title: Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels.
abstract: In this study we have analyzed the effects of a glucoamylase gene fusion on the mRNA levels and protein levels for the human interleukin-6 gene (hil6) and the guar alpha-galactosidase gene (aglA). Previously it was shown that production of nonfused alpha-galactosidase and hIL-6 in Aspergillus awamori was limited at transcriptional and (post)translational 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 transcription 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, whereas with a glaA fusion to the 3' end of the hil6 gene, almost no protein 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 almost 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 optimized Saccharomyces cerevisiae codon usage, full-length mRNA was obtained. Compared to a nonfused synthetic aglA gene, a glaA fusion with the synthetic aglA gene resulted in a 25-fold increase in the mRNA level and, as a consequence, a similar increase in the alpha-galactosidase 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 nucleus proceeded at least 400 bases downstream of the site where the truncation was determined, indicating that transcription elongation or premature termination was not the reason for the generation of truncated mRNAs. As the truncated mRNA also contained a poly(A) tail, truncation most likely occurs by incorrect processing of the aglA mRNA in the nucleus.
authors.affiliation: Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, Rijswijk, The Netherlands.
authors.forename: R J
authors.initials: RJ
authors.lastname: Gouka
authors.forename: P J
authors.initials: PJ
authors.lastname: Punt
authors.forename: C A
authors.initials: CA
authors.lastname: van den Hondel
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D005685:Galactans; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D008351:Mannans; D008969:Molecular Sequence Data; D053149:Plant Gums; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014158:Transcription, Genetic; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D005685:Galactans; D015850:Interleukin-6; D008351:Mannans; D053149:Plant Gums; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C007894:guar gum; D000519:alpha-Galactosidase; D005087:Glucan 1,4-alpha-Glucosidase