Abstract
Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically coupled to the metabolization of gluconate and the designed strain was subjected to laboratory evolution experiments. Evolved strains incorporate up to 24% methanol into core metabolites under a co-consumption regime and utilize methanol at rates comparable to natural methylotrophs. Genome sequencing reveals mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.
Highlights
Methanol represents an attractive substrate for biotechnological applications
Work in the last 50 years has revealed several conserved pathways, including one-carbon dissimilation pathways consisting of nicotinamide adenine dinucleotide (NAD)- or pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases, as well as formaldehyde oxidation pathways that depend on cofactors such as tetrahydromethanopterin or glutathione, and assimilation pathways, i.e., the ribulose monophosphate pathway, the serine cycle, and the Calvin cycle[10, 11]
We used the stoichiometric genome scale model of E. coli iAF126036 into which reactions catalyzed by methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps), and 6-phospho-3-hexuloisomerase (Phi) were integrated
Summary
Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism. We use theoretical considerations as a starting point to force the heterologous host, here E. coli, to utilize methanol as an essential substrate via a synthetic pathway and by coupling co-consumption of methanol to growth. This strategy allows us to harness the power of natural selection to optimize strain performance (Fig. 1a). Using isotopic steady-state labeling experiments, we demonstrate significant incorporation of methanol into core metabolites during exponential growth of the engineered and evolved strains (24% into hexose 6-phosphate) and show a yield increase of approximately 30% due to methanol a
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