Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine
Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine
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DOI：
10.1038/73773
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发表时间：
2000-03-01
影响因子：
46.9
通讯作者：
Arnold, FH
中科院分区：
文献类型：
--
作者：
May, O;Nguyen, PT;Arnold, FH
利用定向进化，我们改进了在大肠杆菌中生产 L-蛋氨酸 (L-met) 的乙内酰脲酶工艺。这是通过反转对映选择性并增加全细胞催化剂中关键酶的总活性来实现的。所有已知的乙内酰脲酶对 D-5-(2-甲硫基乙基)乙内酰脲 (D-MTEH) 相对于 L-对映体的选择性会导致中间体的积累并降低 L-氨基酸的生产率。我们使用随机诱变、饱和诱变和筛选来转化来自节杆菌属的 D-选择性乙内酰脲酶。 DSM 9771 转化为 L-选择性酶，并将其总活性提高了五倍。表达进化的 L-乙内酰脲酶、L-N-氨基甲酰化酶和乙内酰脲消旋酶的完整大肠杆菌细胞在不到 2 小时内从 100 mMD,L-MTEH 产生 91 mM L-met。改进的乙内酰脲酶将生产率提高了五倍，底物转化率超过 90%。与野生型途径的细胞相比，不需要的中间体 D-氨基甲酰基-甲硫氨酸的积累减少了四倍。还发现了高度 D-选择性乙内酰脲酶突变体。通过定向进化快速优化对映选择性酶并将其引入多酶途径可能会改进全细胞催化剂，以有效生产手性化合物。
Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.