Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production

Abstract

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^-1) 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. ¹³C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.

Keywords: 1-butanol; Biofuel; C1 metabolism; Escherichia coli; Ethanol; Metabolic engineering; Methanol; Synthetic biology.