Synthetic biology toolkit of Ralstonia eutropha (Cupriavidus necator).

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g.

Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations reveals the presence of a fucose salvage pathway.

Process engineering of biotechnological productions can benefit greatly from comprehensive analysis of microbial physiology and metabolism. Ralstonia eutropha (syn. Cupriavidus necator) is one of the best studied organisms for the synthesis of biodegradable polyhydroxyalkanoate (PHA).

Enhanced isobutanol production by co-production of polyhydroxybutyrate and cofactor engineering.

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.

Mechanical and biological properties of chitin/polylactide (PLA)/hydroxyapatite (HAP) composites cast using ionic liquid solutions.

This research investigates the potential development of lobster shell waste-derived chitin reinforced with poly(lactic acid) (PLA) and nano-hydroxyapatite (nHAP) into new materials with potentially superior mechanical and thermal properties for biomedical applications. The ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was used as a solvent to prepare chitin/PLA/nHAP composites. The effect of variation of the polymer concentrations on the conduct of the resulting composite was explored.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolymer production from volatile fatty acids using engineered Ralstonia eutropha.

One of the advantages of microbial synthesis of polyhydroxyalkanoates (PHAs) is the production of diverse polymers with different properties by the addition of different monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). Considering the number of possible variables, terpolymers can have more variations than copolymers. In this study, we aimed to synthesize the terpolymer P(3HB-co-3HV-co-3HHx) from volatile fatty acids such as propionate and butyrate using the recombinant Ralstonia eutropha strain (Re2133/pCB81), containing deletions in the phaB1, phaB2, and phaB3 genes, and overexpression of synthetic PHA operon (phaC2, phaA, phaJ).

Draft Genome Sequence of sp. Strain SL1, a Putative Polyhydroxyalkanoate-Producing Halophile.

Halomonas sp. strain SL1, a halophilic gammaproteobacterium, was isolated from samples from the Great Salt Lake in Utah. We report here the draft genome sequence of SL1, which has an estimated total sequence length of 3.

Enhanced microbial lipid production by Cryptococcus albidus in the high-cell-density continuous cultivation with membrane cell recycling and two-stage nutrient limitation.

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.

Fabrication of porous chitin membrane using ionic liquid and subsequent characterization and modelling studies.

The application of green chemistry principles for the processing of biopolymers is a steadily increasing field of research. Chitin membranes were successfully prepared by using the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Cmim][OAc]) as solvent media. The resulting materials were thoroughly characterized, revealing that freeze drying produced membranes that were highly porous.

Solvent production by engineered Ralstonia eutropha: channeling carbon to biofuel.

Microbial production of solvents like acetone and butanol was a couple of the first industrial fermentation processes to gain global importance. These solvents are important feedstocks for the chemical and biofuel industry. Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H and CO under aerobic conditions.

Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47.

Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16.

In this study, we constructed a set of H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (), (p)ppGpp synthase (), and/or polyhydroxybutyrate (PHB) depolymerase ( or ) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the and genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase.

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC.

Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha.

Isobutanol (IBT) has attracted much attention from researchers as a next generation drop-in biofuel. Ralstonia eutropha is a gram-negative bacterium which naturally produces polyhydroxybutyrate (PHB), and has been reported to produce IBT after metabolic engineering. Similar to other microbes, R.

Draft Genome Sequence of Ralstonia sp. MD27, a Poly(3-Hydroxybutyrate)-Degrading Bacterium, Isolated from Compost.

Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth.

Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here.

Starch based polyhydroxybutyrate production in engineered Escherichia coli.

Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer.

Draft Genome Sequences of Pseudomonas fluorescens Strains SF39a and SF4c, Potential Plant Growth Promotion and Biocontrol Agents.

Pseudomonas fluorescens SF4c and SF39a, strains isolated from wheat rhizosphere, have potential applications in plant growth promotion and biocontrol of fungal diseases of crop plants. We report the draft genome sequences of SF4c and SF39a with estimated sizes of 6.5 Mb and 5.

Lipid production by microalgae Chlorella protothecoides with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment.

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation.

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD).

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs), a promising family of bio-based polymers, are considered to be alternatives to traditional petroleum-based plastics. Copolymers like poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) have been shown to exhibit favorable physical and mechanical properties, due to decreased crystallinity resulting from the presence of medium-chain-length 3-hydroxyhexanoate (3HHx) monomers. In this study, we produced P(HB-co-HHx) using engineered Ralstonia eutropha strains containing deletions of the acetoacetyl-CoA reductase (phaB) genes and replacing the native PHA synthase with phaC2 from Rhodococcus aetherivorans I24 and by using butyrate, a short-chain organic acid, as the carbon source.

Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16.

Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium's capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle.

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance.

Effects of intracellular poly(3-hydroxybutyrate) reserves on physiological-biochemical properties and growth of Ralstonia eutropha.

Microbial polyhydroxyalkanoates (PHAs), because of their well studied complex physiology and commercial potential, are vehicles for carbon and potential storage reduction for many microbial species. Even with the wealth of studies about microbial PHAs in the scientific literature, polymer accumulation and degradation are still not comprehensively understood. Poly(3-hydroxybutyrate) (P3HB) granule formation and polymer mobility were studied here in the bacterium Ralstonia eutropha strain B5786 in autotrophic cultures.