Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Feb;63(2):488–497. doi: 10.1128/aem.63.2.488-497.1997

Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels.

R J Gouka 1, P J Punt 1, C A van den Hondel 1
PMCID: PMC168339  PMID: 9023927

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.

Full Text

The Full Text of this article is available as a PDF (3.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bergkamp R. J., Kool I. M., Geerse R. H., Planta R. J. Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. Curr Genet. 1992 Apr;21(4-5):365–370. doi: 10.1007/BF00351696. [DOI] [PubMed] [Google Scholar]
  2. Broekhuijsen M. P., Mattern I. E., Contreras R., Kinghorn J. R., van den Hondel C. A. Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein. J Biotechnol. 1993 Nov;31(2):135–145. doi: 10.1016/0168-1656(93)90156-h. [DOI] [PubMed] [Google Scholar]
  3. Carrez D., Janssens W., Degrave P., van den Hondel C. A., Kinghorn J. R., Fiers W., Contreras R. Heterologous gene expression by filamentous fungi: secretion of human interleukin-6 by Aspergillus nidulans. Gene. 1990 Oct 15;94(2):147–154. doi: 10.1016/0378-1119(90)90381-z. [DOI] [PubMed] [Google Scholar]
  4. Contreras R., Carrez D., Kinghorn J. R., van den Hondel C. A., Fiers W. Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6. Biotechnology (N Y) 1991 Apr;9(4):378–381. doi: 10.1038/nbt0491-378. [DOI] [PubMed] [Google Scholar]
  5. Fellinger A. J., Verbakel J. M., Veale R. A., Sudbery P. E., Bom I. J., Overbeeke N., Verrips C. T. Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha. Yeast. 1991 Jul;7(5):463–473. doi: 10.1002/yea.320070505. [DOI] [PubMed] [Google Scholar]
  6. Fowler T., Berka R. M. Gene expression systems for filamentous fungi. Curr Opin Biotechnol. 1991 Oct;2(5):691–697. doi: 10.1016/0958-1669(91)90036-5. [DOI] [PubMed] [Google Scholar]
  7. Gouka R. J., Hessing J. G., Punt P. J., Stam H., Musters W., Van den Hondel C. A. An expression system based on the promoter region of the Aspergillus awamori 1,4-beta-endoxylanase A gene. Appl Microbiol Biotechnol. 1996 Aug;46(1):28–35. doi: 10.1007/s002530050779. [DOI] [PubMed] [Google Scholar]
  8. Gouka R. J., Hessing J. G., Stam H., Musters W., van den Hondel C. A. A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori. Curr Genet. 1995 May;27(6):536–540. doi: 10.1007/BF00314444. [DOI] [PubMed] [Google Scholar]
  9. Gouka R. J., Punt P. J., Hessing J. G., van den Hondel C. A. Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains. Appl Environ Microbiol. 1996 Jun;62(6):1951–1957. doi: 10.1128/aem.62.6.1951-1957.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guo Z., Sherman F. 3'-end-forming signals of yeast mRNA. Mol Cell Biol. 1995 Nov;15(11):5983–5990. doi: 10.1128/mcb.15.11.5983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hessing J. G., van Rotterdam C., Verbakel J. M., Roza M., Maat J., van Gorcom R. F., van den Hondel C. A. Isolation and characterization of a 1,4-beta-endoxylanase gene of A. awamori. Curr Genet. 1994 Sep;26(3):228–232. doi: 10.1007/BF00309552. [DOI] [PubMed] [Google Scholar]
  12. Humphrey T., Proudfoot N. J. A beginning to the biochemistry of polyadenylation. Trends Genet. 1988 Sep;4(9):243–245. doi: 10.1016/0168-9525(88)90028-5. [DOI] [PubMed] [Google Scholar]
  13. Irniger S., Egli C. M., Braus G. H. Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3060–3069. doi: 10.1128/mcb.11.6.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jeenes D. J., Mackenzie D. A., Archer D. B. Transcriptional and post-transcriptional events affect the production of secreted hen egg white lysozyme by Aspergillus niger. Transgenic Res. 1994 Sep;3(5):297–303. doi: 10.1007/BF01973589. [DOI] [PubMed] [Google Scholar]
