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

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 (PhbC Ps ), β-ketothiolase (PhbA Ps ), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbB Ps ) were found. The genetic organization showed a putative promoter region, followed by phbB Ps - phbA Ps - phbC Ps . Upstream from phbB Ps was found the phbR Ps gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbR Ps gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbR Ps in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptional phb promoter- lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbR Ps is a positive regulatory protein controlling the transcription of phbBAC Ps in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1 Ps and PhaC2 Ps ) were flanked by a PHA depolymerase gene ( phaZ Ps ), and two adjacent open reading frames (ORF1 and phaD Ps ), and the gene order was ORF1, phaC1 Ps , phaZ Ps , phaC2 Ps , and phaD Ps . 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.