Abstract
For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeastHansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase ( (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using theMOX promoter ofH. polymorpha and the mating factor α secretion signal ofS. cerevisiae. Multiple integrants of a transformang vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression inH. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.
Similar content being viewed by others
References
Hollenberg, C. P. and G. Gellissen (1997) Production of recombinant proteins by methylotrophic yeasts.Curr. Opinion in Biotechnol. 8: 554–560.
Hollenberg, C. P. and Z. A. Janowicz (1987) DNA Molecules encoding for FMDH control region.European. Patent Application. EPA No. 0299108.
Gatzke, R. U., U. Weydemann, Z. A. Janowicz, and C. P. Hollenberg (1995) Stable muticopy integration of vector sequences inHansenula polymorpha.Appl. Microbiol. Biotechnol. 43: 844–849.
Kim, C. H., H. W. Seo, H. Y. Kim, J. H. Sohn, E. S. Choi, and S. K. Rhee (1998) Production of recombinant hirudin inHansenula polymorpha: Variation of gene expression level depends on methanol oxidase and fermentation strategies.J. Ind. Microbiol. Biotechnol. 21: 1–5.
Roggenkamp, R. O., H. Hansen, M. Eckart, Z. A. Janowicz, and C. P. Hollenberg (1986) Transformation of the methylotrophic yeastHansenula polymorpha by autonomous replication and integration vectors.Mol. Gen. Genet. 202: 302–308.
Weydemann, U., P. Keup, M. Pionitek, A. W. M. Strasser, J. Schweden, G. Gellissen, and Z. A. Janowicz (1995) Highlevel secretion of hirudin byHansenula polymorpha— Authentic processing of three different preprohirudins.Appl. Microbiol. Biotechnol. 44: 377–385.
Janowicz, Z. A., K. Melber, A. Merckelbach, E. Jacobs, N. Harford, M. Comberbach, and C. P. Hollenberg (1991) Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast,Hansenula polymorpha.Yeast 7: 431–443.
Gellisen, G., C. P. Hollenberg, and Z. A. Janowicz, 1994. Gene expression in recombinant yeast. pp. 195–239. In: A. Smith, (ed.),Gene Expression in Recombinant Microorganisms, Marcel Dekker, New York.
Sohn, J. H., E. S. Choi, H. A. Kang, M. O. Agaphonov, M. D. Teavanesyan, J. S. Rhee, and S. K. Rhee (1999) A dominant selection system designed for the copy-number controlled gene integration inHansenula polymorpha DL-1.Appl. Microbiol. Biotechnol. 51: 800–807.
Agaphonov, M. O., P. M. Trushkina, J. H. Sohn, E. S. Choi, S. K. Rhee, and M. D. Teavanesyan (1999) Vectors for rapid selection of intergrants with different plasmid copy numbers in the yeastHansenula polvmorpha DL1.Yeast 15: 541–555.
Harvey, R. P., E. Degryse, L. Stefani, F. Schamber, J.-P. Cazenave, M. Courtney, P. Tolstoshev, and J.-P. Lecocq (1986) Cloning and expression of a cDNA encoding for the anticoagulant hirudin from the bloodsucking leech,Hirudo medicinalis.Proc. Natl. Acad. Sci. U.S.A. 83: 1084–1088.
Hill, J., K. A. G. Donald, and D. E. Griffiths (1991) DMSO-enhanced whole cell yeast transformation.Nucleic Acid Res. 19: 5791.
Inoue, H., H. Nojima, and H. Okayama (1990) High efficiency transformation ofEscherichia coli with plasmids.Gene 96: 23–28.
Sambrook, J., F. Fritsch, and T. Maniatis (1989)Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A.
Johnston, J. R. (1988) Yeast genetics, molecular aspects. pp. 107–123. In: I. Campbell and J. H. Duffus (ed.),Yeast, a Practical Approach, IRL Press, Oxford.
Sohn, J. H., S. K. Lee, E. S. Choi, and S. K. Rhee (1991) Gene expression and secretion of the anticoagulant hirudin inSaccharomyces cerevisiae.J. Microbiol. Biotechnol. 1: 266–273.
Sohn, J. H., E. S. Choi, H. A. Kang, J. S. Rhee, and S. K. Rhee (1999) A family of telomere-associate ARSs and their funchings for targeted recombination inHansenula polymorpha DL-1.J. Bacteriol. 181: 1005–1013.
Degryse, E. (1990) Influence on the second and third codon on the expression of recombinant hirudin inE. coli.FEBS Lett. 269: 244–246.
Ejdeback, M., S. Young, A. Samuelsson, and C. Karlsson (1997) Effects of codon usage and vectohost combination on the expression of spinach plastocyanin inE. coli.Protein Exp. Pur. 11: 17–25.
Hoekema, A., R. A. Kastelein, M. Vasser, and H. A. De Boer (1987) Codon replacement in the PGK1 gene ofSaccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.Mol. Cell. Biol. 7: 2914–2924.
Looman, A. C., M. Laude, and U. Stahl (1991) Influence of the codon following the initial codon on the expression of thelacZ gene inSaccharomyces cerevisiae.Yeast 7: 157–165.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kim, H.Y., Sohn, J.H., Kim, C.H. et al. Rapid selection of multiple gene integrant for the production of recombinant hirudin inHansenula polymorpha . Biotechnol. Bioprocess Eng. 5, 1–6 (2000). https://doi.org/10.1007/BF02932344
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02932344