Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei

The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the beta-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetylCoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential beta-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 alpha and PhaA beta) and encoded by the cotranscribed HFX_1023 (phaA alpha) and HFX_1022 (phaA beta) 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 alpha and BktB beta) and encoded by cotranscribed HFX_6004 (bktB alpha) and HFX_6003 (bktB beta). BktB alpha and PhaA alpha 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-HisCys." BktB beta and PhaA beta both contained an oligosaccharide-binding fold domain, which was essential for the beta-ketothiolase activity. Interestingly, BktB beta and PhaA beta were functionally interchangeable, although PhaA beta preferred functioning with PhaA alpha. 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 beta-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.