Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations.

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2 g L-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4 g L-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7 g L-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1 g L-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

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

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

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

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

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production.

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Migration of Polyphosphate Granules in Agrobacterium tumefaciens.

Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle.

Biosynthesis of Poly(3HB-co-3HP) with Variable Monomer Composition in Recombinant Cupriavidus necator H16.

Polyhydroxyalkanoates are attractive alternatives to traditional plastics. However, although polyhydroxybutyrate (PHB) is produced in large quantities by Cupriavidus necator H16, its properties are far from ideal for the manufacture of plastic products. These properties may be improved through its coproduction with 3-hydroxypropionate (3HP), which leads to the formation of the copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (poly(3HB-co-3HP). To achieve this, a pathway was designed to enable C. necator H16 to convert ß-alanine to 3HP. The initial low levels of incorporation of 3HP into the copolymer were overcome by the overproduction of the native propionyl-CoA transferase together with PHA synthase from Chromobacterium sp. USM2. Following optimization of 3HP incorporation into the copolymer, the molar fraction of 3HP could be controlled by cultivation in medium containing different concentrations of ß-alanine. Between 0 and 80 mol % 3HP could be achieved. Further supplementation with 2 mM cysteine increased the maximum 3HP molar fraction to 89%. Additionally, the effect of deletions of the phaA and phaB1 genes of the phaCAB operon on 3HP molar fraction were investigated. A phaAB1 double knockout resulted in a copolymer containing 91 mol % 3HP without the need for cysteine supplementation.

Isopropanol production with reutilization of glucose-derived CO2 by engineered Ralstonia eutropha.

Chemolithoautotrophic bacterium Ralstonia eutropha is a versatile host for production of various useful compounds including polyhydroxyalkanoates (PHAs) under both heterotrophic and autotrophic conditions. In this bacterium, Calvin-Benson-Bassham (CBB) cycle is functional even under heterotrophic conditions on sugars and reutilizes CO2 emitted through sugar metabolisms into PHA, leading to increase in yield of the storage polyester. This study focused on isopropanol production from glucose by engineered strains of R. eutropha. The isopropanol-producing strains were constructed by introduction of codon-optimized genes of acetoacetate decarboxylase (adc) and primary-secondary alcohol dehydrogenase (adh) from clostridia into glucose-utilizing and PHA-negative (ΔphaC1) strain of R. eutropha. Several genetic modifications showed that high expression of the isopropanol synthesis genes by using a strong synthetic promoter and deletion of NAD+-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase genes (paaH1 and had) in addition to NADPH-dependent acetoacetyl-CoA reductase genes (phaB1 and phaB3) were effective for improving isopropanol production with low by-production of acetone. Isopropanol titer of 4.13 g/L was achieved by two-stage cultivation of the strain IP-007/pBj5c2-adh-adc, corresponding to overall yield of 0.6 mol mol-glucose-1. The fixation of sugar-derived CO2 during isopropanol synthesis was evaluated by 13C-labelling of the isopropanol produced from [1-13C]-glucose. The 13C-abundance in isopropanol synthesized by the engineered strain was significantly increased up to 4.8%, demonstrating actual reassimilation of CO2 emitted from glucose moiety by decarboxylation and potential contribution towards increase in the carbon yield of isopropanol on glucose.

Trehalose production by Cupriavidus necator from CO2 and hydrogen gas.

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 H2/CO2/O2-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.

Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and phaJ genes in Cupriavidus necator.

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

Autotrophic biosynthesis of polyhydroxyalkanoate by Ralstonia eutropha from non-combustible gas mixture with low hydrogen content.

OBJECTIVES: To autotrophically produce polyhydroxyalkanoate (PHA) by Ralstonia eutropha without the risk of gas explosion, the feasibility of using a non-combustible gas mixture with low hydrogen content was investigated. RESULTS: A non-combustible gas mixture (H2: O2: CO2: N2 = 3.6: 7.6: 12.3: 76.5) was used for a 144-hour flask cultivation of two R. eutropha strains. Initially, using strain H16, the production conditions for poly(3-hydroxybutyrate) [P(3HB)] were explored by examining nutrient deficiency. Of these, a nitrogen source-deficient culture medium yielded the highest polymer content of 70 wt% in cells. Next, to produce PHA copolymer, the recombinant strain 1F2 was cultured under the nitrogen source-deficient autotrophic condition. As a result, the accumulation of 3HB-based copolymer containing of 1.2 mol% 3-hydroxyvalerate unit and 1.2 mol% 3-hydroxy-4-methylvalerate unit was observed with 57 wt% of the cell content. CONCLUSIONS: The use of a non-combustible gas with low hydrogen content is beneficial for PHA production in eliminating the risk of explosion due to hydrogen leakage.

