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Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells

机译：通过不生长的大肠杆菌细胞了解和改善NADPH依赖性反应

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We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g epsilon-caprolactone/L(.)h) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2002, 18, 262-268). This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from baker's yeast (Saccharomyces cerevisiae). Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L-. and a final product titer of 15.8 g/L using a biocatalyst: substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 +/- 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway.
机译：我们已经表明，在非生长条件下（Walton，AZ; Stewart，JD），过表达NADPH依赖的环己酮单加氧酶的整个大肠杆菌细胞以高体积生产率（0.79 g epsilon-己内酯/L(.h））进行了模型的拜耶-维利格氧化。生物技术进展，2002，18，262-268）。这比使用相同菌株的生长细胞的反应的时空产率高约20倍。在这里，我们表明环己酮单加氧酶的细胞内稳定性和底物跨细胞膜的运输速率分别是对总体反应持续时间和速率的关键限制。在生物过程条件下直接测量细胞内烟酰胺辅助因子的水平表明，如果鉴定出更合适的酶-底物对，则大肠杆菌细胞可以支持更有效的NADPH依赖性生物转化。这是通过用大肠杆菌菌株的全细胞还原乙酰乙酸乙酯来证明的，该菌株过度表达了来自面包酵母（Saccharomyces cerevisiae）的NADPH依赖性短链脱氢酶。在葡萄糖喂养的非生长条件下，这种还原以2.0 g / L-的时空产率进行。使用生物催化剂：底物比例（g / g）仅为0.37的最终产品滴定度为15.8 g / L。这些值明显高于以前获得的值。此外，化学计量学联系了酮的减少和葡萄糖的消耗（2.3 +/- 0.1），表明在我们的工艺条件下，柠檬酸循环提供了大部分细胞内NADPH。通过代谢工程策略（可增加通过戊糖磷酸途径的碳通量），该信息甚至可用于进一步提高葡萄糖利用效率。

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