生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (5): 31-.doi: 10.3969/j.issn.2095-1736.2025.05.031

• 合成生物学专题 • 上一篇    下一篇

基于关键酶和底盘菌株筛选的色氨酸高产菌株构建

章无怨1， 梁泽宇1， 李　敏1， 范舜骅1， 郭淑元1,2， 马晓焉1,2， 孙丽超1,2,3， 霍毅欣1,2   

  1. 1. 北京理工大学 分子医学与生物诊疗重点实验室， 北京 100081；
    2. 北京理工大学唐山研究院， 唐山 063000； 3. 北京理工大学 郑州智能科技研究院， 郑州 450000
  • 出版日期:2025-10-18 发布日期:2025-10-14
  • 通讯作者: 孙丽超，博士，副研究员，研究方向为合成生物学、代谢工程、基因编辑、碳中和与酶工程，E-mail：sunlichao@bit.edu.cn；霍毅欣，博士，教授，研究方向为合成生物学、碳中和、基因编辑、代谢工程、酶工程和生物信息学，E-mail：huoyixin@bit.edu.cn；孙丽超和霍毅欣为共同通信作者
  • 作者简介:章无怨，硕士，研究方向为大肠杆菌代谢工程，E-mail：1793808978@qq.com
  • 基金资助:
    国家重点研发计划项目（2019YFA0906500）； 河北省自然科学基金项目（C2023105022）； 北京市自然科学基金项目（2222026）； 国家自然科学基金项目（32370095）

Construction of tryptophan overproducers via key enzyme and chassis strain selection

ZHANG Wuyuan1, LIANG Zeyu1, LI Min1, FAN Shunhua1, GUO Shuyuan1,2,MA Xiaoyan1,2, SUN Lichao1,2,3, HUO Yixin1,2   

  1. 1. Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology,
    Beijing 100081, China; 2. Tangshan Research Institute, Beijing Institute of Technology, Tangshan 063000, China;
    3. Zhengzhou Research Institute, Beijing Institute of Technology, Zhengzhou 450000, China
  • Online:2025-10-18 Published:2025-10-14

摘要： 为了快速构建色氨酸高产菌株，分别对色氨酸生产途径关键酶及底盘菌株进行高通量筛选。通过优化摇菌管发酵体系，使其产量与摇瓶发酵产量保持了较高一致性。利用色氨酸与盐酸萘乙二胺的显色反应，建立了适配摇菌管发酵水平的色氨酸定量检测方法，检测范围为50~1000 mg/L。利用该方法，从转酮醇酶TktA的随机突变库中分离出优势突变体TktA2。与野生型TktA相比，TktA2具有更紧凑的二聚体结构和更大的底物结合口袋，这些结构变化有助于提高酶的稳定性和底物进出速度。同时，从MG1655基因组简化菌株库中分离出优势底盘菌株T51，在T51中过表达TktA2后，获得了色氨酸高产菌株T51A2ΔtrpRΔtnaB，3 L发酵罐中色氨酸产量达31.37 g/L，相较出发菌株提高80.49%，糖酸转化率达19.01%。利用色氨酸快检方法，在摇菌管水平实现了色氨酸合成关键酶和底盘菌株的低成本高效筛选，优势整合后的色氨酸生产菌株在发酵规模放大后仍可复现其高产性能，为色氨酸高产菌株的开发奠定了基础。

关键词: 色氨酸高产菌株, 摇菌管发酵, 底盘筛选, 酶筛选, 色氨酸比色法

Abstract: To rapidly develop tryptophan overproducers, high-throughput screening was performed on key enzyme in the tryptophan biosynthetic pathway and the chassis strains. The tube fermentation system was optimized to maintain high production consistency with flask fermentation. A colorimetric method compatible with the tube fermentation system was established for tryptophan quantification, using the reaction of tryptophan with naphthylethylenediamine dihydrochloride, providing a detection range of 50 mg/L to 1000 mg/L. Using this method, a superior enzyme mutant, TktA2, was successfully identified from a random mutagenesis library of transketolase TktA. Compared to the wild-type TktA, TktA2 exhibited a more compact dimer structure and a larger substrate-binding pocket, which contributed to the enhanced enzyme stability and substrate turnover rate. Additionally, a dominant chassis strain, T51, was isolated from a genome-reduced strain library of MG1655. Overexpression of TktA2 in T51 yielded the tryptophan overproducer, T51A2ΔtrpRΔtnaB, producing 31.37 g/L tryptophan in a 3 L bioreactor, at a sugar-to-acid conversion rate of 19.01% and an 80.49% higher titer than that of the parental strain. This employed a rapid tryptophan detection method for cost-effective and efficient screening of key enzyme and chassis strains at the tube fermentation level. The integrated high-yielding tryptophan producer maintained high-performance even after fermentation scale-up, laying a foundation for the development of tryptophan overproduction strains.

Key words: tryptophan overproducers, tube fermentation, chassis screening, enzyme screening, tryptophan-colorimetric method

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