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Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

ABSTRACT: Methanol is a liquid with high energy storage capacity that holds promise as an alternative substrate to replace sugars in the biotechnology industry. It can be produced from CO₂ or methane and its use does not compete with food and animal feed production. However, there are currently only limited biotechnological options for the valorization of methanol, which hinders its widespread adoption. Here, we report the conversion of the industrial platform organism Escherichia coli into a synthetic methylotroph that assimilates methanol via the energy efficient ribulose monophosphate cycle. Methylotrophy is achieved after evolution of a methanol-dependent E. coli strain over 250 generations in continuous chemostat culture. We demonstrate growth on methanol and biomass formation exclusively from the one-carbon source by ¹³C isotopic tracer analysis. In line with computational modeling, the methylotrophic E. coli strain optimizes methanol oxidation by upregulation of an improved methanol dehydrogenase, increasing ribulose monophosphate cycle activity, channeling carbon flux through the Entner-Doudoroff pathway and downregulating tricarboxylic acid cycle enzymes. En route towards sustainable bioproduction processes, our work lays the foundation for the efficient utilization of methanol as the dominant carbon and energy resource.

SUBMITTER: Keller P

PROVIDER: S-EPMC9448777 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

Keller Philipp P Reiter Michael A MA Kiefer Patrick P Gassler Thomas T Hemmerle Lucas L Christen Philipp P Noor Elad E Vorholt Julia A JA

Nature communications 20220906 1

Methanol is a liquid with high energy storage capacity that holds promise as an alternative substrate to replace sugars in the biotechnology industry. It can be produced from CO<sub>2</sub> or methane and its use does not compete with food and animal feed production. However, there are currently only limited biotechnological options for the valorization of methanol, which hinders its widespread adoption. Here, we report the conversion of the industrial platform organism Escherichia coli into a s ...[more]

PMID: 36068201

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Project description:The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol. The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol.

Project description:There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Dataset Information

Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

Publications

Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

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