Analysis And Optimization Of Key Processes Of Squalene Accumulation In Saccharomyces Cerevisiae

Squalene is an important polyunsaturated triterpenoid widely used in food,cosmetics,pharmaceutical,and other industries.The traditional method of squalene production is extracted from animals and plants.Compared with it,the microbial fermentation method by using synthetic biology technology has the advantages of being low cost and environmentally friendly,etc.So it has broad industrial application prospects.The current methods using microbial fermentation production of squalene still need to be improved to increase yield and reduce production costs.This study aims to effectively improve the production of squalene by strengthening key rate-limiting enzymes,strengthening precursor supply,compartmentalized construction,ARTP mutagenesis combined with flow cytometry screening,and fermentation optimization.This research provides efficient modification strategies for the synthesis of many other terpenoids which has important application prospects.The main research contents are as follows:（1）The metabolic pathway for squalene production was optimized to achieve efficient accumulation of squalene.Metabolic engineering strategies were used to overexpress key genes and strengthen the MVA pathway.ERG1 promoter was optimized to block the downstream metabolic pathway.By jointly strengthening theβ-oxidation pathway and ethanol metabolism pathway,the accumulation of acetyl-Co A can be realized.A diploid strain with a yield of 1.5g·L^-1 was constructed by hybridizing the cytoplasm-engineered haploid strain and peroxisome-compartmentalized haploid strain,achieving dual regulation of cytoplasm and peroxisome.（2）Multiple rounds of mutagenesis and high-throughput screening have been operated on squalene-producing S.C by ARTP and Flow Cytometer.A good linear relationship between fluorescence intensity and intracellular squalene content has been established to achieve rapid sorting of high yield strains by adjusting the condition of Nile red staining.After several rounds of mutagenesis and screening,an excellent strain with an 8.2%increase in squalene yield was obtained.（3）The yield of squalene was improved by optimizing the inoculum size,dissolved oxygen,initial sugar concentration,feeding method,and C/N of feeding medium.The results show that when the inoculum size is 2%,the dissolved oxygen is 30%,the initial sugar concentration is 40 g·L^-1,and the feed medium C/N=40:1.High cell growth and squalene level were obtained under two-stage fermentation which glucose was used as the carbon source during 12-60 h and ethanol were used as the carbon source during 60-120 h.The yield of squalene finally reached 6.8 g·L^-1 at 120 h in a 20-L fermenter.