Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-β-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium
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Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-β-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-β-hydroxybutyrate.
KeywordsOxaloacetate Pyruvate Carboxylase Biotin Carboxylase Methanosarcina Barkeri Pyruvate Carboxylase Activity
This work was supported by grant 36276-N from CONACyT. We thank S. Moreno and R. Nájera for technical support. The experiments comply with the current laws of México.
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