The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
J Biotechnol. 2022 Jan 10;343:7-14. doi: 10.1016/j.jbiotec.2021.11.002. Epub 2021 Nov 9.
Transaminases catalyze the transfer of an amino group from a donor to a keto group of an acceptor substrate and are applicable to the asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). Here, the important residue sites (C390, I22, V52, R141, Y138 and D239) of transaminase from Salmonella enterica (SeTA) were modified at the adjacency of the substrate-binding pocket to improve the enzyme activity. Among the constructed mutant library, the SeTA-Y138F mutant displayed higher activity than the wild-type enzyme. Compared to the wild-type, SeTA-Y138F showed improved catalytic efficiency with a 4.36-fold increase. The K and k of SeTA -Y138F toward 4-(hydroxy(methyl) phosphoryl)-2-oxobutanoic acid (PPO) were 26.39 mM and 34.28 s, respectively. Subsequently, the three-enzyme co-expression system of E. coli BL21 (DE3)/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH was developed by combining a alanine dehydrogenase (AlaDH) to recycle the byproduct of amino donor, a glucose dehydrogenase (BsGDH) for cofactor recycling. Under the optimized conditions, an excellent L-PPT yield of 90.8% was achieved by the whole-cell biotransformation with 500 mM PPO. It exhibited the tri-enzymatic coupling system was potential for effective production of target L-PPT.
转氨酶催化供体的氨基基团向受体底物的酮基团的转移，适用于除草剂 L-草丁膦（L-PPT）的不对称合成。在这里，对沙门氏菌（Salmonella enterica）转氨酶（SeTA）的重要残基位点（C390、I22、V52、R141、Y138 和 D239）在紧邻底物结合口袋的位置进行修饰，以提高酶活性。在构建的突变文库中，SeTA-Y138F 突变体的活性高于野生型酶。与野生型相比，SeTA-Y138F 表现出更高的催化效率，比活提高了 4.36 倍。SeTA-Y138F 对 4-（羟甲基）膦酰基-2-氧代丁酸（PPO）的 K 和 k 值分别为 26.39 mM 和 34.28 s。随后，通过组合丙氨酸脱氢酶（AlaDH）来回收氨基供体的副产物，葡萄糖脱氢酶（BsGDH）来回收辅因子，构建了大肠杆菌 BL21（DE3）/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH 的三酶共表达系统。在优化条件下，通过 500 mM PPO 的全细胞生物转化，获得了 90.8%的优异 L-PPT 产率。该结果表明，该三酶偶联系统有望有效生产目标 L-PPT。
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