemical p oduc s in use could
be made h ough he e men a ion o suga s ob ained
om seeds (i.e., co n, whea , and o he s), plan esidues,
o ganic was e and lignin [37–40]. The OECD and o he
agencies a e aiming o ansi ion 30% o he o al pe o-
chemical indus y o enewable sou ces by 2050 [41–44].
Se e al esea ch g oups ha e pu g ea e o in o
de eloping Pseudomonas pu ida as a chassis o he bio-
chemical p oduc ion o a oma ic compounds because o
i s me abolic p ope ies, and i s abili y o ole a e s ess
and high concen a ions o sol en s [24, 33, 45–49]. Ou
speci ic o e all aim is he cons uc ion o sol en - ole -
an Pseudomonas s ains ha can pe o m op imally and
achie e high yields o indus ially aluable chemicals,
such as 2-PE, ans-cinnamic acid and s y ene. We ha e
es ima ed ha hese cell ac o ies can educe CO2 emis-
sions by abou 90% compa ed o cu en pe ochemical-
based me hods.
In his s udy, he molecula basis o 2-PE p oduc ion
when Pseudomonas is used as a chassis we e analyzed.
P oduc ion o 2-PE can be conside ed o ake place in
wo se s o eac ions: one ha leads o syn hesis o he
p ima y subs a e, L-Phe, and a second se ha uses
he Eh lich pa hway o p oduce 2-PE. I was ound ha ,
whe he in acellula ly made o exogenously supplied,
L-Phe is s oichiome ically con e ed on 2-PE. The genes
o he Eh lich pa hway we e exp essed in pPE-1 plasmid
om he egula ed Pm p omo e [50, 51], whose exp es-
sion is d i en by XylS in he p esence o alkylbenzoa es
in plasmid pPE-1, o om a cons i u i ely exp essed lac
p omo e in plasmid pPE-2 (no shown). Ou unpub-
lished esul s e ealed ha he egula ed exp ession o
he Eh lich pa hway yielded mo e consis en p oduc ion
han he cons i u i e exp ession, p obably due o he gen-
e a ion o ins abili y, as p oduc ion dec eased wi h ime
(P. Godoy, unpublished esul s).
The limi ing ac o in he 2-PE p oduc ion p ocess was
linked o he p oduc ion o he p ima y subs a e, phe-
nylalanine (L-Phe). The limi ed lux owa d he shiki-
ma e syn hesis pa hway seems o be he main bo leneck
o he p oduc ion o a oma ic chemicals, because i is
highly egula ed a ansc ip ional and enzyma ic le els
[25, 52]. A key ea u e in a wide ange o l-phenylalanine
p oduce s is he need o o e come he eedback inhibi-
ion by l-Phe o he bi unc ional cho isma e mu ase/
p ephena e dehyd a ase (PheA) enzyme [24, 25]. This
ac has been con i med in his s udy, as he exp ession
o he PheA b a ian lacking he R-domain exp essed in
plasmid pPHE1 [24] led o signi ican inc eases in L-phe-
nylalanine accumula ion in he medium. Ano he s ep is
o inc ease he a ailabili y o shikima e p ecu so s phos-
phoenolpy u a e (PEP) and e y h ose-4-phospha e (E4P)
[25, 53–57]. This s udy shows ha xylose is a less e i-
cien C-sou ce o l-Phe p oduc ion han glucose, which
is p obably ela ed o he low a ailabili y o PEP in cells
g owing on xylose, which limi s he C low owa ds he
shikima e pa hway. In addi ion, while he Eh lich pa h-
way in e media es a e s oichiome ically ans o med
in o 2-PE, we obse ed ha pa o shikima e made in
he cells may di e o dead-end 3-hyd oxyshikima e.
This was indeed con i med when clones able o p oduce
highe amoun s o L-Phe we e isola ed upon a ound o
andom chemical mu agenesis ha esul ed in he iden-
i ica ion o a mu a ion in shikima e dehyd ogenase ha
p e en s 3-hyd oxyshikima e accumula ion, and limi ed
me abolism h ough he phenylace ic acid deg ada ion
pa hway.
Lignocellulose om ag icul u al esidues o om
u ban o ganic was e a e expec ed o ep esen a ele an
aw ma e ial o he p oduc ion o chemicals. Thus, P.
pu ida DOT-T1E and he l-Phe o e p oducing s ains
we e enginee ed o bea he machine y in ol ed in xylose
up ake and ca abolism and also o he 2-PE p oduc-
ion genes. Howe e , low p oduc ion o l-Phe and 2-PE
om xylose was ound, in spi e xylose being me abolized
h ough he pen ose phospha e pa hway ha gene -
a es E4P, which is a p ecu so o he shikima e pa hway
(see Fig.1). Due o he ac ha when l-Phe o Eh lich
pa hway in e media es we e added o cells g own o
xylose, hey we e s oichiome ically con e ed o 2-PE
by CM12-5 de i a i es; channeling xylose o shikima e
seems o be a key limi ing s ep in 2G con e sion o C5
suga s in o 2-PE. Fu he mo e, PEP is also limi ing due o
i s channeling o py u a e ha is e en ually ans o med
in o ace yl-CoA and en e s he TCA cycle [25, 56, 57].
