4. Discussion
The heterologous biosynthesis of flavonoids has attracted increasing
researchers and significant progress has been achieved (Shah et al.,
2019). However, sakuranetin is one natural flavonoid, and few reports
about its microbial synthesis were found in the previous publication.
Only Escherichia coli was reported as the chassis cell for
sakuranetin synthesis with low yield. Few reports were found on the
synthesis of sakuranetin in S. cerevisiae . Here, the biosynthetic
pathway of sakuranetin from glucose was constructed in S.
cerevisiae and the yield of sakuranetin was improved through a
multi-modules strategy.
We first constructed a complete sakuranetin synthetic pathway from
glucose (AtPAL2, AtC4H , AtATR2 , At4CL1 ,PhCHS , MsCHI , and OsNOMT ) in S. cerevisiae .
The resultant strain of YHS01 successfully achieved de novo biosynthesis
of sakuranetin. Furthermore, we compared the ability of the
constitutive regulation system and modified GAL regulation system
to synthesize sakuranetin. The result showed that the yield of
sakuranetin in GAL regulation system YHS02 (9.19 mg/L) was two
times higher than that of constitutive regulation system YHS01 (4.28
mg/L), which showed the potential of the GAL regulation system in
sakuranetin production.
In module 1, the pathway genes of sakuranetin synthesis (AtPAL2 ,AtC4H , HaTAL , PhCHS , and OsNOMT ) were
enhanced to further strengthen the metabolic flow. The sakuranetin yield
of the engineered strain YHS07 is 25.37 mg/L, which is 2.76 times
compared with YHS02. It was confirmed that increasing the copy number of
key enzyme genes makes a significant improvement in sakuranetin
production.
Optimizing the synthetic pathway of aromatic amino acids can introduce
more carbon flux into the downstream pathway (Liu et al., 2019). In
module 2, we knocked out the bypass metabolic flux genes of ARO10and PDC5 and further removed the rate-limiting factors to enhance
the supply of the precursor p -coumaric acid, including enhancing
the expression of endogenous genes ARO4K229L ,ARO7G141S , ARO1 , ARO2 ,PHA2, exogenous genes MtPDH1 and EcaroL . The
accumulation of sakuranetin reached 43.82 mg/L in strain YHS16, which
increased by 72.7% compared with YHS07 (25.37 mg/L). This result
confirmed that introducing more carbon flux into the downstream pathway
could improve the yield of sakuranetin, but the improvement effect is
limited. This may result from the weakness of the downstream metabolic
flow of the sakuranetin synthesis pathway.
Another crucial precursor of flavonoids is malonyl-CoA, which is of
great significance for flavonoid production (Zhang et al., 2021). In
module 3, we aimed to enhance the supply of precursor malonyl-CoA by
deleting YPL062W and introducingACC1S659A, S1157A . The yield of sakuranetin in
the resultant strain YHS18 increased to 50.62 mg/L, suggesting the
supply of malonyl-CoA was beneficial to sakuranetin production. However,
the production of p -coumaric acid and naringenin accumulated
significantly, resulting in the inhibition of conversion to sakuranetin
effectively.
The sakuranetin pathway genes were transformed into Escherichia
coli via plasmids and the resultant recombinant strains produced 40.1
mg/L (Kim et al., 2013) and 79 mg/L (Wang et al., 2020) sakuranetin in
shaking flask and a 2.5-L bioreactor, respectively. Whereas, these could
result in issues like plasmid instability, excessive metabolic pressure,
the requirement of selective burden, and the synthesis of sakuranetin inS. cerevisiae has not been reported so far. In comparison to
prior publications on engineering Escherichia coli for the
systhesis of sakuranetin, we realized de novo synthesis of sakuranetin
with the pathway genes inserted into the genome of S. cerevisiae, which was stable without selective pressure. Using the strategy of
metabolic engineering of multiple modules, the yield of sakuranetin
increased by 10.8-fold. The best-performing mutant strain exhibited
enhanced sakuranetin production at a titer of 50.62 mg/L in shaking
flask cultures and 158.65 mg/L in a 1-L bioreactor, which is the highest
reported sakuranetin production in microbial cell factories. This study
also established the foundation for the biosynthesis of sakuranetin and
its derived metabolites.