Metabolic Engineering Of Ergosterol Synthesis Pathway For Vitamin D3 Precursor Cholesta-5,7,24-trienol Production In Saccharomces Cerevisiae

Cholesterol and its relatives serve a myriad of biological function and it is supposed to be the most highly decorated small molecule in biology with increasing amount of studies on its biosynthetic technology.Herein,the ergosterol synthetic pathway of yeast Saccharomyces cerevisiae was proposed to be metabolically engineered for the production of vitamin D₃ precursor.In order to convert accumulated ergosterol to cholesta-5,7,24-trienol,erg5 and erg6 genes invovled in the postsqualene formation steps were disrupted lead to yeast strains that accumulate high amounts of compounds that are otherwise only precursors of ergosterol,e.g.zymosterol and cholesta-5,7,24-trienol??erg6?,ergosta-5,7-dienol??erg5?and cholesta-5,7,24-trienol??erg5/6?,which was barely detectable in wild type strain by GC-MS analysis of free sterols.Additionaly,deletion of erg6 gene significantly induced growth inhibition and diminished capacity for genetic transformation.Moreover,the supposed rate-limiting enzymes including tHMG1,ERG1 and ERG11 were over-expressed.Compared with wild type strain,no obvious increase of sterol was found and significant inhibition of biomass was observed in tHMG1 over-expressed in?erg5 strain.Overexpression of Hmg1p led to a significant accumulation of squalene,and induction of Erg1p/Erg11p expression raised the yield of both total sterols and ergosta-5,7-dienol with no obvious changes in growth behavior.Furthermore,the transcription factor allele upc2-1 was overexpressed to explore the effect of combined induction of rate-limiting enzymes.Compared with an obviously enhanced yield of ergosterol in the wild-type strain,decreases of both the ergosta-5,7-dienol levels and the total sterol yield were found in?erg5-upc2-1,probably due to the unbalanced NADH/NAD⁺ ratio observed in the erg5 knockouts,suggesting the whole-cell redox homeostasis was also vital for end-product biosynthesis.The data obtained in this study can be used as reference values for the production of sterol-related intermediates involved in the post-squalene biosynthetic pathway in food-grade S.cerevisiae strains.