Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-beta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium.
Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-beta-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene ( pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-beta-hydroxybutyrate.
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Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, 62250, Cuernavaca, Morelos, Mexico.
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D000105:Acetyl Coenzyme A / Q000378:metabolism; D001360:Avidin / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D002952:Citric Acid Cycle; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004251:DNA Transposable Elements; D023061:Gene Order / Q000235:genetics; D005798:Genes, Bacterial; D006885:Hydroxybutyrates / Q000378:metabolism*; D008854:Microscopy, Electron; D016254:Mutagenesis, Insertional; D011091:Polyesters / Q000378:metabolism*; D021122:Protein Subunits; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D000094122:Polyhydroxybutyrates
D000105:Acetyl Coenzyme A; D001360:Avidin; D003470:Culture Media; D004251:DNA Transposable Elements; D006885:Hydroxybutyrates; D011091:Polyesters; D021122:Protein Subunits; D011766:Pyruvate Carboxylase; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates