Methanol production by reversed methylotrophy constructed in Escherichia coli.

Tomoyuki Takeya,Hiroya Yurimoto,Miyabi Yamakita,Kento Fujisawa,Yasuyoshi Sakai,Daisuke Hayashi

doi:10.1080/09168451.2020.1715202

Tomoyuki Takeya, Hiroya Yurimoto + Show 4 more

Open Access

https://doi.org/10.1080/09168451.2020.1715202

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Abstract

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

Highlights

Much attention has been paid to methanol as an alternative carbon resource to replace fossil fuels, because methanol can be derived from various carbon sources including methane, CO2, and biomass, and is a key organic chemical used in the production of many kinds of chemicals, plastic materials and other value-added products [1]
NAD+-dependent methanol dehydrogenase (MDH) requires the activator protein Act, we did not express it because it is not required for the reverse reaction [16,22]
This value was comparable to that of MDH purified from B. methanolicus C1 (19.6 units/mg at 50°C) [16] or the recombinant E. coli strain expressing the MDH gene of strain C1 (3.5 units/mg at 50°C) [23]

Summary

Introduction

Much attention has been paid to methanol as an alternative carbon resource to replace fossil fuels, because methanol can be derived from various carbon sources including methane, CO2, and biomass, and is a key organic chemical used in the production of many kinds of chemicals, plastic materials and other value-added products [1]. A variety of methods for methanol production via chemical processes have been developed [1], no biological production processes for methanol from biomass constituents such as sugar compounds, that is analogous to bioethanol production, are currently available. Construction of a synthetic biological pathway in a heterologous host using enzymes involved in the metabolism of one-carbon (C1) compounds including methanol can be a possible solution to establish a metabolic pathway that produces methanol from biomass-derived sugar compounds. Methylotrophic bacteria, which can use methanol as the sole carbon and energy source, have diverse types of methanol metabolic pathways. Gram-negative methylotrophic bacteria possess MDHs that require pyrroloquinoline quinone (PQQ) as a cofactor [5]

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