Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Abstract

Microbial conversion of methane to high-value bio-based fuels, chemicals, and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense, encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. Given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

Lipid accumulation capability of typical non‐acclimated activated sludge microbial consortia using methane gas as secondary carbon source

“…Genetically modified obligate pure culture methanotrophs can even achieve biomass yields (biomass/CH 4 ) greater than one. 45 To account for the fate of glucose as carbon source, the concentrations of VFAs in the liquid phase were also measured ( Figure 3D). Notably, the methane-charged cultures produced more VFAs than the control.…”

Bioproduction of Isoprenoids and Other Secondary Metabolites Using Methanotrophic Bacteria as an Alternative Microbial Cell Factory Option: Current Stage and Future Aspects

“…The overexpression of this pathway in type I methanotrophs has been successfully used to increase the yield of acetyl-CoA. High carbon flux through the PKT pathway was achieved by the overexpression of two PKT isoforms in M. buryatense; among them, ptkB overexpression showed a two-fold increase in intracellular acetyl-CoA [57]. Therefore, the integration of PKT and MVA pathways in type I methanotrophs could improve isoprenoid production in type I methanotrophs.…”

Biomethane Production from Anaerobic Codigestion of Palm Oil Mill Effluent with Cattle Manure: A Review

“…Biogas production from anaerobic codigestion: ACoD is not only an effective process for biogas yield but also wellused for environmental protection. Moreover, ACoD can develop system steadiness, nutrient stability and lessen greenhouse gas (GHG) radiations also other expensive treatment [47]. Due to direct use of organic constituents, nutritious inequity makes ACoD process difficult to produce biogas efficiently.…”