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The International Space Station currently requires four annual replenishments for food supply, a practice that won't be feasible for deep space missions due to the greater distances. Based on the design of closed ecological life support systems, two waste streams were identified: urea from the crew urine, volatile fatty acids (VFAs) from a first stage of anaerobic digestion of waste. The objective of this study was to assess the ability of bacterium Cupriavidus necator to produce single cell protein on urea and VFAs. Thus, the effect of carbon sources (glucose vs VFAs) and the dilution rate on the biomass composition was determined in continuous cultures. Complete transformation of the carbon source into protein-rich biomass was achieved up to 78 % cell dry weight (CDW). For both carbon sources, the protein content increased from 55.0 %CDW to 78 %CDW with a decrease in the dilution rate. Conversely, the nucleic acid and polyhydroxyalkanoate contents decreased with the dilution rate from 8.8 %CDW to 4.8 %CDW and 9.8 %CDW to 0.6 %CDW respectively. Working at a low dilution rate seems to be a good way to maximize protein content while minimizing unwanted nucleic acids and polyhydroxyalkanoates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The rapid global increase in fossil fuel and energy consumption has resulted in the accumulation of greenhouse gases, especially carbon dioxide (CO 2 ), thus contributing to climate change. Therefore, transforming CO 2 into valuable products could yield beneficial outcomes. In this review, the capabilities of Cupriavidus necator H16, a light-independent chemoautotrophic bacterium, as a host platform for the transformation of CO 2 into diverse products are explored. We begin by examining the progress in synthetic biology toolkits, gas fermentation technologies, and engineering approaches, considering the chemoautotrophic metabolic traits of C. necator to enhance the capacity of the strain for CO 2 fixation. Additionally, recent research focused on the metabolic engineering of C. necator H16 for the conversion of CO 2 into biodegradable plastics, biofuels, bioactive compounds, and single-cell proteins was reviewed. Finally, we address the limitations affecting the advancement and utilization of C. necator H16 strain, such as inefficiencies and the range of product types, and offer several recommendations for enhancement. This review acts as a resource for the development of C. necator H16 cell factories and the industrial manufacture of products derived from CO 2 ., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: The current study does not involve the use of animals. Consent to participate: Not applicable. Consent to publish: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting nonmodel bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in nonmodel bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO 2 and hydrogen. However, its low electroporation efficiency hampers the high-throughput genetic engineering required to develop C. necator into an industrially relevant host organism. Thus, conjugation has often been the method of choice for introducing exogenous DNA, especially when introducing large plasmids or suicide plasmids. We first propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of nonmodel bacteria.

The "knallgas" bacterium Cupriavidus necator is attracting interest due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO 2 via the Calvin-Benson-Bassham (CBB) cycle, and grows on organic acids and sugars. Its tripartite genome is notable for its size and duplications of key genes (CBB cycle, hydrogenases, and nitrate reductases). Little is known about which of these isoenzymes and their cofactors are actually utilized for growth on different substrates. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on succinate, fructose, hydrogen (H 2 /CO 2 ), and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that (i) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. (ii) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. (iii) For hydrogenases, both soluble and membrane-bound enzymes were utilized for lithoautotrophic growth. (iv) Of the six terminal respiratory complexes in C. necator H16, only some are utilized, and utilization depends on the energy source. (v) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necator 's genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium and inspire future engineering strategies.IMPORTANCEThe soil bacterium Cupriavidus necator can grow on gas mixtures of CO 2 , H 2 , and O 2 . It also consumes formic acid as carbon and energy source and various other substrates. This metabolic flexibility comes at a price, for example, a comparatively large genome (6.6 Mb) and a significant background expression of lowly utilized genes. In this study, we mutated every non-essential gene in C. necator using barcoded transposons in order to determine their effect on fitness. We grew the mutant library in various trophic conditions including hydrogen and formate as the sole energy source. Fitness analysis revealed which of the various energy-generating iso-enzymes are actually utilized in which condition. For example, only a few of the six terminal respiratory complexes are used, and utilization depends on the substrate. We also show that the protein cost for the various lowly utilized enzymes represents a significant growth disadvantage in specific conditions, offering a route to rational engineering of the genome. All fitness data are available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/., Competing Interests: The authors declare no conflict of interest.

