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[Display omitted] • Nutrient stresses were applied to Cupriavidus necator growing on carbon dioxide. • Nitrogen starvation induced a PHB accumulation of 54% of total cell dry weight. • The carbon conversion yield into PHB reached a maximum of 85% under nitrogen stress. • Single-stage starvation was more effective than double-stages for PHB production. • Gas transfer enhancement in the fed-batch mode could improve the polymer yield. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cupriavidus necator can grow by consuming CO 2 derived from raw biogas streams. • The choice of growth conditions is crucial for the setup of autotrophic cultivation. • Biogas upgrading is achieved through the effective CO 2 assimilation by C. necator. • Transcriptomics prove the relevance of inoculum adaptation for autotrophic growth. • C. necator adapts to CO 2 changing the respiratory chain and central carbon metabolism. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • More archaea, bacteria, microalgae, and yeasts show potentials as PHA producers. • Feeding strategies and C/N ratio are essential for Cupriavidus necator to produce PHA. • Biomass waste and C1 compounds are the alternatives and economical raw materials. • PHA manufacturing from bench to industrial scale is demonstrated in this review. • Integrative waste utilization and carbon capture is the new opportunity of PHA industry. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Industrial scale PHB production model using various carbon sources was developed. • Techno-economic analysis was used to assess the effect of raw materials on cost. • Cheapest PHB unit production cost was 3.63 $/kg when fructose was consumed. • Sensitivity analysis show that the price of H 2 effects PHB price the most. • Using CO 2 and formate for producing PHB can be feasible in the bioplastic market. 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. [ABSTRACT FROM AUTHOR]

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.)

[Display omitted] • First reported evidence of PHA autotrophic accumulation from biogenic H 2 and CO 2. • Under autotrophic conditions, C. necator DSM 545 increased 3HB accumulation of 42%. • Two reactors: (i) acetogenesis of melon waste; (ii) H 2 and CO 2 fermentation in PHA. • To boost H 2 and CO 2 , methanogenesis was stopped and OLR increased to 20 gCOD/L/d. • 13 gCOD/L of VFAs and alcohols were also produced and converted into 3HB and 3HV. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • 84.9 % of VFA was recovered from landfill leachate by pilot-scale MVR. • 91.3 % of ammonia was removed from landfill leachate by pilot-scale MVR. • The recovered VFA was converted to PHBV in a yield of 96% (g PHBV/g DCW). • The ratio of 3HV to 3HB of PHBV produced from landfill leachate was 45%. 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. [ABSTRACT FROM AUTHOR]

Highlights • Novel two-stage fermentation process for PHB production from grass silage. • Various carbon sources in grass silage used for bioconversion to PHB. • Nutrient-rich PJ as a suitable ready-to-use medium for bacterial growth. • Upstream LAB fermentation of PC hydrolyzate prevents repression effects. • PHB accumulation on consumption of organic acids reached 8.5 g L−1 in 32 h. Abstract Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L−1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L−1 contained 42% PHB. [ABSTRACT FROM AUTHOR]

Industrial manufacturing of polyhydroxyalkanoates (PHAs) requires purification of PHAs granules from high-cell-density cultures. Cells are broken by homogenization and PHAs granules are cleansed and treated to obtain PHAs latexes. However, cell lysis releases large amounts of DNA which results in an increasing viscosity of the medium, hampering the following downstream steps. Drop in viscosity is generally achieved by costly procedures such as heat treatment or the supplementation of hypochlorite and commercially available nucleases. Searching for a cost-effective solution to this issue, a nuclease gene from Staphylococcus aureus has been integrated into two efficient PHAs-producing bacteria: Cupriavidus necator DSM 545 and Delftia acidovorans DSM 39. Staphylococcal nuclease has been proficiently expressed in both microbial hosts without affecting PHAs production. Moreover, the viscosity of the lysates of recombinant C. necator cells was greatly reduced, indicating that the engineered strain is expected to yield large reduction cost in PHAs downstream processing. [ABSTRACT FROM AUTHOR]

