Enhancement Strategies For The Expression Of Key Genes For Synthetic Flavonoids In Saccharomyces Cerevisiae

Flavonoids are a class of secondary metabolites in plants,which have a variety of biological activities and have critical applications in food,medicine,cosmetics,and many other fields.At present,most flavonoids are obtained mainly by plant extraction.Compared with plant extraction,the use of microorganisms to synthesize natural products have the advantages of milder reaction conditions,high safety,strong reproducibility and environmental friendliness.In this study,from the perspective of ubiquitination modification of heterologous enzymes,a system for detecting the ubiquitination level of heterologous enzymes was established in Saccharomyces cerevisiae.In addition,the expression of the synthesis pathway from naringenin to dihydroquercetin was enhanced,and the heterologous synthesis of dihydroquercetin was realized.The details are as follows:（1）Construction of a real-time in situ detection system for ubiquitination levels in S.cerevisiae.Using S.cerevisiae as a platform strain,the synthesis of many high-value natural products,including flavonoids involves the expression of various heterologous enzymes.Ubiquitinated proteins can be degraded by the ubiquitin-proteasome system,which is essential for maintaining physiological activities of cells.Ubiquitination modification may also have a significant impact on the expression of heterologous proteins.In this paper,a real-time in-situ detection system for ubiquitination has been established in S.cerevisiae by using a fluorescence bimolecular complementation approach,and the level of ubiquitination of heterologous proteins was characterized by fluorescence intensity.Taking the proteins involved in naringenin biosynthesis pathway as an example,we obtained mutants with decreased fluorescence intensity,that is,decreased ubiquitination level.（2）Bioinformatics tools were used to predict the potential ubiquitination sites of proteins involved in naringenin synthesis pathway and mutate the site from lysine to arginine to reduce the ubiquitination level.The tyrosine ammonia-lyase（Fj TAL）,chalcone synthase（Sj CHS,Sm CHS）mutants showed decreased fluorescence,suggested that a decreased ubiquitination level.The fermentation results further confirmed that the tyrosine ammonia-lyase Fj TAL mutant Fj TAL^K487R had more advantages in the catalytic production of p-coumaric acid in S.cerevisiae.Compared with the original Fj TAL,the p-coumaric acid production increased by32.3%.The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin.The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin.The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.（3）Further,dihydroquercetin was synthesized from naringenin,and the best combination of Sm F3’H and Sc CPR was obtained by strengthening the adaptability of F3’H and CPR,which increased the titer of eriodictyol to 435.7 mg·L^-1.The potential ubiquitination sites of Sm F3’H were predicted by bioinformatics tools,and the dominant mutant Sm F3’H^K290R was obtained through fermentation experiments,which further increased the titer of eriodictyol.The function of the transmembrane structure of Sm F3’H and Sc CPR was preliminarily explored,and the results showed that the transmembrane structure of Sm F3’H has an important influence on its catalytic efficiency.After fermentation experiment,Cs F3H with higher catalytic efficiency was obtained,and its expression level was optimized by promoter engineering.After 72 h of fermentation with naringenin as substrate,381.2 mg·L^-1 of dihydroquercetin was obtained.