The growing demand for sustainable food packaging and the increasing concerns regarding environmental pollution have driven interest in biodegradable materials. This paper presents an in-depth review of the production of Polyhydroxyalkanoates (PHA), a biodegradable polymer, from food waste. PHA-based bioplastics, particularly when derived from low-cost carbon sources such as volatile fatty acids (VFAs) and waste oils, offer a promising solution for reducing plastic waste and enhancing food packaging sustainability. Through optimization of microbial fermentation processes, PHA production can achieve significant efficiency improvements, with yields reaching up to 87 % PHA content under ideal conditions. This review highlights the technical advancements in using PHA for food packaging, emphasizing its biodegradability, biocompatibility, and potential to serve as a biodegradable alternative to petroleum-based plastics. However, challenges such as high production costs, mechanical limitations, and the need for scalability remain barriers to industrial adoption. The future of PHA in food packaging hinges on overcoming these challenges through further research and innovation in production techniques, material properties, and cost reduction strategies, along with necessary legislative support to promote widespread use., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

The expanding field of synthetic biology requires diversification of microbial chassis to expedite the transition from a fossil fuel-dependent economy to a sustainable bioeconomy. Relying exclusively on established model organisms such as Escherichia coli and Saccharomyces cerevisiae may not suffice to drive the profound advancements needed in biotechnology. In this context, Cupriavidus necator, an extraordinarily versatile microorganism, has emerged as a potential catalyst for transformative breakthroughs in industrial biomanufacturing. This comprehensive book chapter offers an in-depth review of the remarkable technological progress achieved by C. necator in the past decade, with a specific focus on the fields of molecular biology tools, metabolic engineering, and innovative fermentation strategies. Through this exploration, we aim to shed light on the pivotal role of C. necator in shaping the future of sustainable bioprocessing and bioproduct development., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situ to investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator (MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

Cupriavidus necator is a facultative chemolithoautotrophic bacterium able to convert carbon dioxide into poly-3-hydroxybutyrate. This is highly promising as the conversion process allows the production of sustainable and biodegradable plastics. Poly-3-hydroxybutyrate accumulation is known to be induced by nutrient starvation, but information regarding the optimal stress conditions controlling the process is still heterogeneous and fragmentary. This study presents a comprehensive comparison of the effects of nutrient stress conditions, namely nitrogen, hydrogen, phosphorus, oxygen, and magnesium deprivation, on poly-3-hydroxybutyrate accumulation in C. necator DSM545. Nitrogen starvation exhibited the highest poly-3-hydroxybutyrate accumulation, achieving 54% of total cell dry weight after four days of nutrient stress, and a carbon conversion efficiency of 85%. The gas consumption patterns indicated flexible physiological mechanisms underlying polymer accumulation and depolymerization. These findings provide insights into strategies for efficient carbon conversion into bioplastics, and highlight the key role of C. necator for future industrial-scale applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The non-pathogenic β-proteobacterium Cupriavidus necator has the ability to switch between chemoorganotrophic, chemolithoautotrophic and electrotrophic growth modes, making this microorganism a widely used host for cellular bioprocesses. Oxygen usually acts as the terminal electron acceptor in all growth modes. However, several challenges are associated with aeration, such as foam formation, oxygen supply costs, and the formation of an explosive gas mixture in chemolithoautotrophic cultivation with H 2 , CO 2 and O 2 . Bioelectrochemical systems in which O 2 is replaced by an electrode as a terminal electron acceptor offer a promising solution to these problems. The aim of this study was to establish a mediated electron transfer between the anode and the metabolism of living cells, i.e. anodic respiration, using fructose as electron and carbon source. Since C. necator is not able to transfer electrons directly to an electrode, redox mediators are required for this process. Based on previous observations on the extracellular electron transfer enabled by a polymeric mediator, we tested 11 common biological and non-biological redox mediators for their functionality and inhibitory effect for anodic electron transfer in a C. necator-based bioelectrochemical system. The use of ferricyanide at a concentration of 15 mM resulted in the highest current density of 260.75µAcm -2 and a coulombic efficiency of 64.1 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB -4 harboring the gene phaC BP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 (CO 2 ) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO 2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H 2 and O 2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO 2 -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Important improvements have been achieved in developing the coupling of electrochemical CO 2 reduction to formate with its subsequent microbial conversion to polyhydroxybutyrate (PHB) by Cupriavidus necator. The CO 2 based formate electrosynthesis was optimised by electrolysis parameter adjustment and application of Sn based gas diffusion electrodes reaching almost 80 % Faradaic efficiency at 150 mA cm -2 . Thereby, catholyte with the high formate concentration of 441±9 mmol L -1 was generated as feedstock without intermediate downstream processing for semi-automated formate feeding into a fed-batch reactor system. Moreover, microbial formate conversion to PHB was studied further, optimised, and successfully scaled from shake flasks to semi-automated bioreactors. Therein, a PHB per formate ratio of 16.5±4.0 mg g -1 and a PHB synthesis rate of 8.4±2.1 mg L -1  OD -1  h -1 were achieved. By this process combination, an almost doubled overall process yield of 22.3±5.5 % was achieved compared to previous reports. The findings allow a detailed evaluation of the overall CO 2 to PHB conversion, providing the basis for potential technical exploitation., (© 2024 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

