Modularization Design And Synthesis Of7-dehydrocholesterol In Yeast Chassis

7-Dehydrocholesterol （7-DHC） is not only an important steroid intermediate, but also the key precursor of Vitamin D3synthesis, and preparing material for cholesteric liquid crystal, which makes it have significant medicinal effects and broad market prospects. Traditional7-DHC preparation method is chemical synthesis, however, due to long chemical processes, multi-step reaction, low yield, complex byproduct removal process, high energy consumption and serious pollution to the environment, chemical synthesis greatly limits7-DHC production and application. Although there are related reports on enzymatic biochemical reaction to produce7-DHC, but the cost issue has always restricted the industrial application of this technology. Using biotechnology to synthesize7-DHC can solve the above problems, and universities abroad have achieved7-DHC biosynthesis. Whereas, there is no relevant reports about constructing7-DHC artificial synthetic cell and studying on the fitness between functional module and yeast chassis by synthetic biology.This research used synthetic biotechnology to enhance the fitness between functional module and yeast chassis, reduced metabolism stress of yeast cells from functional modules building and chassis optimizing two aspects, for the purpose of increasing7-DHC production. To optimize the chassis in order to accumulate zymosterol, the substrate for7-DHC synthesis, we overexpressed tHMGR and ERG1, which are key genes of yeast endogenous zymosterol biosynthetic pathway. In addition, we knocked out ERG5and ERG6to inhibit the conversion of zymosterol to ergosterol. Altering different promoters, optimized chassis SyBE₀00956is obtained. By introducing heterologous C-24reductase （DHCR24） and overexpressing ERG3and ERG2, we constructed nine7-DHC synthesis functional modules. Thirty-six artificial synthetic cells were built up to study the fitness between functional module and chassis from aspects such as promoters of different strength, different chassis and so on. The result shows that under the same chassis, promoters of different strength have different fitness, from good to poor respectively is TDH3p, PGK1p, TDH1p. The7-DHC production of artificial cell composed of functional module which includes single gene part DHCR24is higher than most artificial cell composed of functional module which includes gene parts ERG3, ERG2and DHCR24. Under the same functional module, artificial cell with chassis SyBE₀00956obtains the most enhanced7-DHC production, which means it has better fitness than others. This study provides evidences for importance of fitness and empirical support for rational design of subsequent researches.