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 aci d 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 g enome sequence. Its coding region therein called phaG(Pa) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The r esulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phuG(Pp)-negati ve mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydod ecanoate, 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 gluconat e was decreased to about 40% of wild-type level, whereas from fatty acids w ild-type level PHA accumulation was obtained. These data suggested that Pha G from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity a nd strongly enhances the metabolic flux from fatty acid de novo synthesis t owards PHA(MCL) synthesis. Therefore, a function could be assigned to the O RF present in the P. aeruginosa genome, and a second PhaG is now known. (C) 2000 Published by Elsevier Science B.V. All rights reserved.