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 (R 2 : 0.935) was superior when compared to the polynomial model (R 2 : 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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The discovery of formate dehydrogenase (Me-FDH1) from Methylorubrum extorquens has provided an avenue for sustainable CO 2 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 CO 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

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 (CO 2 ) 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 CO 2 were used, the breakeven price was 10.30 and 10.24 $/kg PHB due to raw material cost, respectively. Although using formic acid and CO 2 is more expensive, they meet the emerging sustainable needs for plastic production and contribute to the circular economy via CO 2 fixation. This study suggests that the use of formic acid and CO 2 as feedstock for PHB production has potential to become competitive in the bioplastic market with further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are grateful for the financial support received from the Ministry of Science and Technology (MOST 111-2221-E-006-012-MY3) and National Science and Technology Council (NSTC 112-2218-E006-022) in Taiwan., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

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 CO 2 ., 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 CO 2 in the feed industry., (© 2023. The Author(s).)

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., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

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/g inulin 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 M w , 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., (© 2023. The Author(s).)

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator , but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner-Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD 600 of the phaC1AB1 -knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD 600 of phaC1AB1 -knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator , an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator .

Membrane biofilm reactors are a growing trend in wastewater treatment whereby gas-transfer membranes provide efficient bubbleless aeration. Recently, there has been a growing interest in using these bioreactors for industrial biotechnology using microorganisms that can metabolise gaseous substrates. Since gas fermentation is limited by the low solubilities of gaseous substrates in liquid media, it is critical to characterise mass transfer rates of gaseous substrates to enable the design of membrane biofilm reactors. The objective of this study is to measure and analyse mass transfer rates and reaction engineering characteristics for a single tube membrane biofilm reactor using Cupriavidus necator H16. At elevated Reynolds numbers, the dominant resistance for gas diffusion shifts from the liquid boundary layer to the membrane. The biofilm growth rate was observed to decrease after 260 μm at 96 h. After 144 h, some sloughing of the biofilm occurred. Oxygen uptake rate and substrate utilisation rate for the biofilm developed showed that the biofilm changes from a single-substrate limited regime to a dual-substrate-limited regime after 72 h which alters the localisation of the microbial activity within the biofilm. This study shows that this platform technology has potential applications for industrial biotechnology.

The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO 2 and H 2 into PHAs, offer an additional strategy. Typically, the preferred source for CO 2 and H 2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H 2 and CO 2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H 2 (26.7 %) and CO 2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H 2 and CO 2 from fruit waste to produce PHAs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases., Results: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD + -reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose -nitrogen mineral salt medium (FN)., Conclusion: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80., (© 2023. The Author(s).)

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO 2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator, thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 g PHA /g Cells . For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joao Medeiros Garcia Alcantara reports financial support was provided by Horizon Europe., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not from inexpensive carbon sources such as sugars. In previous studies, we demonstrated that engineered strains of a hydrogen-oxidizing bacterium, Cupriavidus necator , synthesized PHBHHx with a high cellular content not only from sugars but also from CO 2 as the sole carbon source in the flask culture. In this study, the highly efficient production of PHBHHx from CO 2 was investigated via pH-stat jar cultivation of recombinant C. necator strains while feeding the substrate gas mixture (H 2 /O 2 /CO 2 = 80:10:10 v / v %) to a complete mineral medium in a recycled-gas, closed-circuit culture system. As a result, the dry cell mass and PHBHHx concentration with the strain MF01/pBPP-ccr Me J Ac -emd reached up to 59.62 ± 3.18 g·L -1 and 49.31 ± 3.14 g·L -1 , respectively, after 216 h of jar cultivation with limited addition of ammonia and phosphate solutions. The 3HHx composition was close to 10 mol%, which is suitable for practical applications. It is expected that the autotrophic cultivation of the recombinant C. necator can be feasible for the mass production of PHBHHx from CO 2 .

