Construction Of Recombinant Escherichia Coli For Production Of 1.4-blutyldiamine Via Biotransformation

Butanediamine,a kind of important chemical raw materials,can polymerize with dibasic acid into a new polymer plastic with better performance and broad application prospect.At present,the synthesis of butanediamine relys on chemical synthesis using non-renewable fossil resources which can result in environmental pollution.Biological synthesis of butanediamine by catalyzing ornithine to butanediamine from renewable carbohydrate is expected to replace chemical synthesis in the future,so which was regarded as a great potential method.This study cloned three kinds of ornithine decarboxylase from Escherichia coli and expressed them in E.coli periplasmic space and analyzed the enzymology properties of them.Further,bio-based conversion from ornithine to butanediamine was done by constructing engineering strains of E.coli which laid a foundation to achieving efficient synthesis of diaminopentane in E.coli.The concrete research content is as follows.Firstly,speF,speCl and speC2 gene were amplificated from E.coli.These three genes were connected to pET20b and pET22b containing pelB signal peptide respectively and transformed to BL21 to construct engineering strains E.coliBL21/pET20b-speF,BL21/pET20b-speCl,BL21/pET20b-speC2,BL21/pET22b-speF,BL21/pET22b-speCl and BL21/pET22b-speC2.Then in shake flask level,we determined the expression of recombinant ornithine decarboxylase as well as the conversion of ornithine in the synthesis of butanediamine in the engineering bacteria.Protein electrophoresis analysis showed that the expression of recombinant enzyme SpeCl and SpeC2 were significantly higher than recombinant enzyme SpeF.The determination of enzyme activity showed that the enzyme activity of recombinant enzyme SpeCl and SpeC2 respectively achieved 17.88 U/mL(17.48 U/mL)and 17.83 U/mL(17.53 U/mL),significantly higher than that of recombinant enzyme SpeF,6.88 U/mL(7.44 U/mL).Through HPLC analysis,yields of diaminopentane in BL21/pET20b-speF,BL21/pET20b-speCl,BL21/pET20b-speC2,BL21/pET22b-speF,BL21/pET22b-speCl and BL21/pET22b-speC2were 1.04 g/L,1.51 g/L,1.42 g/L,1.51 g/L,1.70 g/L and 2.10 g/L,respectively.While in strains BL21/pET20b and BL21/pET22b,there were not shown on map of HPLC.The conversion rates of which were 13.01%,14.02%,17.42%,20.20%,16.46%and 17.22%,respectively.Then enzymology properties of recombinant SpeF,SpeCl and SpeC2 with higher filed were carried out.The optimum pH of SpeF was 5.2 and the optimal temperature was 50?,the optimum pH of SpeCl is 6.6 and the optimal temperature was 46?,the optimum pH of SpeC2 is 6.8 and the optimal temperature was 50?.Under the optimum conditions,the enzyme activity of SpeC2 was highest,while,the enzyme activity of SpeF was lowest.To discrease the amount of ornithineto arginine,the gene argI was knocked out E.coli(the recombinant PUT-A was obtained).Butanediamine production was 0.64 g/L.