Abstract
Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.
Highlights
Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2
To identify gene deletions that result in such strains, we modeled E. coli metabolism using FBA34
We use the strength of flux balance analysis (FBA) to predict growth on a non-native carbon source and the degree to which carbon can be assimilated into various central metabolites
Summary
Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Apart from its potential for biotechnological applications, the generation of a synthetic methylotroph is of value because it would help to uncover and test basic design principles of methylotrophy This engineering challenge of creating a synthetic methylotrophic organism has attracted considerable interest in recent years, with the focus mostly on the naturally occurring ribulose monophosphate (RuMP) cycle due to its energy efficiency[18,19,20,21,22,23,24,25,26,27,28,29,30,31]. Methanol-dependent strains have been described in literature[21,22,28,29], which-due to a compromised RuMP cycle-lacked the potential for evolution towards growth on methanol alone
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