【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-67ΔaroP,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-67ΔaroP/pXAS。通过摇瓶发酵研究PD-67ΔaroP和PD-67ΔaroP/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-67ΔaroP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。更多还原

【Abstract】 [Objective] 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.更多还原