Taking all hese ac s in o conside a ion, i is clea ha i
is necessa y o e-o ganize he PEP-4EP me abolic p o-
cesses when xylose is used as he C-sou ce.
I is known ha 2G hyd olysa es, in addi ion o sug-
a s, con ain a wide ange o oxic chemicals such as
u u al, 5-hyd oxyme hyl u u al and o he s ([30] and
Addi ional ile 4: Table S3). These oxic compounds
a e o igina ed om pa ial deg ada ion o suga s du -
ing s eam-explosion in he in ensi e physicochemical
p e- ea men o lignocellulosic ma e ial [30]. These
compounds a ec he iabili y o some mic oo gan-
isms used o bio ans o ma ion o 2G subs a es [20,
58, 59]. In his con ex , P. pu ida DOT-T1E has been
shown o g ow in he p esence o > 50, 25 and 25mM
o u u al, 3-hyd oxyme hyl u u al and sy ingic acid,
espec i ely, in ag eemen wi h he high ole ance o
his s ain o oxic compounds, al hough hei combi-
na ion enhanced oxici y. DOT-T1E and i s de i a i es
Page 8 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
h i e a maximal a e wi h up o 3% (w/ ) o lignocel-
lulose subs a es, and adap a i e e olu ion assays (ALE)
in ou lab ended wi h e ol ed clones adap ed o ole -
a e up o 11% (w/ ) lignocellulosic subs a es (Juan
L. Ramos, unpublished esul s). We a e ocusing ou
e o s on enhancing he in acellula p oduc ion o l-
Phe as a p ima y subs a e o he syn hesis o a oma ic
compounds and he e olu ion o he s ain o h i e
wi h a wide ange o C-sou ces and ole ance o sub-
s a es, p oduc s and possible oxican s in indus ial
was es o u he iden i y and enginee possible bo -
lenecks o 2-PE p oduc ion. We s ongly suppo he
idea ha P. pu ida DOT-T1E could become an e icien
mic obial cell ac o y o he p oduc ion o a oma ic
compounds om ag o-indus ial was e ma e ial.
Conclusions
Pe ochemicals exace ba e en i onmen al issues by
necessi a ing subs an ial ene gy inpu s o hei p oduc-
ion, con ibu ing o ca bon emissions, and di ec ly o
indi ec ly causing pollu ion. In con as , we ha e iden i-
ied P. pu ida DOT-T1E as a p omising bio e ine y o
he sus ainable and en i onmen ally iendly p oduc ion
o a oma ic compounds, speci ically 2-PE, om a ious
suga s. This app oach s ands in con as o con en ional
chemical me hods.
The s ains CM12-5 and CM12-5Δgcd, designed o
o e p oduce l-phenylalanine, demons a ed he capabil-
i y o con e his amino acid in o 2-PE upon he exp es-
sion o Eh lich pa hway genes om plasmid pPE-1. The
in acellula concen a ion o l-phenylalanine was iden i-
ied as a c i ical limi ing ac o in 2-PE p oduc ion. The
in oduc ion o a mu an a ian o cho isma e mu ase/
p ephena e dehyd a ase, bypassed eedback inhibi ion by
a oma ic amino acids, and signi ican ly enhanced l-phe-
nylalanine p oduc ion and, consequen ly, boos ed 2-PE
yields. Fu he mo e, he applica ion o andom mu agen-
esis led o s ains exhibi ing ele a ed l-phenylalanine
p oduc ion, esul ing in inc eased 2-PE p oduc ion.
In he ealm o indus ial applica ions, we explo ed he
use o ag icul u al was e subs a es such as co n s o e ,
suga cane s aw, and co n sy up as po en ial ca bon
sou ces. Rega dless o he was e sou ce, glucose was he
p e e ed suga o 2-PE biosyn hesis.
To sum up, ou s udy unde sco es he po en ial o
mic obial bio e ine ies, pa icula ly hose employing P.
pu ida, as an en i onmen ally sus ainable al e na i e o
p oducing aluable a oma ic compounds like 2-PE. The
implemen a ion o s ain enginee ing and he u iliza-
ion o ag icul u al was e subs a es a e pi o al s a egies
iden i ied o enhancing p oduc ion e iciency and mi i-
ga ing en i onmen al impac .
