Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol

Chen, Frederic Y.-H.; Jung, Hsin-Wei; Tsuei, Chao-Yin; Liao, James C.

doi:10.1016/j.cell.2020.07.010

Cited by 181 publications

(180 citation statements)

References 40 publications

(52 reference statements)

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“…Autonomous synthetic methylotrophy, wherein growth on methane or methanol does not require additional carbon sources, has been difficult to realize. Only one study to date has reported the successful construction of a true E. coli methylotroph (Chen et al, 2020). In order to achieve this feat, the authors relied on a combination of rational engineering and adaptive laboratory evolution (ALE).…”

Section: Introductionmentioning

confidence: 99%

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

et al. 2021

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“…The ability for EfgA to provide an immediate benefit in dealing with formaldehyde stress may also have more immediate biotechnological benefits. Given that formaldehyde toxicity was also a key challenge in the engineering and evolution of an E. coli strain that can grow on methanol as a sole carbon source [128], this raises the possibility that introducing EfgA would increase the cells’ ability to grow while producing formaldehyde as a high flux intermediate. We perhaps should not be surprised that metabolic pathways that generate toxic intermediates need molecular systems to sense their accumulation and mount responses that either eliminate the toxin, increase the ability to repair such damage, or help the cell avoid making the molecules that the toxin damages in the first place.…”

Section: Discussionmentioning

confidence: 99%

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

Bazurto

Nayak

Ticak

et al. 2020

Preprint

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Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that inhibits translation in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved and may include translational machinery. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and modulates translational activity.

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“…The ability for EfgA to provide an immediate benefit in dealing with formaldehyde stress may also have more immediate biotechnological benefits. Given that formaldehyde toxicity was also a key challenge in the engineering and evolution of an E. coli strain that can grow on methanol as a sole carbon source [132], this raises the possibility that introducing EfgA would increase the cells' ability to grow while producing formaldehyde as a high-flux intermediate. We perhaps should not be surprised that metabolic pathways that generate toxic intermediates need molecular systems to sense their accumulation and mount responses that either eliminate the toxin, increase the ability to repair such damage, or help the cell avoid making the molecules that the toxin damages in the first place.…”

Section: Plos Biologymentioning

confidence: 99%

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde

et al. 2021

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Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

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Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol

Cited by 181 publications

References 40 publications

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde

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