Engineered Yeast For Efficient De Novo Synthesis Of 7-dehydrocholesterol

Vitamin D is a fat-soluble steroid hormone,which plays an important role in balancing metabolism and maintaining health.With the development of synthetic biology and metabolic engineering,the synthesis of vitamin D3 by microbial fermentation is an effective means to solve the current contradiction between supply and demand.7-dehydrocholesterol（7-DHC）is a key precursor of vitamin D3,which is formed by isomerization of 7-DHC after UV irradiation.Currently,7-DHC synthesis by microorganisms has some problems such as unbalanced cell metabolism,slow growth,and accumulation of toxic intermediate metabolites such as terpenoids.Therefore,in this study,Saccharomyces cerevisiae（S.cerevisiae）CEN.PK2-1C was used as the starting strain to reconstruct the synthesis pathway of 7-DHC.The metabolic network model and Clustered Regularly Interspaced Short Palindromic Repeats interference（CRISPRi）system were used to coordinate the relationship between cell growth and product accumulation,and the synthesis amount of 7-DHC was further improved.Finally,a strain with high yield of 7-DHC was obtained.The main results are as follows:（1）The 7-DHC synthesis pathway was constructed in S.cerevisiae by heterogeneously expressing DHCR24 gene from Gallus gallus.The titer of 7-DHC was only 1.4 mg·L^-1.In order to further improve the production of 7-DHC,key genes of MVA pathway and post-squalene pathway（t HMG1,IDI1,ERG20,ERG1,ERG11）were overexpressed,and the promoter of regulatory factor INO2 was replaced with a strong promoter P_PGK1to enhance gene expression.The titer of 7-DHC reached 51.6 mg·L^-1.Knockout of ROX1 gene was beneficial to cell growth and ERG gene expression,and cell OD₆₀₀ was increased to 63.（2）Using metabolic network model to predict knockout gene.The metabolic pathway of7-DHC synthesis was predicted based on the global regulation of S.cerevisiae,and the combined knockout of five predicted genes（GDH1,GDH3,SER2,MAE1 and AAT1）was carried out.It was found that the single knockout of GDH1 gene increased the 7-DHC production to 101.5 mg·L^-1,and theμ_max increased by 28%.（3）Using yeast TY1 transposon to realize multiple copies of genes.DHCR24 and ERG1genes were inserted into TY1 transposons.Then,single colonies with high copy number were screened on a plate using Leu degradation tags.In addition,q PCR was used to observe the relationship between the relative transcriptional level of genes and the yield of 7-DHC.It was found that the copy number of DHCR24 and ERG1 genes was positively correlated with the yield of 7-DHC.The titer of 7-DHC increased by 120%to 223.3 mg·L^-1.（4）Construction of CRISPRi system.In order to verify the feasibility of the system,three plasmids were introduced into the original strain to construct the gene circuit system.GFP and m Cherry proteins were used for fluorescence characterization,and the normal operation of the system was confirmed by flow cytometry results.Subsequently,binding sites of d Cas9 protein were randomly screened,and the inhibition intensity of different sites was characterized by GFP fluorescence protein.The best inhibition sites were applied to the modified strain.It was found that the production of 7-DHC reached 365 mg·L^-1 in shake flask without affecting the cell growth.The production of 7-DHC titer reached 1328 mg·L^-1 in 3 L bioreactor.