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Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH‐regulated promoters and secretion using two different pre‐pro sequences
Author(s) -
Cardoza R. E.,
Gutiérrez S.,
Ortega N.,
Colina A.,
Casqueiro J.,
Martín J. F.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10666
Subject(s) - chymosin , aspergillus awamori , biology , gene , microbiology and biotechnology , plasmid , promoter , gene expression , biochemistry , enzyme
A copy of the bovine chymosin gene ( chy ) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpd A gene and pep B genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self‐processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpd A promoter was constitutive, whereas expression from the pep B promoter was strongly influenced by pH. A very high expression from the pep B promoter was observed during the growth phase. The A. awamori pep B gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 249–259, 2003.