pta基因敲除对L-色氨酸发酵的影响

Effects of gene pta disruption on L-tryptophan fermentation

摘要:

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. coli TRTHΔpta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NH4+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

Abstract:

Abstract: [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 NH4+, 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 NH4+ 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.