The intracellular NADPH insufficient supply is the main bottleneck to the synthesis of chiral alcohols by asymmetric reduction with whole-cell catalysis. Herein, we provide a novel strategy to strengthen intracellular NADPH supply through introducing an NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (gapB from Bacillus subtilis 168) into the Embden-Meyerhof pathway and a NAD kinase (yfjB from E. coli MG1655) to further enhance the NADP(H) pool. A recombinant E. coli (E. coli BL21 (DE3)/pETDuet-1-gapB-yueD&pET28a-yfjB) was constructed to co-express gapB and yfjB with a carbonyl reductase gene yueD together. The result showed that the intracellular NADPH amount increased by 134.4% with the strategy. To the model reaction (asymmetric reduction of acetophenone to S-phenyl ethanol), the yield was 3.7-fold with this strategy compared to the control. This provides a technological route for strengthening the intracellular NADPH supply in E. coli for biocatalysis and biosynthesis.

中文翻译：

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通过修饰EMP和引入NAD激酶增强细胞内NADPH供应促进重组大肠杆菌对前手性酮的不对称还原

细胞内NADPH供应不足是全细胞催化不对称还原合成手性醇的主要瓶颈。在此，我们提供了一种新策略，通过将 NADP+ 依赖性甘油醛 3-磷酸脱氢酶（来自枯草芽孢杆菌 168 的 gapB）引入 Embden-Meyerhof 途径和 NAD 激酶（来自大肠杆菌MG1655 的 yfjB）以进一步加强细胞内 NADPH 供应。增强 NADP(H) 池。构建重组大肠杆菌（大肠杆菌BL21（DE3）/pETDuet-1-gapB-yueD&pET28a-yfjB）以将gapB和yfjB与羰基还原酶基因yueD一起共表达。结果表明，该策略使细胞内NADPH量增加了134.4%。对于模型反应（苯乙酮不对称还原为 S-苯基乙醇），产率为 3。与对照相比，此策略的 7 倍。这为加强大肠杆菌中的细胞内 NADPH 供应提供了一条技术路线，用于生物催化和生物合成。