Metabolic engineering of Methanosarcina acetivorans for lactate production from methane

Abstract

We previously demonstrated anaerobic conversion of the greenhouse gas methane into acetate using an engineered archaeon that produces methyl-coenzyme M reductase (Mcr) from unculturable microorganisms from a microbial mat in the Black Sea to create the first culturable prokaryote that reverses methanogenesis and grows anaerobically on methane. In this work, we further engineered the same host with the goal of converting methane into butanol. Instead, we discovered a process for converting methane to a secreted valuable product, L-lactate, with sufficient optical purity for synthesizing the biodegradable plastic poly-lactic acid. We determined that the 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum is responsible for lactate production. This work demonstrates the first metabolic engineering of a methanogen with a synthetic pathway; in effect, we produce a novel product (lactate) from a novel substrate (methane) by cloning the three genes for Mcr and one for Hbd. We further demonstrate the utility of anaerobic methane conversion with an increased lactate yield compared to aerobic methane conversion to lactate. Biotechnol. Bioeng. 2017;114: 852-861. © 2016 Wiley Periodicals, Inc.

Electricity from methane by reversing methanogenesis

“…In a follow-up study, we further metabolically engineered the methane-consuming M. acetivorans strain to synthesize lactate by adding the gene for the production of a 3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum 32. Furthermore, the archaeal host, M. acetivorans, was adapted to small pulses of oxygen (named air-adapted M. acetivorans (AA)33), making it more robust in terms of biofilm production for potential use in a MFC.…”

In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex

“…This step has been shown to be reversible in both native methanogens 17 and in a methanogen host expressing a Mcr homolog from an anaerobic methanotroph of the clade ANME-1. 8,9 The next two steps in methanol production are catalyzed by the MtaABC complex (Figures 1b,c). Although the individual components of this complex, consisting of MtaA and MtaBC, were previously shown to be reversible, 14,15 the reversibility of the entire MtaABC complex had not been demonstrated prior to this work.…”

“…Since the MtaABC enzyme complex catalyzes the second and final reaction needed to convert methane to methanol during anaerobic methane oxidation, the demonstration of this reaction in vitro represents a significant step toward realizing the potential of converting methane to methanol biologically rather using chemical routes. 20 We envision an engineered strain that combines an effective MCR protein (such as the ANME-1 Mcr) expressed in M. acetivorans 8,9 with the MtaABC complex in a suitable host and then provide culture conditions that would promote the reaction from methane to methanol. That would include high methane concentrations under pressure to increase the effective soluble concentration of methane to which the cells would be exposed, combined in a syntrophic co-culture with an organism that uses methanol effectively and with very high affinity (i.e., a very low overall effective K m value for methanol).…”

“…production from methane. 8 Further engineering of this strain resulted in lactate production from methane, 9 demonstrating the feasibility of biological production of valuable chemicals from methane. Methylotrophic methanogens catalyze the formation of CH 3 -CoM from methanol with the methanol:CoM methyltransferase complex (MtaABC).…”

Microbial Methylotrophic Metabolism: Recent Metabolic Modeling Efforts and Their Applications In Industrial Biotechnology

“…The native products of reverse methanogenesis in A. acetivorans were determined to be acetate and CO 2 . Using the same engineered host, McAnulty et al produced lactate yielding 0.59 g per gram of methane. This is an order of magnitude greater than the previously reported yield of lactate on methane in an aerobic process …”