Rational Design of Tryptophan Hydroxylase-2 for Improving 5-Hydroxytryptophan Production in Escherichia coli
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 con-taining 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 un-changed,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 produc-tion 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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