One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modeling, we designed a new route that “mixed and matched” two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput ¹³C-labeling experiments. The highest level of incorporation, 22% of labeled methanol carbon into the multi-carbon compound PEP, was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.

中文翻译：

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混合和匹配甲基营养酶以在大肠杆菌中设计新的甲醇利用途径

一碳（C1）化合物（例如甲醇）作为微生物生物过程的替代低成本和非食品原料，最近受到关注。因此，目前大量的研究工作集中在通过将甲醇同化途径转移到已建立的细菌生产宿主中来生成合成甲基营养生物。在这项研究中，我们使用了干法和湿法的迭代组合来设计，实现和优化最常见底盘大肠杆菌中的这种代谢特性。通过计算机模拟，我们设计了一种新的途径，可以“混合和匹配”两种甲基营养酶：一种来自细菌的细菌甲醇脱氢酶（Mdh）和一种二羟基丙酮合酶（Das）。确定引入大肠杆菌的酶的最佳组合，我们建立了266个途径变体的文库，其中包含Mdh和Das同源物的不同组合，并使用高通量¹³ C标记实验对其进行了筛选。使用由gerneri产的Mdh和密码子优化版本的P. angusta组成的变体，可获得最高水平的掺入，即多碳化合物PEP中22％的标记甲醇碳。达斯 最后，通过使用组学和建模方法确定工程策略性代谢目标，可以进一步改善这种新的合成途径的活性。最终的合成菌株在细胞内代谢产物和蛋白原氨基酸中的甲醇同化率比起始菌株高1.5至5.9倍。拓宽甲醇同化途径的范围是在大肠杆菌中向合成甲基营养迈进的一步。