Ralstonia eutropha strain H16 is a Gram-negative non-pathogenic betaproteobacterium ubiquitously found in soils and has been the subject of intensive research for more than 50 years. Due to its remarkable metabolically versatility, it utilizes a broad range of renewable heterotrophic resources. The substrate utilization range can be further extended by metabolic engineering as genetic tools are available. It has become the best studied "Knallgas" bacterium capable of chemolithoautotrophic growth with hydrogen as the electron donor and carbon dioxide as the carbon source. It also serves as a model organism to study the metabolism of poly(β-hydroxybutyrate), a polyester which is accumulated within the cells for storage of both carbon and energy. Thermoplastic and biodegradable properties of this polyhydroxyalkanoate (PHA) have attracted much biotechnical interest as a replacement for fossil resource-based plastics. The first applications of R. eutropha aimed at chemolithoautotrophic production of single cell protein (SCP) for food and feed and the synthesis of various PHAs. The complete annotated genome is available allowing systematic biology approaches together with data provided by available omics studies. Besides PHAs, novel biopolymers of 2-hydroxyalkanoates and polythioesters or cyanophycin as well as chemicals such as alcohols, alkanes, alkenes, and further interesting value added chemicals significantly recently extended the range of products synthesized by R. eutropha. High cell density cultivations can be performed without too much effort and the available repertoire of genetic tools is rapidly growing. Altogether, this qualifies R. eutropha strain H16 to become a production platform strain for a large spectrum of products.
-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single-and multiple-deletion mutants were generated to investigate the influence of these -ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16⌬2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16⌬2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth -ketothiolase or a combination of several of the other -ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.Polyhydroxyalkanoates (PHAs) are naturally occurring polyoxoesters that are synthesized and accumulated as cytoplasmic inclusions by diverse bacteria. Poly(3-hydroxybutyrate) [poly(3HB)] was detected in 1926 by Maurice Lemoigne as an intracellular compound of Bacillus megaterium (16). Generally the accumulation of PHAs proceeds under unbalanced cultivation conditions when a carbon source is available in excess and if another macroelement like nitrogen, phosphorus, or oxygen is limiting growth at the same time (36,44). Ralstonia eutropha strain H16, a Gramnegative facultative chemolithoautotrophic hydrogen-oxidizing bacterium, accumulates poly(3HB) as insoluble granules as a storage compound for carbon and energy in the cytoplasm. The genome of R. eutropha H16 harbors the PHA operon, which comprises three genes encoding a -ketothiolase (phaA...
Ralstonia eutropha H16 is an interesting candidate for the biotechnological production of polyesters consisting of hydroxy- and mercaptoalkanoates, and other compounds. It provides all the necessary characteristics, which are required for a biotechnological production strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic resources into diverse valuable compounds. High cell density fermentations of the non-pathogenic R. eutropha can be easily performed. Furthermore, this bacterium is accessible to engineering of its metabolism by genetic approaches having available a large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metabolism and allowing a systematic biology approach. However, high production costs for bacterial large-scale production of biomass and biotechnologically valuable products are still an economic challenge. The application of inexpensive raw materials could significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial by-products and residual compounds like glycerol, and substrates containing high carbon content per weight like palm, soybean, corn oils as well as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were intensively studied.
In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct(Re)) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct(Re) was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct(Re). In addition to already known CoA acceptors for Pct Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct(Re). Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg⁻¹ min⁻¹). K(m) values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct(Re) in biotechnological polyester production.
In this study, a 9-fold mutant deficient in nine -ketothiolase gene homologues (phaA, bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, and pcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short-or long-chain and even-or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-type R. eutropha and the mutants. In contrast, during the growth on oleate of a multiple mutant lacking phaA, bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The -ketothiolase activity toward acetoacetyl-CoA in H16⌬phaA and all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents. P olyhydroxyalkanoates (PHAs) are naturally occurring polyoxoesters that are synthesized and accumulated as cytoplasmic inclusions by diverse bacteria. Ralstonia eutropha strain H16, a Gram-negative facultatively chemolithoautotrophic hydrogenoxidizing betaproteobacterium, accumulates poly(3-hydroxybutyrate) [poly(3HB)] in the form of insoluble granules as a storage compound for carbon and energy in the cytoplasm. The genome is composed of one megaplasmid and two chromosomes, whose nucleotide sequences were published in 2003 and 2006, respectively (26, 34). R. eutropha H16 harbors the PHA operon, which comprises three genes encoding a -ketothiolase (phaA), an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase (phaB), and a PHA synthase (phaC) (33). The -ketothiolase (PhaA) condenses two acetyl-CoA molecules to acetoacetyl-CoA, and a stereospecific acetoacetyl-CoA reductase (PhaB) reduces the latter to R-(Ϫ)-3-hydroxybutyryl-CoA (24). Finally, the PHA synthase (PhaC) polymerizes the 3-hydroxybutyrate moieties of 3HB-CoA to poly(3HB). R. eutropha possesses two PHA synthases, of which only PhaC1 seems to contribute to the polymerization of the monomers (25,33). PhaC1 belongs to the type I PHA synthases, which are known to produce short-chain-length PHAs (PHA SCL ) with 3 to 5 carbon atoms. However, during cultivation on fatty acids and in the presence of acrylate, which suppresses -oxidation, this PHA synthase is also capable of incorporating small amounts of 3-hydroxyhexanoate (3HHx) and even 3-hydroxyoctanoate (3HO) into the polyester (7, 9). In addition to the enzymes mentioned above, granule-associated phasin proteins are crucial for the poly(3HB) metab...
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