Global Metabolic Regulation Of Naringenin Synthesis In Saccharomyces Cerevisiae

Naringenin is one of the essential skeletons of flavonoids.Naringenin-based derivatives have been developed and applied in the pharmaceutical,food,and nutritional industries.Currently,naringenin is produced primarily by extracting it from orange peels,but this method has a low production efficiency,a high extraction cost,and geographical restrictions,which do not conform to the concept of green development.In this study,Saccharomyces cerevisiae was selected as the host.To address the issues of the lengthy naringenin biosynthesis pathway,the poor water solubility of fermentation products,and insufficient supply of precursors,a one-step multi-copy integration method of naringenin metabolic pathway genes and a naringenin glycosylation hydrolysis strategy were established to enhance product synthesis and improve its solubility,and a strategy of multi-pathway synergy and subcellular organelle carbon flux regulation was proposed to achieve sufficient supply and balanced utilization of naringenin precursors.Through the use of multi-copy integration,glycosyl modification,multi-pathway coordination,and carbon metabolic flux regulation,the metabolic regulation and optimization of the system were accomplished,and the best engineered strain was able to produce a high titre of naringenin efficiently.This study offers excellent chassis cells for the microbial heterologous synthesis of flavonoids,which is crucial to produce flavonoids in industrial quantities.The specific work includes the following:(1)Construction of multi-copy integration engineering strains of naringenin heterologous metabolic pathway.Single-copy site-dependent integration methods have been used for the heterologous synthesis of flavonoids,but the yield of target products is limited by gene copy number.A one-step multi-copy integration method of the naringenin biosynthesis pathway was designed based on the r DNA multi-copy site of S.cerevisiae.The efficiency of pathway gene integration into the r DNA multi-copy site was 93.7%.The supply of p-coumaric acid was enhanced by eliminating the feedback inhibition of shikimate pathway genes on tyrosine and downregulating the expression of competing pathway genes.By optimizing the transfection concentration of the donor DNA,an engineered strain capable of producing 149.8 mg/L naringenin was screened.The copy number and expression level of the key genes Pc4 CL and Ph CHS in the metabolic pathway of naringenin were analyzed,and it was clear that the production of naringenin was proportional to the copy number and expression level.Multi-copy integration of metabolic pathway genes contributes to enhanced naringenin production.(2)Glycosylation modification promotes the synthesis of naringenin.Due to the poor water solubility and strain effect of naringenin,the heterologous microbial synthesis of naringenin is hindered.Based on the principle that increasing the glucosyl group can improve the water solubility of the compound,the 7-position of naringenin was modified by glucosylation to improve its water solubility.Knockout of the endogenous glycoside hydrolase genes EXG1 and SPR1 in S.cerevisiae and co-expression of the heterologous glucosyltransferase Ugt733c6 achieved the synthesis of the naringenin-7-O-glucoside.By optimizing the supply of UDP-glucose and strengthening the shikimate pathway,the synthesis of naringenin-7-O-glucoside was further improved,and it was found that the glycosylation product could be completely secreted into the extracellular space.The 5-L bioreactor was used for fermentation,hydrochloric acid was used to hydrolyze the fermentation product,and the naringenin titer reached 1184.1 mg/L.The glycosylation modification strategy is effective for the efficient heterologous synthesis of naringenin using S.cerevisiae.(3)Reconstructed S.cerevisiae carbon metabolism to enhance naringenin synthesis.An adequate supply of precursors significantly impacts the synthesis of naringenin by microbial cell factories.Aiming at the problem of insufficient supply of coumaric acid from precursors,the synthetic pathway of aromatic amino acids was optimized,and the effect of pathway gene expression on naringenin production was clarified.The TAL pathway alone cannot achieve an adequate supply of p-coumaric acid.Using the synergistic effect of the TAL pathway and the PAL pathway,the synthesis of aromatic amino acids to p-coumaric acid was improved.Combined with the gene optimization of the upstream metabolic pathway,the engineered strain at the shake flask level could produce 347.5 mg/L of naringenin.The central carbon metabolism of S.cerevisiae was reconstructed by introducing the heterophos photransketoase pathway,which increased naringenin production to 391.8 mg/L.Replacing the endogenous gene TSC13 with Md ECR significantly alleviated the accumulation of by-products.The production of naringenin production reached 1671.8 mg/L after fermentation in a 5-L bioreactor,and the accumulation of p-coumaric acid was 1096.5 mg/L.This strategy relieves the insufficient supply of p-coumaric acid.(4)Subcellular carbon flux regulation strategy enhances malonyl-Co A synthesis.MalonylCo A is another essential precursor in naringenin synthesis.To solve the problems of insufficient supply of malonyl-Co A and unbalanced utilization of precursors,the production of naringenin was increased by 10.9% by strengthening the endogenous malonyl-Co A synthesis pathway and knocking out the gene affecting the accumulation of p-coumaric acid.The introduction of the heterologous acylated acetaldehyde dehydrogenase gene Dz ADA increased the malonyl-Co A supply,and the naringenin titer reached 492.5 mg/L.The connection of carbon metabolic flow between subcellular and cytoplasmic was achieved by expressing the citrate lyase gene ACL.Further enhancement of citric acid synthesis by subcellular organelles and overexpression of genes related to citric acid transport proteins achieved high levels of naringenin synthesis in S.cerevisiae.The naringenin titer in the 5-L bioreactor increased to 3420.6 mg/L.