Abstract
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.
Highlights
There is great interest in utilizing methane and methanol from natural gas reserves as industrial feedstocks (Haynes and Gonzalez, 2014)
E. coli formate by S-hydroxymethylglutathione dehydrogenase (frmA) integration host factor subunit α (ihfA) + pUD9 and frmA + pUD9 + pCrp were serially passaged in LB medium supplemented with increasing methanol concentrations in a similar manner as before (Supplementary Figure S6)
To assess whether a non-methylotrophic E. coli strain could achieve improved methanol tolerance, we serially passaged E. coli frmA in LB medium supplemented with increasing methanol concentrations in a similar manner as before without chemical mutagenesis
Summary
There is great interest in utilizing methane and methanol from natural gas reserves as industrial feedstocks (Haynes and Gonzalez, 2014). Emphasis has been placed on the development and utilization of synthetic methylotrophic organisms that are engineered to utilize methane and methanol (Whitaker et al, 2015; Bennett et al, 2018b). In the last several years, there have been multiple approaches to engineer synthetic methylotrophs, with most work done in E. coli (Price et al, 2016; Bennett et al, 2018a, 2020c; Chen et al, 2018; Gonzalez et al, 2018; Meyer et al, 2018; Woolston et al, 2018a; Zhang et al, 2018; Rohlhill et al, 2020), Corynebacterium glutamicum (Lessmeier et al, 2015; Witthoff et al, 2015; Tuyishime et al, 2018), and Saccharomyces cerevisiae (Dai et al, 2017). The ribulose monophosphate (RuMP) pathway is the most often employed pathway for synthetic methylotrophy This pathway is made up of two enzymes, Hps (hexulose phosphate synthase) and Phi (phosphohexulose isomerase). Together with Mdh (methanol dehydrogenase), this pathway oxidizes methanol to formaldehyde (via Mdh), which is fixed with ribulose 5phosphate to produce hexulose 6-phosphate (via Hps), which is converted to fructose 6-phosphate (via Phi)
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