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.