Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. However, attempts to engineer Escherichia coli to metabolize methanol have achieved limited success. Here, we provide a rigorous systematic analysis of several potential pathway bottlenecks. We show that regeneration of ribulose 5-phosphate in E. coli is insufficient to sustain methanol assimilation, and overcome this by activating the sedoheptulose bisphosphatase variant of the ribulose monophosphate pathway. By leveraging the kinetic isotope effect associated with deuterated methanol as a chemical probe, we further demonstrate that under these conditions overall pathway flux is kinetically limited by methanol dehydrogenase. Finally, we identify NADH as a potent kinetic inhibitor of this enzyme. These results provide direction for future engineering strategies to improve methanol utilization, and underscore the value of chemical biology methodologies in metabolic engineering.

中文翻译：

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甲醛消耗量的增加推动了工程化大肠杆菌中甲醇的吸收。

由于糖价波动，食品与燃料的争论以及最近天然气供应的增加，甲醇已成为生物基经济的有前途的原料。然而，工程改造大肠杆菌以代谢甲醇的尝试取得了有限的成功。在这里，我们对几种潜在的途径瓶颈进行了严格的系统分析。我们表明，大肠杆菌中5-磷酸核糖的再生不足以维持甲醇同化，并通过激活核糖单磷酸途径的七庚二糖双磷酸酶变异体克服了这一问题。通过利用与氘化甲醇相关的动力学同位素效应作为化学探针，我们进一步证明，在这些条件下，总途径通量在动力学上受到甲醇脱氢酶的限制。最后，我们确定NADH是该酶的有效动力学抑制剂。这些结果为提高甲醇利用率的未来工程策略提供了方向，并强调了化学生物学方法在代谢工程中的价值。