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Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16.
- Source :
-
Journal of bacteriology [J Bacteriol] 2010 Oct; Vol. 192 (20), pp. 5319-28. Date of Electronic Publication: 2010 Aug 20. - Publication Year :
- 2010
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Abstract
- The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.
- Subjects :
- Alcohol Oxidoreductases classification
Bacterial Proteins classification
Bacterial Proteins genetics
Culture Media chemistry
Cupriavidus necator classification
Cupriavidus necator genetics
Gene Expression Regulation, Bacterial physiology
Gene Expression Regulation, Enzymologic genetics
Gene Expression Regulation, Enzymologic physiology
Genetic Complementation Test
Genome, Bacterial
Genotype
Mutation
Alcohol Oxidoreductases metabolism
Bacterial Proteins metabolism
Cupriavidus necator metabolism
Hydroxybutyrates metabolism
Polyesters metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1098-5530
- Volume :
- 192
- Issue :
- 20
- Database :
- MEDLINE
- Journal :
- Journal of bacteriology
- Publication Type :
- Academic Journal
- Accession number :
- 20729355
- Full Text :
- https://doi.org/10.1128/JB.00207-10