Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis
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Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography
Candida boidinii NAD + -dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source ‘Turkish DeLight’. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
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Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in Escherichia coli
Microbial bioproduction using one-carbon (C1) feedstocks has the potential to decarbonize the manufacturing of materials, fuels, and chemicals. Formate is a promising C1 feedstock, and the realization of industrial, formatotrophic platform organisms is a key goal for C1-based bioproduction. So far, a major limitation for synthetic formatotrophy has been slow energy supply due to slow formate dehydrogenase activity. Here, we implement a fast, metal-dependent formate dehydrogenase complex in a synthetic formatotrophic Escherichia coli utilizing the reductive glycine pathway. After a short-term evolution, we demonstrate formatotrophic growth of E. coli with a doubling time of less than 4.5 h, comparable to the fastest natural formatotrophs. To further explore the potential of a formate-based bioeconomy, this strain is engineered to produce mevalonate, as well as the terpenoid and aviation fuel precursor isoprenol, using formate we generate directly from the electrochemical reduction of CO2. This work demonstrates an improvement in bioproduct titer from formate, achieving the production of 3.8 g/L of mevalonate. Additionally, the abundant and recalcitrant polymer lignin is chemically decomposed into a formate-rich mixture of small organic acids and subsequently bioconverted into mevalonate. Overall, the described fast-growing, formatotrophic bioproduction strain demonstrates that a sustainable formate bioeconomy is within reach.
Cowan, Aidan E↗
Heterologous expression of formate dehydrogenase enables photoformatotrophy in the emerging model microalga, Picochlorum renovo
Rising global greenhouse gas emissions and the impacts of resultant climate change necessitate development and deployment of carbon capture and conversion technologies. Amongst the myriad of bio-based conversion approaches under evaluation, a formate bio-economy has recently been proposed, wherein CO 2 -derived formate serves as a substrate for concurrent carbon and energy delivery to microbial systems. To date, this approach has been explored in chemolithotrophic and heterotrophic organisms via native or engineered formatotrophy. However, utilization of this concept in phototrophic organisms has yet to be reported. Herein, we have taken the first steps to establish formate utilization in Picochlorum renovo, a recently characterized eukaryotic microalga with facile genetic tools and promising applied biotechnology traits. Plastidial heterologous expression of a formate dehydrogenase (FDH) enabled P. renovo growth on formate as a carbon and energy source. Further, FDH expression enhanced cultivation capacity on ambient CO 2 , underscoring the potential for bypass of conventional CO 2 capture and concentration limitations. This work establishes a photoformatotrophic cultivation regime that leverages light energy-driven formate utilization. The resultant photosynthetic formate platform has widespread implications for applied phototrophic cultivation systems and the bio-economy at large.
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How a Formate Dehydrogenase Responds to Oxygen: Unexpected O 2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters
The reversible two-electron interconversion of formate and CO 2 is catalyzed by both nonmetallo- and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenum- or tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl (Sec) ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe–4S] clusters for facilitating long-distance electron transfer. However, an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O 2 . Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O 2 lability of their metal centers, studies dating as far back as the 1930s emphasize that some of these systems exhibit formate oxidase (FOX) activity, coupling formate oxidation to O 2 reduction. Therefore, to reconcile these conflicting views, we explored context-dependent functional linkages between metallo-FDHs and their cognate electron acceptors within the same organism vis-à-vis catalysis under atmospheric O 2 . Here, we report the discovery and characterization of an O 2 -insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe–4S] clusters. By advancing a robust expression platform for its recombinant production, we eliminate both the requirement of nitrate or azide during purification and reductive activation with thiols and/or formate prior to catalysis. Because the distinctive spectral signatures of formate-reduced DvH-FDH2 remain invariant under anaerobic and aerobic conditions, we benchmarked the enzyme activity in air, identifying CO 2 as the catalytic product. Full reaction progress curve analysis discloses a high catalytic efficiency when probed with a high-potential artificial electron acceptor. Furthermore, we show that DvH-FDH2 enables near-stoichiometric hydrogen peroxide production without superoxide release to achieve O 2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O 2 reveals that metal-based electron bifurcation is operational in this system. Taken together, our work proves the co-occurrence of redox bifurcated FDH and FOX activities within a metalloenzyme scaffold. These findings set the stage for uncovering previously unknown O 2 -insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O 2 -stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.
