Impaired polyhydroxybutyrate biosynthesis from glucose in Pseudomonas sp. 14-3 is due to a defective beta-ketothiolase gene.
Pseudomonas sp. 14-3 accumulates polyhydroxybutyrate (PHB) from octanoate, but not from glucose. To elucidate this unusual phenotype, genes responsible for the synthesis of PHB were cloned and analyzed. A PHB polymerase gene (phaC) was found downstream from genes coding for a beta-ketothiolase (phaA), an acetoacetyl-coenzyme A reductase (phaB) and a putative transcriptional regulator (phaR). All genes were similar to pha genes from several related species, but differences were observed in the distal region of phaA. Complementation with heterologous beta-ketothiolase genes from Azotobacter sp. FA8 or Pseudomonas putida GPp104 restored the capability of Pseudomonas sp. 14-3 to synthesize PHB from glucose, demonstrating that its beta-ketothiolase was nonfunctional. Analysis of the genome sequences of other Pseudomonas species has revealed the existence of putative beta-ketothiolase genes. The functionality of one of these thiolase genes, belonging to P. putida GPp104, was experimentally demonstrated. Pseudomonas sp. 14-3 is the first natural phaA mutant described, that despite this mutation accumulates high amounts of PHB when growing on fatty acids.
Ayub
Nicolás D
ND
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
Julia Pettinari
M
M
Méndez
Beatriz S
BS
López
Nancy I
NI
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002210:Caprylates / Q000378:metabolism; D003001:Cloning, Molecular; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D011091:Polyesters / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D000094122:Polyhydroxybutyrates
D000102:Acetyl-CoA C-Acyltransferase; D001426:Bacterial Proteins; D002210:Caprylates; D005947:Glucose; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C031492:octanoic acid