Metabolic Engineering For L-Valine Production

Based on the basic principles of the metabolic engineering, L-valine biosynthesis was enhanced by plasmid pDXW-8-ilvEBNrC, which can effectively improve the production of L-valine; and then the supply of precursor for L-valine biosynthesis was regulated, accumulation of by-products were minimized, the problem of by-products accumulation involved was successfully resolved. Main contents and conclusions are as follows:B. flavum ATCC14067 was engineered for L-valine production by over-expression of different ilv genes with pDXW-8(tac ; KmR), the ilvEBNrC genes (AHAS was feedback-resistant for L-valine, L-Leucine,L-Isoleucine) from B. flavum NV128 was the best candidate for L-valine production. In traditional fermentation, L-valine production reached 30.08±0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L-valine production and conversion efficiency based on the optimum temperatures of L-valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was enhanced to 34°C, 37°C, 40°C respectively. As a result, higher metabolic rate and L-valine biosynthesis enzymes activity were obtained at high temperature, maximum L-valine production, conversion efficiency and specific L-valine production rate reached 38.08±1.32 g/L, 0.241 g/g and 0.133 g/g/h respectively at 37°C in 48 h fermentation. Plasmid pDXW-8-ilvEBNrC is an effective tool to enhance L-valine production, the strategy for enhancing L-valine production with over-expression the genes of key enzymes in thermotolerance strain may provide an alternative approach to enhance branched-chain amino acids production with other strains.C.glutamicum ATCC13032 and B. flavum JV16 (auxotrophic for L-Leucine, L-Isoleucine, L-Methionine) were engineered for L-valine production by over-expressing ilvEBNrC genes at 31°C in 72 h fermentation. Different strategies were carried out to reduce the by-products in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C.glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products formation were recorded, indicating 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To remove L-alanine, alaT and/or avtA was inactivated in C.glutamicum and B.flavum respectively. Compared with high concentration of L-alanine accumulated in alaT inactivated strains with over-expression of ilvEBNrC genes, C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBNrC produced 31.15±1.032 g/L L-valine with 0.18±0.002 g/L L-alanine, meanwhile, B. flavum JV16avtA::Cm pDXW-8-ilvEBNrC produced 38.82±1.974 g/L L-valine with 0.22±0.002 g/L L-alanine. The results suggest that avtA gene should be disrupted when L-valine synthesis was enhanced. This study provides combined strategies to improve L-valine yields by minimization of by-products production.