Expanding chemistry through in vitro and in vivo biocatalysis.

Living systems contain a vast network of metabolic reactions, providing a wealth of enzymes and cells as potential biocatalysts for chemical processes. The properties of protein and cell biocatalysts-high selectivity, the ability to control reaction sequence and operation in environmentally benign conditions-offer approaches to produce molecules at high efficiency while lowering the cost and environmental impact of industrial chemistry. Furthermore, biocatalysis offers the opportunity to generate chemical structures and functions that may be inaccessible to chemical synthesis. Here we consider developments in enzymes, biosynthetic pathways and cellular engineering that enable their use in catalysis for new chemistry and beyond.

Engineering nonphotosynthetic carbon fixation for production of bioplastics by methanogenic archaea.

The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.