Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA.
Metab Eng. 2020 Jul;60:45-55. doi: 10.1016/j.ymben.2020.03.003. Epub 2020 Mar 13.
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 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and C-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 C-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.
人工合甲基营养旨在通过工程改造大肠杆菌等平台宿主中的甲烷和甲醇利用途径，用于天然气和沼气的工业生物加工。虽然最近尝试构建人工甲醇营养缺陷型的尝试取得了成功，但这些研究集中在稀缺和昂贵的共底物上。在这里，我们通过删除葡萄糖 6-磷酸异构酶（pgi）、磷酸葡萄糖酸脱水酶（edd）和核酮糖 5-磷酸异构酶（rpiAB），使大肠杆菌能够在葡萄糖这种丰富且廉价的共底物上依赖甲醇生长。由于亲本菌株在最小培养基中不能以葡萄糖为唯一碳源生长，我们首先通过添加氨基酸实现了甲醇依赖型生长，并使用这种培养基来进化该菌株以使其能够在葡萄糖最小培养基中依赖甲醇生长。进化后的菌株在含甲醇的葡萄糖最小培养基中的最大生长速率为 0.15 h，与其他人工甲醇营养缺陷型相当。全基因组测序和 C 代谢通量分析揭示了进化菌株中的致突变。磷酸转移酶系统酶 I 基因（ptsI）的突变导致葡萄糖摄取率降低，以维持底物利用的一比一摩尔比。e14 噬菌体 DNA 区的缺失导致异柠檬酸脱氢酶（icd）基因中的两个非同义突变，从而降低三羧酸循环的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中，甲醇依赖型丙酮生产导致丙酮中 C-甲醇的平均碳标记为 22%，远远超过之前用甲基营养型大肠杆菌 Δpgi 获得的最佳值（2.4%）。这项研究解决了需要为构建人工甲醇营养缺陷型确定合适的共底物的问题，并为朝着工业一碳生物加工的下一步提供了基础。
