Controlled Expression and Secretion of Bovine Chymosin in Aspergillus Nidulans

Abstract

To test the ability of the filamentous fungus Aspergillus nidulans to secrete bovine prochymosin, four plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. niger glucoamylase gene were functionally coupled to either prochymosin or preprochymosin cDNA. Three plasmid constructions involved the in–frame fusion of prochymosin coding sequences to glucoamylase sequences at 1) the glucoamylase signal peptide cleavage site, 2) the glucoamylase propeptide cleavage site, or 3) after 11 codons of the mature glucoamylase. In a fourth construction, preprochymosin was directly fused to the glucoamylase promoter. In all four constructions, the glucoamylase terminator was fused to the 3′ end of the prochymosin coding sequence. Secretion of polypeptides enzymatically and immunologically indistinguishable from bovine chymosin was achieved following transformation of A. nidulans with each of these plasmids. In all cases the primary translation product was partially processed to a polypeptide having a molecular weight similar to bovine chymosin. Synthesis of the chymosin polypeptides was induced in a medium that contained starch as the sole carbon source, whereas little or no expression was detected when xylose was the sole carbon source. Immunological assays indicated that the majority (>90%) of chymosin was extracellular. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences, and for some transformants, multiple copies were observed.