The Study Of Expression Of Protein In The L-oleandose Biosynthesis In E.coli

Avermectin contain 8 components: A1 a,A2a,B1 a,B2a,A1 b,A2b,B1 b,B2b.B1 a component showed the highest insecticidal activity in the eight avermectin component,disaccharide composed of two ?-linked L-oleandrose is linked to avermectin aglycone by ?-1,4 glycosidic bond.The gene avrCDEFGHI is involved in the biosynthesis of oleandrose in avermectin,then the glycosyltransferase(AvrB)catalyzes the iterative glycosylation,complete avermectin molecules is got.Genes of avrC,avrD,avrE,avrF,avrG,avrI do not express in E.coli BL21(DE3)and Rosseta 2(DE3)pLysS.Methyltransferasegene encoded by avrH is soluble in E.coli BL21(DE3).Glycosyltransferase encoded by gene avrB forms inclusion bodies.In this study.we optimize codon of avrF and avrI,then soluble protein AvrF in E.coli BL21(DE3)is obtained,soluble protein AvrI in ArcticExpress(DE3)is obtained.Genes of rmlA,rmlB,rmlD,kijB1,tylC1,kijD10 are synthesis and soluble protein of RmlA,RmlB,Rml D,KijB1,TylC1,KijD10 in E.coli BL21(DE3)are got.TDP-L-olivose which is possible substrate of AvrH protein was synthesized,in vitro,AvrH can convert the TDP-L-olivose into something.After TDP-L-oleandrose biosynthesis pathway in Escherichia coli was determined,we preliminarily attempt to construction a plasmid by GoldenBraid method.The plasmid was transformed into Escherichia coli,this Escherichia coli can biosynthesis TDP-L-oleandrose.The standardized vector rmlA-PBSK,rmlD-PBSK,tylC1-PBSK,avrF-PBSK,avrH-PBSK,avrB-PBSK,avrI-PBSK,avrE-PBSK,kijB1-PBSK,KiJD10-PBSK are got by one-step cloning and gene mutation.pau23-pET28 a and pau22-pET28 a are obtained by gene synthesis.Acceptor vectors of p-sfp-1,p-sfp-2,PBSK-sfp-1,PBSK-sfp-2 and RGPA-sfp are Obtained by one-step cloning method.P-pau23-pau22,p-avrF-rmlD,p-avrH-gfp,p-avrH-avrB,PBSK-avrF-rmlD-avrH-gfp and PBSK-avrF-rmlD-avrH-avrB were obtained by linking standardized vectors and acceptor vectors via GoldenBraid.Due to deletion mutation between genes kijB1 and tylC1,the vector of p-kijB1-tylC1 was not got.Therefore,standardized vector of PBSK-tylC1-1 and acceptor vectors of RGPA-sfp-1 were constructed.p-avrH-tylC1,PBSK-avrF-rmlD-avrH-tylC1 and RGPA-avrF-rmlD-avrH-tylC1 were obtained by linking standardized vectors and acceptor vectors via GoldenBraid.RGPA-avrF-rmlD-avrH-tylC1 and kijB1-pET28 a were transformed into Escherichia coli BL21(DE3),in this recombianat strain the TDP-L-oleandrose was not detected by LC-MS.