Research To The Production Of 2,3-oxidosqualene By Escherichia Coli

Squalene and 2,3-oxidosqualene are important pharmaceutical intermediates,also are precursors of the terpenoids synthesized in biological creatures.The production of ginsenosides is now mainly dependent on plant tissue and cell culturing,plant cultivating,bioconversion,which production efficiency can’t satisfy the demand of the market.In recent years,the development of synthetic biology,especially the successful application on microbial synthesis of plant terpenoids,can provide a feasible reference plan for the biosynthesis of ginsenosides.Escherichia coli is an outstanding expression vector for foreign genes because of its clear genetic background,simple operation in technology,simple culture conditions,and economic large scale fermentation.Isoprenoid pathway in E.coli can provide precursors for terpenoids,therefore constructing engineering strains to synthetic plant terpenoids by E.coli as chassis cells has certain advantages.At first,this paper constructed an engineering E.coli bacteria which had squalene synthase expression module,and then to improve the accumulation of precursor farnesyl diphosphate(FPP),some relative genes such as farnesyl diphosphate synthase gene(ispA),isopentenyl diphosphate isomerase gene(idi)are overexpressed.Meanwhile the fermentation conditions are optimized,then the fermentation medium chose glycerol as carbon source,yeast powder as nitrogen source,which provided a good platform for squalene synthesis.Then the field of squalene reached to 6.73mg/L.After getting a certain amount of squalene,squalene epoxidase gene was needed to import in E.coli to product 2,3-oxidosqualene.However the processes of squalene epoxidase oxidizing squalene required the participation of NADPH-P450 reductase.This article selected the squalene epoxidase gene from methylococcus and yeast as two oxidase genes,selection two NADPH-P450 reductase genes from arabidopsis and yeast as reductase,through different squalene epoxidase gene combination with reductase,this article constructed the expression vector for the synthesis of epoxidation.Import of biological module has great influence on the chassis cells,besides exogenous eukaryotic genes have transmembrane structure at the same time,and cannot act effectively in E.coli.So this article optimized the codon of the squalene epoxidase gene from methylococcus so that it could have a higher transcription levelin E.coli;then to remove the transmembrane structure in the exogenous eukaryotic genes,this article truncated yeast squalene epoxidase gene(C terminal truncated 28 aa and 55aa),arabidopsis NADPH-P450 reductase gene(N terminal truncated 45aa),yeast NADPH-P450 reductase gene(N terminal truncated 33aa).Therefor it could achieve the further adaptation between module and chassis cell,then make it play an efficient role in cytoplasm of E.coli,eventually raised the synthesis level of2,3-oxidosqualene.Finally,the field of 2,3-oxidosqualene reached about 4.64mg/L.