title: 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 CO2lost 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.
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, MS 3323, Golden, CO 80401, United States.
authors.forename: Calvin A
authors.initials: CA
authors.lastname: Henard
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, MS 3323, Golden, CO 80401, United States.
authors.forename: Holly K
authors.initials: HK
authors.lastname: Smith
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, MS 3323, Golden, CO 80401, United States. Electronic address: Michael.Guarnieri@nrel.gov.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Guarnieri
mesh_terms: D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D018533:Biomass; D008055:Lipids / Q000096:biosynthesis* / Q000235:genetics; D008697:Methane / Q000378:metabolism*; D008772:Methylococcaceae / Q000235:genetics / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D008055:Lipids; D000446:Aldehyde-Lyases; C022367:phosphoketolase; D008697:Methane
keywords: Biogas; Greenhouse gas mitigation; Methane biocatalysis; Methanotroph; Phosphoketolase