A GENERAL-METHOD FOR IDENTIFICATION OF POLYHYDROXYALKANOIC ACID SYNTHASE GENES FROM PSEUDOMONADS BELONGING TO THE RIBOSOMAL-RNA HOMOLOGY GROUP-I

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyal kanoic 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 1 9.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to ac cumulate PHA of medium-chain-length 3-hydoxyalkanoic acids (HA(MCL)) f rom 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 octanoat e but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated t hat strain GP4BH1 possesses three different functionally active PHA sy nthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PH A synthase, preferentially incorporating hydroxyalkanoic acids of shor t chain length (HA(SCL)), and the synthases were expressed in either G Pp104 and Alcaligenes eutrophus H16-PHB(-)4, respectively. The PHA syn thase encoded by the third fragment (6.5 kbp) led to the incorporation of HA(MCL) and was expressed in GPp104 but not in PHB(-)4.