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。
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.[JP+1]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.
作者
章无怨
梁泽宇
李敏
范舜骅
郭淑元
马晓焉
孙丽超
霍毅欣
ZHANG Wuyuan;LIANG Zeyu;LI Min;FAN Shunhua;GUO Shuyuan;MA Xiaoyan;SUN Lichao;HUO Yixin(Key Laboratory of Molecular Medicine and Biotherapy,School of Life Science,Beijing Institute of Technology,Beijing 100081,China;Tangshan Research Institute,Beijing Institute of Technology,Tangshan 063000,China;Zhengzhou Research Institute,Beijing Institute of Technology,Zhengzhou 450000,China)
出处
《生物学杂志》
北大核心
2025年第5期31-39,共9页
Journal of Biology
基金
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)。
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
tryptophan overproducers
tube fermentation
chassis screening
enzyme screening
tryptophan-colorimetric method
