Biological carbon capture from biogas streams: Insights into Cupriavidus necator autotrophic growth and transcriptional profile.

Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator, known for converting carbon dioxide (CO2) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H2 and O2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO2-adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications.

Heterotrophic and autotrophic production of L-isoleucine and L-valine by engineered Cupriavidus necator H16.

Advancement in commodity chemical production from carbon dioxide (CO2) offers a promising path towards sustainable development goal. Cupriavidus necator is an ideal host to convert CO2 into high-value chemicals, thereby achieving this target. Here, C. necator was engineered for heterotrophic and autotrophic production of L-isoleucine and L-valine. Citramalate synthase was introduced to simplify isoleucine synthesis pathway. Blocking poly-hydroxybutyrate biosynthesis resulted in significant accumulation of isoleucine and valine. Besides, strategies like key enzymes screening and overexpressing, reducing power balancing and feedback inhibition removing were applied in strain modification. Finally, the maximum isoleucine and valine titers of the best isoleucine-producing and valine-producing strains reached 857 and 972 mg/L, respectively, in fed-batch fermentation using glucose as substrate, and 105 and 319 mg/L, respectively, in autotrophic fermentation using CO2 as substrate. This study provides a feasible solution for developing C. necator as a microbial factory to produce amino acids from CO2.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator.

Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

Gold nanoparticles activate hydrogenase synthesis and improve heterotrophic growth of Ralstonia eutropha H16.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

Toward the production of block copolymers in microbial cells: achievements and perspectives.

The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaCAR, enabled the direct synthesis of PHA-PHA type block copolymers in microbial cells. PhaCAR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaCAR can synthesize poly(D-lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. KEY POINTS: • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties.

Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Impact of Acetic Acid Supplementation in Polyhydroxyalkanoates Production by Cupriavidus necator Using Mixture-Process Design and Artificial Neural Network.

The trend in bioplastic application has increased over the years where polyhydroxyalkanoates (PHAs) have emerged as a potential candidate with the advantage of being bio-origin, biodegradable, and biocompatible. The present study aims to understand the effect of acetic acid concentration (in combination with sucrose) as a mixture variable and its time of addition (process variable) on PHA production by Cupriavidus necator. The addition of acetic acid at a concentration of 1 g l-1 showed a positive influence on biomass and PHA yield; however, the further increase had a reversal effect. The addition of acetic acid at the time of incubation showed a higher PHA yield, whereas maximum biomass was achieved when acetic acid was added after 48 h. Genetic algorithm (GA) optimized artificial neural network (ANN) was used to model PHA concentration from mixture-process design data. Fitness of the GA-ANN model (R2: 0.935) was superior when compared to the polynomial model (R2: 0.301) from mixture design. Optimization of the ANN model projected 2.691 g l-1 PHA from 7.245 g l-1 acetic acid, 12.756 g l-1 sucrose, and the addition of acetic acid at the time of incubation. Sensitivity analysis indicates the inhibitory effect of all the predictors at higher levels. ANN model can be further used to optimize the variables while extending the bioprocess to fed-batch operation.

Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability.

The pursuit of carbon neutrality goals has sparked considerable interest in expanding bioplastics production from microbial cell factories. One prominent class of bioplastics, polyhydroxyalkanoates (PHA), is generated by specific microorganisms, serving as carbon and energy storage materials. To begin with, a native PHA producer, Cupriavidus necator (formerly Ralstonia eutropha) is extensively studied, covering essential topics such as carbon source selection, cultivation techniques, and accumulation enhancement strategies. Recently, various hosts including archaea, bacteria, cyanobacteria, yeast, and plants have been explored, stretching the limit of microbial PHA production. This review provides a comprehensive overview of current advancements in PHA bioproduction, spanning from the native to diversified cell factories. Recovery and purification techniques are discussed, and the current status of industrial applications is assessed as a critical milestone for startups. Ultimately, it concludes by addressing contemporary challenges and future prospects, offering insights into the path towards reduced carbon emissions and sustainable development goals.

Maximization of 3-hydroxyhexanoate fraction in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) using lauric acid with engineered Cupriavidus necator H16.

As polyhydroxybutyrate (P(3HB)) was struggling with mechanical properties, efforts have been directed towards increasing mole fraction of 3-hydroxyhexanoate (3HHx) in P(3HB-co-3HHx) to improve the properties of polyhydroxyalkanoates (PHAs). Although genetic modification had significant results, there were several issues related to cell growth and PHA production by deletion of PHA synthetic genes. To find out easier strategy for high 3HHx mole fraction without gene deletion, Cupriavidus necator H16 containing phaC2Ra-phaACn-phaJ1Pa was examined with various oils resulting that coconut oil gave the highest 3HHx mole fraction. When fatty acid composition analysis with GC-MS was applied, coconut oil was found to have very different composition from other vegetable oil containing very high lauric acid (C12) content. To find out specific fatty acid affecting 3HHx fraction, different fatty acids from caproic acid (C6) to stearic acid (C18) was evaluated and the 3HHx mole fraction was increased to 26.5 ± 1.6 % using lauric acid. Moreover, the 3HHx mole fraction could be controlled from 9 % to 31.1 % by mixing bean oil and lauric acid with different ratios. Produced P(3HB-co-3HHx) exhibited higher molecular than P(3HB-co-3HHx) from phaB-deletion mutant. This study proposes another strategy to increase 3HHx mole fraction with easier way by modifying substrate composition without applying deletion tools.

Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16.

The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC-MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

Eliminating genes for a two-component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions.

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single-stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

Process modelling for industrial scale polyhydroxybutyrate production using fructose, formic acid and CO2: Assessing carbon sources and economic viability.

Polyhydroxybutyrate (PHB) is a biodegradable polymer that has potential to replace petroleum-derived plastics. However, the commercialisation of PHB is hindered by high production costs. In this study, the material flow and economics of an industrial scale PHB production process using fructose, formic acid and carbon dioxide (CO2) as carbon sources were simulated and analysed. The lowest breakeven price of 3.64 $/kg PHB was obtained when fructose was utilized as carbon source. When formic acid and CO2 were used, the breakeven price was 10.30 and 10.24 $/kg PHB due to raw material cost, respectively. Although using formic acid and CO2 is more expensive, they meet the emerging sustainable needs for plastic production and contribute to the circular economy via CO2 fixation. This study suggests that the use of formic acid and CO2 as feedstock for PHB production has potential to become competitive in the bioplastic market with further research.

Production of polyhydroxybutyrate by coupled saccharification-fermentation of inulin.

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/ginulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of Mw, and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization.

Development of Cupriavidus necator H16 as a host for heterologous production of formate dehydrogenase I of Methylorubrum extorquens: Possibilities and limitations.

The discovery of formate dehydrogenase (Me-FDH1) from Methylorubrum extorquens has provided an avenue for sustainable CO2 fixation and utilization. However, the mass production of Me-FDH1 is challenging due to the presence of its unique tungsto-bis-metalopterin guanine dinucleotide (W-bis-MGD) cofactor, limiting its practical applications. In this study, C. necator H16 is proposed as a host for the large-scale production of Me-FDH1, utilizing fructose as a carbon source and its inherent machinery for cofactor synthesis. In a minimal salt medium, C. necator H16 could produce active Me-FDH1, which exhibited a specific activity of 80 to 100 U/mg for CO2 conversion to formate. In fed batch bioreactor experiments, approximately 50 g CDW/L (cell dry weight/L) and 10,000 U/L Me-FDH1 were achieved within 50 h. This study highlights C. necator H16 as the recombinant host for Me-FDH1, paving the way for the future development of efficient mass-production methods for this crucial enzyme.

Gas fermentation combined with water electrolysis for production of polyhydroxyalkanoate copolymer from carbon dioxide by engineered Ralstonia eutropha.

A recycled-gas closed-circuit culture system was developed for safe autotrophic cultivation of a hydrogen-oxidizing, polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha, using a non-combustible gas mixture with low-concentration of H2 supplied by water electrolysis. Automated feedback regulation of gas flow enabled input of H2, CO2, and O2 well balanced with the cellular demands, leading to constant gas composition throughout the cultivation. The engineered strain of R. eutropha produced 1.71 g/L of poly(3-hydroxybutyrate-co-12.5 mol% 3-hydroxyhexanoate) on a gas mixture of H2/CO2/O2/N2 = 4:12:7:77 vol% with a 69.2 wt% cellular content. Overexpression of can encoding cytosolic carbonic anhydrase increased the 3HHx fraction up to 19.6 mol%. The yields of biomass and PHA on input H2 were determined to be 72.9 % and 63.1 %, corresponding to 51.0 % and 44.2 % yield on electricity, respectively. The equivalent solar-to-biomass/PHA efficiencies were estimated to be 2.1-3.8 %, highlighting the high energy conversion capability of R. eutropha.

Redox Characterization of the Complex Molybdenum Enzyme Formate Dehydrogenase from Cupriavidus necator.

The oxygen-tolerant and molybdenum-dependent formate dehydrogenase FdsDABG from Cupriavidus necator is capable of catalyzing both formate oxidation to CO2 and the reverse reaction (CO2 reduction to formate) at neutral pH, which are both reactions of great importance to energy production and carbon capture. FdsDABG is replete with redox cofactors comprising seven Fe/S clusters, flavin mononucleotide, and a molybdenum ion coordinated by two pyranopterin dithiolene ligands. The redox potentials of these centers are described herein and assigned to specific cofactors using combinations of potential-dependent continuous wave and pulse EPR spectroscopy and UV/visible spectroelectrochemistry on both the FdsDABG holoenzyme and the FdsBG subcomplex. These data represent the first redox characterization of a complex metal dependent formate dehydrogenase and provide an understanding of the highly efficient catalytic formate oxidation and CO2 reduction activity that are associated with the enzyme.