北京棒杆菌芳香族氨基酸转运蛋白基因敲除对L-色氨酸积累的影响

摘要

[目的]为了减少北京棒杆菌PD-67(Corynebacterium pekinense PD-67)从细胞外吸收色氨酸,降低细胞内色氨酸库的浓度,从而使色氨酸的反馈控制作用减弱,增加胞外L-色氨酸的积累量,构建北京棒杆菌PD-67的芳香族氨基酸转运蛋白基因aroP敲除的菌株,研究aroP基因敲除对菌株L-色氨酸积累的影响.并进一步研究在aroP敲除菌株中表达邻氨基苯甲酸合成酶(AS)基因对L-色氨酸积累的影响.[方法]运用PCR技术扩增aroP基因,与整合质粒连接后,用限制性内切酶法构建带有内部片段缺失的aroP基因的敲除载体.利用同源重组技术,敲除北京棒杆菌PD-67的aroP基因,构建菌株PD-67 ΔaroP,并用带有aroP基因的表达载体对PD-67ΔaroP进行互补验证.采用PCR技术扩增AS基因,与表达载体连接构建重组质粒.将重组质粒转入菌株PD-67ΔaroP,构建工程菌株PD-67 ΔaroP/pXAS.通过摇瓶发酵研究PD-67 AaroP和PD-67 ΔaroP/pXAS的发酵特性.[结果]经PCR验证获得了aroP基因缺陷的菌株.摇瓶发酵结果表明,与出发菌株相比,PD-67ΔaroP的L-色氨酸的积累量提高了65％.酶活分析结果表明,AS基因在菌株PD-67 △aroP中得到表达.AS基因表达使工程菌单位菌体产酸率提高了25.6％.[结论]北京棒杆菌PD-67中芳香族氨基酸转运蛋白基因arop的敲除能够提高胞外L-色氨酸的积累量.在arop基因敲除菌中表达AS基因,可以进一步提高工程菌的产酸率.%A Corynebacterium pekinense PD-67 mutant with aromatic amino acid transport system gene (aroP) in-frame deletion was constructed to decrease the uptake of L-tryptophan and reduce the intracellular pool of L-tryptophan, further to deregulate the feedback regulation of L-tryptophan and increase the extracellular accumulation . The effects of aroP knock-out as well as anthranilate synthetase (EC4. 1. 3.27; AS) gene overexpression on L-tryptophan accumulation of the mutant were investigated. [Methods] The aroP gene was cloned from C. pekinense PD-67 chromosome and ligated to integration vector, and then deleted about 600bp fragment by restriction endonuclease digestion. The mutant C. pekinense PD-67-ΔaroP was screened by homologous recombination. The mutant phenotype can be reversed by complementation with aroP gene from the expression vector. AS gene was cloned and ligated to expression vector to construct a recombinant plasmid. The plasmid was transformed into PD-67ΔaroP to generate the engineering strain PD-67ΔaroP/pXAS. The fermentation characteristics of the mutant and the engineering strain were investigated. [Results] The aroP gene in-frame deletion was screened and confirmed by PCR analysis and the AS gene expression was confirmed by determination of enzyme activity. The aroP knock-out resulted in increase of L-tryptophan accumulation by 65% compared with that of the parent strain, while the expression of AS gene resulted in increase of L-tryptophan yield on cell mass by 25. 6% in engineered strain. [ Conclusion] The aroP gene knock-out of the strain PD-67 improved L-tryptophan accumulation. The expression of AS gene could further improve L-tryptophan yield on cell mass in engineered strain.