7-氯-色氨酸(7-Cl-Trp)具有重要生物活性,被广泛用于制药、化学和农业领域。前期研究中,通过在E.coli中同时表达L-氨基酸脱氨酶(L-amino acid deaminase,L-AAD)和卤化酶构建全细胞催化剂,合成7-Cl-Trp,并通过L-AAD和卤化酶表达水平调节和加强辅酶供应提高7-Cl-Trp合成量。但是7-Cl-Trp合成仍受限于底物色氨酸降解副反应及卤化酶催化效率低。该研究中,首先通过敲除色氨酸降解基因tnaA阻断副反应提高色氨酸利用率,7-Cl-Trp合成量提高了15.8%;然后通过结合定向进化结合定点突变对卤化酶进行分子改造,三点突变体F104Y/T278C/N346E全细胞催化7-Cl-Trp合成量提高了54.9%,动力学分析表明,三点突变体的Kcat增加了50.0%,Km减少了9.2%,整体催化效率提高了65.2%;最后,对全细胞催化条件进行优化,在最优催化条件下(40 ℃,pH 7.4,400 mg/L色氨酸,10 μmol/L黄素腺嘌呤二核苷酸)下,12 h后,7-Cl-Trp合成量为105.7 mg/L。该研究为全细胞催化合成卤化物提供了新思路。
7-chlorine-tryptophan (7-Cl-Trp) has important biological activities and is widely used in pharmaceutical, chemical and agricultural fields.In the previous study, a whole-cell catalyst was constructed to synthesize 7-Cl-Trp by expressing L-amino acid deaminase (L-AAD) and halogenase in E.coli.The amount of 7-Cl-Trp synthesis was increased by regulation of expression levels of L-AAD and halogenase and strengthening the coenzyme supply.However, 7-Cl-Trp synthesis is still limited by the substrate tryptophan degradation side reaction and the low catalytic efficiency of halogenase.In this study, tryptophan utilization was first improved by knocking down the gene, tnaA, which increased the 7-Cl-Trp synthesis by 15.8%.Then, by combination with directed evolution and site-directed mutagenesis, whole-cell-catalyzed amount of 7-Cl-Trp synthesis of the triple mutant was increased by 54.9%.Kinetic analysis showed that the three-point mutant increased Kcat by 50.0%, Km by 9.2%, and the overall catalytic efficiency by 65.2%.Finally, the whole-cell catalytic conditions were optimized to produce 105.7 mg/L of 7-Cl-Trp after 12 h (40 ℃, pH 7.4, 400 mg/L tryptophan, 10 μmol/L FAD).This study provides a new idea for whole-cell catalytic synthesis of halides.
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