Metabolic Modification Of Saccharomyces Cerevisiae To Synthesize Steroid Derivatives

Ergosterol and 7-dehydrocholesterol are important steroid compounds.Ergosterol is widely present in fungal cell membranes.Derivatives of ergosterol have significant anti-tumor and anti-HIV activities,and ergosterol can also be used to produce drugs such as cortisone and progesterone.7-dehydrocholesterol is a sterol widely present in animals,which can be directly converted into vitamin D₃ after ultraviolet irradiation.Vitamin D₃ can not only be used for the prevention and treatment of skeletal diseases such as rickets,but also improve the body’s immune system and reduce the risk of cardiovascular diseases.At present,the method of industrial production of ergosterol is microbial fermentation.7-dehydrocholesterol can be obtained through chemical catalysis or fermentation by Saccharomyces cerevisiae.Currently,the main method is chemical synthesis.In recent years,due to environmental and production safety reasons,microbial production of ergosterol and 7-dehydrocholesterol has received widespread attention.The bottleneck issues of industrial production of ergosterol and 7-dehydrocholesterol include the complex metabolic pathway of ergosterol,resulting in multiple rate limiting genes;Excessive pathway leads to low precursor conversion rate and high accumulation of intermediate sterols;Ultimately,it leads to low production capacity of Saccharomyces cerevisiae and high production costs.Previous studies have mainly addressed the above issues by increasing the supply of precursor acetyl Co A,modifying the Upc2p transcription factor,and high-density fermentation.This article starts from two aspects:rational transformation of the metabolic pathway of Saccharomyces cerevisiae and fermentation optimization,to solve the problem of yeast’s ability to produce ergosterol and 7-dehydrocholesterol,and obtains two high-yield ergosterol and 7-dehydrocholesterol strains.The main results of this study are as follows:（1）Knocking out bypass genes and coordinating metabolic flux to increase the accumulation of ergosterol.Knocking out four non-pathway genes ROX1,YJL064W,YPL062W,and DOS2 in Saccharomyces cerevisiae enhanced precursor supply,and the accumulation of ergosterol increased from 55.0 mg·L^-1 to 161.1 mg·L^-1.By overexpressing pathway genes,key speed limiting genes were identified as t HMG1,IDI1,ERG2,ERG3,and ERG4.By utilizing strategies such as genome multi copy integration to overexpress the key speed limiting step genes,the ergosterol production of the engineered strain further increased to 292.6 mg·L^-1,achieving coordinated expression of pathway genes.By replacing the promoter of the speed limiting gene and optimizing the ethanol pathway,the accumulation of ergosterol at the shake flask level was 537.8 mg·L^-1.（2）Construction of 7-dehydrocholesterol pathway,expansion of organelle and regulation of gene expression of pathway.Constructing a synthesis pathway for 7-dehydrocholesterol in Saccharomyces cerevisiae,introducing the DHCR24 gene and blocking the endogenous ergosterol pathway to achieve the biosynthesis of 7-dehydrocholesterol,with a yield of 118.5 mg·L^-1.ERG2 was identified as the rate limiting gene of 7-dehydrocholesterol.The expression of pathway genes was enhanced through metabolic engineering and endoplasmic reticulum engineering.The 7-dehydrocholesterol production was further increased to 459.3 mg·L^-1 by adopting the strategy of"pushing forward and pulling back".（3）Fermentation optimization of engineering strains for producing ergosterol and 7-dehydrocholesterol.By optimizing the composition of the culture medium,carbon source types,feeding mode,and metal ions,efficient accumulation of ergosterol and 7-dehydrocholesterol was achieved at the 5-L fermentation tank level.Among them,the titer of ergosterol and 7-dehydrocholesterol achieved to 3.4 g·L^-1 and 4.2 g·L^-1,respectively.Both are the highest reported accumulation levels.