Dataset Information

Haloarchaeal-type ?-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.

ABSTRACT: The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the ?-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential ?-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaA? and PhaA?) and encoded by the cotranscribed HFX_1023 (phaA?) and HFX_1022 (phaA?) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktB? and BktB?) and encoded by cotranscribed HFX_6004 (bktB?) and HFX_6003 (bktB?). BktB? and PhaA? were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad "Ser-His-His" was distinct from the bacterial "Cys-His-Cys." BktB? and PhaA? both contained an oligosaccharide-binding fold domain, which was essential for the ?-ketothiolase activity. Interestingly, BktB? and PhaA? were functionally interchangeable, although PhaA? preferred functioning with PhaA?. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific ?-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.

SUBMITTER: Hou J

PROVIDER: S-EPMC3753943 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Haloarchaeal-type β-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.

Hou Jing J Feng Bo B Han Jing J Liu Hailong H Zhao Dahe D Zhou Jian J Xiang Hua H

Applied and environmental microbiology 20130621 17

The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complement ...[more]

PMID: 23793631

Similar Datasets

Project description:Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] and poly(hydroxybutyrate-co-hydroxyvalerate-co-hydroxyhexanoate) [P(HB-co-HV-co-HHx)] demonstrate interesting mechanical and thermal properties as well as excellent biocompatibility, making them suitable for multiple applications and notably biomedical purposes. The production of such polymers was described in Rhodospirillum rubrum, a purple nonsulfur bacteria in a nutrient-lacking environment where the HHx synthesis is triggered by the presence of hexanoate in the medium. However, the production of P(HB-co-HHx) under nutrient-balanced growth conditions in R. rubrum has not been described so far, and the assimilation of hexanoate is poorly documented. In this study, we used proteomic analysis and a mutant fitness assay to demonstrate that hexanoate assimilation involve β-oxidation and the ethylmalonyl-coenzyme A (CoA) (EMC) and methylbutanoyl-CoA (MBC) pathways, both being anaplerotic pathways already described in R. rubrum. Polyhydroxyalkanoate (PHA) production is likely to involve the de novo fatty acid synthesis pathway. Concerning the polymer composition, HB is the main component of the polymer, probably as acetyl-CoA and butyryl-CoA are intermediates of hexanoate assimilation pathways. When no essential nutrient is lacking in the medium, the synthesis of PHA seems to help maintain the redox balance of the cell. In this framework, we showed that the fixation of CO2 is required to sustain the growth. An increase in the proportion of HHx in the polymer was observed when redox stress was engendered in the cell under bicarbonate-limiting growth conditions. The addition of isoleucine or valerate in the medium also increased the HHx content of the polymer and allowed the production of a terpolymer of P(HB-co-HV-co-HHx). IMPORTANCE The use of purple bacteria, which can assimilate volatile fatty acids, for biotechnological applications has increased, since they reduce the production costs of added-value compounds such as PHA. P(HB-co-HHx) and P(HB-co-HV-co-HHx) have demonstrated interesting properties, notably for biomedical applications. In a nutrient-lacking environment, R. rubrum is known to synthesize such polymers when hexanoate is used as the carbon source. However, their production in R. rubrum in non-nutrient-lacking growth conditions has not been described so far, and the assimilation of hexanoate is poorly documented. As the carbon source and its assimilation directly impact the polymer composition, we studied under non-nutrient-lacking growth conditions the assimilation pathway of hexanoate and PHA production in R. rubrum. Proteomic analysis and mutant fitness assays allowed us to explain PHA production and composition. An increase in HHx content of the polymer and production of P(HB-co-HV-co-HHx) was possible using the knowledge gained on metabolism under hexanoate growth conditions.

Dataset Information

Haloarchaeal-type ?-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.

Publications

Haloarchaeal-type β-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets