S. cerevisiae
In S. cerevisiae , a novel sakuranetin biosynthetic pathway was
designed and constructed to synthesize sakuranetin from glucose, which
contained two parallel branching pathways (Fig. 1). One is the formation
of p -coumaric acid from the
L-tyrosine branch catalyzing by tyrosine ammonia lyase, codon-optimizedHaTAL from Hemiphyllodactylus aurantiacus . Another is from
the L-phenylalanine branch, which is catalyzed by phenylalanine
ammonia-lyase from Arabidopsis thaliana (AtPAL2) to generate
cinnamic acid, and then cinnamic acid is catalyzed by
cinnamate-4-hydroxylase from Arabidopsis thaliana (AtC4H), P450
reductase (AtATR2), and yeast native cytochrome b5 (CYB5), to synthesizep -coumaric acid.
Then, for sakuranetin synthesis starting from p -coumaric acid,
4-coumarate-CoA ligase from A. thaliana (At4CL1), chalcone
synthase from Petunia hybrida (PhCHS), chalcone isomerase fromMedicago sativa (MsCHI) and naringenin-7-O-methyltransferase fromOryza sativa (OsNOMT) were chosen as the target genes to
integrate into S. cerevisiae . The gene cluster of the
abovementioned four enzymes under four constitutive promoters (Fig. 2A),
was introduced into strain YT02 (Xiao et al., 2022) (Table 1). The
resulting strain YHS01 (YT02, X3 ::PGK1p-At4CL1-HXT7t-TPI1p-PhCHS-TPI1t-ENO2p-MsCHI-PGK1t-TEF1p-OsNOMT-TEF1t )
produced 4.28 mg/L sakuranetin (Fig. 2D), indicating that the synthesis
pathway of sakuranetin was successfully constructed inS. cerevisiae. To improve
the expression of target genes responsible for the transformation
process from p -coumaric acid to sakuranetin and the yield of
sakuranetin, the effect of galactose promoters instead of constitutive
promoters was evaluated. However, it is necessary to eliminate the
dependence of galactose promoter on expensive inducer galactose, while
maintaining a high expression of target genes under a GALregulation system (Xie et al., 2015). Thus, GAL80 was deleted in
strain YT02. Then, a series of expression cassettes were constructed and
introduced into the YT02 mutant strain which GAL80 was deleted
for the expression of the above four genes (At4CL1, PhCHS, MsCHI,
OsNOMT ) under the control of GAL promoters (Fig. 2B). The resultant
strain YHS02
(YT02,GAL80 ::ADH1t-At4CL1-GAL10p-GAL7p-PhCHS-TPS1t-PGK1t-MsCHI-GAL2p-GAL1p-OsNOMT-CYC1t )
exhibited increased productivity of sakuranetin (Fig. 2C, D, E), which
titer (9.19 mg/L) was twice higher than that of strain YHS01. These
results suggested that the yield of sakuranetin modified by GALregulation system is higher than that by constitutive regulation system.
Although ΔGAL80 does not require galactose for induction, these
strains still require the utilization of galactose,
ΔGAL1 /7 /10 can eliminate the utilization of
galactose (Westfall et al., 2012). Here, the resulting strain YHS05
(YHS04, ΔGAL1/7/10 ) produced 13.59 mg/L sakuranetin (Fig. 3B)
without improvement.
3.2 Enhanced sakuranetin biosynthesis by adjusting the number of gene
copies
Although the constructed strain YHS02 can de novo synthesize
sakuranetin from glucose, the yield is low. To improve the yield of
sakuranetin, increasing the number of gene copies of heterologous genes
is a feasible strategy for pathway optimization (Lyu et al., 2017).
Firstly, one more gene copy of phenylalanine ammonia-lyase (AtPAL2) was
introduced into strain YHS02. The resultant strain was named strain
YHS03 (YHS02, HO-1 ::GPM1p-AtPAL2-ADH1t ), in which
sakuranetin titer was 8.24 mg/L. YHS03 exhibited no significant
improvement in sakuranetin production. Meanwhile, more carbon flux can
flow to p -coumaric acid. However, when one more copy of the
tyrosine ammonia lyase (HaTAL) was integrated into strain YHS03, the
resultant strain YHS04 (YHS03, XVI1 :: GAL7p-HaTAL-TPS1t )
produced 17.97 mg/L of sakuranetin (Fig. 3A), which increased by 95.5%
compared with YHS02.
After one more copy of chalcone synthase (PhCHS) and
naringenin-7-O-methyltransferase (OsNOMT) was introduced into YHS04, the
resultant strain YHS07 (YHS04, X5 ::TPI1p-PhCHS-TPI1t-TEF1p-OsNOMT-TEF1t ) produced 25.37 mg/L
sakuranetin, 41.2% higher than strain YHS04(17.97 mg/L)(Fig. 3A),
which indicated increasing copy number of PhCHS and OsNOMTcan improve the yield of sakuranetin efficiently.
3.3 Optimizing the synthetic pathway of aromatic amino
acids(L-Phe/L-Tyr)
Phenylalanine and tyrosine, are the direct precursor of sakuranetin
biosynthesis. Therefore, relieving feedback inhibition of tyrosine
synthesis may release more carbon flux into the sakuranetin pathway.ARO4 and ARO7 are feedback inhibition genes in the
tyrosine synthesis pathway, it has been reported that
feedback-insensitive DAHP synthase (ARO4K229L) and
chorismate mutase (ARO7G141S) can alleviate the
feedback inhibition regulation (Liu et al., 2019). Unexpectedly, the
resulting strain YHS09 (YHS07, TRP1 ::TEF1p-ARO4K229L-TEF1t-PGK1p-ARO7G141S-HXT7t )
produced 20.51 mg/L sakuranetin by enhancing the expression level ofARO4K229L andARO7G141S , decreased by 19.1% compared with
YHS07 (25.37 mg/L) (Fig. 3B), we speculated that there may be a
bottleneck in the synthesis of sakuranetin due to the weak metabolic
flux in the downstream pathway. Meanwhile, knocking out of the bypass
metabolic flux genes of phenylpyruvate decarboxylase (ARO10) and
pyruvate decarboxylase (PDC5), can increase carbon flux into aromatic
amino acids (Koopman et al., 2012; Rodriguez et al., 2015). Then,ARO10 and PDC5 were deleted respectively in YHS09 and
YHS10. The resultant strains, YHS10 (YHS09, △ARO10 ) and YHS11
(YHS10, △PDC5 ) produced 24.91 mg/L and 26.65 mg/L of sakuranetin
after 72 h culture (Fig.3B), which indicated no significant improvement
in production.
Endogenous genes ofARO2/ARO1/PHA2 , MtPDH1 (tyrosine prephenate dehydrogenase fromMedicago truncatula ), and EcaroL (shikimate kinase from E.
coli) have been demonstrated beneficial to release more carbon flux
into aromatic amino acid pathway (Liu et al., 2019). These genes were
introduced into strain YHS11 one by one via a series of the expression
cassette, the sakuranetin production was improved to 33.05 mg/L, 25.96
mg/L, 26.23 mg/L, 23.89 mg/L, and 43.82 mg/L in the resultant strains
YHS12 (YHS11, XII5 :: GPDp-ARO2-CYC1t ), YHS13 (YHS11,ARO1p :: GPDp-ARO2-CYC1t-ENO2p ), YHS14 (YHS13,III1 :: GPM1p-PHA2-ADH1t ), YHS15 (YHS14, X2 ::FBA1p-MtPDH1-CYC1t ), and YHS16 (YHS15, HO-2 ::PDC1p-EcaroL-ADH3t ), respectively (Fig. 3B). The strain YHS16
produced 43.82 mg/L sakuranetin, 64.4% higher than that of YHS11 (26.65
mg/L), which suggested that introducing more carbon flux into the
downstream pathway of sakuranetin synthesis could significantly improve
the production of sakuranetin.
3.4 Enhancing malonyl-CoA biosynthetic pathway
In a previous publication, it was reported that the supply of
malonyl-CoA is of great importance for flavonoid production in S.
cerevisiae (Leonard et al., 2007; Zhang et al., 2021). Thus, the
effect of malonyl-CoA supply on sakuranetin synthesis was evaluated by
two methods. One method is to delete YPL062W in S.
cerevisiae , which can decrease the transcription of downstream geneALD6 and increase acetyl-CoA accumulation (Chen et al., 2019;
Leonard et al., 2007). However, when YPL062W was deleted, the
resultant strain YHS17 (YHS16, △YPL062W ) produced 35.38 mg/L
sakuranetin, which decreased by 19.2% compared to YHS16 (Fig. 4). This
result suggested that the excessive accumulation of acetyl-CoA and the
weak transformation to downstream malonyl-CoA might occur. Another
method is to convert more acetate-CoA to malonyl-CoA. Acetyl-CoA
carboxylase (ACC1) can catalyze acetyl-CoA to malonyl-CoA in S.
cerevisiae , expression of a double-point mutant at Ser659 and Ser1157
(ACC1S659A, S1157A ) has been verified to
increase manlony-CoA production (Ferreira et al., 2018; Sun et al.,
2019). Thus, one copy of ACC1S659A, S1157A was
further introduced into the biosynthetic pathway to convert more
acetate-CoA to malonyl-CoA, YHS18 (YHS16, △YPL062W ::PGK1t-ACC1S659A, S1157A-GAL2p ) produced
50.62 mg/L of sakuranetin,
increase by 15.5% compared with YHS16 (Fig. 4). Our results confirmed
that enhancing the supply of the
precursor malonyl-CoA was beneficial for sakuranetin production.
3.5 Production of sakuranetin in a 1-L bioreactor fermentation
To scale up sakuranetin production, the engineered strain YHS16 was
selected for fermentation in a 1-L bioreactor with 500 mL YPD medium. As
shown in Fig. 5, the cell densities increased gradually with the
prolongation, and the broth OD660 reached a maximum of
25.7 after 46 h fermentation, while sakuranetin titer increased
continuously and finally reached 158.65 mg/L after 70 h. To our
knowledge, it is the highest titer of sakuranetin via microbial cell
factories method among all in the past publications.