[Display omitted] • Conversion of CO 2 to GlcNAc was achieved for the first time by engineered C. necator. • GlcNAc biosynthetic pathway was constructed by expression of Cegna1 *. • PHB synthesis pathway and ED pathway disruption increased the production of GlcNAc. • The maximum GlcNAc titer from CO 2 reached 75.3 mg/L. 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.. [ABSTRACT FROM AUTHOR]

Glycerol is a by-product of many industrial processes and huge amounts of it are generated in the form of waste, thereby necessitating a search for the method of its disposal. An interesting solution is the valorization of crude glycerol into value added product such as polyhydroxyalkanoates (PHAs). The feasibility of producing PHAs by Cupriavidus necator was evaluated using crude glycerol (WG). Various cultivation strategies were designed for the production of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) copolymer by adding different organic acids as precursors at different concentrations levels. Batch cultivation of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) production showed accumulation of 6.76 g/L biomass containing 4.84 g/L copolymer on WG with a maximum 3-hydroxyvalerate content of 24.6 mol%. PHAs extraction using a non-toxic and recyclable solvent, 1,2 propylene carbonate, showed the highest recovery yield (90%) and purity (93%) at 120 °C temperature and 30 min incubation. This is the first report on jatropha based glycerol valorization for poly(3-hydroxybutyrate- co -3-hydroxyvalerate) production coupled with extraction using non-toxic solvent. [ABSTRACT FROM AUTHOR]

[Display omitted] • Heterotrophic and autotrophic myo -inositol production was achieved in C. necator. • Myo -inositol biosynthetic pathway was constructed by expression of Sc IPS and Ec IMP. • Central metabolic network modification enhanced the myo -inositol production. • The production level of myo -inositol (1054.8 mg/L) from CO 2 set up the record. Bioconversion of sustainable feedstocks to commodity chemicals is considered as an effective solution for transforming the fossil-based economy into a carbon–neutral model. Here, the CO 2 -fixing bacterium Cupriavidus necator H16 was exploited for myo -inositol production from renewable substrates. First, by introducing the glucose transportation system, the glucose consumption route was established. Second, two key enzymes involved in myo -inositol biosynthesis were screened and evaluated. A myo -inositol-producing strain was constructed via overexpression of myo -inositol-3-phosphate synthase from Saccharomyces cerevisiae and inositol monophosphatase from Escherichia coli. Finally, carbon flux redirection was achieved through disruption of Entner-Doudoroff pathway and poly(3-hydroxybutyrate) synthesis pathway, resulting in a final myo -inositol production of 520.2, 1076.3 and 1054.8 mg/L from glucose, glycerol and CO 2 , respectively. The myo -inositol production level from CO 2 achieved here set up the record. This study underlines the potential of C. necator to be utilized as microbial factory for upcycling the renewable feedstocks and CO 2 to high-value biochemicals. [ABSTRACT FROM AUTHOR]

Poly-3-hydroxybutrate (PHB) is a biodegradable polymer synthesised via bacterial fermentation as a means of storing carbon and energy under unbalanced growth conditions. The production cost of petroleum-based plastics is currently lower than that for biopolymers, and the carbon source is the most significant contributor to biopolymer production cost. A feasibility study to assess the suitability of using a date seed derived media as an alternative for PHB production under various stress conditions was investigated. Results include fructose extraction from date seeds and a mass transfer model to describe the process, demonstrating that the high nutrient content of date seeds makes them a promising raw material for microbial growth and that a meaningful amount of PHB can be produced without supplementation. Maximum dry cell weight and PHB concentrations were 6.3 g/l and 4.6 g/l respectively, giving a PHB content of 73%, when an initial fructose concentration of 10.8 g/l was used. [ABSTRACT FROM AUTHOR]

