CLONING, CHARACTERIZATION AND OVEREXPRESSION OF THE PHYTASE-ENCODING GENE (PHYA) OF ASPERGILLUS-NIGER

Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosp hate) to myo-inositol and inorganic phosphate. A gene (phyA) of Asperg illus niger NRRL3135 coding for extracellular, glycosylated phytase wa s isolated using degenerate oligodeoxyribonucleotides deduced from phy tase amino acid (aa) sequences. Nucleotide (nt) sequence analysis of t he cloned region revealed the presence of an open reading frame coding for 467 aa and interrupted once by an intron of 102 bp in the 5' part of the gene. The start codon is followed by a sequence coding for a p utative signal peptide. Expression of phyA is controlled at the level of mRNA accumulation in response to inorganic phosphate levels. After cell growth in low-phosphate medium, a transcript of about 1.8 kb was visualized. Transcription of phyA initiates at at least seven start po ints within a region located 45-25 nt upstream from the start codon. I n transformants of A. niger, expression of multiple copies of phyA res ulted in up to more than tenfold higher phytase levels than in the wil d-type strain.