过表达hom基因对谷氨酸棒杆菌发酵L-异亮氨酸的影响

Effects of Overexpression of Homoserine Dehydratase on L-Isoleucine Production in Corynebacterium glutamicum

谷氨酸棒杆菌(Corynebacterium glutamicum)中hom基因编码的高丝氨酸脱水酶为L-异亮氨酸合成过程的关键酶,本研究通过在L-异亮氨酸生产菌C.glutamicum YILW中过表达高丝氨酸脱水酶,考察过表达高丝氨酸脱水酶对发酵L-异亮氨酸产量的影响.以L-异亮氨酸生产菌C.glutamicum YILW基因组为模板克隆hom基因,将hom基因与表达载体pXMJ19连接构建出重组质粒pXMJ19-hom,再转入C.glutamicum YILW构建C.glutamicumYILW(pXMJ19-hom).通过5,L罐发酵研究重组质粒对工程菌的生长、耗糖、L-异亮氨酸产量及副产物积累等方面的影响.结果显示,重组酶的表达使菌株对L-苏氨酸的抗反馈抑制作用得到加强.最终L-异亮氨酸和L-蛋氨酸积累量分别为36.5,g/L和2.8,g/L,分别较出发菌株提高7%和33%,同时L-赖氨酸合成量仅为2.1,g/L,较出发菌株降低了63.8%.

Homoserine dehydratase, a key enzyme of L-isoleucine synthesis process, is encoded by the horn gene in Cory- nebacterium glutamicum. The effect of overexpressed homoserine dehydratase in C. glutamicum YILW on the production of L-isoleucine was studied. The horn gene was PCR amplified using C. glutamicum YILW genomic DNA as template and then inserted into a multicopy plasmid pXMJ19 to construct a recombined expression plasmid pXMJ19-hom. The recombined plasmid was then transformed into C. glutamicum YILW to construct recombined C. glutamicum YILW （pXMJ 19-horn）. The effects of pXMJ19-hom on cell growth, glucose consumption rate, yield of L-isoleucine and accumulation of byproducts were investigated by using 5 L batch fermentation. Enzyme studies showed that the recombined homoserine dehydratase expressed in C. glutamicum YILW （pXMJ 19-horn） strain was much less sensitive to threonine inhibition. Compared with the original strain, the yields of L-isoleucine and L-methionine by using C. glutamicum YILW （pXMJ 19-hom）were 36.5 g/L and 2.8 g/L, which indicated on increase of 7% and 33% respectively, but the yield of L-lysine was only 2.1 g/L, decreased by 63.8%.