Blank Lars M, Ebert Birgitta E, Bühler Bruno, Schmid Andreas
Laboratory of Chemical Biotechnology, TU Dortmund, D-44221 Dortmund, Germany.
Biotechnol Bioeng. 2008 Aug 15;100(6):1050-65. doi: 10.1002/bit.21837.
Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.
全细胞氧化还原生物催化依赖于微生物宿主进行氧化还原辅因子再生。在此，我们基于大肠杆菌代谢网络应用通量平衡分析来估计最大NADH再生速率。基于此优化标准，模拟显示在高葡萄糖分解代谢速率下磷酸戊糖途径被专门使用，这种通量分布通常在野生型细胞中未发现。在计算机模拟中，基因扰动表明NADH产量和形成速率强烈依赖于基础代谢网络结构。重组中心碳代谢突变体的实测环氧化活性与葡萄糖摄取速率之间的线性相关性以及实测活性与模拟NADH再生速率之间的线性相关性意味着细胞内NADH短缺。对计算预测的NADH再生和实验环氧化速率的定量比较表明，可实现的生物催化活性由代谢和酶学限制决定，包括非最优通量分布、高维持能量需求、能量泄漏、副产物形成和解偶联。在生物转化过程的细胞优化背景下讨论了这些结果，并且可能指导将微生物细胞设计为氧化还原生物催化剂的先验设计。
