Department of Chemical and Biomolecular Engineering, University of California, 420 Westwood Plaza, Los Angeles, CA 90095, United States.
Department of Chemical and Biomolecular Engineering, University of California, 420 Westwood Plaza, Los Angeles, CA 90095, United States; Institute of Biological Chemistry, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan.
Metab Eng. 2018 Sep;49:257-266. doi: 10.1016/j.ymben.2018.08.010. Epub 2018 Aug 30.
Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy". Our best strains were able to utilize methanol for growth at a rate of 0.17 ± 0.006 (h) with methanol and xylose co-assimilation at a molar ratio of approximately 1:1. Genome sequencing and reversion of mutations indicated that mutations on genes encoding for adenylate cyclase (cyaA) and the formaldehyde detoxification operon (frmRAB) were necessary for the growth phenotype. The methanol auxotrophic strain was further engineered to produce ethanol or 1-butanol to final titers of 4.6 g/L and 2.0 g/L, respectively. C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.
甲醇作为一种有潜力的化学物质生物生产的底物，因为天然气和沼气衍生的甲烷丰富。为了将甲醇作为唯一的碳源加以利用，我们对大肠杆菌菌株进行了工程改造，使其能够将甲醇利用与五碳（C5）糖的生长结合起来。通过敲除戊糖磷酸途径中用于戊糖利用的必需基因，并表达核酮糖-1,5-二磷酸（RuMP）途径中的异源酶，我们构建了一种不能在木糖或核糖基础培养基上生长的菌株，除非利用甲醇，从而产生了一种被称为“合成甲醇营养缺陷型”的表型。我们最好的菌株能够以 0.17±0.006（h）的速度利用甲醇进行生长，并且可以以大约 1:1 的摩尔比同时利用甲醇和木糖进行共吸收。基因组测序和突变回复表明，编码腺苷酸环化酶（cyaA）和甲醛解毒操纵子（frmRAB）的基因上的突变对于生长表型是必需的。进一步对甲醇营养缺陷型菌株进行工程改造，以分别达到 4.6 g/L 和 2.0 g/L 的最终乙醇和 1-丁醇产量。C 追踪表明，分别有 43%和 71%的乙醇和 1-丁醇产物的标记碳来自甲醇。
