Methanotrophic bacteria use methane, a greenhouse gas and cheap carbon feedstock, as a sole source of carbon and energy, and can convert it to value-added chemicals like isoprenoids which has generated interest as many of them are precursors of ultraperformance fuels, such as jet fuel. Methylomicrobium album BG8 is a methanotrophic bacterium notable for its fast and robust growth and rich genetic potential, making it an excellent candidate for development of a microbial bioproduction platform. Production of isoprenoid from methane would require controlled expression of the Methyl Erythritol Phosphate [MEP] pathway involved in the production of isopentenyl pyrophosphate [IPP] and dimethylallyl pyrophosphate [DMAPP] in M.album BG8 , two precursors of all isoprenoids. In this context, development of functional genetic tools can enable strategies for increased productivity of isoprenoids. We determined broad host range plasmids of IncP, IncQ and pBBR1 family can be conjugated and propagated successfully in M.album BG8. We tested four constitutive promoters PMMO (Particulate methane monooxygenase), PMMA (Mxa type methanol dehydrogenase), PGAP (Glyceraldehyde 3-phosphate dehydrogenase) and PPYC (Pyruvate carboxylase) and two inducible promoters PXOX (Xox type methanol dehydrogenase induced with Lanthanum) and PTet (Tetracycline promoter) using GFP expression. GFP expression data shows PMMO and PMMA to be high expression while PGAP to be a low expression promoter. Of the two inducible promoters PTet shows tightly controlled expression while Pxox shows to be partially inducible. We validate the kit by controlled expression of an esterase under PMMO and PGAP. This work will lay the basis for use of M. album BG8 as a microbial platform suitable for industrial conversion of methane to a vast array of isoprenoids of interest.

Developing genetic tools for Methylomicrobium album BG8: A versatile microbial platform for conversion of methane to isoprenoids

ACS Fall 2021

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