| Bile acid is an important component of animal bile and plays an important role in lipid metabolism.At the same time,bile acid can also serve as a metabolic signal molecule,participating in the regulation of glucose metabolism,endocrine metabolism,energy balance and immune response,and is closely related to the occurrence of metabolic syndrome,tumor,inflammation,cholestasis,intestinal microecology and atherosclerosis.Bile acid is one of the main functional components of traditional Chinese medicine bezoar.Natural bezoar is scarce and expensive,while artificial bezoar is a substitute of natural bezoar prepared artificially,which can meet people’s demand for medicine to a certain extent.Among them,bile acid is mainly extracted from the bile of animals.This process has complicated process route and will cause damage to animals.However,the production of bile acid by microbial cell factory is a green and economic method.Saccharomyces cerevisiae has become a common host cell due to its advantages of simple genetic manipulation,biosafety and availability of cheap carbon sources.Cholesterol can be formed into bile acids through a series of enzymatic reactions,so the construction of yeast cells that can biosynthesize cholesterol from scratch is the prerequisite for the artificial biosynthesis of bile acids.The biosynthesis of cholesterol in S.cerevisiae takes zymosterol,which is the endogenous sterol of yeast and the precursor of ergosterol synthesis.,as the precursor.Ergosterol is not only involved in a variety of biological functions of cell membrane,but also an important regulatory factor of a variety of biological processes in the cells.It is also the main raw material of vitamin D2 and steroid drugs,and can be used in the production of hydrocortisone and progesterone.Therefore,it is important to increase ergosterol production in S.cerevisiae.In this study,S.cerevisiae was used as the chassis cells to construct artificial biosynthetic cells that could biosynthesize cholesterol using homologous recombination,a highly efficient and stable method of recombination,in order to lay the foundation for the construction of recombinant strains that could biosynthesize bile acids.At the same time,this paper optimized the fermentation conditions for ergosterol production of a S.cerevisiae strain in the laboratory,in order to improve the yield of ergosterol.The specific research contents and results are as follows:1.Construction of recombinant yeast strain for cholesterol biosynthesisIn order to realize cholesterol biosynthesis in S.cerevisiae,a cholesterol biosynthesis pathway was introduced in yeast cells by homologous recombination.Firstly,the exogenous genes encoding sterol C-7 reductase and sterol C-24 reductase were identified through database search and literature investigation,which were rattus norvegicus DHCR7 and Homo sapiens DHCR24,respectively.Then,yeast screening tag URA3,exogenous genes DHCR7 and DHCR24 were simultaneously introduced into BY4741 by homologous recombination method to construct the recombinant strain.After fermentation,the extracted products were detected and analyzed,but the target product cholesterol was not detected in the fermentation products of the recombinant strain.It is speculated that there is a competitive relationship between the endogenous ergosterol pathway in yeast and the constructed cholesterol synthesis pathway,and the expression level of DHCR7 and DHCR24 in S.cerevisiae is not high,resulting in a low metabolic flow to cholesterol.2.Disruption of the endogenous ergosterol biosynthetic pathway in yeastIn order to improve cholesterol production,the ERG5 and ERG6 genes in ergosterol synthesis pathway were knocked out in this experiment to direct the carbon flux to flow into the cholesterol biosynthesis pathway.After knocking out ERG6,cholesterol was detected in the fermentation product.Compared with the wild type,the growth speed of the ΔERG6mutant was similar to the BY4741 wild-type strain,indicating that knocking out ERG6 did not affect the growth and development of yeast strains.3.Optimization of fermentation conditions for ergosterol production by yeastIn this experiment,a S.cerevisiae strain CEN-1 was used to produce ergosterol,and the fermentation conditions of ergosterol production were optimized,such as glucose concentration,peptone concentration,yeast extract concentration,initial fermentation p H,and the fermentation time.Production of ergosterol was increased significantly after the optimization.Conclusions:1.Through homologous recombination,the DHCR7 encoding the sterol C-7 reductase(Rattus norvegicus)and the DHCR24 encoding the sterol C-24 reductase(Homo sapiens)were integrated at YPRCΔ15 of S.cerevisiae BY4741,using URA3 as the screening marker.The recombinant strain Y-BY-0 was constructed,however,no cholesterol was produced in the fermentation broth.2.The ERG6 locus of S.cerevisiae BY4741 was replaced with DHCR7 and DHCR24,using URA3 as the screening marker.The recombinant strain Y-BY-1 was constructed to produce cholesterol.When the fermentation time was 96 h,the cholesterol yield of the recombinant strain Y-BY-1 was the highest,which was 0.81 mg/L.3.The fermentation conditions of ergosterol production by the yeast strain CEN-1 were studied.The best fermentation conditions were found as follows: 7.5 g/L glucose concentration,15 g/L peptone concentration,20 g/L yeast extract concentration,initial fermentation p H 6.5,and the best fermentation time of 3 days.The yield of ergosterol was30.76 mg/L under the optimal fermentation conditions. |
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