Cloning Of Phytase PhyA Gene From Aspergillus Niger N-2 And Its Overexpression In Pichia Pastoris

Phytate (phytic acid, myo-inositol hexakisphosphate) is the primarystorage form of phosphorus in plant feeds. However, monogastric animals,such as swine and poultry, are unable to metabolize phytic acid due to lowlevels of phytase activity in their digestive tracts. Thus, nearly all of thedietary phytate phosphorus is excreted into the environment, resulting inphosphorus pollution. Furthermore, phytic acid acts as an antinutrient factorby chelating essential minerals and possibly proteins, therefore decreases thebioavailability of these nutrients. Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8)can catalyze the hydrolysis of phytate to the mono-,di-,tri-,tetra- andpentaphosphates of myo-inositol and inorganic phosphate, they belong to thefamily of histidine acid phosphatases. A broad range of microorganisms,including bacteria, filamentous fungi and yeasts can produce phytases.Phytases can be used as a feed additive that improves the bioavailability ofphytate phosphorus in plant feeds and mineral uptake in monogastric animals,and reduces phosphorus pollution of animal waste. Recently, there have beenseveral reports on the cloning and heterologous expression of fungal phyAphytase from Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus,Emericella nidulans, Myceliophthora thermophila, Thermomyces lanuginosusand so on. In this paper, we established techniques to overexpress recombinantphytase in the Pichia pastoris expression system and obtained bioactiveextracellular phytase. The results are as follows: The phyA gene encoding an extracellular phytase from the filamentousfungus Aspergillus niger N-2 was amplified by polymerase chain reaction 3吴静: Aspergillus niger N-2 植酸酶 phyA 基因的克隆及其在 Pichia pastoris 中的高效表达(PCR), the amplified fragment was cloned into pMD18-T vector andsequenced. The results show that the coding region comprises 1347nucleotides without the intron and putative signal sequence, it encodes apolypeptide of 448 amino acids. Comparision with phytase gene phyA of A.niger NRRL3135 (GenBank Accession No. Z16414), it shows 92.5%homology in nucleotides and 95.3% in amino acids sequence. It has the mostconservative active-site amino acids sequence RHGARYP (RHGXRXP, X isany amino acid) of microbial phytase. The phyA gene sequence has beenaccessed by GenBank, the accession number is AY615712. The sequenced phyA fragment was cloned into Pichia secretiveexpression vector pPIC9K and spliced with the a-factor secretion signalpeptide sequence in open reading frame correctly, then the recombinantplasmid pPIC9K/phyA was constructed successfully. The phytase recombinantexpression plasmid pPIC9K/phyA was transformed into chromosome of Pichiapastoris GS115 strain by electroporation after being linearized with Bpu1102 I.After His+Mut+ phenotype screening and in vivo multicopy integrantsscreening with G418. Fifteen G418 reistence transformants were culturedinducedly by methanol (0.5%) and the phytases activity of secretionsupernatant were assayed. They all showed the measurable phytase activities,particularly, the transformant GS-NP-3 showed the highest expression, itsphytase activity in 144 hours induction reached 62221.43U/mL, which wasmore than 3700 times of that of the original strain Aspergillus niger N-2(16.723U/mL), the specific activity of expressed phytase was 93348.08U/mgprotein. The genetic stability of engineering yeasts is quite good. SDS-PAGErevealed that the apparent molecular weight of the recombinant expressedphytase is about 76kDa. The assay results revealed that the recombinantphytase gene was expressed highly and secreted effectively in Pichia pastoris,and the expressed product had the normal bioactivity. The enzymatic properties assay shows that the expressed phytase PHYAhas optimum reaction pH at 5.0~5.5 and pH 2.5, the recombinant phytase is 4