Development of a novel Aspergillus uracil deficient expression system and its application in expressing a cold-adapted alpha-amylase gene from Antarctic fungi Geomyces pannorum

摘要

Filamentous fungi have increasingly been used as hosts for heterologous protein production because of their high secretion capability and ability to add eukaryotic post-translational modifications. In this study, a novel uracil-deficient Aspergillus transformation system, which was based on an orotate phosphoribosyltransferase (pyrF) nutritional selection marker, was discovered. Additionally, a universal, purify-able vector that directed genes into the Aspergillus host strain was engineered. A genomic DNA segment encoding a novel a-amylase was isolated from the psychrotolerant fungus Geomyces pannorum and the open reading frame was determined, deduced 497 amino acids. G. pannorum alpha-amylase was then expressed in the newly constructed Aspergillus oryzae system, with an amylase activity reaching 958 U/ml. It was purified to electrophoretic homogeneity and has a molecular mass of approximately 54 kDa. The enzyme exhibited an optimal activity at pH 5.0 and 40 degrees C and retained over 20% of maximal activity over the temperature range 0-20 degrees C. To our knowledge, this report is the first of the heterologous expression of a cold-adapted enzyme in filamentous fungi. G. pannorum alpha-amylase is an economical amylase with many potential applications. (C) 2015 Elsevier Ltd. All rights reserved.