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.

Formation of an Organic-Inorganic Biopolymer: Polyhydroxybutyrate-Polyphosphate.

A considerable variety of different biopolymers is formed by the entirety of organisms present on earth. Most of these compounds are organic polymers such as polysaccharides, polyamino acids, polynucleotides, polyisoprenes or polyhydroxyalkanoates (PHAs), but some biopolymers can consist of solely inorganic monomers such as phosphate in polyphosphates (polyPs). In this contribution, we describe the formation of an organic-inorganic block copolymer consisting of poly(3-hydroxybutyrate) (PHB) and polyP. This was achieved by the expression of a fusion of the polyP kinase gene (ppk2c) with the PHB synthase gene (phaC) of Ralstonia eutropha in a polyP-free and PHB-free mutant background of R. eutropha. The fusion protein catalyzed both the formation of polyP by its polyP kinase domain and the formation of PHB by its PHB synthase domain. It was also possible to synthesize the polyP-PHB polymer in vitro with purified Ppk2c-PhaC, if the monomers, adenosine triphosphate (ATP) and 3-hydroxybutyryl-CoA (3HB-CoA), were provided. Most likely, the formed block copolymer (polyP-protein-PHB) turns into a blend of polyP and PHB after release from the enzyme.

Synthesis of Formate from CO2 Gas Catalyzed by an O2-Tolerant NAD-Dependent Formate Dehydrogenase and Glucose Dehydrogenase.

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Siderophore-Mediated Iron Acquisition Enhances Resistance to Oxidative and Aromatic Compound Stress in Cupriavidus necator JMP134.

Many bacteria secrete siderophores to enhance iron uptake under iron-restricted conditions. In this study, we found that Cupriavidus necator JMP134, a well-known aromatic pollutant-degrading bacterium, produces an unknown carboxylate-type siderophore named cupriabactin to overcome iron limitation. Using genome mining, targeted mutagenesis, and biochemical analysis, we discovered an operon containing six open reading frames (cubA-F) in the C. necator JMP134 genome that encodes proteins required for the biosynthesis and uptake of cupriabactin. As the dominant siderophore of C. necator JMP134, cupriabactin promotes the growth of C. necator JMP134 under iron-limited conditions via enhanced ferric iron uptake. Furthermore, we demonstrated that the iron concentration-dependent expression of the cub operon is mediated by the ferric uptake regulator (Fur). Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and resistance to oxidative and aromatic compound stress in C. necator JMP134. In conclusion, we identified a carboxylate-type siderophore named cupriabactin, which plays important roles in iron scavenging, bacterial motility, biofilm formation, and stress resistance.IMPORTANCE Since siderophores have been widely exploited for agricultural, environmental, and medical applications, the identification and characterization of new siderophores from different habitats and organisms will have great beneficial applications. Here, we identified a novel siderophore-producing gene cluster in C. necator JMP134. This gene cluster produces a previously unknown carboxylate siderophore, cupriabactin. Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and oxidative stress resistance. Most notably, this system also plays important roles in increasing the resistance of C. necator JMP134 to stress caused by aromatic compounds, which provide a promising strategy to engineer more efficient approaches to degrade aromatic pollutants.

Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens.

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene (phaCPf) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (phaCPf+, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (phaCPf+) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaCPf, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Production of (3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer from coffee waste oil using engineered Ralstonia eutropha.

Polyhydroxyalkonate (PHA) is a type of polymer that has the potential to replace petro-based plastics. To make PHA production more economically feasible, there is a need to find a new carbon source and engineer microbes to produce a commercially valuable polymer. Coffee waste is an inexpensive raw material that contains fatty acids. It can act as a sustainable carbon source and seems quite promising with PHA production in Ralstonia eutropha, which is a well-known microbe for PHA accumulation, and has the potential to utilize fatty acids. In this study, to make poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)), which has superior properties in terms of biodegradability, biocompatibility, and mechanical strength, engineered strain Ralstonia eutropha Re2133 overexpressing (R)-specific enoyl coenzyme-A hydratase (phaJ) and PHA synthetase (phaC2) with deletion of acetoacetyl Co-A reductases (phaB1, phaB2, and phaB3) was used to produce PHA from coffee waste oil. At a coffee oil concentration of 1.5%, and C/N ratio of 20, the R. eutropha Re2133 fermentation process results in 69% w/w of DCW PHA accumulation and consists of HB (78 mol%) and HHx (22 mol%). This shows the feasibility of using coffee waste oil for P(HB-co-HHx) production, as it is a low-cost fatty acid enriched waste material.