Bioconversion of methane to lactate by an obligate methanotrophic bacterium

Abstract

Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resulted in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

“…New genetic tools for engineering methanotrophs have been developed, 11,31 and robust strains of methanotrophs have been discovered and characterized. 32–34 Methanotrophs have been engineered to produce butanol, lactate, and carotenoids, 20,35–38 catalytically active MMO-containing polymers have been developed, 39 and novel enzymes and new biochemical pathways for forming C–C bonds from methanol and formate have been designed. 40,41 In addition, genes for methylotrophy have been successfully incorporated into Escherichia coli , 42 and strains of methane producing archaea known as methanogens have been engineered to catalyze AOM, thus reversing methane synthesis.…”

Recent Advances in the Genetic Manipulation of Methylosinus trichosporium OB3b

“…These protocols have been used to engineer methanotrophs for biofuel production (de la Torre et al, 2015; Henard et al, 2016; Kalyuzhnaya, Puri, & Lidstrom, 2015) and have also enabled studies of lanthanide-dependent methanol oxidation (Chu & Lidstrom, 2016), ectoine biosynthesis (Mustakhimov et al, 2009), and fatty acid biosynthesis (Demidenko, Akberdin, Allemann, Allen, & Kalyuzhnaya, 2017). This chapter describes the incorporation of these newer techniques developed for Type I methanotrophs into the traditional mutagenesis protocol for Ms. trichosporium OB3b (Murrell, 1994; Lloyd, Finch, Dalton, & Murrell, 1999; Smith & Murrell, 2011).…”

A Prospective Study on the Fermentation Landscape of Gaseous Substrates to Biorenewables Using Methanosarcina acetivorans Metabolic Model

“…The biological routes of methane utilization, have received renewed interest because of process simplicity (Lopez et al, 2013), selectivity toward targeted pathways (Haynes and Gonzalez, 2014; Mueller et al, 2015), and recent advancements in the characterization and genetic tools of methanotrophic microbes enabling direct transformation of methane into valuable chemicals and fuel molecules (Coleman et al, 2014; Fei et al, 2014; Strong et al, 2015; Henard et al, 2016). Much of the current industrial applications of methane utilization have been devoted to the use of aerobic methanotrophic bacteria (Fei et al, 2014).…”