Ma e ials andme hods
Chemicals
l-Phenylalanine (99%), 2-phenyle hanol (99%), phe-
nylace aldehyde (90%), shikimic acid (99%), sodium
phenylpy u a e (powde ), cho ismic acid ba ium
sal (≥ 80%), o- oluic acid (99%), m- oluic acid (99%),
p- luo o-dl-phenylalanine (FPA) (98%), ViscozymeR
l-celluloly ic enzyme mix u e and e hylme hanesul ona e
(EMS) we e pu chased om Sigma-Ald ich (Me ck).
Ace oni ile (HPLC g ade), d-( +)-xylose and d-( +)-glu-
cose we e p o ided by VWR Chemicals (F ance).
S ains, plasmids andg ow h condi ions
S ains and plasmids used in his s udy a e shown in
Table 4. Pseudomonas DOT-T1E is a sol en - ole an
s ain ha was o iginally isola ed om a was ewa e
ea men plan [8]. Pseudomonas pu ida CM12-5 is a
DOT-T1E de i a i e ha p oduced phenylalanine and i
was p e iously desc ibed [24]. To make P. pu ida CM12-5
able o use xylose as a C-sou ce, a de i a i e o his s ain,
bea ing and xylE genes om E. coli ha anspo s xylose
in o he cell and he xylAB, genes ha encode enzymes o
con e xylose in o xylose-5 phospha e, which is me abo-
lized h ough he pen ose phospha e pa hway, was con-
s uc ed [45]. E icien u iliza ion o xylose equi ed he
inac i a ion o he glucose/xylose dehyd ogenase (Gcd)
o a oid he mis ou ing o xylose o dead-end xylona e
[33]. A de i a i e o CM12-5 wi h Δgcd and bea ing he
xylAB and xylE genes was also a ailable. Esche ichia coli
DH5α was used o he cloning expe imen s and p opa-
ga ion o plasmids (Table4).
Esche ichia coli was ou inely g own a 37ºC in LB
medium, while he Pseudomonas pu ida s ains we e
g own a 30ºC in LB medium o in M9 minimal medium
[60] wi h 5g L−1 glucose as he sole ca bon sou ce. When
indica ed M8 minimal medium was used, his is iden i-
cal o M9 medium excep ha ammonium chlo ide was
eplaced by 10mM KNO3. Fo liquid cul u es 100mL
conical lasks seeded wi h 20mL cul u e medium we e
incuba ed in a Kühne he mos a incuba o wi h agi a-
ion (200 pm, 30ºC). G ow h was moni o ed by ollow-
ing he u bidi y o he cul u es a 660nm (OD660) in a
UV–VIS spec opho ome e Shimadzu 1900i (Kyo o,
Japan).
Fo es ing cells assays, he cells we e g own in M9
minimal medium wi h 5g L−1 glucose as he sole ca bon
sou ce, un il he cul u e eached OD660 = 1.0. Then, he
cells we e cen i uged and washed wi h 1xM9 and e-sus-
pended in he app op ia e medium a OD660 = 10.
P e ea ed suga cane s aw (PSCS) and co n s o e
(PCS) we e sou ced om he Abengoa Bioene gy Bio-
mass Pilo Plan in Yo k, Neb aska, USA. The inal d y
ma e con en was de e mined o be 39.9% o PSCS
Page 9 o 12
Godoye al. Bio echnology o Bio uels and Biop oduc s (2024) 17:51
and 43.3% o PCS, as epo ed in he composi ional
cha ac e iza ion by Rocha-Ma ín e al. [30]. Following
acid- and s eam-explosion p e ea men s, he soluble lig-
nocellulosic subs a es exhibi ed u u al con en s ang-
ing om 0.31% o 1.1%, and 5’-hyd oxyme hyl u u al
con en s anging om 0.23% o 0.28%, as documen ed
in he same s udy (30). PCS and PSCS we e hen hyd o-
lyzed using VISCOZYME (Sigma-Ald ich) as desc ibed
by Godoy e al. [45]. The suga con en o hese hyd o-
lysa es was 1.7 ± 0.1 g L−1 glucose and 2.8 ± 0.1 g L−1
xylose o PSC and 1.5 ± 0.1g L−1 glucose and 1.4 ± 0.06g
L−1 xylose o PSCS. The s ains we e p e-cul u ed o e -
nigh in M8 minimal medium wi h 10mM KNO3, as he
N sou ce, hen, he cul u es we e dilu ed a 0.1 OD660nm
in M8 minimal medium wi h 10mM KNO3 as N sou ce
wi hou glucose and 3% (w/ ) acid- and hea - p e- ea ed
co n s o e (PCS) o acid- and hea - p e- ea ed suga
cane s aw (PSCS) [30]. No e ha ni a e was used in
hese assays, because he pH o he cul u e medium
emain along he e men a ion es abo e 6.1, a which
g ow h o P. pu ida akes place, while when ammonium
was he N sou ce, he pH o he cul u e medium d opped
o 4.5–5.3, which comp omised he su i al o he s ain.
