Metabolic Engineering For Modifying Corynebacterium Glutamicum To Produce L-malate

This research took Corynebacterium glutamicum ATCC 13032 as modle strain,the high-yield L-malic acid strain was constructed through metabolic engineering strategy.Further,the carboxylation pathway of phosphoenolpyruvate and pyruvate to oxaloacetate was enhanced for increasing yield of L-malic acid.The main results were described as follows:(1)L-malic acid producing strain was constructed.Homologous recombination method was established by plasmid pK19 mobsacB for knocking out gene in Corynebacterium glutamicum.In order to block the metabolic pathway of pyruvate to acetic acid and lactic acid as well as the pathway of L-malate anaplerotic reaction to pyruvate,the phosphoacetyltransferase-acetate kinase encoding gene pta-ack,lactate dehydrogenase encoding gene ldh,pyruvate quinone oxidoreductase encoding gene pqo and malic enzyme encoding gene male were knocked out continuously,which constructed L-malic acid producing strain A04 successfully.The production of L-malic acid and pyruvate were 7.48 g/L and 6.704 g/L respectively after fermental cultivation of strain A04.(2)The production of L-malic acid increased by strengthening carboxylation pathway.The phosphoenolpyruvate carboxylaseencoding gene ppc and the pyruvate carboxylase encoding gene pyc were overexpressed by replacing strong promoter Psod,and then the carboxylation pathway was strengthened in the L-malic caid synthesis.Further the carbon flux to L-malic caid increased,and the ability of the strain to produce L-malic caid was improved.Accordingly,with the initially optimized fermentation conditions the maximum production of L-malic caid was 14.932 g/L and the yield was 0.761 mol/mol glucose.(3)Comparison of ppc metabolic pathway and pyc metabolic pathway.By analyzing the enzyme activity of ppc and pyc and the yield of organic acid of different engineering strains.The results indicated that the ppc metabolic pathway and the pyc metabolic pathway could both synthesize oxaloacetate first and then L-malic acid during anaerobic fermentation.Howover,the carbon flux in the ppc metabolic pathway was much higher than that in pyc metabolic pathway.So,the low carbon flow rate in the pyc metabolic pathway was one of the key factors for pyruvate accumulating in engineering strains and then to prevent the strains from utilizing glucose to synthesize L-malic caid efficiently.