Cloning and expression of fungal phytases in genetically modified strains of Aspergillus awamori

• Published in: Journal of industrial microbiology & biotechnology 2003-09, Vol.30 (9), p.568-576
• Main Authors: MARTIN, Judith A, MURPHY, Richard A, POWER, Ronan F. G
• Format: Article
• Language: English
• Subjects: 6-Phytase - biosynthesis; 6-Phytase - genetics; Aspergillus - drug effects; Aspergillus - enzymology; Aspergillus - genetics; Aspergillus awamori; Aspergillus fumigatus; Biological and medical sciences; Biotechnology; Biotechnology - methods; Blotting, Northern; Cloning; Cloning, Molecular; Culture Media; Electrophoresis, Polyacrylamide Gel; Enzymes; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genes; Glucose - pharmacology; Hydrogen-Ion Concentration; Nitrogen - pharmacology; Polymerase Chain Reaction; Proteins; Starch - pharmacology; Transformation, Genetic
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-003-0083-8

In an effort to produce phytases cost-effectively, and to determine the efficiency of a novel expression system, the genes for Aspergillus awamori ( phyA) phytase and Aspergillus fumigatus ( phyA) phytase (a putative thermostable enzyme) were cloned and overexpressed in A. awamori. Regulation of phytase expression was achieved by separately placing the genes under the transcriptional control of the glucoamylase A ( glaA) promoter of A. awamori. A gene fusion strategy was employed that involved the insertion of a hexapeptide Kex-2 protease cleavage site between the native glucoamylase and heterologous proteins and allowed for the efficient secretion and processing of the resultant chimeric proteins produced in this system by an endogenous Kex-2 protease. The genes for both of the above-mentioned phytases have already been cloned; however, this is the first report of either of the two phytases being fused with the glucoamylase gene, placed under the transcriptional control of the glaA promoter and overexpressed in A. awamori. Following transformation of A. awamori with separate expression vectors (one for each phytase), induction of phytase expression in submerged culture was effected by utilisation of a starch-containing medium. Optimisation of heterologous protein production in small shake-flask cultures involved changes in medium constituents. Maximum phytase expression levels of 200 phytase units (PU) ml(-1) and 62 PU ml(-1) for recombinantly expressed phytases from A. awamori and A. fumigatus, respectively, were obtained in crude fermentation extracts. Subsequent process scale-up to 4 l batch fermentation yielded phytase production levels comparable to those obtained on small scale. The enzyme yields herein reported demonstrate that the expression system developed and the host strain utilised were capable of expressing phytase at levels comparable to, or exceeding, previously reported data.