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. 1996 Jun;62(6):1951–1957. doi: 10.1128/aem.62.6.1951-1957.1996

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

R J Gouka 1, P J Punt 1, J G Hessing 1, C A van den Hondel 1
PMCID: PMC167973  PMID: 8787393

Abstract

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

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Selected References

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  1. Atwater J. A., Wisdom R., Verma I. M. Regulated mRNA stability. Annu Rev Genet. 1990;24:519–541. doi: 10.1146/annurev.ge.24.120190.002511. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Dunn-Coleman N. S., Bloebaum P., Berka R. M., Bodie E., Robinson N., Armstrong G., Ward M., Przetak M., Carter G. L., LaCost R. Commercial levels of chymosin production by Aspergillus. Biotechnology (N Y) 1991 Oct;9(10):976–981. doi: 10.1038/nbt1091-976. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Frenken L. G., Egmond M. R., Batenburg A. M., Bos J. W., Visser C., Verrips C. T. Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues. Appl Environ Microbiol. 1992 Dec;58(12):3787–3791. doi: 10.1128/aem.58.12.3787-3791.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Haas I. G., Wabl M. Immunoglobulin heavy chain binding protein. Nature. 1983 Nov 24;306(5941):387–389. doi: 10.1038/306387a0. [DOI] [PubMed] [Google Scholar]
  12. Hentze M. W. Determinants and regulation of cytoplasmic mRNA stability in eukaryotic cells. Biochim Biophys Acta. 1991 Nov 11;1090(3):281–292. doi: 10.1016/0167-4781(91)90191-n. [DOI] [PubMed] [Google Scholar]
  13. 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]
  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. Knittler M. R., Dirks S., Haas I. G. Molecular chaperones involved in protein degradation in the endoplasmic reticulum: quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1764–1768. doi: 10.1073/pnas.92.5.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Metwally M., el Sayed M., Osman M., Hanegraaf P. P., Stouthamer A. H., van Verseveld H. W. Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures of Aspergillus niger. Antonie Van Leeuwenhoek. 1991 Jan;59(1):35–43. doi: 10.1007/BF00582117. [DOI] [PubMed] [Google Scholar]
  18. 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]
  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. Punt P. J., van den Hondel C. A. Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol. 1992;216:447–457. doi: 10.1016/0076-6879(92)16041-h. [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. Shamu C. E., Cox J. S., Walter P. The unfolded-protein-response pathway in yeast. Trends Cell Biol. 1994 Feb;4(2):56–60. doi: 10.1016/0962-8924(94)90011-6. [DOI] [PubMed] [Google Scholar]
  25. Van Snick J., Cayphas S., Vink A., Uyttenhove C., Coulie P. G., Rubira M. R., Simpson R. J. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9679–9683. doi: 10.1073/pnas.83.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Verdoes J. C., Calil M. R., Punt P. J., Debets F., Swart K., Stouthamer A. H., van den Hondel C. A. The complete karyotype of Aspergillus niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies. Mol Gen Genet. 1994 Jul 8;244(1):75–80. doi: 10.1007/BF00280189. [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]

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