以黄色短杆菌MH-1000 为出发菌株，使用PCR技术克隆ilvBN 与ilvC 基因，对ilvBN 进行定点突变，获得解除L-缬氨酸对乙酰羟酸合酶反馈抑制突变型基因ilvBN'． 对基因ilvC 进行点突变，获得乙酰羟酸变位酶突变基因ilvC'． 通过重叠延伸PCR方法，将基因片段ilvBN'和ilvC'拼接为ilvBN'C'，进而连接至穿梭载体pXMJ19 获得重组质粒pXMJ19-ilvBN'C'． 该重组质粒转化至出发菌株获得工程菌株MH-1032． 50 L 分批补料发酵结果显示: MH-1000 发酵72 h L-缬氨酸质量浓度为35． 2 g /L，MH-1032 发酵72 h L-缬氨酸质量浓度为38． 4 g /L，增长9． 1%，糖酸转化率从21． 7%提高到25． 8%．

Brevibacterium flavum 1000 was used as the original strain and site-specific mutagenesis was performed in its ilvBN gene，resulting in an anti-feedback inhibition gene ilvBN'． The gene ilvC' encoding acetohydroxyacid isomeroreductase was obtained by site-directed mutagenesis from ilvC． The gene ilvBN'C' was obtained by overlap extension PCR from ilvBN' and ilvC，and then inserted into E． coli-B． flavum shuttle expression vector pXMJ19 to construct a recombinant plasmid pXMJ19-ilvBN'C'． The recombinant plasmid was subsequently transformed into B． flavum MH-1000，resulting in the strain MH-1032． The results from fed-batch fermentation experiments in 50 L fermenter indicated that the L-valine productivity of MH-1032 ( 38． 4 g /L) was increased by 9． 1% in contrast to that of MH1000 ( 35． 2 g /L) in 72 hours，and the conversion of glucose to L-val increased from 21． 7% to 25． 8%．