摘要
目的:探究NH4+浓度对大肠杆菌E.coli TRTH发酵生产L-色氨酸的影响。方法:通过外源添加试验,利用30 L发酵罐进行分批补料发酵试验,考察E.coli TRTH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、耗糖速率、发酵液中NH4+浓度及质粒稳定性变化。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发酵中后期代谢网络的代谢流分布。结果:发酵结果显示,利用NaOH和氨水混合补料,控制NH4+浓度在120 mmol/L以下,菌体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12.16%和19.80%。随着NH4+浓度的增加,发酵液中丙酮酸、乳酸及乙酸浓度均略有增加,细胞质粒稳定性下降。控制NH4+浓度在120 mmol/L以下,E.coli TRTH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7.31%,PP途径的代谢流量增加7.14%,TCA循环的代谢流量降低22.04%。结论:高浓度的NH4+导致菌体生长提前结束,耗糖速率降低,产酸受阻,控制NH4+浓度在120 mmol/L以下,解除了NH4+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,实现了高密度发酵培养的目的。
Objective：To study the effects of NH4＋ on biosynthesis of L-tryptophan.Methods：Fed-batch fermentation of E.coli TRTH with addition of ammonium sulfate carried out in 30-Liter fermentor and biomass,yield of L-tryptophan,consumption rate of glucose,plasmid stability were measured.The concentration of acetate,NH4＋,lactate and pyruvate were investigated by BioProfile 300A Nova and high performance liquid chromatography（HPLC）.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 TRTH were determined with the linear program planted in MATLAB software.Results：During the fed-batch culture,E.coli TRTH was able to maintain higher growth rate at exponential phase,finally,the biomass and the yield of L-tryptophan were increased by 51.07% and 46.54% respectively by controlling the concentration of NH4＋ less than 120 mmol/L.As the increase of the concentration of NH4＋,the concentration of pyruvate,lactate and acetate were slightly increased,but cell plasmid stability was decreased.Data indicated that EMP pathway and TCA cycle were decreased by 7.31% and 22.04% respectively,PP pathway was increased by 7.14% compared with addition 5g/L of ammonium sulfate at 14h during the fed-batch culture.Conclusion：High concentrations of NH4＋ terminated the cell growth,decreased the consumption rate of glucose and inhibited the L-tryptophan production.Controlled the concentration of NH4＋ less than 120 mmol/L,the biomass and the yield of L-tryptophan were significantly increased,which accessed high cell density cultivation.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2011年第3期55-60,共6页
China Biotechnology
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)资助项目