13C NMR↗
The air-inactivation of formate dehydrogenase FdsDABG from Cupriavidus necator
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Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator
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Reply to “Comment on ‘How a Formate Dehydrogenase Responds to Oxygen: Unexpected O 2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters’”
For the past 15 years, researchers have asserted that the ability to split a pair of electrons by an enzyme-bound redox cofactor called flavin can occur only in a particular way. In addition, they have claimed that this process known as flavin-based electron bifurcation (FBEB) was only discovered in 2008. Here, my laboratory's work not only demonstrates that these claims are untrue, but also illustrates how the research community has failed to responsibly reference key precedents for FBEB dating back to the early 1970s. Furthermore, our research places EB in the proper context, thus allowing for significant new bifurcating systems to be discovered and characterized.
59 BASIC BIOLOGICAL SCIENCES↗
Recent Advances in Picochlorum renovo Strain Development
Microalgae are amongst the most efficient phototrophs for the reduction of CO2 to industrially relevant products and product intermediates. We have recently identified a novel species in the genus Picochlorum (Picochlorum renovo) that demonstrates high areal productivity (>30 g/m2/day), high temperature and salinity tolerances, and rapid growth rate (-2 hour doubling time): attributes essential for economically viable industrial scale deployment for low value commodities (e.g. fuel and chemical precursors). Following screening of >300 algal isolates to identify Picochlorum renovo, we developed baseline genetic tools necessary for genetic engineering of the nucleus and chloroplast, and further built upon these tools to establish a protein secretion system. We have also deployed genetic engineering tools to establish the utilization of phosphite as a selectable marker in the chloroplast and nuclear genomes, while concurrently acting as a potential crop protection strategy. We have further implemented these tools to enable the utilization of formate as a novel carbon source for phototroph cultivation. Explicitly, formate toxicity was assayed, followed by introduction of a formate dehydrogenase to allow formate utilization. Cultures supplemented with formate grew to a higher density when cultivated under ambient CO2, highlighting the potential for this strategy to increase growth. Additionally, we have established a synthetic transcription factor and core promoter system which lays the foundation for tunable, high expression engineering in the nuclear genome, and potentially can be applied for universal algal genetic engineering. Finally, current work has focused on direct photosynthetic production of chemicals, and generation of mutant libraries. Combined, our work has established a robust genetic toolbox for Picochlorum renovo, which we have deployed for strain development purposes to introduce biotechnologically relevant traits.
algae↗
Formate dependent heterodisulfide reduction in a Methanomicrobiales archaeon
Hydrogenotrophic methanogens produce CH 4 using H 2 as an electron donor to reduce CO 2 . In the absence of H 2 , many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H 2 , but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus. Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus co-purified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr associated hydrogenase, and H 2 could not function as an electron donor, even with Hdr purified from cells grown on H 2 . Here, we found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales.
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Biocatalytic Membranes for Carbon Capture and Utilization
Innovative carbon capture technologies that capture CO 2 from large point sources and directly from air are urgently needed to combat the climate crisis. Likewise, corresponding technologies are needed to convert this captured CO 2 into valuable chemical feedstocks and products that replace current fossil-based materials to close the loop in creating viable pathways for a renewable economy. Biocatalytic membranes that combine high reaction rates and enzyme selectivity with modularity, scalability, and membrane compactness show promise for both CO 2 capture and utilization. This review presents a systematic examination of technologies under development for CO 2 capture and utilization that employ both enzymes and membranes. CO 2 capture membranes are categorized by their mode of action as CO 2 separation membranes, including mixed matrix membranes (MMM) and liquid membranes (LM), or as CO 2 gas–liquid membrane contactors (GLMC). Because they selectively catalyze molecular reactions involving CO 2 , the two main classes of enzymes used for enhancing membrane function are carbonic anhydrase (CA) and formate dehydrogenase (FDH). Small organic molecules designed to mimic CA enzyme active sites are also being developed. CO 2 conversion membranes are described according to membrane functionality, the location of enzymes relative to the membrane, which includes different immobilization strategies, and regeneration methods for cofactors. Parameters crucial for the performance of these hybrid systems are discussed with tabulated examples. Progress and challenges are discussed, and perspectives on future research directions are provided.
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