The Reconstruction And Optimization Of Ectoine Synthesis Pathway In E.Coli

Genes encoding the enzymes for the biosynthesis of ectoines are arranged as ectABC and ectABCask in nature. In this experiment, Ectoine synthesis pathway in Escherichia coli was reconstructed and optimizated using biosynthetic methods.The main research results are as follows:(1) In this experiment, ectABC genes from Bacillus halodurans、Halomonas elongata、 Thermomonospora fusca、Methylomicrobium alcaliphilum20Z and Methylophaga thalassica have been cloned successfully.(2) Among recombinant plasmids pET21a-Bh/HeEctABC, the ectA、ectB、ectC genes could be expressed simultaneously in BL21(DE3), and ectA、ectB、ectC genes could be expressed simultaneously in BW25113among pBAD-MaEctAB and pBAD-MtEctABC.(3) Ectoine can be synthesized among pET21a-HeEctABC and pBAD-MaEctABC.(4) Basing on the expression of enzymes for the biosynthesis of ectoines from pET21a-Bh/He/TfEctABC, recombinant plasmid pET21a-TfABhBC and pET21a-HeABhBC were constructed. Moreover,amount of ectoine can be improved inpET21a-TfABhBC.(5) The ectABCask genes from Methylomicrobium alcaliphilum20Z and Methylophaga thalassica have also been cloned successfully.(6) Recombinant plasmids pBAD-MaEctABCAsk and pBAD-MtEctABCAsk have been constructed. In pBAD-MaEctABCAsk recombinant plasmids, the ectA、ectB、 ectC、ask genes could be expressed simultaneously in BW25113. However, The results of HPLC showed the amount of ectoine between pBAD-MaEctABCAsk and pBAD-MaEctABC appeared to have changed little.(7) The recombinant plasmid pBAD-MtRbsM was constructed by converting RBS of ectB to RBS of ask in pBAD-MtEctABCAsk. The ectA、ectB、ectC、ask genes could be expressed simultaneously in BW25113. The results of HPLC showed the amount of ectoine in pBAD-MtRbsM has been improved5-fold compared with pBAD-MtEctABC.The successful construction of ectoine biosynthetic pathway and improvement of amount of ectoine in E.coli provide a basic method on the optimized synthesis of ectoine in E.coli...