A NEW METABOLIC LINK BETWEEN FATTY-ACID DE-NOVO SYNTHESIS AND POLYHYDROXYALKANOIC ACID SYNTHESIS - THE PHAG GENE FROM PSEUDOMONAS-PUTIDA KT2440 ENCODES A 3-HYDROXYACYL-ACYL CARRIER PROTEIN COENZYME-A TRANSFERASE

To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it enc oded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the p rimary structure of the rhlA gene product, which is involved in the rh amnolipid biosynthesis in Pseudomonas aeruginosa PG201. S-1 nuclease p rotection assay identified the transcriptional start site 239 base pai rs upstream of the putative translational start codon, Transcriptional induction of phaG was observed when gluconate was provided, and PHA s ynthesis occurred from this carbon source. No complementation of the r hlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressing ph aG gene under lac promoter control, was obtained. Heterologous express ion of phaG in Pseudomonas oleovorans, which is not capable of PHA syn thesis from gluconate, enabled PHA synthesis on gluconate as the carbo n source, Native recombinant PhaG was purified by native polyacrylamid e gel electrophoresis from P. oleovorans-harborinag plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3 -hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exh ibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.