Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3

Abstract

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

FIG. 1

Organization of phb and pha loci in Pseudomonas sp. strain 61-3 and DNA fragments including the phb or pha locus on the broad-host-range vector used in this study. (A) Restriction map of the 6.0-kb HindIII-ApaI region and organization of phbB_Ps, phbA_Ps, phbC_Ps, and phbR_Ps. (C) Restriction map of the 6.0-kb EcoRI-PstI region and organization of ORF1, phaC1_Ps, phaZ_Ps, phaC2_Ps, and phaD_Ps. (B and D) DNA fragments including the phb and pha loci used in this study, respectively. A, ApaI; B, BamHI; Bg, BglII; E65, EcoO65I; H, HindIII; Pv, PvuI; S, ScaI; Sc, SacI; Sp, SpeI. Translational stop codons (▾▾▾) are present in all three reading frames upstream from the GalK start codon to prevent translational initiation elsewhere on the plasmid from traversing the galK gene and interfering with translation initiation at the galK start codon (22).