The inhibitory effect of limonene on polyhydroxybutyrate (PHB) production in Cupriavidus necator H16 was studied. Firstly, results demonstrate the feasibility of using orange juicing waste (OJW) as a substrate for PHB production. An intracellular PHB content of 81.4% (w/w) was attained for a total dry matter concentration of 9.58 g L −1 , when the OJW medium was used. Later, a mineral medium designed to mimic the nutrient levels found in the complex medium derived from OJW was used to study the effect of limonene on the production of PHB. Results showed a drop in specific growth rate ( μ ) of more than 50% when the initial limonene concentration was 2% (v/v) compared to the limonene free medium. This work highlights the importance of a limonene recovery stage prior to fermentation, to maintain levels below 1% (v/v) in the medium, adding value to the OJW and enhancing the fermentation process productivity. [ABSTRACT FROM AUTHOR]

[Display omitted] • The metabolically engineered C. necator can convert CO 2 to glucose directly. • The conversion process is independent of chemical or electrochemical CO 2 fixation. • Deletion of glk gene generated a glucose-producing strain. • The maximum glucose titer from CO 2 reached 253.3 mg/L. Artificial synthesis of glucose, the monomer of starch, from renewable resources and CO 2 is a promising method for addressing food crisis and alleviating climate change. Here, the construction of a microbial biocatalyst for glucose production from renewable resources and CO 2 was reported. Initially, blocking the glucose catabolic pathway via deletion of glk gene generated a glucose-producing strain of Cupriavidus necator with titers of 24.7, 47.5 and 180.1 mg/L from fructose, glycerol and CO 2 , respectively. Subsequently, the Entner-Doudoroff pathway and polyhydroxybutyrate biosynthesis pathway were disrupted to further increase glucose accumulation. The maximum glucose titer and yield on biomass from CO 2 reached 253.3 mg/L and 91.6 mg/L/OD 600 , respectively. Finally, the phosphatases that mediate the dephosphorylation of phosphorylated glucose were identified. Overexpression of HAD1 and cbbY2 could enhance glucose titer by 5.5-fold when fructose was used as sole carbon source. This study demonstrates a feasible route for microbial-based synthesis of glucose from CO 2. [ABSTRACT FROM AUTHOR]

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.)

Polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [P(3HB- co -3HV)] production was developed in bioreactor cultures using the strain Cupriavidus necator DSM 7237 cultivated on crude glycerol, sunflower meal (SFM) hydrolysates and levulinic acid as the sole fermentation feedstocks. Bacterial growth and PHB production was influenced significantly by the free amino nitrogen and inorganic phosphorus content of the SFM hydrolysate. Fed-batch bioreactor fermentations led to the production of 27 g L −1 PHB with an intracellular content of 72.9% (w/w). Continuous feeding of levulinic acid led to the production of up to 23.4 g L −1 P(3HB- co -3HV) with an intracellular content of 66.4% (w/w) and a 3HV content of 22.5 mol%. A maximum 3HV content of 31 mol% was achieved at earlier fermentation time (53 h). Thus, levulinic acid could be combined with biodiesel industry by-products for the production of high P(3HB- co -3HV) concentration, intracellular content and industrially useful 3HV content. [ABSTRACT FROM AUTHOR]

Highlights: [•] NaCl is a low cost and sustainable external stress that can enhance PHA production. [•] NaCl addition at 9g/l resulted in 30% more and an earlier PHA accumulation. [•] Addition of 12 and 15g/l NaCl were found to inhibit the production of PHA. [•] A dielectric spectroscopy probe was able to indicate PHA accumulation in real-time. [•] The window for maximum accumulation was detected by the probe avoiding polymer loss. [ABSTRACT FROM AUTHOR]

Highlights: [•] Oil from spent coffee grounds was extracted with supercritical carbon dioxide. [•] An oil extraction yield of 90% was achieved at a CO2/SCG mass ratio of 35kgkg−1. [•] Cupriavidus necator was cultivated with SCG oil as sole carbon source. [•] The cells accumulated up to 78% (w/w) poly-3-hydroxybutyrate. [•] The polymer exhibited similar properties to other P(3HB) biopolymers. [ABSTRACT FROM AUTHOR]

