Impact of Multiple β-Ketothiolase Deletion Mutations in
            <i>Ralstonia eutropha</i>
            H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers

Lindenkamp, Nicole; Peplinski, Katja; Володина, Елена; Ehrenreich, Armin; Steinbüchel, Alexander

doi:10.1128/aem.01058-10

Cited by 38 publications

(28 citation statements)

References 50 publications

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“…It was determined that a deletion mutation of the A1528 ␤-ketothiolase gene did not have an effect on acetoacetyl-CoA biosynthesis and thus PHB production. Based on these findings, the authors of this study postulate astutely that the A1528 gene product may be involved in fatty acid degradation (26).…”

Section: Discussionmentioning

confidence: 98%

Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression

Brigham¹,

Budde²,

Holder³

et al. 2010

J Bacteriol

110

109

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Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid ␤-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single ␤-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.

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Section: Discussionmentioning

confidence: 98%

Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression

Brigham¹,

Budde²,

Holder³

et al. 2010

J Bacteriol

110

109

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“…However, the disclosure of the genome sequence in 2006 revealed in total 14 homologues in addition to phaA (26). In a recent study, several R. eutropha mutants lacking ␤-ketothiolases, which accumulate less poly(3HB) than the wild type, were generated (20). We obtained experimental evidence that the ␤-ketothiolase H16_A0170, besides PhaA and BktB, plays an important role in poly(3HB) synthesis in R. eutropha (20).…”

mentioning

confidence: 81%

“…Another homologue deleted at that time, H16_A1528, which showed no effect on poly(3HB) storage behavior, was recently identified as a component of one of the two ␤-oxidation operons present in R. eutropha (4). PcaF, a ␤-ketoadipyl CoA thiolase/ acetyl-CoA acyltransferase, was still active in an 8-fold multiple mutant accumulating poly(3HB) to only 20% (wt/wt) of the dry weight of the cell, while genomewide transcriptome analyses of the wild type did not reveal this gene (20).…”

mentioning

confidence: 99%

“…In a recent study, several R. eutropha mutants lacking ␤-ketothiolases, which accumulate less poly(3HB) than the wild type, were generated (20). We obtained experimental evidence that the ␤-ketothiolase H16_A0170, besides PhaA and BktB, plays an important role in poly(3HB) synthesis in R. eutropha (20). In that previous study, we observed that mutants deficient in the three ␤-ketothiolases mentioned above, cultivated on sodium gluconate, accumulated poly(3HB) to only 30% (wt/wt) of the dry weight of the cell.…”

mentioning

confidence: 99%

“…Since the complete genome sequence of R. eutropha has become available, its metabolism can be exactly modified or engineered, and new features can be designed (5,20). Since R. eutropha accumulates poly(3HB) to a level as high as 90% (wt/wt) of the dry weight of its cell, it is an interesting organism for large-scale production of PHAs and tailor-made polyester production by metabolic engineering (1,18).…”

mentioning

confidence: 99%

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Genetically Modified Strains of Ralstonia eutropha H16 with β-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents

Lindenkamp

Володина

Steinbüchel

2012

Appl Environ Microbiol

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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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Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha

Raberg

Heinrich

Steinbüchel

2015

Springer Protocols Handbooks

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Impact of Multiple β-Ketothiolase Deletion Mutations in Ralstonia eutropha H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers

Cited by 38 publications

References 50 publications

Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression

Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression

Genetically Modified Strains of Ralstonia eutropha H16 with β-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents

Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha

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