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Brevibacteriumflavum ATCC14067 was engineered for L-valine production by over-expression of the ilvEBNrC genes from B.flavum NV128.In traditional fermentation,L-valine production reached 30.08±0.92 g/L at 31℃ 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℃) and the thermotolerance of B.flavum,the fermentation temperature was enhanced to 40℃.As a result,maximum L-valine production,conversion efficiency and specific L-valine productivity reached 38.08 ±1.32 g/L,0.241 g/g and 0.133 g/g/h respectively at 37℃ in 48h fermentation.The strategy for enhancing L-valine production with over-expression key enzymes in thermotolerance strain may provide an altemative approach to enhance branchedchain amino acids production with other strains.