Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter P_frm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under P_frm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of ¹³C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo ¹³C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.

大肠杆菌是构建能够利用非天然底物甲醇作为碳源和能源的合成甲基营养菌的理想选择。当前所有基于大肠杆菌的合成甲基营养生物都需要共底物。由于缺乏对生物合成途径的天然调节控制，它们显示出可变水平的甲醇-碳掺入，因为大肠杆菌尽管将其分解代谢，却无法将甲醇识别为适当的底物。在这里，使用大肠杆菌甲醛诱导型启动子P _frm，我们响应于甲醇氧化产生的甲醛，实现了选定的戊糖磷酸酯基因的动态表达控制。P _frm基因对照在甲醇上生长期间表现出8到30倍的转录上调。甲醛诱导的甲醇芽孢杆菌rpe和tkt基因的游离表达涉及甲醛固定所需的核糖5-磷酸的再生，从而显着改善了甲醇同化成细胞内代谢产物的过程，包括将¹³ C-甲醇转化为谷氨酸的2倍增加。通过删除大肠杆菌NAD依赖性苹果酸脱氢酶基因maldh，使用简单的氧化还原扰动策略，我们证明了在低浓度酵母提取物存在下生长在甲醇上的细胞的生物量形成提高了5倍。通过适应性实验室进化以及异源rpe和tkt表达，可以进一步提高甲醇利用率。短期体内 ¹³ C-甲醇标记测定法用于确定Δmaldh菌株的甲醇同化活性，并证明细胞内代谢产物的标记明显更高，其中rpe /的甘氨酸标记增加了6倍和1.8倍。tkt和进化株。甲醛控制的戊糖磷酸途径表达和氧化还原扰动与maldh敲除相结合，极大地改善了甲醇和甲醇碳掺入细胞内代谢产物的生长效益。