title: Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.
abstract: In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan; UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, China. Electronic address: me_abrarkhan@yahoo.com.
authors.forename: Ibrar
authors.initials: I
authors.lastname: Khan
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
authors.forename: Sadia
authors.initials: S
authors.lastname: Qayyum
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
authors.forename: Shehzad
authors.initials: S
authors.lastname: Ahmed
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
authors.forename: Farhana
authors.initials: F
authors.lastname: Maqbool
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
authors.forename: Isfahan
authors.initials: I
authors.lastname: Tauseef
authors.affiliation: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
authors.forename: Kashif Syed
authors.initials: KS
authors.lastname: Haleem
authors.affiliation: UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, China.
authors.forename: Zhen-Ming
authors.initials: ZM
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D059001:Aquatic Organisms; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D007526:Isoelectric Point; D008293:Malates / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D016366:Open Reading Frames; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D026723:Polyadenylation; D011401:Promoter Regions, Genetic; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D005656:Fungal Proteins; D008293:Malates; D011994:Recombinant Proteins; D001710:Biotin; C030298:malic acid; D011766:Pyruvate Carboxylase
keywords: Biotin; Calcium malate; Corn steep liquor; Genome walking; Penicillium viticola 152; Pyruvate carboxylase