A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I

Abstract

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydoxyalkanoic acids (HAMCL) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HASCL), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB-4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HAMCL and was expressed in GPp104 but not in PHB-4.