黄色短杆菌ilvBN和ilvC基因定点突变对L-缬氨酸发酵的影响

Site-directed mutagenesis of ilvBN and ilvC in Brevibacterium flavum for improved production of L-valine

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%.