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Metabolic engineering of Saccharomyces
cerevisiae for enhanced production of caffeic acid
Pingping Zhou ( ppzhou@yzu.edu.cn )
Yangzhou University https://orcid.org/0000-0001-5162-1198
Chunlei Yue
Yangzhou University
Bin Shen
Zhejiang University
Yi Du
Yangzhou University
Nannan Xu
Yangzhou University
Lidan Ye
Zhejiang University
Research
Keywords: Caffeic acid, Saccharomyces cerevisiae, De novo biosynthesis, Controllable pathway
expression, Tyrosine-induced feedback inhibition
Posted Date: June 22nd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-36569/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Version of Record: A version of this preprint was published at Applied Microbiology and Biotechnology on
July 20th, 2021. See the published version at https://doi.org/10.1007/s00253-021-11445-1.
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Abstract
Background
As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and
acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical
synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently,
Saccharomyces cerevisiae
has been reported sporadically for biosynthesis of caffeic acid via free
plasmid‐mediated pathway assembly. However, the production was far from satisfactory and even relied
on the addition of precursor.
Results
In this study, we rst established a controllable caffeic acid pathway by employing a modied
GAL
regulatory system in
S. cerevisiae
and realized
de novo
biosynthesis of 313.8mg/L caffeic acid from
glucose. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback
inhibition, deleting genes involved in competing pathways and overexpressing rate-limiting enzymes led
to about 2.5-fold improvement in the caffeic acid production, reaching up to 769.3mg/L in shake-ask
cultures. To our knowledge, this is the highest ever reported titer of caffeic acid
de novo
synthesized by
engineered yeast.
Conclusions
Caffeic acid production in
S. cerevisiae
strain was successfully improved by adopting a glucose-regulated
GAL
system and comprehensive metabolic engineering strategies. This work showed the prospect for
microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of
its derived metabolites.
Background
Caffeic acid, also known as 3,4-dihydroxy cinnamic acid, has attracted increasing attention due to its
antioxidant [1], antivirus [2], anticancer [3, 4] and anti-inammatory biological properties [5]. Moreover,
caffeic acid is an important precursor of plant-originated aromatic chemicals like rosmarinic acid,
chlorogenic acid and caffeic acid phenethyl ester [6–8]. Therefore, it shows great potential in nutritional,
pharmaceutical and cosmetics industries [9]. Considering the environmental and economic benets,
biosynthesis of caffeic acid via engineering model microbes such as
Escherichia coli
and
Saccharomyces cerevisiae
provides a promising alternative to chemical synthesis or plants extraction
[10].
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The biosynthesis of caffeic acid starts from L-phenylalanine or L-tyrosine through the endogenous
shikimate pathway [11]. In plant, the deamination of L-phenylalanine is catalyzed by phenylalanine
ammonia lyase (PAL) to produce cinnamic acid. The sequential two-step hydroxylation at the 4- and 3-
positions of the benzyl ring of cinnamic acid is executed by two cytochrome P450 monooxygenases,
cinnamate-4-hydroxylase (C4H) and
p
-coumarate 3-hydroxylase (Coum3H) [12], forming caffeic acid
through
p
-coumaric acid. In recent years, reports have sporadically emerged regarding metabolic
engineering for heterogeneous caffeic acid production in
E. coli
. However, the plant-originated P450
enzymes are dicult to express in microbial systems [13]. Alternatively, tyrosine containing a 4-hydroxyl
group could be directly converted to
p
-coumaric acid by microbial tyrosine ammonia lyase (TAL) [14]. For
further hydroxylation of
p
-coumaric acid, the
sam5
-encoded Coum3H from the actinomycete
Saccharothrix espanaensis
[15, 16] or the cytochrome P450 CYP199A2 from the bacteria
Rhodopseudomonas palustris
[17, 18] could be used, enabling caffeic acid formation in
E. coli
. By
introducing
RgTAL
from
Rhodotorula glutinis
into
E. coli
together with expressing endogenous 4-
hydroxyphenylacetate 3-hydroxylase (4HPA3H) and increasing the intracellular supply of tyrosine by
overexpression of PEP synthase (encoded by ppsA) and transketolase (encoded by tktA) and feedback
inhibition resistant 3-deoxy-
D
-arabino-heptulosonate-7-phosphate synthase (encoded by
aroG
fbr
) and
chorismate mutase-prephenate dehydrogenase (encoded by
tyrA
fbr
), the highest caffeic acid production
reached 766.7mg/L from simple carbon sources in shake asks [19]. However, the cell growth and
caffeic acid production still relied on phenylalanine supplement.