Serious global problems faced due to many petroleum-based materials in the last century, which is called the plastic age, constitute the main motivation of this research. Considering wastewater treatment from this perspective, both the recovery of organic acids from wastewater and their conversion into bioplastics are extremely important in terms of reducing petroleum dependency. In this study, while the treatment of landfill leachate was provided with biological process integrated into Mechanical Vapor Recompression (MVR), simultaneously PHBV production was carried out with 84.9% recovered VFA as carbon source. The effects of C/N/P ratio and feeding regime on PHBV storage were investigated by Cupriavidus necator. PHBV storage of 96% (g PHBV/g DCW) was maximized by 2-stage feeding and nitrogen restriction. The ratio of 3HV to 3HB of PHBV was 45%. In addition, extracted PHBV was compared with standard PHA in terms of thermal and chemical properties with FTIR, XRD, TGA and DSC analyses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ahmet Karagunduz reports financial support was provided by Scientific and Technological Research Council of Turkey (TUBITAK)., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO 2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO 2 up to concentrations of 11 mg L -1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO 2 . In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

Polyhydroxyalkanoate (PHA)-producing bacteria represent a powerful synthetic biology chassis for waste bioconversion and bio-upcycling where PHAs can be produced as the final products. In this study, we present a seamless plasmid construction for orthogonal expression of recombinant PET hydrolase (PETase) in model PHA-producing bacteria P. putida and C. necator . To this end, this study described seamless cloning and expression methods utilizing SureVector (SV) system for generating pSV-Ortho-PHA (pSVOP) expression platform in bioengineered P. putida and C. necator . Genetic parts specifically Trc promoter, pBBR1 origin of replication, anchoring proteins and signal sequences were utilized for the transformation of pSVOP-based plasmid in electrocompetent cells and orthogonal expression of PETase in both P. putida and C. necator . Validation steps in confirming functional expression of PETase activity in corresponding PETase-expressing strains were also described to demonstrate seamless and detailed methods in establishing bioengineered P. putida and C. necator as whole-cell biocatalysts tailored for plastic bio-upcycling.•Seamless plasmid construction for orthogonal expression in PHA-producing bacteria.•Step-by-step guide for high-efficiency generation of electrotransformants of P. putida and C. necator. •Adaptable methods for rapid strain development (Design, Build, Test and Learn) for whole-cell biocatalysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

The synthesis of PHA was first investigated using WFOs obtained from smoked-sprat heads, substandard fresh sprats, and fresh mackerel heads and backbones. All the WFOs ensured the growth of the wild-type strain Cupriavidus necator B-10646 and the synthesis of PHA, regardless of the degree of lipid saturation (from 0.52 to 0.65) and the set and ratio of fatty acids (FA), which was represented by acids with chain lengths from C14 to C24. The bacterial biomass concentration and PHA synthesis were comparable (4.1-4.6 g/L and about 70%) when using WFO obtained from smoked-sprat heads and fresh mackerel, and it was twice as high as the bacterial biomass concentration from the fresh sprat waste. This depended on the type of WFO, the bacteria synthesized P(3HB) homopolymer or P(3HB- co -3HV- co -3HHx) copolymer, which had a lower degree of crystallinity (C x 71%) and a lower molecular weight (M n 134 kDa) compared to the P(3HB) (M n 175-209 kDa and C x 74-78%) at comparable temperatures (T melt and T degr of 158-168 °C and 261-284 °C, respectively). The new types of WFO, studied for the first time, are suitable as a carbon substrates for PHA synthesis. The WFOs obtained in the production of canned Baltic sprat and Baltic mackerel can be considered a promising and renewable substrate for PHA biosynthesis.