Highlights: [•] Carbon distribution of propionic acid between 3HB and 3HV monomers was investigated. [•] Limiting phosphorus feeding leads to a maximum of 33% (Mole.Mole−1) of 3HV. [•] Phosphorus deficiency leads to an instant production of up to 100% of 3HV. [•] But the increase in phosphorus limitation degree decreases PHA productivity. [•] Mechanisms defining PHA composition differed depending on the limitation (N or P). [Copyright &y& Elsevier]

Highlights: [•] A complementary modeling and experimental approach for PHB production was used. [•] The anabolic requirements control NADPH regeneration pathways and availability. [•] Sustaining a residual growth does not alter PHB production yield. [•] Sustaining a residual growth improves PHB specific production rate. [Copyright &y& Elsevier]

Abstract: Two low structured mathematical models for fed-batch production of polyhydroxybutyrate and poly[hydroxybutyrate-co-hydroxyvalerate] by Cupriavidus necator DSM 545 on renewable substrates (glycerol and fatty acid methyl esters-FAME) combined with glucose and valeric acid, were established. The models were used for development/optimization of feeding strategies of carbon and nitrogen sources concerning PHA content and polymer/copolymer composition. Glycerol/glucose fermentation featured a max. specific growth rate of 0.171h−1, a max. specific production rate of 0.038h−1 and a PHB content of 64.5%, whereas the FAME/valeric acid fermentation resulted in a max. specific growth rate of 0.046h−1, a max. specific production rate of 0.07h−1 and 63.6% PHBV content with 4.3% of 3-hydroxyvalerate (3HV) in PHBV. A strong inhibition of glycerol consumption by glucose was confirmed (inhibition constant k i,G =4.28×10−4 gL−1). Applied concentration of FAME (10–12gL−1) positively influenced on PHBV synthesis. HV/PHBV ratio depends on applied VA concentration. [Copyright &y& Elsevier]

Abstract: Utilization of by-products from oilseed-based biodiesel production (crude glycerol, rapeseed meal hydrolysates) for microbial polyhydroxyalkanoate (PHA) production could lead to the replacement of expensive carbon sources, nutrient supplements and precursors for co-polymer production. Batch fermentations in shake flasks with varying amounts of free amino nitrogen led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) with a 2.8–8% 3HV content. Fed-batch fermentations in shake flasks led to the production of 10.9g/L P(3HB-co-3HV) and a 55.6% P(3HB-co-3HV) content. NaCl concentrations between 2 and 6g/L gradually became inhibitory to bacterial growth and PHA formation, whereas in the case of K2SO4, the inhibitory effect was observed only at concentrations higher than 20g/L. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and nuclear magnetic resonance (13C NMR) demonstrated that the incorporation of 3HV into the obtained P(3HB-co-3HV) lowered glass transition temperature, crystallinity and melting point as compared to polyhydroxybutyrate. Integrating PHA production in existing oilseed-based biodiesel plants could enhance the viability and sustainability of this first generation biorefinery. [Copyright &y& Elsevier]

Abstract: The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. 1H NMR and 13C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties. [Copyright &y& Elsevier]

Abstract: Cupriavidus necator DSM 545 is a well-known polyhydroxyalkanoates (PHAs) producer, but unable to grow on lactose. The aim of this study was to construct a recombinant strain of C. necator that can use lactose-containing waste material such as cheese whey, to produce PHAs. One of the intracellular PHA depolymerases (phaZ1) of C. necator was chosen to insert the lacZ, lacI and lacO genes of Escherichia coli. This would have the effect to allow polymer production on lactose and, at the same time, to remove part of the PHA intracellular degradation system. Disruption of phaZ1 was achieved by gene replacement after isolating a fragment of this gene and interrupting it with a cartridge containing the lac genes and a synthetic promoter. Growth and polymer production studies of the genetically modified (GM) strain mRePT in lactose, whey permeate and hydrolyzed whey permeate as carbon sources, were performed. Lower PHA degradation and higher yields were obtained compared to the wild-type strain. Inactivation of the putative depolymerase gene phaZ3 on mRePT recombinant strain was also reported. [Copyright &y& Elsevier]

Abstract: The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL−1) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0–23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (M w) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M w compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (T m), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate. [Copyright &y& Elsevier]

Abstract: Solid-state fermentation (SSF) has recently been proposed as an alternative to submerged fermentation for the production of poly(hydroxyalkanoates). In the present work, X-ray diffraction, differential scanning calorimetry, nuclear magnetic resonance and infrared spectroscopy were employed to investigate the chemical structure, as well as the thermal properties and the crystalline morphology of poly(3-hydroxybutyrate) samples produced by SSF, using as raw material either soy cake or soy cake supplemented with 2.5% (m/m) sugarcane molasses. The results obtained showed that the biopolymer obtained by SSF presented the same properties as commercial PHB, except for the higher molar mass and the lower degree of crystallinity that were observed. Thus, the present data indicate that solid-state fermentation is an interesting alternative for the production of PHB, allowing the production of biopolymers with adequate properties from low-cost, renewable resources. [Copyright &y& Elsevier]

[Display omitted] • Cupriavidus necator shows high potential in managing wastes as alternative feedstocks. • C. necator has been designed to use various renewable biomass and biomass-based wastes. • C1 gases have emerged as promising resources that can be utilized by C. necator. • C. necator has been successfully engineered to produce value-added polymers and chemicals. Cupriavidus necator , a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy. [ABSTRACT FROM AUTHOR]

• The disaccharide trehalose is naturally produced by Cupriavidus necator H16. • Trehalose can be secreted by the sugar efflux transporter A from E. coli. • Trehalose production from synthesis gas by C. necator H16. • Trehalose production from glycerol by C. necator H16. In this work, the hydrogen-oxidizing bacterium Cupriavidus necator H16 was engineered for trehalose production from gaseous substrates. First, it could be shown that C. necator is a natural producer of trehalose when stressed with sodium chloride. Bioinformatic investigations revealed a so far unknown mode of trehalose and glycogen metabolism in this organism. Next, it was found that expression of the sugar efflux transporter A (setA) from Escherichia coli lead to a trehalose leaky phenotype of C. necator. Finally, the strain was characterized under autotrophic conditions using a H 2 /CO 2 /O 2 -mixture and other substrates reaching titers of up to 0.47 g L−1 and yields of around 0.1 g g−1. Taken together, this process represents a new way to produce sugars with high areal efficiency. With further metabolic engineering, an application of this technology for the renewable production of trehalose and other sugars, as well as for the synthesis of 13C-labeled sugars seems promising. [ABSTRACT FROM AUTHOR]

Acetate is a potential non-food carbon source for industrial production, coping with the shortage of food-based feedstocks. (R)-3-hydroxybutyric acid (R-3HB) can be used as an important chiral intermediate in the fine chemical and pharmaceutical industry. In this study, the R-3HB biosynthesis pathway was successfully constructed when genes of β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) from Ralstonia eutropha, and propionyl-CoA transferases (pct) from Clostridium beijerinckii 8052 were introducedinto Escherichia coli. The effects of host E. coli strains, different propionyl-CoA transferases, and post-induction temperatures were investigated. The final concentration of R-3HB reached 0.86 g/L using acetate as the sole carbon source. Subsequently, a kind of culture broth containing the syngas-derived acetate was used to produce 1.02 g/L of R-3HB with a yield of 0.26 g/g. Inthis study, the engineered E. coli strain could efficiently utilize syngas-derived acetate to synthesize R-3HB., (Copyright © 2021 Elsevier Ltd. All rights reserved.)