Research paper Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis Ibrar Khan a,b, ⁎, Sadia Qayyum a , Shehzad Ahmed a , Farhana Maqbool a , Isfahan Tauseef a , Kashif Syed Haleem a , Zhen-Ming Chi b a Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan b UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, China abstract article info Article history: Received 21 August 2016 Received in revised form 12 December 2016 Accepted 29 December 2016 Available online 03 January 2017 In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from ma- rine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582 bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757 kDa. A putative promoter (intronless) of the gene was lo- cated at -666 bp and contained a TATA box, several CAAT boxes, the 5′-SYGGRG-3′ and a 5′-HGATAR-3′ se- quences. A consensus polyadenylation site (AATAAA) was also observed at + 10 bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four func- tional 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 bi- otin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5 U/mg) than in MA medium supplemented with biotin (0.3 U/mg), suggesting that op- timal 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. © 2016 Elsevier B.V. All rights reserved. Keywords: Pyruvate carboxylase Genome walking Calcium malate Biotin Corn steep liquor Penicillium viticola 152 1. Introduction Malic acid is a four-carbon dicarboxylic acid and an intermediate in the tricarboxylic acid (TCA) cycle. It has many applications in the bever- age and food industry as an acidulant and flavor enhancer, and in metal cleaning, textile finishing, pharmaceuticals and agriculture. In the chemical industries, it is also used as a feedstock for chemical synthesis of polymalic acid (PMA) (Fu et al., 2016). The salts and esters of malic acid are known as malates, which plays a key role in the citric acid, or Krebs cycle, which is a series of metabolic reactions in organisms that produce high-energy phosphate compounds. Malic acid, produced as malates using various minerals such as calcium (Calcium malate), which can enhance pharmaceutical stability and improve absorption (Branson et al., 2002). It can be synthesized by chemical means through the hydration of fumaric acid under high temperature and pressure, yielding a racemic mixture of D- and L-malate or by enzymatic ways that transform fumaric acid to L-malate using the purified fumarase or microbial cells containing fumarase (Presecki and Vasic-Racki, 2005). However, these processes have many disadvantages including; low con- centration of acid production and high product recovery cost. In recent years, L-malic acid production from glucose by one-step fermentation has received increasing attention and many microorganisms have been found to be able to produce L-malate as it can be produced from sustainable and eco-friendly sugars, not from petroleum-based feed- stocks where the source is being depleted and price is increasing. Fur- thermore, three metabolic pathways for the production of L-malic acid from glucose including; non-oxidative pathway, oxidative pathway and L-malic acid from the glyoxylate cycle have been identified (Chi et al., 2016). Whereas, formation of malate from two molecules of acetyl- CoA via the glyoxylate cycle is the main pathway for malic acid synthesis (Fig. 1)(Zelle et al., 2008). There were several native strains and Gene 605 (2017) 81–91 Abbreviations: Amp, ampicillin; bp, base pair; TCA, tricarboxylic acid cycle; rRNA, ribosomal ribonucleic acid; rDNA, ribosomal deoxyribonucleic acid; ITS, the internal transcribed spacer; gDNA, genomic deoxyribonucleic acid; CSL, corn steep liquor; kb, kilobase; MS, mass spectrometry; PYC, pyruvate carboxylase; Rpm, revolutions per minute; Taq, Thermus aquaticus (DNA polymerase); U, unit; SP Primer, specific primers; MA, malic acid; BCCP, biotin carboxyl carrier protein. ⁎ Corresponding author at: Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan. E-mail address: me_abrarkhan@yahoo.com (I. Khan). http://dx.doi.org/10.1016/j.gene.2016.12.036 0378-1119/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene