Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.
A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.
Fukui
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Polymer Chemistry Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan.
Doi
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D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004746:Enoyl-CoA Hydratase / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D004269:DNA, Bacterial; D004746:Enoyl-CoA Hydratase; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C060970:poly-beta-hydroxybutyrate polymerase