pta基因敲除对L-色氨酸发酵的影响  

Effects of gene pta disruption on L-tryptophan fermentation

作　　者：黄静[1] 史建明[1] 刘倩[1] 徐庆阳[1] 谢希贤[1] 温廷益[2] 陈宁[1] 

机构地区：[1]天津科技大学生物工程学院,教育部工业微生物重点实验室,天津300457 [2]中国科学院微生物研究所,北京100101

出　　处：《微生物学报》2011年第4期480-487,共8页Acta Microbiologica Sinica

基　　金：国家“重大新药创制”科技重大专项课题(2008ZX09401-05);“十一五”国家科技支撑计划重点项目(2008BAI63B01)~~

摘　　要：【目的】探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响。【方法】运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTHΔpta。利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHΔpta发酵生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH 4+浓度及变化。建立了大肠杆菌合成L-色氨酸的代谢流平衡模型,应用MATLAB软件计算出E.coli TRTH及其pta缺失株发酵中后期代谢网络的代谢流分布。【结果】发酵结果显示,pta缺失株能够以较长时间和较高比生长速率保持对数生长,最终菌体生物量和L-色氨酸产量分别提高了52.7%和46.8%。有机酸含量分析结果表明,pta缺失株乙酸含量显著降低,只有2.5 g/L,仅为亲株的19.5%;丙酮酸和乳酸的含量有一定增加。多参数分析结果显示,pta缺失株发酵过程中NH 4+浓度较亲株降低33.2%。pta缺失株发酵生产L-色氨酸的代谢流量分析结果表明,EMP途径的代谢流量降低7.4%,PP途径的代谢流量增加8.4%,TCA循环的代谢流量降低32.2%。【结论】pta基因的缺失导致在发酵生产L-色氨酸过程中代谢流发生变化,乙酸含量的显著降低解除了乙酸对菌体生长和产物生成的抑制,使得菌体生物量和L-色氨酸产量大幅提高。[Objective]To study the effects of gene pta disruption on biosynthesis of L-tryptophan.[Methods]The pta gene of the L-tryptophan producing strain E.coli TRTH was disrupted by Red recombination technology and a pta mutant E.coli TRTHΔpta was constructed.Fed-batch fermentation of E.coli TRTHΔpta was carried out in 30-Liter fermentor to investigate the biomass,L-tryptophan production,organic acid content and the concentration of NH 4＋,lactate,pyruvate and succinate.The metabolic flux balance model of L-tryptophan synthesis by E.coli was established.Based on this model,the practical metabolic flux distribution of E.coli and its pta mutant were determined with the linear program planted in MATLAB software.[Results]Compared with E.coli TRTH,the pta mutant was able to maintain higher growth rate at exponential phase,the final biomass and the L-tryptophan production were increased by 52.7% and 46.8% respectively.Meanwhile,the data analysis of organic acids accumulated during fed-batch culture showed that the concentration of acetate was decreased to 2.5 g /L,which was only 19.5% of that of the parental strain;as the decreased concentration of succinate,the accumulation of pyruvate and lactate was increased.The concentration of Na ＋,K ＋,PO43-were consistent with E.coli TRTH during the fed-batch culture,the concentration of NH 4＋ was decreased by 33.2%.The metabolic flux analysis indicated that EMP pathway and TCA cycle were reduced by 7.4% and 32.2% respectively,but PP pathway was increased by 8.4% compared with E.coli TRTH during the middle and late period of the fed-batch culture.[Conclusion]In the process of L-tryptophan fermentation,pta gene deletion in E.coli TRTH led to change in metabolic flux and acetate content,which derepressed its inhibition on cell growth and production of L-tryptophan and finally made a substantial increase of bacterial biomass and L-tryptophan production.

关 键 词：Red重组技术 pta基因 基因敲除 乙酸 L-色氨酸 

分 类 号：TQ922.9[轻工技术与工程—发酵工程]