Construction And Application Of The Engineering Strain Of Escherichia Coli For The Synthesis Of Geraniol

Traditional geraniol extraction and chemical synthesis methods have this certain limitation,and it is difficult to meet the market demand for natural geraniol.Therefore,a green and environmentally friendly biosynthesis method has emerged as the times require.With the advancement of synthetic biology technology,the production of geraniol by the fermentation of cheap and renewable microorganisms has become a development trend.E.coli biosynthesis has the advantages of high catalytic efficiency,strong product specificity,mild reaction conditions,short fermentation time,and green economy.In this study,by using E.coli as a chassis cell,a plasmid vector was designed based on the yeast metabolic pathway and the genes involved.Finally,nine related genes on the pathway were linked to three target vectors and transformed into E.coli BL21(DE3).In competent cells,the ability to ferment geraniol was investigated.Through continuous optimization,a low-carbon,efficient and safe way to produce industrialized geraniol was explored,laying a theoretical basis for industrial production.The main findings of this paper are divided into the following aspects:1.Design of geraniol synthesis pathway.Genes required for the production of geraniol in E.coli cell plants(co-expression of acetoacetyl-CoA thiolase gene ERG10,hydroxymethylglutaryl-CoA synthetase gene ERG13,hydroxymethylglutarate-CoA reductase Genes tHMG1,mevalonate kinase gene ERG12,mevalonate kinase gene ERG8,decarboxylase gene ERG19,isopentenyl pyrophosphate isomerase gene IdI1,geranyl pyrophosphate synthase gene GPPS2,and geraniol The codon optimization and synthesis of the synthetase gene GES(a total of 9 genes),each gene has independent promoter and terminator sequences.2.Construction of recombinant plasmid.The acetoacetyl-CoA thiolase gene ERG10,the hydroxymethylglutaryl coenzyme A synthetase gene ERG13 and the hydroxymethylglutaryl coenzyme A reductase gene tHMG1 were ligated to the plasmid pCOLADuet-1 to obtain a recombinant plasmid 1;The mevalonate kinase gene ERG12,the phosphate mevalonate kinase gene ERG8 and the decarboxylase gene ERG19 were ligated to the plasmid pTrcHis2 B to obtain a recombinant plasmid 2;the isopentenyl pyrophosphate isomerase gene IdI1,geranyl pyrophosphate The synthetase gene GPPS2 and the geraniol synthase gene GES were ligated to the plasmid pACYCDuet-1 to obtain a recombinant plasmid 3.3.Introduce 3 recombinant plasmids into E.coli BL21(DE3)to obtain E.coli engineered strains.Biosynthesis of geraniol from glucose was induced under aerobic conditions.4.Optimization of shake flask fermentation medium and fermentation conditions.Single factor experiments were used to optimize fermentation conditions: the effects of fermentation temperature,induction temperature,rotation speed,inducer concentration,and different carbon and nitrogen sources on geraniol production were studied.In this experiment,the yield of geraniol was 58 mg/L in 24 hours.After optimization of the culture medium and culture conditions,the yield can reach 150 mg/L.It can be predicted that the E.coli engineering strain SZL used in this paper will be necessary in the future production.Moreover,the use of E.coli expression strain SZL to produce geraniol has the advantages of reliable quality,economical products,and green production,and the application prospect is very good.This study laid the foundation for the large-scale industrial production of geraniol,which has important economic and social benefits.