Producing Glycolate At High Yield And Titer In Escherichia Coli By Metabolic Engineering

Glycolic acid is the most simple alpha-hydroxy acids which is widely used in in various of industries,such as chemical cleaning,biodegradation,daily chemical industry,textile industry and so on.In rencent years,many researchers have succeeded in obtaining glycolic acid accumulation in Escherichia coli and that laid the foundation for biosynthesis of glycolic acid,further solving the problems of serious environmental pollution,complex preparation conditions and difficulties in downstream extraction and separation in traditional chemical synthesis methods.However,these methods has a low yield of glycolic acid,rendering them can not meet the needs of industrial production.Therefore,based on the previous studies,we obtained the glycolate synthase strains with high yield of glycolic acid by regulating the expression level of key enzymes in glycolic acid biosynthesis pathway,and knocking out the competition pathway of glycolic acid synthesis and glycolic acid metabolic pathway.And,the production of glycolic acid was further improved through the optimization of fermentation strategy.Isocitrate lyase?aceA?,glyoxylic acid reductase?ycd W?and isocitrate dehydrogenase kinase/phosphorylase?aceK?were the key enzymes in the biosynthesis of glycolic acid.The expression of these genes is very important for the production of glycolate.In order to regulate the expression level of key genes in glycolic acid biosynthesis pathway,we chosed three expression plasmids with diffrent copy number and another two pasmids with diffrent promoter to adjust the expression level of the target genes.Fermentation results of recombinant strains with different copy plasmids as expression vectors showed that the recombinant strain Mgly12 with intermediate-copy expression plasmid obtained the highest yield of glycolate which is 0.258g·?g glucose?^-1.Meanwhile expression level and enzyme activity of key genes in Mgly12 were higher than that of the low copy or high copy expression plasmid;and comparing the T7 promoter,the pTet promoter and the pTrc promoter for the expression of the key genes,the yield of glycolic acid is Significantly increased to0.385 g·?g glucose?^-1 using the expression plasmid containing the pTrc promoter.Further more,we found another enzyme that plays a key role in the biosynthesis of glycolic acid,that is citrate synthase?gltA?.The gene gltA was overexpressed and the yield of glycolate in recombinant strain Mgly145 was raised to 0.504 g·?g glucose?^-1.In order to enhance the glycolic acid synthesis pathway and reduce the glycolic acid metabolism,malate synthase genes?glcB,aceB?of the glyoxylate shunt were firstly knocked out in the host strain Mgly1?MG1655?DE3???ldhA)to reduce metabolic of glyoxylate and further to enhance the accumulation of glycolic acid.And in turn,the glycolate dehydrogenase gene?ald A?and the glycolate oxidase gene?glcDEF?were knocked out to cut down the metabolic of glycolic acid.After the malate synthase genes were knocked out,the yield of glycolic acid in the engineered strain Mgly345 was 0.750 g·?g glucose?^-1,reaching88.2%of the theoretical yield and increased 49%than Mgly145.After knocking out glycolate dehydrogenase gene?aldA?,the yield of glycolic acid of engineered strain Mgly445 was further raised to 0.79 g·?g glucose?^-1,reaching 92.9%of the theoretical yield.After knocking down the glycolate oxidase gene?glcDEF?,the yield of glycolic acid of the engineered strain Mgly545 was only 0.678 g·?g glucose?^-1.Based on the above results,Mgly445 was selected as the starting strain for subsequent studies.To improve cell growth,increase the production of glycolic acid,The optimum fermentation medium was determined by optimizing the nitrogen source and C/N ratio of fermentation medium:6.78 g·L^-1 Na₂HPO₄,3 g·L^-11 KH₂PO₄,0.5 g·L^-1 NaCl,2.64 g·L^-1?NH4?₂HPO₄,2.986 g·L^-1 yeast,0.24 g·L^-1 MgSO₄,0.011 g·L^-1 CaCl₂.And The optimal induction concentration is 0.1 mmol·L^-1 IPTG.The titer of glycolic acid in batch fermentation was 4.639 g·L^-1.Compared with the intermittent fed-batch fermention,the glucose-stat fed-batch fermention effectively controlled the glucose content in the fermentation broth,so that the content of acetic acid in the whole fermentation process was kept in low lever,and the production of glycolic acid reached 16.663 g·L^-1.