NH<sub>4</sub><sup>+</sup>对L-色氨酸发酵的影响

中国生物工程杂志

2011, Vol. 31

Issue (03): 55-60

研究报告

NH₄⁺对L-色氨酸发酵的影响

黄静¹, 史建明¹, 霍文婷¹, 徐庆阳¹, 谢希贤¹, 温廷益², 陈宁¹

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

The Effects of NH₄⁺ on L-tryptophan Fermentation

HUANG Jing¹, SHI Jian-ming¹, HUO Wen-ting¹, XU Qing-yang¹, XIE Xi-xian¹, WEN Ting-yi², CHEN Ning¹

1. College of Biological Engineering, Tianjin University of Science & Technology, Key Laboratory of Industrial Microbiology, Tianjin 300457, China;
2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

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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。

关键词： 大肠杆菌; NH₄⁺; L-色氨酸; 高密度发酵

Abstract:

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.

Key words: E. coli NH₄⁺ L-Tryptophan High cell density cultivation

收稿日期: 2010-11-24 出版日期: 2011-04-01

ZTFLH:

Q815

基金资助:

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

通讯作者: 陈宁 E-mail: ningch@tust.edu.cn

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引用本文:

黄静, 史建明, 霍文婷, 徐庆阳, 谢希贤, 温廷益, 陈宁. NH₄⁺对L-色氨酸发酵的影响[J]. 中国生物工程杂志, 2011, 31(03): 55-60.

HUANG Jing, SHI Jian-ming, HUO Wen-ting, XU Qing-yang, XIE Xi-xian, WEN Ting-yi, CHEN Ning. The Effects of NH₄⁺ on L-tryptophan Fermentation. China Biotechnology, 2011, 31(03): 55-60.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I03/55

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