We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

中文翻译：

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通过在大肠杆菌中构建的反向甲基营养而产生甲醇。

我们构建了一条反向的甲基营养途径，该途径可以从果糖6磷酸酯（F6P）中产生甲醇，甲醇是生产有用化合物的有前途的原料，可以通过合成生物学方法通过生物量衍生的糖（包括葡萄糖）的分解代谢来提供甲醇。使用大肠杆菌作为表达宿主，我们异源表达了编码甲基营养细菌甲醇利用酶的基因，即甲醇芽孢杆菌S1的NAD +依赖性甲醇脱氢酶（MDH）和3-己糖-6-磷酸合酶和6的人工融合酶。来自胃分枝杆菌MB19（HPS-PHI）的-磷酸-3-己糖异构酶。我们证实，这些酶可以催化甲醇氧化和甲醛固定的逆反应。工程化的E. 共表达MDH和HPS-PHI基因的大肠杆菌菌株在静止细胞反应中不仅从F6P而且从葡萄糖产生甲醇。我们成功地将反向甲基营养授予了大肠杆菌，我们的结果为从生物质衍生的糖化合物生产生物甲醇提供了概念证明。