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In Komagataella phaffii (Pichia pastoris), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self‐induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF‐C1 metabolism, and it cannot be converted into carbon dioxide in a FdhKO strain. Under non‐repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF‐C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for a FdhKO strain on sorbitol and a non‐disrupted strain on sorbitol‐methanol. Considering a K. Phaffii FdhKO strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol‐free processes in K. phaffii. Formate from THF‐C1 metabolism induces pAOX1 in FDH formate dehydrogenase‐deficient P. pastoris under derepressed conditions.

Nitrification, the sequential aerobic oxidation of ammonia via nitrite to nitrate, is a key process of the biogeochemical nitrogen cycle and catalyzed by two aerobic microbial guilds (nitrifiers): ammonia oxidizers and nitrite-oxidizing bacteria (NOB). NOB are generally considered as metabolically restricted and dependent on ammonia oxidizers. Here, we report that, surprisingly, key NOB of many ecosystems ( Nitrospira ) convert urea, an important ammonia source in nature, to ammonia and CO 2 . Thus, Nitrospira supply urease-negative ammonia oxidizers with ammonia and receive nitrite produced by ammonia oxidation in return, leading to a reciprocal feeding interaction of nitrifiers. Moreover, Nitrospira couple formate oxidation with nitrate reduction to remain active in anoxia. Accordingly, Nitrospira are unexpectedly flexible and contribute to nitrogen cycling beyond nitrite oxidation. Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira . Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO 2 . Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Electrocatalytic carbon dioxide (CO 2 ) reduction by CO 2 reductases is a promising approach for biomanufacturing. Among all known biological or chemical catalysts, hydrogen-dependent carbon dioxide reductase from Thermoanaerobacter kivui ( Tk HDCR) possesses the highest activity toward CO 2 reduction. Herein, we engineer Tk HDCR to generate an electro-responsive carbon dioxide reductase considering the safety and convenience. To achieve this purpose, a recombinant Escherichia coli Tk HDCR overexpression system is established. The formate dehydrogenase is obtained via subunit truncation and rational design, which enables direct electron transfer (DET)-type bioelectrocatalysis with a near-zero overpotential. By applying a constant voltage of −500 mV ( vs . SHE) to a mediated electrolytic cell, 22.8 ± 1.6 mM formate is synthesized in 16 h with an average production rate of 7.1 ± 0.5 μmol h −1 cm −2 , a Faradaic efficiency of 98.9% and a half-cell energy efficiency of 94.4%. This study provides an enzyme candidate for high efficient CO 2 reduction and opens up a way to develop paradigm for CO 2 -based bio-manufacturing. Thermoanaerobacter kivui -drived CO 2 reductase ( Tk HDCR) requires hydrogen as substrate, which can lead to safety issue. Here, the authors engineered Tk HDCR into an electro-responsive carbon dioxide reductase to harvest electrons from either an external mediator or a polarized electroactive surface.

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 CO 2 . 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. Low formate dehydrogenase (FDH) activity limits formate assimilation via the synthetic reductive glycine pathway. In this study, the authors introduce a faster FDH in synthetic formatotrophic E. coli , which boosts growth rates and bioproduction titers from formate as sole carbon source.

NAD-dependent formate dehydrogenase (FDH) from Candida boidinii (CbFDH) has been widely used in various CO2-reduction systems but its practical applications are often impeded due to low CO2-reducing activity. In this study, we demonstrated superior CO2-reducing properties of FDH from Thiobacillus sp. KNK65MA (TsFDH) for production of formate from CO2 gas. To discover more efficient CO2-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO2-reducing activities were evaluated. All FDHs including CbFDH showed better CO2-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO2 reduction reaction. In particular, the FDH from Thiobacillus sp. KNK65MA (TsFDH) exhibited the highest CO2-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO2 reduction to formate oxidation in catalytic efficiency (kcat/KB) compared to CbFDH. Formate was produced from CO2 gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO2-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO2 reduction systems.

Background The methylotrophic yeast Komagataella phaffii is a premier host for recombinant protein (rProt) production, which traditionally relies on methanol induction of the alcohol oxidase 1 promoter (P AOX1 ). However, the flammability and associated industrial limitations of methanol have motivated the search for methanol-free induction systems. We recently demonstrated that disruption of formate dehydrogenase gene (FDH) in K. phaffii allows endogenous formate derived from tetrahydrofolate (THF)-mediated C1 metabolism to induce P AOX1 without the addition of external inducers. Therefore, we hypothesized that increasing intracellular formate production by enhancing serine biosynthesis could further improve promoter induction and rProt productivity. Results Overexpression of SER3 , encoding 3-phosphoglycerate dehydrogenase, the rate-limiting enzyme in serine synthesis, significantly increased P AOX1 -driven expression of an intracellular reporter protein (eGFP) and secreted glucose oxidase (Gox) from Aspergillus niger without compromising cell fitness. Enhanced formate accumulation and stronger P AOX1 induction were observed in both microbioreactor and bioreactor cultivations using sorbitol or glycerol-sorbitol mixtures. In GOX- m SER3 strain grown in bioreactor, SER3 overexpression led to a 30% increase in specific Gox activity compared with that of the parental FdhKO strain. Conclusions This study provides a cost-effective metabolic engineering strategy for methanol-free, self-inducible expression systems in K. phaffii based on P AOX1 , enabling safer and more sustainable industrial rProt production.

Shewanella oneidensis MR-1 is a potent hydrogen producer in the deficiency of exogenous electron acceptors. The electron transfer pathway for hydrogen production remains unclear, although enzymes for hydrogen production have been identified in S. oneidensis MR-1. In this study, we investigated the electron transfer pathway from formate to hydrogen, given that formate is commonly a key chemical for bacterial hydrogen production. We revealed that two formate dehydrogenases FdhA1B1C1 and FdhA2B2C2, rather than FdnGHI, played a dominant role in formate-driven hydrogen production. Menaquinone was indispensable for the electron transfer from formate to hydrogen, which excluded the presence of formate hydrogen-lyase in S. oneidensis MR-1. A previously proposed formate dehydrogenase subunit HydC was identified as a menaquinone-binding subunit of [FeFe] hydrogenase HydAB, and the hydABC operon is conserved in bacteria living in diverse environments. A formate exporter FocA and transcriptional regulator FhlA were identified for their effect on formate metabolism and hydrogen production. FhlA positively affected the metabolism of formate and hydrogen by regulating the expression of fdhA2B2C2, fdnGHI, focA, and dld-II. Overall, the electron transfer pathway deciphered in this work will facilitate the improvement of biohydrogen production by S. oneidensis MR-1.Key Points• The electron transfer pathway from formate to hydrogen in MR-1 is deciphered.• Menaquinone is indispensable for hydrogen production.• A cytochrome b subunit transfers electrons from menaquinone to [FeFe] hydrogenase.

The world is at a critical stage to switch from fossil and agriculture feedstocks with sustainable alternatives for the production of biobased chemicals of everyday use. This has spurred interest in using carbon one compounds; methanol and formate as a substrate or cosubstrate for microbial-based production. However, considering that native methylotrophs and formatotrophs utilize methanol and formate respectively, their capabilities to efficiently produce high value-added chemicals are limited. Therefore, shifting from these native C1 microbes to metabolically engineered non-native C1 model strains has attracted increasing attention thanks to many advantages such as the availability of well-established tools and strategies for metabolic engineering, and in addition to its high cell growth rate. Herein, we discussed recent trends in developing synthetic methylotrophs and formatotrophs for methanol and formate-based biomanufacturing. Finally, we highlighted barriers and provided broad prospects on possible avenues for optimizing synthetic methylotrophic and formatotrophic strains with respect to the recent advances in biology.

Carbon dioxide (CO₂) is a kinetically and thermodynamically stable molecule. It is easily formed by the oxidation of organic molecules, during combustion or respiration, but is difficult to reduce. The production of reduced carbon compounds from CO₂ is an attractive proposition, because carbon-neutral energy sources could be used to generate fuel resources and sequester CO₂ from the atmosphere. However, available methods for the electrochemical reduction of CO₂ require excessive overpotentials (are energetically wasteful) and produce mixtures of products. Here, we show that a tungsten-containing formate dehydrogenase enzyme (FDH1) adsorbed to an electrode surface catalyzes the efficient electrochemical reduction of CO₂ to formate. Electrocatalysis by FDH1 is thermodynamically reversible-only small overpotentials are required, and the point of zero net catalytic current defines the reduction potential. It occurs under thoroughly mild conditions, and formate is the only product. Both as a homogeneous catalyst and on the electrode, FDH1 catalyzes CO₂ reduction with a rate more than two orders of magnitude faster than that of any known catalyst for the same reaction. Formate oxidation is more than five times faster than CO₂ reduction. Thermodynamically, formate and hydrogen are oxidized at similar potentials, so formate is a viable energy source in its own right as well as an industrially important feedstock and a stable intermediate in the conversion of CO₂ to methanol and methane. FDH1 demonstrates the feasibility of interconverting CO₂ and formate electrochemically, and it is a template for the development of robust synthetic catalysts suitable for practical applications.

Oxygen tolerant complex metal-dependent formate dehydrogenases hold potential for biotechnological applications. In this work, we report the functional expression of the complex, molybdenum-dependent soluble formate dehydrogenase encoded by the fdsGBACD operon from Cupriavidus necator (CnFDH) in Escherichia coli. Expression of the operon from plasmids or from a copy integrated in the chromosome enabled growth of an energy-auxotrophic selection strain on formate as sole energy source under aerobic conditions. Growth could be accelerated in turbidostat, leading to a drop of the generation time of 1 hour. While no mutation was found in the operon of evolved isolates, genome sequencing revealed non-synonymous point mutations in the gene focA coding for a bidirectional formate transporter carried in all isolates sequenced. Reverting the mutations led to a drop in the growth rate demonstrating the focA gene as principal target of continuous culture adaptation. A member of the oxygen-tolerant subclass of complex FDH showed stable formate oxidation activity when expressed in the heterologous host E. coli, a model organism of biotechnology. The integration of the operon in the chromosome offers the possibility of structure/function studies and activity enhancements through in vivo mutagenesis, which can also be applied to CO2 reduction in appropriate selection hosts.