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.
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 gpdA gene and pepB 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 gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.
Cardoza
R E
RE
Institute of Biotechnology of León (INBIOTEC), Science Park of León, León, Spain.
Gutiérrez
S
S
Ortega
N
N
Colina
A
A
Casqueiro
J
J
Martín
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000172:deficiency; D001230:Aspergillus / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D014170:Transformation, Genetic
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C034675:preprorennin; C029094:aspergillopepsin I; D012085:Chymosin