The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region therein called phaG(Pa) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phuG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol%, 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation From gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known. (C) 2000 Published by Elsevier Science B.V. All rights reserved.