The bacterial molybdenum (Mo)-containing formate dehydrogenase (FdsDABG) from Cupriavidus necator is a soluble NAD + -dependent enzyme belonging to the DMSO reductase family. The holoenzyme is complex and possesses nine redox-active cofactors including a bis(molybdopterin guanine dinucleotide) (bis-MGD) active site, seven iron-sulfur clusters, and 1 equiv of flavin mononucleotide (FMN). FdsDABG catalyzes the two-electron oxidation of HCOO - (formate) to CO 2 and reversibly reduces CO 2 to HCOO - under physiological conditions close to its thermodynamic redox potential. Here we develop an electrocatalytically active formate oxidation/CO 2 reduction system by immobilizing FdsDABG on a glassy carbon electrode in the presence of coadsorbents such as chitosan and glutaraldehyde. The reversible enzymatic interconversion between HCOO - and CO 2 by FdsDABG has been realized with cyclic voltammetry using a range of artificial electron transfer mediators, with methylene blue (MB) and phenazine methosulfate (PMS) being particularly effective as electron acceptors for FdsDABG in formate oxidation. Methyl viologen (MV) acts as both an electron acceptor (MV 2+ ) in formate oxidation and an electron donor (MV +• ) for CO 2 reduction. The catalytic voltammetry was reproduced by electrochemical simulation across a range of sweep rates and concentrations of formate and mediators to provide new insights into the kinetics of the FdsDABG catalytic mechanism.

Bacterial flagella are assembled with ∼30 different proteins in a defined order via diverse regulatory systems. In gram-negative bacteria from the Gammaproteobacteria and Betaproteobacteria classes, the transcription of flagellar genes is strictly controlled by the master regulator FlhDC. In Gammaproteobacteria species, the FlhDC complex has been shown to activate flagellar expression by directly interacting with the promoter region in flagellar genes. To obtain the DNA-binding mechanism of FlhDC and determine the conserved and distinct structural features of Betaproteobacteria and Gammaproteobacteria FlhDCs that are necessary for their functions, we determined the crystal structure of Betaproteobacteria Cupriavidus necator FlhDC (cnFlhDC) and biochemically analyzed its DNA-binding capacity. cnFlhDC specifically recognized the promoter DNA of the class II flagellar genes flgB and flhB. cnFlhDC adopts a ring-like heterohexameric structure (cnFlhD 4 C 2 ) and harbors two Zn-Cys clusters, as observed for Gammaproteobacteria Escherichia coli FlhDC (ecFlhDC). The cnFlhDC structure exhibits positively charged surfaces across two FlhDC subunits as a putative DNA-binding site. Noticeably, the positive patch of cnFlhDC is continuous, in contrast to the separated patches of ecFlhDC. Moreover, the ternary intersection of cnFlhD 4 C 2 behind the Zn-Cys cluster forms a unique protruding neutral structure, which is replaced with a charged cavity in the ecFlhDC structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO 2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse β-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical., Competing Interests: Declaration of competing interest F.C., A.K. and F.K. are employed by b.fab GmbH, a German biotech company aiming for the biomanufacturing of proteins and chemicals from C1 feedstocks. The other authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cupriavidus necator H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear β -hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from C. necator B-4383 grown under iron limitation. The characterization of B-4383 cupriachelins is based on MS/MS fragmentation analysis, which was confirmed by 1D- and 2D-NMR for the most abundant analog ( 1 ). The cupriachelin congeners distinguish these two strains with differences in the preferred lipid tail; however, our rigorous metabolomic investigation also revealed minor analogs with changes in the peptide core, hinting at a potential mechanism by which these siderophores may reduce biologically unavailable ferric iron ( 4-6 ). Antifungal screening of the C. necator B-4383 supernatant extract and the isolated cupriachelin analog ( 1 ) revealed inhibitory activity against Cryptococcus neoformans , with IC 50 values of 16.6 and 3.2 μg/mL, respectively. This antifungal activity could be explained by the critical role of the iron acquisition pathway in the growth and pathogenesis of the C. neoformans fungal pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ahmed, Tripathi and Boudreau.)

The conversion of CO 2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO 2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner-Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO 2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO 2 under normal conditions.., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O 2 -tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H 2 ), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD 600 4.0) and H 2 -oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H 2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H 2 yields of ~ 5 mmol/L and cumulative H 2  ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable β-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable β-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H 2 production was shown in a genetically modified strain of Escherichia coli., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Here a multiple heavy metal and antibiotic resistant bacterium Cupriavidus necator C39 ( C. necator C39) was isolated from a Gold-Copper mine in Zijin, Fujian, China. C. necator C39 was able to tolerate intermediate concentrations of heavy metal(loid)s in Tris Minimal (TMM) Medium (Cu(II) 2 mM, Zn(II) 2 mM, Ni(II) 0.2 mM, Au(III) 70 μM and As(III) 2.5 mM). In addition, high resistance to multiple antibiotics was experimentally observed. Moreover, strain C39 was able to grow on TMM medium containing aromatic compounds such as benzoate, phenol, indole, p-hydroxybenzoic acid or phloroglucinol anhydrous as the sole carbon sources. The complete genome of this strain revealed 2 circular chromosomes and 1 plasmid, and showed the closest type strain is C. necator N-1 T based on Genome BLAST Distance Phylogeny. The arsenic-resistance ( ars ) cluster GST-arsR-arsICBR-yciI and a scattered gene encoding the putative arsenite efflux pump ArsB were identified on the genome of strain C39, which thereby may provide the bacterium a robust capability for arsenic resistance. Genes encoding multidrug resistance efflux pump may confer high antibiotic resistance to strain C39. Key genes encoding functions in degradation pathways of benzene compounds, including benzoate, phenol, benzamide, catechol, 3- or 4-fluorobenzoate, 3- or 4-hydroxybenzoate and 3,4-dihydroxybenzoate, indicated its potential for degrading those benzene compounds.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial copolymer in the polyhydroxyalkanoates (PHAs) family, a next-generation bioplastic. Our research team recently engineered a newly P(3HB-co-3HHx)-producing bacterial strain, Cupriavidus necator PHB - 4/pBBR_CnPro-phaC Rp . This strain can produce P(3HB-co-2 mol% 3HHx) using crude palm kernel oil (CPKO) as a sole carbon substrate. However, the improvement of P(3HB-co-3HHx) copolymer production by this strain has not been studied so far. Thus, this study aims to enhance the production of P(3HB-co-3HHx) copolymers containing higher 3HHx monomer compositions using response surface methodology (RSM). Three significant factors for P(3HB-co-3HHx) copolymers production, i.e., CPKO concentration, sodium hexanoate concentration, and cultivation time, were studied in the flask scale. As a result, a maximum of 3.6 ± 0.4 g/L of P(3HB-co-3HHx) with 4 mol% 3HHx compositions was obtained using the RSM optimized condition. Likewise, the higher 3HHx monomer composition (5 mol%) was obtained when scaling up the fermentation in a 10L-stirrer bioreactor. Furthermore, the produced polymer's properties were similar to marketable P(3HB-co-3HHx), making this polymer suitable for a wide range of applications., (© 2023. The Author(s).)

Bacterial biomasses are suitable and inexpensive biosorbents for the removal of metal ions. The Gram-negative betaproteobacterium Cupriavidus necator H16 is found in soil and freshwater environments. In this study, C. necator H16 was used to remove chromium (Cr), arsenic (As), aluminum (Al), and cadmium (Cd) ions from water. Minimum inhibition concentration (MIC) values of C. necator to Cr, As, Al, and Cd were found as 76, 69, 341, and 275 mg/L, respectively. The highest rates of Cr, As, Al, and Cd bioremoval were 45, 60, 54, and 78%, respectively. pH levels between 6.0 and 8.0 and an average temperature of 30 °C were optimum for the most efficient bioremoval. Scanning electron microscopy (SEM) images of Cd-treated cells showed that the morphology of the cells was significantly impaired compared to the control. Shifts in the Fourier transform infrared spectroscopy analysis (FTIR) spectra of the Cd-treated cell walls also confirmed the presence of active groups. As a result, it can be said that C. necator H16 has a moderate bioremoval efficiency for Cr, As, and Al and a high bioremoval efficiency for Cd.

Microorganisms have the potential to produce nutrient-rich products that can be consumed as food or feed. The protein-rich powder derived from heat treatment of the whole-cell biomass of polyhydroxybutyrate-deficient Cupriavidus necator, a metabolically versatile organism that uses elements found in the air, is an example of such a product. To assess the safety of the protein powder for use as a nutritional ingredient in human food, in accordance with internationally accepted standards, its genotoxic potential and repeated-dose oral toxicity were investigated. A bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test were performed. No evidence of mutagenicity or genotoxicity was found. Additionally, a 90-day repeated-dose oral toxicity study in rats was completed, in which a total of 100 male and female Wistar rats were exposed by gavage to daily doses of 1000, 2000, or 3000 mg/kg bw/day of the test material. Following 90 days of continuous exposure, no mortality or treatment-related adverse effects were observed and no target organs were identified. Therefore, a no observed adverse effect level was determined at 3000 mg/kg bw/day, the highest dose tested., (© 2023 John Wiley & Sons Ltd.)

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO 2 + H 2 , although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon ( cydA2B2 ), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wickham-Smith, Malys and Winzer.)

Converting anthropogenic CO 2 to value-added products using renewable energy has received much attention to achieve a sustainable carbon cycle. CO 2 electrolysis has been extensively investigated, but the products have been limited to some C 1-3 products. Here, we report the integration of CO 2 electrolysis with microbial fermentation to directly produce poly-3-hydroxybutyrate (PHB), a microbial polyester, from gaseous CO 2 on a gram scale. This biohybrid system comprises electrochemical conversion of CO 2 to formate on Sn catalysts deposited on a gas diffusion electrode (GDE) and subsequent conversion of formate to PHB by Cupriavidus necator cells in a fermenter. The electrolyzer and the electrolyte solution were optimized for this biohybrid system. In particular, the electrolyte solution containing formate was continuously circulated through both the CO 2 electrolyzer and the fermenter, resulting in the efficient accumulation of PHB in C. necator cells, reaching a PHB content of 83% of dry cell weight and producing 1.38 g PHB using 4 cm 2 Sn GDE. This biohybrid system was further modified to enable continuous PHB production operated at a steady state by adding fresh cells and removing PHB. The strategies employed for developing this biohybrid system will be useful for establishing other biohybrid systems producing chemicals and materials directly from gaseous CO 2 .

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L -1 could be achieved. When the isopropanol concentration has reached about 8 g. L -1 , cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L -1 of IPA compared to the reference strain (8 g. L -1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L -1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L - 1 ., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guillouet reports financial support was provided by French Ministry of Research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A promising strategy to alleviate the plastic pollution from traditional petroleum-based plastics is the application of biodegradable plastics, in which polyhydroxyalkanoates (PHAs) have received increasing interest owing to their considerable biodegradability. In the PHAs family, poly(3-hydroxybutyrate-co-3-hydroxvalerate) (PHBV) has better mechanical properties, which possesses broader application prospects. With this purpose, the present study adopted Cupriavidus necator to synthesize PHBV utilizing volatile fatty acids (VFAs) as sole carbon sources. Results showed that the concentration and composition of VFAs significantly influenced the production of PHAs. Especially, even carbon VFAs (acetate and butyrate) synthesized only poly(3-hydroxybutyrate) (PHB), while the addition of odd carbon VFAs (propionate and valerate) resulted in PHBV production. The 3-hydroxyvalerate (3HV) contents in PHBV were directly determined by the specific VFAs compositions, in which valerate was the preferred substrate for 3HV accumulation. After optimization by response surface methodology, the highest PHBV accumulation achieved 79.47% in dry cells, and the conversion efficiency of VFAs to PHBV reached 40%, with the PHBV production of 1.20 ± 0.05 g/L. This study revealed the metabolic rule of VFAs converting into PHAs by C. necator and figured out the optimal VFAs condition for PHBV accumulation, which provides a valuable reference for developing downstream strategies of PHBV production in industrial applications in future., (© 2022 Wiley-VCH GmbH.)

