ThePseudomonas aeruginosa phaGgene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

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Abstract
We recently identified the phaGPp 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 (herein called phaGPa) 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 phaGPp-negative mutant P. putida PhaGN-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 phaGPa knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaGPa 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 PHAMCL synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.

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