Walton Adam Z, Stewart Jon D
Department of Chemistry, 127 Chemistry Research Building, University of Florida, Gainesville, Florida 32611-7200, USA.
Biotechnol Prog. 2004 Mar-Apr;20(2):403-11. doi: 10.1021/bp030044m.
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.h 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.
我们已经表明，过表达NADPH依赖的环己酮单加氧酶的全大肠杆菌细胞在非生长条件下能够进行模型拜耳-维利格氧化反应，具有高体积产率（0.79 gε-己内酯/（L·h））（Walton，A. Z.；Stewart，J. D. Biotechnol. Prog. 2002，18，262 - 268）。这比使用同一菌株生长细胞的反应的时空产率高约20倍。在此，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转运速率分别是对整个反应持续时间和速率的关键限制因素。在生物过程条件下直接测量细胞内烟酰胺辅因子的水平表明，如果鉴定出更合适的酶-底物对，大肠杆菌细胞可以支持更高效的NADPH依赖的生物转化。这通过用过量表达来自酿酒酵母（Saccharomyces cerevisiae）的NADPH依赖的短链脱氢酶的大肠杆菌菌株的全细胞还原乙酰乙酸乙酯得到了证明。在葡萄糖喂养的非生长条件下，使用仅0.37的生物催化剂：底物比（g/g），该还原反应的时空产率为2.0 g/（L·h），最终产物滴度为15.8 g/L。这些值明显高于先前获得的值。此外，将酮还原与葡萄糖消耗联系起来的化学计量比（2.3±0.1）表明，在我们的过程条件下，柠檬酸循环提供了大部分细胞内的NADPH。这些信息可用于通过增加通过磷酸戊糖途径的碳通量的代谢工程策略进一步提高葡萄糖利用效率。