S. cerevisiae
as GRAS (generally regarded as safe) organism with well-characterized genetic background,
superior stress tolerance and excellent fermentation properties becomes an attractive microbial host for
caffeic acid production. However, neither Coum3H nor CYP199A2 could enable caffeic acid biosynthesis
in yeast [11]. The bacterial 4-hydroxyphenylacetate 3-hydroxylases (4HPA3H) complex encoded by
HpaB
and
HpaC
was also found to effectively catalyze p-coumaric acid to caffeic acid (Fig.1) [13]. Expression
of HpaB and HpaC from
E. coli
in
S. cerevisiae
led to caffeic acid production [11], and replacement of the
E. coli
enzymes with the combination of HpaB from
Pseudomonas aeruginosa
and HpaC from
Salmonella enterica
signicantly improved the caffeic acid yield by 45.9-fold, leading to the highest
production of caffeic acid (about 289.4mg/L) in yeast [11]. However, this process still relied on feeding
of exogenous L-tyrosine as the precursor. Li Y
et al
reported that simultaneous expression of RcTAL from
Rhodobacter capsulatus
and the P450-dependent monooxygenase C3H together with its associated
cytochrome P450 reductase CPR1 from
Arabidopsis thaliana
could enable
de novo
biosynthesis of
caffeic acid from glucose in
S. cerevisiae
. However, low caffeic acid production (about 11.432mg/L) was
obtained, ascribed to the low activity of C3H [20]. In both studies, episomal vectors were used for
expression of caffeic acid pathway genes in
S. cerevisiae
. Considering that the yeast transformants
harboring several plasmids are genetically unstable, integrating the caffeic acid pathway into the yeast
genome may create a more stable cell factory.
As found with other tyrosine-derived products [21–23], the shortage of precursor supply may be another
limiting factor of caffeic acid biosynthesis in
S. cerevisiae
. The critical step of shikimate pathway is the
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condensation of two starter units named phosphoenolpyruvate (PEP) and 4-erythritol phosphate (E4P) by
isoenzymes Aro3 and Aro4 to produce 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) [23, 24]. In
addition, chorismite, the last common intermediate for three aromatic amino acids, is transformed by the
chorismate mutase Aro7 to generate prephenate, which is further divided into two branches, one towards
L-phenylalanine and the other towards L-tyrosine (Fig.1). In this pathway, the activity of Aro4 and Aro7
are feedback-inhibited by the end product tyrosine. Feedback-insensitive variant Aro4
K229L
and Aro7
G141S
have been created by rational design [25–27]. Either individual overexpression Aro4
K229L
or simultaneous
expression of Aro4
K229L
and Aro7
G141S
could effectively increase intracellular shikimate, phenylalanine
and tyrosine concentrations [24, 27]. On the other hand, the prephenate dehydrogenase (Tyr1) which
catalyzes prephenate to α-keto acid 4-hydroxyphenylpyruvate (4HPP), the direct precursor of L-tyrosine, is
transcriptionally inhibited by phenylalanine. Replacement of the native Tyr1 promoter with a constitutive
one or expression of the feedback-insensitive cyclohexadienyl dehydrogenase TyrC from
Zymomonas
mobilis
could both improve the production of tyrosine and its derivatives [28, 29]. Meanwhile,
decarboxylation of 4HPP catalyzed by phenylpyruvate decarboxylases (encoded by
Aro10
,
Pdc1
,
Pdc5
,
and
Pdc6
) would decrease the ux towards tyrosine, among which Pdc5 and Aro10 showed stronger
decarboxylation activity than the others [30]. Deletion of
Aro10
and
Pdc5
increased the intracellular
tyrosine production by 5.7 folds [21] .
Taken together, alleviation of feedback inhibition and removal of competitive branches could improve the
precursor supply and thus contribute to further enhancement of caffeic acid synthesis. In this study, we
rst constructed a controllable caffeic acid biosynthetic pathway in
S. cerevisiae
by employing a modied
GAL
system. The precursor supply was then strengthened by eliminating the feedback inhibition of
aromatic amino acid and down-regulating the competitive pathways, and the rate-limiting enzyme TyrC
was overexpressed to enhance the ux towards caffeic acid production.
Results And Discussion
Construction of a glucose-regulated caffeic acid biosynthetic pathway in S. cerevisiae
Biosynthesis of caffeic acid could be derived from tyrosine, while
S. cerevisiae
cells do not possess the
pathway downstream of tyrosine. For heterologous biosynthesis of caffeic acid,
RgTAL
( KF765779.1)
from
R. glutinis
,
HpaB
(PHSS01000001.1) from
Pseudomonas aeruginosa
and
HpaC
from
S
.
enterica
reported with excellent performance [11, 31] were chosen as the target genes for pathway construction.
To avoid the expensive galactose addition, the modied
GAL
system constructed by knocking out
GAL80
(encoding a repressor of Gal4, which confers repression in the absence of galactose) was employed
herein, leading to dynamic expression of the target genes in response to glucose concentration (Fig.2b).
The codon-optimized
OHpaB
together with
HpaC
under
GAL1
/
GAL10
promoters were integrated into
GAL1-7
genomic loci of the
GAL80
deletion strain YXWP-113 [32] while the codon- optimized
ORgTAL
was localized in
DPP1
site. The metabolites of the resulting strain YCA113-2B were analyzed by high
performance liquid chromatography (HPLC). A new peak with the same retention time as the caffeic acid
standard was observed when comparing the spectra to those of the starting strain YXWP-113 (Fig.2a).
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Time courses of YCA113-2B showed that caffeic acid production started upon depletion of glucose and
nally accumulated to 313.8mg/L (38.5mg/g DCW) in the medium after 72h shake-ask culture
(Fig.2c). This yield was slightly higher than the previous highest production (about 289.4mg/L) reported
in
S. cerevisiae
by employing same species originated HpaB and HpaC with this study [11] and much
higher than the production reported by Li Ye
et al
[20]. The differences in promoters, terminators,
functional genes and fermentation media between the study of Li Y
et al
[20] and ours might be
responsible for the different caffeic acid production in
S. cerevisiae
.
Elimination of feedback inhibition of DAHP synthases and chorismate mutase
The biosynthesis of tyrosine, the direct precursor of caffeic acid, is strictly regulated in
S. cerevisiae
.
Therefore, relieving feedback inhibition of tyrosine may lead to improved caffeic acid production. After
deletion of
Aro3
gene inhibited by phenylalanine, little effect on caffeic acid accumulation and cell
growth was observed (Fig.3). Thus,
Aro3
can be used as a target locus for gene integration.
Subsequently, tyrosine feedback inhibition insensitive mutant
Aro4
K229L
under
GAL1
promoter was
integrated into
Aro3
site by using the linearized PUMRI-Δ
Aro3
-
Aro4
K229
L
vector. The resulting strain
YCA113-4B produced 537.7mg/L of caffeic acid, which was about 79.5% higher before
Aro4
K229
L
overexpression (Fig.3). However, the biomass of YCA113-4B decreased by 14.7% compared with
YCA113-3B.
In addition, chorismate mutase Aro7 is another key enzyme feedback-inhibited by tyrosine. Therefore,
Aro7
G141S
which abolished the tyrosine feedback inhibition properties was overexpressed in YCA113-4B
strain, which slightly increased the caffeic acid level in the resulting strain YCA113-5B (551.8mg/L). The
production improvement after alleviation of feedback inhibition regulation was not as signicant as
anticipated, which may be ascribed to existing downstream bottlenecks.
Deletion of competing pathway genes and overexpression of rate-limiting caffeic acid synthetic enzyme
To further improve the production of caffeic acid, the rate-limiting steps in the synthetic pathway were
eliminated by overexpression of the corresponding enzymes, and the carbon ux was redirected to the
target pathway by deletion of the competitive pathways. Phenylpyruvate decarboxylase (Aro10) is a key
enzyme responsible for channeling tyrosine ux to tyrosine degradation pathway. Thus,
Aro10
gene was
deleted in YCA113-5B strain to reduce the competitive consumption of 4-HPP. The resulting strain
YCA113-6B produced about 625.0mg/L of caffeic acid, which was improved by 13.3% compared with
YCA113-5B, suggesting that 4-HPP is indeed a key intermediate in the caffeic acid biosynthetic pathway.
The transcription of the native
Tyr1
gene is tightly regulated by phenylalanine concentration [28], which
limited the conversion of prephenate to 4-HPP. Therefore, feedback-insensitive TyrC from
Z. mobilis
was
integrated into the
Aro10
genomic loci of YCA113-5B to deregulate this reaction. The resulting strain
YCA113-8B produced 769.3mg/L caffeic acid, which was 23.1% and 145.2% higher than those of
YCA113-6B strain and YCA113-2B, respectively (Fig.4). In addition, we also tested the impact of deleting
the 4-HPP-consuming pyruvate decarboxylase (encoded by
Pdc5
) in strain YCA113-8. However, no
