文章摘要
色氨酸羟化酶2理性设计提高大肠埃希菌5-羟基色氨酸产量
Rational Design of Tryptophan Hydroxylase-2 for Improving 5-Hydroxytryptophan Production in Escherichia coli
  
DOI：doi:10.3969/j.issn.1005-7021.2024.04.003
中文关键词: 色氨酸羟化酶2  理性设计  定点突变  5-羟基色氨酸  细胞工厂
英文关键词: tryptophan hydroxylase-2  rational design  site-directed mutagenesis  5-HTP (5-hydroxytryptophan)  cell factory
基金项目:河北省重点研发计划项目(22372403D)
作者单位
李春辉 河北大学生命科学学院 河北省微生物多样性研究与应用实验室，河北 保定 071002 
杨志彬 2.河北维达康生物科技有限公司，河北 保定 072152；3.保定保利瑞合生物科技有限公司，河北 保定 072152 
崔金旺 2.河北维达康生物科技有限公司，河北 保定 072152；3.保定保利瑞合生物科技有限公司，河北 保定 072152 
郝晋博 河北大学生命科学学院 河北省微生物多样性研究与应用实验室，河北 保定 071002 
田俊波 2.河北维达康生物科技有限公司，河北 保定 072152；3.保定保利瑞合生物科技有限公司，河北 保定 072152 
胡江林 河北维达康生物科技有限公司，河北 保定 072152 
吕志堂 河北大学生命科学学院 河北省微生物多样性研究与应用实验室，河北 保定 071002 
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中文摘要:
      色氨酸羟化酶(TPH)可催化色氨酸羟化为5-羟基色氨酸(5-HTP)，是5-HTP生物合成过程中的关键酶。为了提高大肠埃希菌细胞工厂合成5-HTP的效率，通过酶的理性设计和定点突变技术构建了人色氨酸羟化酶2（TPH2）的系列突变体，并在高产L-色氨酸且含有TPH辅酶四氢生物蝶呤(BH4）合成与再生模块的大肠埃希菌中表达。发现适当降低酶和色氨酸、酶和BH4间结合的氢键数目有助于提高5-HTP产量；酶和底物色氨酸结合情况不变，辅酶BH4与酶间氢键数目越多，5-HTP产量越低。5-HTP产量最高的细胞工厂是由突变酶V195A/V197I构建的，5-HTP产量较野生酶构建的细胞工厂产量提高54%，1 L补料摇床发酵48 h，5-HTP产量达16.17 g/L。理性设计是提高TPH2酶活，突破5-HTP合成限速步骤的有效途径, TPH2和底物色氨酸、辅酶BH4间氢键连接太多或太少都不利于其催化活性的发挥。
英文摘要:
      Tryptophan hydroxylase (TPH) can catalyze the hydroxylation of tryptophan to 5-hydroxytryptophan (5-HTP), which is a key enzyme in the biosynthesis of 5-HTP. In order to improve the efficiency of Escherichia coli cell factory synthesis of 5-HTP. A series of mutant of human tryptophan hydroxylase-2 (TPH2) was constructed by rational enzyme design and site-directed mutagenesis technique, and expressed in E. coli with high L-tryptophan yield and containing TPH coenzyme tetrahydrobiopterin (BH4) synthesis and the expression in E. coli regeneration module. It was found that duly reducing the number of hydrogen bonds between enzyme and tryptophan, enzyme and BH4 was conducive to increase the yield of 5-HTP. Keep the binding condition of the enzyme to the substrate tryptophan remains unchanged, the higher the number of hydrogen bonds between coenzyme BH4 and enzyme, the lower the yield of 5-HTP to a certain extent. The cell factory with the highest production of 5-HTP was constructed by mutant enzyme V195A/V197I, and the production of 5-HTP was 54% higher than that of wild enzyme constructed cell factory, and the production of 5-HTP reached to 16.17 g/L 48 h after fermentation in 1 L feeding shaker. Rational design is an effective way to improve TPH2 enzyme activity and break through the limiting step of 5-HTP synthesis. Too much or too little hydrogen bond linkage between TPH2 with tryptophan and coenzyme BH4 is not conducive to its catalytic activity.
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