Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16.

TitleRoles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16.
Publication TypeJournal Article
Year of Publication2010
AuthorsBudde, CF, Mahan, AE, Lu, J, Rha, CK, Sinskey, AJ
JournalJ Bacteriol
Date Published2010 Oct
KeywordsAlcohol Oxidoreductases, Bacterial Proteins, Culture Media, Cupriavidus necator, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Genetic Complementation Test, Genome, Bacterial, Genotype, Hydroxybutyrates, Mutation, Polyesters

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.

Alternate JournalJ Bacteriol
Citation Key94
PubMed ID20729355
PubMed Central IDPMC2950492
Grant ListR01 GM049171 / GM / NIGMS NIH HHS / United States
T32 GM081081 / GM / NIGMS NIH HHS / United States
GM-049171 / GM / NIGMS NIH HHS / United States
T32GM081081 / GM / NIGMS NIH HHS / United States