乳糖发酵短杆菌lysC突变对L-赖氨酸积累的影响

The Effect of LysC on L-Lysine Accumulation in Brevibacterium lactofermentum

DOI：

中文关键词: 乳糖发酵短杆菌 天冬氨酸激酶 抗反馈抑制 发酵 L-赖氨酸

英文关键词: Brevibacterium lactofermentum aspartate kinase feedback inhibition fermentation L-lysine

基金项目:国家863计划项目(2007AA02Z229和2007AA02Z230)

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中文摘要:

从一株乳糖发酵短杆菌L-异亮氨酸生产菌中克隆出天冬氨酸激酶AK-1的编码基因ly-sC1,经DNA测序并与来自谷氨酸棒杆菌ATCC13032的野生型lysC比对发现其中有2个核苷酸1186G和1187C缺失,造成翻译提前终止。AK-1还有下列2个有效突变位点:Ala 279 Thr和Ser317 Ala。lysC1在大肠杆菌BL21中的诱导表达量约为lysC的1/4,其表观比酶活也较低,但对L-苏氨酸和L-赖氨酸协同反馈抑制不敏感。用大肠杆菌-黄色短杆菌穿梭表达载体pDXW-8将ly-sC1在野生型乳糖发酵短杆菌ATCC13869中诱导型表达,经摇瓶发酵积累L-赖氨酸7.4 g/L,在3 L发酵罐上达40 g/L。

英文摘要:

The gene lysC1 encoding aspartate kinase AK-1 from a Brevibacterium lactofermentum L-isoleucine producer was sequenced.Compared with the lysC from Corynebacterium glutamicum ATCC13032,there were deletions of two nucleotides 1186G and1187C,leading to the pre-termination of translation.In addition,the following two mutated sites were also identified in lysC1: Ala 279 Thr and Ser 317 Ala.In Escherichia coli BL21,the expression level of lysC1 was lower than that of lysC from B.lactofermentum ATCC13869.The AK-1 had a lower apparent value of specific activity than the wild type AK,but it was not sensitive to the feedback inhibition by the mixture of L-lysine and L-threonine.lysC1 was cloned into an E.coli-Brevibacterium flavum shuttle expression vector pDXW-8 and transformed into wild type B.lactofermentum ATCC13869.The recombinant strain ATCC13869/pDXW-8-lysC1 accumulated 7.5 g/L L-lysine after 90 h flask fermentation and 40 g/L after 72 h batch fermentation.These results would be very useful for constructing a genetically identified L-lysine producing strain with minimum genetic alterations.

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