The high costs of bioplastics' production may hinder their commercialization. Development of new processes with high yields and in biorefineries can enhance diffusion of these materials. This work evaluated the production of polyhydroxybutyrate (PHB) from the combination of milled corn starchy fraction hydrolysate and crude glycerol as substrates by the strain Cupriavidus necator LPB 1421. After optimization steps, maximum accumulation of 62 % of PHB was obtained, which represents 11.64 g.L -1 and productivity of 0.162 g.Lh -1 . In a stirred tank bioreactor system with 8 L of operational volume, 70 % of PHB accumulation was reported, representing 14.17 g.L -1 of the biopolymer with 0.197 g.Lh -1 productivity. PHB recovery was conducted using a chemical digestion method, reaching >99 % purity. Therefore, the potential application of milled corn as substrate for PHB production was confirmed. The developed bioplastic process could be coupled to a bioethanol producing unit creating the opportunity of a sustainable and economic biorefinery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The number of genes encoding β-oxidation enzymes in Cupriavidus necator H16 (synonym, Ralstonia eutropha H16) is high, but only the operons A0459-A0464 and A1526-A1531, each encoding four genes for β-oxidation enzymes, were expressed during growth with long-chain-length fatty acids (LCFAs). However, we observed that C. necator ΔA0459-A0464 ΔA1526-A1531 and C. necator H16 showed the same growth behavior during growth with decanoic acid and shorter FAs. The negative effect of the deletion of these two operons increased with an increasing chain length of the utilized FAs. Transcriptome sequencing (RNA-Seq) revealed the expression profiles of genes involved in the catabolism of medium-chain-length fatty acids (MCFAs) in C. necator H16. Operon A0459-A0464 was expressed only during growth with nonanoic acid, whereas operon A1526-A1531 was highly expressed during growth with octanoic and nonanoic acid. The gene clusters B1187-B1192 and B0751-B0759 showed a log2 fold change in expression of up to 4.29 and 4.02, respectively, during growth with octanoic acid and up to 8.82 and 5.50, respectively, with nonanoic acid compared to sodium gluconate-grown cells. Several acyl-CoA ligases catalyze the activation of MCFAs with coenzyme A (CoA), but fadD3 (A3288), involved in activation of LCFAs, was not detected. The expression profiles of C. necator strain ΔA0459-A0464 ΔA1526-A1531 showed that the growth with nonanoic acid resulted in the expression of further β-oxidation enzyme-encoding genes. Additional insights into the transport of FAs in C. necator H16 revealed the complexity and putative involvement of the DegV-like protein encoded by A0463 in the transport of odd-chain-length FAs and of siderophore biosynthesis in the transport mechanism. IMPORTANCE Although Cupriavidus necator H16 has been used in several studies to produce polyhydroxyalkanoates from various lipids, the fatty acid metabolism is poorly understood. The β-oxidation of long-chain-length FAs has been investigated, but the tremendous number of homologous genes encoding β-oxidation enzymes hides the potential for variances in the expressed genes for catabolism of shorter FAs. The catabolism of medium-chain-length FAs and connected pathways has not been investigated yet. As more sustainable substrates such as lipids and the production of fatty acids and fatty acid derivates become more critical with the dependency on fossil-based substances, understanding the complex metabolism in this highly diverse workhorse for biotechnology, C. necator, is inevitable. For further metabolic engineering and construction of production strains, we investigated the metabolism during growth on medium-chain-length FAs by RNA-Seq.

Because of its application potential and biodegradability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate;PHBV), a member of the polyhydroxyalkanoates (PHA) biopolymer family, is one of the most extensively studied PHA. High PHBV productivity with a significant amount of hydroxyvalerate (HV) content is very appealing for commercial scale production. The goal of this study was to investigate the efficiency of various defined limitation strategies, namely nitrogen, phosphorus, and oxygen-limitation, for high yield PHBV production by Cupriavidus necator H16 with increased HV unit using waste frying vegetable oil (WFO) and propionic acid (PA) in a high cell density culture (5 L bioreactor). With optimized WFO and PA feeding, highest PHBV harvest (121.7 ± 2.59 g/L; HV 13.9 ± 0.44% (w/w)) and volumetric productivity (2.03 ± 0.04 gPHBV/L·h) were obtained in oxygen-limited operation, while highest HV content (19.8 ± 0.28 wt%) and yield coefficient (0.43 ± 0.017 gHV/gPA) were observed during phosphorus-limited cultivation. Although nitrogen limitation is widely applied in the production of PHA, nitrogen-limited cultivation had the lowest cell dry matter, PHBV production, volumetric productivity, oil-to-HB and PA-to-HV yield coefficients for the given conditions. The results of the present study demonstrate the highest PHBV yield together with the highest HV content using WFO as main carbon source and PA as the HV precursor ever reported in the literature.