Pathway Optimization For Methionine Biosynthesis By Escherichia Coli

Methionine is significantly industrial valuable,which has been widely used in feed production,food,medical care and so on.Methionine is mainly produced by chemical method,but there is a huge consumption of chemical materials,big waste disposal problems and other issues.Fermentation is low consumption of materials and environmentally friendly,however,the complex regulatory mechanisms and the low yield of methionine make it difficult to produce in large-scale.The methionine biosynthesis-related research in recent years was concluded,the mechanism of methionine anabolic regulation was analyzed and the gene in metabolic pathways in E.coli was modified in this study.The main results are as follows:1)The effect of methionine pathway and related pathways of methionine synthesis.Overexpression of the key enzyme gene?metA,metC?of methionine biosynthesis and cysteine biosynthesis acetyltransferase gene?cysE?can promote the synthesis of methionine.The accumulation of methionine was achieved by knocking of repressor protein gene metJ,strengthening of the methionine cycle,weakening of the threonine methionine biosynthesis pathway through genetic engineering.2)Study on the methionine transporter protein indicated that transport proteins YeaS,YieH have an effective accumulation of methionine.3)methionine biosynthesis was optimized and recombinant strains S13 MG1655?metJ?pDHC29::metA::metC::yeaS?and S14 MG1655?metJ?pTrc99a::metA::cysE?were built.The S13,S14 were cultivated respectively in three sulfur substrate?K₂SO₄,taurine,SDS?fermentation broths,strain S13 cultivated in K₂SO₄fermentation broth containing the highest concentration of methionine 0.396 g/L,which is the highest yield in directed transformation in E.coli.According to the results of different strategy to build strains,further optimization of strain strategies has been offered:improvement of the transportation of inorganic sulfur into intracellular;modification of the methionine transport system?metN,metI,metQ?by genetically operation in E.coli;optimization of culture conditions.