Abstract
BackgroundMethanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Biological conversion of methanol using synthetic methylotrophs has thus gained worldwide attention.ResultsHerein, to endow Escherichia coli with the ability to utilize methanol, an artificial linear methanol assimilation pathway was assembled in vivo for the first time. Distinct from native cyclic methanol utilization pathways, such as ribulose monophosphate cycle, the linear pathway requires no formaldehyde acceptor and only consists of two enzymatic reactions: oxidation of methanol into formaldehyde by methanol dehydrogenase and carboligation of formaldehyde into dihydroxyacetone by formolase. After pathway engineering, genome replication engineering assisted continuous evolution was applied to improve methanol utilization. 13C-methanol-labeling experiments showed that the engineered and evolved E. coli assimilated methanol into biomass.ConclusionsThis study demonstrates the usability of the linear methanol assimilation pathway in bioconversion of C1 resources such as methanol and methane.
Highlights
Methanol is regarded as a biorenewable platform feedstock because most bioresources can be converted into methanol through syngas
Of linear methanol utilization pathway in E. coli The linear methanol utilization pathway consists of two steps: oxidation of methanol into formaldehyde and carboligation of formaldehyde into DHA, which can be phosphorylated to dihydroxyacetone phosphate by dihydroxyacetone kinase and enter lower glycolysis (Fig. 1a)
To assemble the linear pathway in vivo, NAD+-dependent Methanol dehydrogenase (MDH) from Bacillus methanolicus and artificial FLS were overexpressed in E. coli
Summary
Methanol is regarded as a biorenewable platform feedstock because most bioresources can be converted into methanol through syngas. Native methylotrophs are capable of using C1 resources including methanol as carbon and energy sources, they are more challenging to engineer than genetically tractable hosts due to inefficient genetic-transfer systems and editing tools (Whitaker et al 2015). Synthetic methylotrophs were constructed by introducing native methanol assimilation pathways into non-native methylotrophs such as Escherichia coli (Dai et al 2017; Leßmeier et al 2015; Müller et al 2015; Rohlhill et al 2017; Whitaker et al 2017; Witthoff et al 2015). Ribulose monophosphate (RuMP) cycle that utilizes ribulose-5-phosphate (Ru5P) as a formaldehyde acceptor is the only pathway used for synthetic methylotrophs. An artificial linear methanol assimilation pathway based
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