The Construction Of Artificial Yeast Strain For The Production Of 7-DHC And The Study Of Post-squalene Pathway

7-dehydrocholesterol(7-DHC),a crucial precursor of vitamin D3,has extensive applications in medical and clinical fields.In this study,We successfully constructed a 7-dehydrocholesterol producing strain by incorporating heterologous C-24 reductase gene(DHCR24)to Saccharomyces cerevisiae.Introducing DHCR24 into ERG6(sterol C24-methyltransferase)and ERG5(sterol C22-dehydrogenase)knocked out strains respectively,we got 7-DHC artificial production strains.The overexpression of endogenous gene tHMGR can lead to an accumulation of squalene,making the production increased.ERG6 knockout strain can directly increase metabolic substrate zymosterol and keep sterols content in a high level,which is more suitable for the generation of product.Final chassis cells and module matching result in a yield of 3.4 mg/L.Further,we adjusted endogenous genes in the post-squalene part of the 7-DHC pathway.The key endogenous genes were searched by the change of the free sterols contents in cells.Fermentation results showed that the yield of 7-DHC slightly improved by the overexpression of ERG2-ERG3 module.Because ERG2-ERG3 module catalysed the reaction after zymosterol and pushed the synthesis of 7-DHC.Through the difference of productswe deduced a series of unknown sterols substances andthe non-specificity to substrate of DHCR24 was get proved.Combined with PCA analysis,the change of all kinds of material in relative contents confirmed that postsqualene endogenous gene module do have effects on the variation of the relative contents of total sterols.We got the consistent conclusion: The overexpression of post-squalene gene modules will cause obvious sterol composition differences,and the most significant impact iscaused by ERG11 single gene and ERG2-ERG3 combination module.They are the key modules of 7-DHC synthetic pathway.In view of the complex organisms' substrates and the difficulty of product purification such we try to explore new biological expression.Started with two known genes LXYL-P1-2 and DBAT as an example,the test of cell free PURE System(transcription/translation together)has been tried.Proteins have been detectedsuccessfully after the additionof DNA fragments.At the same time,the two genes were expressed in E.coli,and we directly used cell lysis solution as the enzymatic system.BacatinIII was generated after the catalysis of DBAT.It showed the well prospect of enzymatic reaction in vitro,and provided new ways for the synthesis of sterols and other natural products.