(188q) Engineering a Synthetic Methanol Utilization Pathway in Escherichia coli for Examining Metabolic Bottlenecks Associated with Developing Synthetic Methylotrophs

Methylotrophy is the ability of microorganisms to utilize reduced one carbon compounds such as methane and methanol for growth and energy generation. The recent discovery of abundant natural gas reserves has prompted considerable interest in utilizing these compounds as feedstocks in industrial fermentations for production of liquid fuels and chemicals. Increased product yields are expected from these compounds since they are more reduced than lignocellulosic sugars. Native methylotrophs are not ideal industrial host organisms since many are strict aerobes, produce few metabolites, and lack extensive genetic engineering tools. Therefore, the development of synthetic methylotrophs, or non-methylotrophic platform host organisms engineered to utilize reduced one carbon compounds, is of considerable interest. Here, the development of a synthetic Escherichia coli methylotroph engineered for methanol utilization is described.

Engineering a synthetic methanol utilization pathway in E. coli involves incorporation of two synthetic modules: 1) a methanol oxidation module, and 2) a formaldehyde assimilation module. Establishment of these two modules via expression of heterologous methanol dehydrogenase and ribulose monophosphate pathway enzymes successfully imparts E. coli with the ability to utilize methanol for growth and energy generation. This novel ability is evidenced by probing central carbon metabolism for labeled signatures resulting from assimilation of a ¹³C-methanol tracer.

To further improve the synthetic methanol utilization pathway, additional genetic engineering was performed to establish a reliance on methanol utilization for growth. Termed ‘methanol-dependent’ growth, this strategy allows the complete utilization of lignocellulosic sugars only in the presence of methanol. In the absence of methanol, growth does not occur due to incomplete metabolic pathways. Via fine-tuning heterologous gene expression and additional genetic perturbations, further improvements of methanol-dependent growth phenotypes were achieved.

Finally, the application of these synthetic, methanol-dependent E. coli strains will be detailed. Due to the reliance on methanol utilization for growth, screening gene or metagenomic libraries, transcriptionally optimized variants, and mutagenized or evolved strains for improved methylotrophic phenotypes becomes readily accessible. After each round of the design-build-test cycle concludes, identification of potential metabolic bottlenecks and subsequent alleviation of them in the following round is performed to continually improve the methylotrophic phenotypes and provide insight into establishing synthetic methanol utilization pathways in non-methylotrophic platform host organisms.

This work was supported by the US DOE ARPA-E agency through contract no. DE-AR0000432.