Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.

[1]  M. Takano,et al.  The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases , 1999, Applied and Environmental Microbiology.

[2]  S. Zeilinger,et al.  Expression of Two Major Chitinase Genes of Trichoderma atroviride (T. harzianum P1) Is Triggered by Different Regulatory Signals , 1999, Applied and Environmental Microbiology.

[3]  K. Takahashi,et al.  Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes , 1998, Applied Microbiology and Biotechnology.

[4]  R. Cannon,et al.  Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae , 1998, Archives of Microbiology.

[5]  K. Kitamoto,et al.  Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding α-glucosidase , 1996, Current Genetics.

[6]  C. Kubicek,et al.  Molecular cloning and expression of the nag1 gene (N-acetyl-β-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1 , 1996, Current Genetics.

[7]  E. Espeso,et al.  The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid‐ and alkaline‐expressed genes by ambient pH. , 1995, The EMBO journal.

[8]  H. Dalbøge,et al.  A novel method for efficient expression cloning of fungal enzyme genes , 1994, Molecular and General Genetics MGG.

[9]  R. Cannon,et al.  Molecular cloning and expression of the Candida albicans beta-N-acetylglucosaminidase (HEX1) gene , 1994, Journal of bacteriology.

[10]  C. Scazzocchio,et al.  Two different, adjacent and divergent zinc finger binding sites are necessary for CREA‐mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. , 1994, The EMBO journal.

[11]  G. Gooday,et al.  What are the roles of chitinases in the growing fungus , 1992 .

[12]  M. Ueda,et al.  Purification and Some Properties of β-N-Acetylglucosaminidase from Aeromonas sp. 10S-24 , 1992 .

[13]  P. Deininger.  Molecular cloning: A laboratory manual , 1990 .

[14]  C. Vázquez,et al.  Beta-N-acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis. , 1989, FEMS microbiology letters.

[15]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[16]  W. Timberlake,et al.  A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Shepherd,et al.  The secretion of N-acetylglucosaminidase during germ-tube formation in Candida albicans. , 1984, Journal of general microbiology.

[18]  O. P. Bahl,et al.  Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 2. Kinetic studies on the active site. , 1984, Archives of biochemistry and biophysics.

[19]  W. Timberlake,et al.  Transformation of Aspergillus nidulans by using a trpC plasmid. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[20]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[21]  A. Upshall.  Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans , 2004, Current Genetics.

[22]  H. Fujimoto,et al.  Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains , 2004, Glycoconjugate Journal.

[23]  A. Ohta,et al.  Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. , 1998, Bioscience, biotechnology, and biochemistry.

[24]  C. Bulawa.  Genetics and molecular biology of chitin synthesis in fungi. , 1993, Annual review of microbiology.

[25]  S. Unkles.  Gene organization in industrial filamentous fungi , 1992 .

[26]  E. Cabib,et al.  The synthesis and degradation of chitin. , 1987, Advances in enzymology and related areas of molecular biology.

[27]  K. Gomi,et al.  Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. , 1987 .

[28]  G. von Heijne,et al.  A new method for predkting signal sequence cleavage sites , 2022 .