过表达pps基因和aroG^(fbr)基因对北京棒杆菌L-色氨酸合成的影响

Effect of pps and aroG^(fbr) overexpression on L-tryptophan production in Corynebacterium pekinense

【目的】通过增加北京棒杆菌(Corynebacterium pekinense)PD-67芳香族氨基酸合成的前体物质磷酸烯醇式丙酮酸(PEP)的供应,解除终产物对芳香族氨基酸合成途径中第一个酶同时也是关键酶3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶(DS)的反馈抑制并提高抗反馈抑制的DS的活力,使碳流更多地流向芳香族氨基酸合成途径,从而积累更多L-色氨酸。【方法】运用PCR技术扩增北京棒杆菌PD-67磷酸烯醇式丙酮酸合酶基因pps,与表达载体连接构建重组质粒pXPS;运用重叠PCR技术定点突变大肠杆菌(Escherichia coli)受苯丙氨酸调控的DS基因aroG,使相应的编码氨基酸序列发生突变:Leu175Asp,新的基因命名为aroGfbr,与表达载体连接构建重组质粒pXA;构建pps和aroGfbr的共表达重组质粒pXAPS。将3个重组质粒分别转入菌株PD-67,构建工程菌株PD-67/pXPS、PD-67/pXA和PD-67/pXAPS。通过摇瓶发酵研究工程菌株的发酵特性。【结果】酶活分析结果表明,pps基因和aroGfbr基因在北京棒杆菌PD-67中均实现了表达。工程菌株PD-67/pXA粗酶液DS抗反馈抑制分析表明,AroGfbr已解除酪氨酸和苯丙氨酸的反馈抑制。过表达pps基因和aroGfbr基因分别使工程菌L-色氨酸产量提高12.1%和26.8%,双基因共表达可使工程菌的产酸量提高35.9%。【结论】北京棒杆菌PD-67pps基因的过表达以及大肠杆菌来源的解除反馈抑制的aroGfbr的过表达均有助于增加PD-67 L-色氨酸的合成,而双基因的共表达可以进一步提高L-色氨酸的积累量。

[Objective]In order to redirect carbon flows into aromatic amino acids biosynthesis pathway and further improve the production of L-tryptophan in Corynebacterium pekinense PD-67,two schemes were implemented.First,the supply of phosphoenolpyruvate （PEP）,one of precursors of L-tryptophan biosynthesis,was increased. Second,the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase（DS）,a key enzyme in the aromatic amino acids biosynthesis,was relieved and the activity of DS was increased.[Methods]The phosphoenolpyruvate synthase gene （pps） was cloned from C. pekinense PD-67 chromosome by PCR and inserted into expression vector to construct a recombinant plasmid pXPPS; the aroG gene encoding DS isozymes was cloned from Escherichia coli chromosome by PCR and the mutation of Leu175Asp was introduced by site-directed mutagenesis using sequence-overlap extension PCR.The mutated gene named as aroGfbr was cloned to expression vector to construct a recombinant plasmid pXA; and the recombinant plasmid pXAPS co-expressing pps and aroGfbr was constructed.The three recombinant plasmids were transformed into PD-67 to generate the engineering strains PD-67 /pXPS,PD-67/pXA and PD-67/pXAPS,respectively.The fermentation characteristics of the three engineering strains were investigated.[Results]The expression of pps and aroGfbr was confirmed by enzyme activity assays.The deregulation of feedback inhibition of AroGfbr was confirmed by determining DS activity in the presence of three aromatic amino acids.The overexpression of pps and aroGfbr resulted in an increase of L-tryptophan biosynthesis by12. 1% and 26. 8%,respectively,while the co-expression of two genes increased the production of L-tryptophan by 35.9% in the engineering strain PD-67/pXAPS. [Conclusion] Both of the overexpressions of the pps gene and aroGfbr gene can increase L-tryptophan biosynthesis,while the production was further improved by the co-expression of the two genes.