G ow h o he s ains in his cul u e was moni o ed by
de e mining CFU mL−1 in solid M9 medium.
Co n sy up was also used as a sou ce o ca bon as indi-
ca ed in he Resul s sec ion. The co n sy up (pu chased
om Biosan, Ta agona, Spain) con ained abou 410g
L−1 glucose, 550g L−1 uc ose and 40g L−1 suc ose.
Isola ion o l‑phenylalanine o e p oduce P. pu ida
mu an s
In Pseudomonas pu ida, l-phenylalanine is p oduced
h ough he shikima e pa hway (Fig. 1). Mu an s o
CM12-5 and CM12-5 (pPHE1) s ains ha o e p oduce
l-phenylalanine we e gene a ed using e hylme hane-
sul ona e (EMS) and selec ion o clones able o g ow in
he p esence o > 3 mg mL−1 p- luo -dl-phenylalanine
(FPA), a oxic analogue o l-phenylalanine [61]. B ie ly,
cells we e g own in M9 minimal medium wi h glucose
un il he mid-exponen ial g ow h phase was eached.
Then, 10 aliquo s o 100 μL o liquid cul u e we e sp ead
Table 4 S ains and plasmids used in his s udy
CmR, esis ance o chlo amphenicol; GmR, esis ance o gen amycin; KmR, esis ance o kanamicyn; Ri R, esis ance o i ampicin; SmR, esis ance o s ep omycin; SpR,
esis ance o spec inomycin. FPA, p- luo o-DL-phenylalanine
S ains/plasmids Geno ype/ ele an ea u es Re e ences
S ains
Esche ichia coli
DH5α Cloning hos : F-λ-endA1 glnX44(AS) hiE1 ecA1 elA1 spoT1 gy A96(NalR) bC1 deoR nupG Φ80(lacZΔM15)
Δ(a gF-lac)U169 hsdR17( K–mK+)[35]
Pseudomonas pu ida
DOT-T1E P o o oph, CmR, Ri R[8]
DOT-T1EΔgcd DOT-T1E mu an gene a ed by inse ional inac i a ion o DOT-T1E_2882, KmR[34]
DOT-T1E (xylABE) Wild ype ha bo ing plasmid pSEVA633_xylABE, GmR[34]
DOT-T1EΔgcd (xylABE) DOT-T1E Δgcd mu an ha bo ing plasmid pSEVA633_xylABE, KmR, GmR[34]
CM12-5 DOT-T1E mu an ha p oduces L-phenylalanine, Ri R[24]
CM12-5Δgcd CM12-5 mu an gene a ed by inse ional inac i a ion o DOT-T1E_2882, KmR[34]
CM12-5 (xylABE) CM12-5 ha bo ing plasmid pSEVA633_xylABE, GmR[34]
CM12-5Δgcd (xylABE) CM12-5 Δgcd ha bo ing plasmid pSEVA633_xylABE, GmR[34]
PG1E CM12-5 de i a i e ha g ows in he p esence o 10 mg mL−1 o FPA, Ri RThis s udy
PG2E CM12-5 (pPHE1) de i a i e ha g ows in he p esence o 10 mg mL−1 o FPA, Ri R KmRThis s udy
Plasmids
pSEVA238 Exp ession ec o : o iV(pBBR1),
xylS/ Pm, KmR[67]
pPHE1 pSEVA238 de i a i e ca ying pheA b gene om P. pu ida DOT-T1E [24]
pSEVA633 Exp ession ec o : o iV(pBBR1), lacZα-pUC18, GmR[67]
pSEVA633_xylABE pSEVA633 wi h xylABE genes exp essed om he EM7 p omo e , GmR[34]
pSEVA438 Exp ession ec o : o iV(pBBR1), xylS/Pm, Sm/SpR[67]
pPE-1 pSEVA438 de i a i e ca ying PP_0968 om P. pu ida KT2440, kdc om Rhodospi illum ub um,
and T1E_5478 om P. pu ida DOT-T1E, SmR/SpR[24]
pSEVA433 Exp ession ec o : o iV(pBBR1), lacZα-pUC18, SmR/SpR[67]
pPE-2 pSEVA433 de i a i e ca ying PP_0968 om P. pu ida KT2440, kdc om Rhodospi illum ub um,
and T1E_5478 om P. pu ida DOT-T1E, SmR/SpRThis s udy