过量表达苏氨酸脱水酶对谷氨酸棒杆菌合成L–异亮氨酸的影响

Effect of Overexpression of Feedback-Resistant Threonine Dehydratases on L-Isoleucine Production in Corynebacterium glutamicum

考察过量表达解除反馈抑制的苏氨酸脱水酶对谷氨酸棒杆菌发酵生产L–异亮氨酸的影响.将解除反馈抑制的苏氨酸脱水酶基因ilvA(F383V)连接大肠杆菌/谷氨酸棒杆菌穿梭质粒pXMJ19,过量表达于L–异亮氨酸生产菌株Corynebacterium glutamicum YILW.经5 L发酵罐分批补料发酵产酸实验,与出发菌株C.glutamicum YILW相比,耗糖高峰期滞后,L–异亮氨酸产量增加了10.3%,副产物L–蛋氨酸、L–赖氨酸含量分别降低了33.3%2、6.5%,发酵液中没有L–苏氨酸的累积,乳酸的累积量增加了41.7%.对发酵稳定期的代谢流分布研究表明,过量表达解除反馈抑制的苏氨酸脱水酶使HMP途径流量增加了31.7%,L–异亮氨酸生物合成途径的代谢流提高了8.5%.

The effect of overexpressing ilvA（F383V）gene encoding feedback-resistant threonine dehydratase in a L-isoleucine producing strain C.glutamicum YILW was studied.A fragment of ilvA（F383V）gene was proliferated by polymerase chain reaction using chromosomal DNA of C.glutamicum YILW as the template.The obtained fragment was inserted into E.coli/C.glutamicum shuttle vector pXMJ19 to construct an expression plasmid pXMJ19-ilvA（F383V）.The recombinant plasmid pXMJ19-ilvA（F383V）and empty plasmid pXMJ19 were respectively transformed into C.glutamicum YILW by electroporation.Fed-batch cultures of C.glutamicum YIMJ383 were carried out in 5 L fermentor.The results showed that the yield of L-isoleucine by the recombinant was increased by 10.3% without any L-threonine as a by-product.The concentrations of L-methionine and L-lysine were decreased by 33.3% and 26.5%r,espectively.The accumulation of lactic acid was 41.7% higher than that of the parent strain.In order to study the effects of overexpressing ilvA（F383V）gene on the metabolic flux distributionst,he metabolic flux analysis of the L-isoleucine production at pseudo steady state was conducted.The research indicated that HMP pathway flux increased by 31.7%,L-isoleucine biosynthesis pathway flux increased by 8.5%.