[背景]大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌.[目的]敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株.[方法]利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysCfbr、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中.[结果]以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶III (AKIII)的基因lysC、磷酸果糖激酶II基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35 g/L,比出发菌株增加9.9％.随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysCfbr)、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株.该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20％,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L.[结论]单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法.%[Background] Escherichia coli is often used to produce threonine because of its excellent growth performance and clear genetic background. [Objective] In order to construct a high-yield strain of threonine, the nonessential genes of the E. coli THR in threonine biosynthetic pathway were knocked out while the key enzymes essential for threonine synthesis were heterologously expressed. [Methods] The lysC, pfkB and sstT of E. coli THR were deleted by using the FLP/FRT recombinant enzyme system. In addition, the recombinant plasmid with the genes lysCfbr and thrE from Corynebacterium glutamicum and the gene gapC from Clostridium acetobutylicum was constructed. Then this plasmid was introduced into different host strains. [Results] The target strain E. coli THR3 as obtained from the parental strain E. coli THR by deleting its threonine synthesis pathway: aspartate kinase III-coding gene lysC and phospho fructose kinase II-coding gene pfkB as well as the threonine absorption protein-coding gene sstT. The threonine production of strain E. coli THR3 as up to 75.64±0.35 g/L, was 9.9％ higher than that of the parental stain E. coli THR (68.7 g/L). In addition, heterogonous expression of threonine secretory transporter-coding gene thrE and aspartic kinase-coding lysCfbr from C. glutamicum as well as heterologous expression of NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase coding gapC from Clostridium acetobutylicum were beneficial to increase the threonine production. The recombinant strain E. coli THR6 could produce 105.3±0.5 g/L of threonine with a biomass of 18.26 g DCW/L and the threonine yield (g/g) from glucose increased by 43.20％ and the acid production capacity per unit increased to 5.76 g/g DCW in fed-batch culture accumulation. [Conclusion] Knocking out a gene alone or modifying a pathway does not allow large amounts of threonine to be synthesized and accumulated. Co-transformation of multiple metabolic pathways is the most effective way to construct threonine engineered strains.
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