Heterologous Biosynthesis And Optimization Of Carnosic Acid In Engineered Saccharomyces Cerevisiae

Carnosic acid is a tricyclic diterpenoid phenolic substance,which has good pharmacological and biological activities such as anti-inflammatory,anti-cancer,anti-obesity,anti-diabetes and anti-neurodegenerative diseases.At present,the main way to obtain it is to extract from plants,chemical synthesis pollutes the environment,and the heterologous synthesis of carnosic acid by microorganisms is a research hotspot.In this paper,the exogenous pathway of carnosic acid was introduced into Saccharomyces cerevisiae,and the pathway was optimized by means of synthetic biology and metabolic engineering to improve the yield of carnosic acid.In this study,Saccharomyces cerevisiae 3HP-F was used as the original strain,and the yield of miltiradiene reached 10.98 mg/L by constructing the t Sm CPS-linker1-t Sm KSL fusion system.At the same time,the expression sequence of miltiradiene synthase was determined.In order to further improve the yield,We constructe a Saccharomyces cerevisae chassis by fusing endogenous BTS1～ERG20（F96C）to synthesize miltiradiene efficiently,reaching 172.77 mg/L.Then,carnosic acid synthase was introduced to produce carnosic acid.Three CPRs from different sources were selected for compatibility study,the optimal CPR was Sm CPR from Salvia miltiorrhiza,and the yield of carnosic acid was 52.5μg/L.The expression of ferruginol synthase CYP76AH1 and cytochrome Sp Cytb5increased the carnosic acid yield by 4.27 times.The optimal combination of carnosic acid was screened and synthesized by different fusion strategies of CYP76AH1,Sm CPR and Sp Cytb5 expression,and the yield of carnosic acid reached 6.2 mg/L by further expression through multiple copy sites.Overexpression of different catalases alleviated the effect of H₂O₂ on cells,and it was found that overexpression of Sc CTT1had the best effect,and the carnosic acid yield reached 8.84 mg/L.Finally,the endoplasmic reticulum was modified to knock out the phospholipid phosphatase gene PAH1,but the carnosic acid yield was not improved.Overexpression of the gene INO2,which can activate phospholipid synthesis,resulted carnosic acid yield of 11.42 mg/L.The carnoic acid yield was increased to 24.65mg/L by overexpression of heme-encoding rate-limiting enzyme HEM3,NADH kinase POS5 and endoplasmic reticulum folding promoter HAC1.After the optimized shaker fermentation,the maximum carnosic acid yield was 31.04 mg/L by batch fermentation,and the maximum carnosic acid yield was 75.18 mg/L after two-stage feeding fermentation in 5 L with glucose and ethanol as the supplementary carbon source.In this study,the ability of artificial yeast cells to synthesize carnosic acid was significantly improved through a variety of strategies,and provided a basis for microbial synthesis of diterpenoids.