Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.

合成甲基营养素旨在设计平台宿主（如大肠杆菌）中甲烷和甲醇的利用途径，以进行天然气和沼气的工业生物处理。尽管最近尝试制造合成甲醇营养缺陷剂的尝试已经成功，但这些研究集中在稀缺且昂贵的共底物上。在这里，我们通过缺失6-磷酸葡萄糖异构酶（pgi），磷酸葡萄糖酸脱水酶（edd）和核糖5-磷酸异构酶（rpiAB），针对大肠杆菌在葡萄糖上的甲醇依赖性生长工程化了大肠杆菌。）。由于亲本菌株在基本培养基中对葡萄糖没有表现出甲醇依赖性生长，因此我们首先通过氨基酸添加实现了甲醇依赖性生长，并使用该培养基在葡萄糖基本培养基中进化出了甲醇依赖性生长菌株。进化的菌株在含甲醇的葡萄糖最小培养基中表现出最大的生长速率为0.15 h ^-1，这与其他合成甲醇营养缺陷型的生长速率相当。全基因组测序和¹³ C-代谢通量分析揭示了进化菌株中的致病突变。磷酸转移酶系统酶I基因（ptsI）导致降低的葡萄糖吸收速率以维持底物利用率的一对一摩尔比。删除e14噬菌体DNA区域导致异柠檬酸脱氢酶（icd）基因发生两个非同义突变，从而降低了TCA循环碳通量以维持内部氧化还原状态。在高细胞密度葡萄糖补料分批发酵，甲醇依赖性丙酮生产导致丙酮的22％的平均碳标签从¹³ C-甲醇，这远远超过以前的最好的（2.4％），该发现的甲基大肠杆菌Δ PGI。这项研究满足了为工程合成甲醇营养缺陷物质确定合适的共底物的需要，并为下一步工业化一碳生物处理提供了基础。