  15. Kolar M., Punt P. J., van den Hondel C. A., Schwab H. Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene. 1988;62(1):127–134. doi: 10.1016/0378-1119(88)90586-0. [DOI] [PubMed] [Google Scholar]
  16. Manley J. L. Polyadenylation of mRNA precursors. Biochim Biophys Acta. 1988 May 6;950(1):1–12. doi: 10.1016/0167-4781(88)90067-x. [DOI] [PubMed] [Google Scholar]
  17. Nyyssönen E., Keränen S. Multiple roles of the cellulase CBHI in enhancing production of fusion antibodies by the filamentous fungus Trichoderma reesei. Curr Genet. 1995 Jun;28(1):71–79. doi: 10.1007/BF00311884. [DOI] [PubMed] [Google Scholar]
  18. Nyyssönen E., Penttilä M., Harkki A., Saloheimo A., Knowles J. K., Keränen S. Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei. Biotechnology (N Y) 1993 May;11(5):591–595. doi: 10.1038/nbt0593-591. [DOI] [PubMed] [Google Scholar]
  19. Overbeeke N., Fellinger A. J., Toonen M. Y., van Wassenaar D., Verrips C. T. Cloning and nucleotide sequence of the alpha-galactosidase cDNA from Cyamopsis tetragonoloba (guar). Plant Mol Biol. 1989 Nov;13(5):541–550. doi: 10.1007/BF00027314. [DOI] [PubMed] [Google Scholar]
  20. Overbeeke N., Termorshuizen G. H., Giuseppin M. L., Underwood D. R., Verrips C. T. Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis. Appl Environ Microbiol. 1990 May;56(5):1429–1434. doi: 10.1128/aem.56.5.1429-1434.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Proudfoot N. Poly(A) signals. Cell. 1991 Feb 22;64(4):671–674. doi: 10.1016/0092-8674(91)90495-k. [DOI] [PubMed] [Google Scholar]
  22. Roberts I. N., Jeenes D. J., MacKenzie D. A., Wilkinson A. P., Sumner I. G., Archer D. B. Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme. Gene. 1992 Dec 1;122(1):155–161. doi: 10.1016/0378-1119(92)90043-o. [DOI] [PubMed] [Google Scholar]
  23. Roberts I. N., Oliver R. P., Punt P. J., van den Hondel C. A. Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Curr Genet. 1989 Mar;15(3):177–180. doi: 10.1007/BF00435503. [DOI] [PubMed] [Google Scholar]
  24. Romanos M. A., Makoff A. J., Fairweather N. F., Beesley K. M., Slater D. E., Rayment F. B., Payne M. M., Clare J. J. Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA. Nucleic Acids Res. 1991 Apr 11;19(7):1461–1467. doi: 10.1093/nar/19.7.1461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Romanos M. A., Scorer C. A., Clare J. J. Foreign gene expression in yeast: a review. Yeast. 1992 Jun;8(6):423–488. doi: 10.1002/yea.320080602. [DOI] [PubMed] [Google Scholar]
  26. Scorer C. A., Buckholz R. G., Clare J. J., Romanos M. A. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene. 1993 Dec 22;136(1-2):111–119. doi: 10.1016/0378-1119(93)90454-b. [DOI] [PubMed] [Google Scholar]
  27. Ward M., Wilson L. J., Kodama K. H., Rey M. W., Berka R. M. Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Biotechnology (N Y) 1990 May;8(5):435–440. doi: 10.1038/nbt0590-435. [DOI] [PubMed] [Google Scholar]
  28. Ward P. P., Piddington C. S., Cunningham G. A., Zhou X., Wyatt R. D., Conneely O. M. A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Biotechnology (N Y) 1995 May;13(5):498–503. doi: 10.1038/nbt0595-498. [DOI] [PubMed] [Google Scholar]
  29. Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci. 1990 Jul;15(7):277–281. doi: 10.1016/0968-0004(90)90054-f. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES