GLUCOAMYLASE GENE FUSIONS ALLEVIATE LIMITATIONS FOR PROTEIN-PRODUCTION IN ASPERGILLUS-AWAMORI AT THE TRANSCRIPTIONAL AND (POST)TRANSLATIONAL LEVELS

In this study we have analyzed the effects of a glucoamylase gene fusi on on the mRNA levels and protein levels for the human interleukin-6 g ene (hil6) and the guar alpha-galactosidase gene (aglA). Previously it was shown that production of nonfused alpha-galactosidase and hIL-6 i n Aspergillus awamori was limited at transcriptional and (post)transla tional levels, respectively (R. J. Gouka, P. J. Punt, J. G. M. Hessing , and C. A. M. J. J. van den Hondel, Appl. Environ. Microbiol. 62:1951 -1957, 1996). Vectors were constructed which contained either the hil6 or aglA gene fused to the Aspergillus niger glucoamylase gene (glaA) under control of the efficient 1,4-beta-endoxylanase A promoter and tr anscription terminator, For comparison, the vectors were integrated in a single copy at the pyrG locus of A. awamori. A glaA fusion to the 5 ' end of the hil6 gene resulted in a large increase in hIL-6 yield, wh ereas, with a glaA fusion to the 3' end of the hi16 gene, almost no pr otein was produced, Nevertheless, the steady-state mRNA levels of both fusions were very similar and not clearly increased compared to those of a strain expressing nonfused hIL-6. Fusions of glaA to the 5' end of the wild-type guar aglA gene resulted in truncated mRNA lacking alm ost 900 bases (>80%) of the agLA sequence. When the coding sequence of the wild-type aglA gene was replaced by a synthetic aglA gene with op timized Saccharomyces cerevisiae codon usage, full length mRNA was obt ained, Compared to a nonfused synthetic aglA gene, a glaA fusion with the synthetic aglA gene resulted in a 25-fold increase in the mRNA lev el and, as a consequence, a similar increase in the alpha-galactosidas e protein level. The truncated transcripts derived from the wild-type aglA gene were further analyzed by nuclear run-on transcription assays , These experiments indicated that transcription elongation in the nuc leus proceeded at least 400 bases downstream of the site where the tru ncation was determined, indicating that transcription elongation or pr emature termination was not the reason for the generation of truncated mRNAs. As the truncated mRNA also contained a poly(A) tail, truncatio n most likely occurs by incorrect processing of the nglA mRNA in the n ucleus.