Abstract
Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar α-galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular α-galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of α-galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.
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References
Bergkamp RJM, Kool IM, Geerse RH, Planta RJ (1992) Multiple-copy integration of — the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. Curr Genet 21:365–370
Bulpin PV, Gidley MJ, Jeffcoat R, Underwood DR (1990) Development of a biotechnological process for the modification of galactomannan polymers with plant α-galactosidase. Carbohydr Res 12:155–168
Dickson RC, Riley MI (1989) The lactose-galactose regulon of Kluyveromyces lactis. In: Barr PJ, Brake AJ, Valenzuela P (eds) Yeast genetic engineering. Butterworth, Boston, pp 19–40
Fellinger AJ, Verbakel, JMA, Veale RA, Sudbery PE, Bom IJ, Overbeeke N, Verrips CT (1991) Expression of the α-galactosidase from Cyamopsis tetragonoloba (Guar) by Hansenula polymorpha. Yeast 7:463–473
Fleer R, Chen XJ, Amellal N, Yeh P, Fournier A, Guinet F, Gault N, Faucher D, Folliard F, Fukuhara H, Mayaux J (1991a) High-level secretion of correctly processed recombinant human interleukin-1β in Kluyveromyces lactis. Gene 107:285–295
Fleer R, Yeh P, Amellal N, Maury I, Fournier A, Bacchetta F, Baduel P, Jung G, L'Hôte H, Becquart J, Fukuhara J, Mayaux JF (1991b) Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts. Biotechnol 9:968–975
Giuseppin MLF, Almkerk JW, Heistek JC, Verrips CT (1993) Comparative study on the production of guar α-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous culture. Appl Environ Microbiol 59:52–59
Goodey AR (1993) The production of heterologous plasma proteins. Trends Biotechnol 11:430–433
Innis MA (1989) Glycosylation of heterologous proteins in Saccharomyces cerevisiae. In: Barr PJ, Brake JA, Valenzuela P (eds) Yeast genetic engineering. Butterworth, Boston, pp 233–246
Kingsman SM, Kingsman AJ, Mellor J (1987) The production of mammalian proteins in Saccharomyces cerevisiae. Trends Biotechnol 5:53–57
LaRue TA, Spencer JFT (1968) The utilization of purines and pyrimidines by yeasts. Can J Microbiol 14:79–86
Lopes TS, Klootwijk J, Veenstra AE, Van der Aar PC, Van Heerikhuizen H, Raué HA, Planta RJ (1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79:199–206
Martinez E, Morales J, Aguiar J, Pineda Y, Izguiredo M, Ferbeyre G (1992) Cloning and expression of hepatitis b surface antigen in the yeast Kluyveromyces lactis. Biotechnol Lett 14:83–86
Meier H, Reed JSG (1982) Reserve polysaccharides other than starch in higher plants. In: Loewus FA, Tanner W (eds) Encyclopedia of plant physiology, New Series 13A. Springer Verlag, New York, pp 418–471
Overbeeke N, Termorshuizen GHM, Giuseppin MLF, Underwood DR, Verrips CT (1990) Secretion of the α-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis. Appl Environ Microbiol 56:1429–1434.
Petrik M, Käppeli O, Fiechter A (1983) An expanded concept for the glucose effect in Saccharomyces uvarum: involvement of short-term and long-term regulation. J Gen Microbiol 129:43–49
Postma E, Scheffers WA, Van Dijken JP (1988) Adaptation of the kinetics of glucose transport to environmental conditions in the yeast Candida utilis CBS 621: a continuous culture study. J Gen Microbiol 134:1109–1116
Roels JA (1983) Energetics and kinetics in biotechnology, Elsevier Biomedical Press, Amsterdam, The Netherlands
Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review Yeast 8:423–488
Rossolini GM, Riccio ML, Gallo E, Galeotti CL (1992) Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination. Gene 119:75–81
Sierkstra LN, Verbakel JMA, Verrips CT (1991) Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha. Curr Genet 119:81–87
Stark MJR, Milner JS (1989) Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histon H3. Yeast 5:35–50
Stouthamer AH, van Verseveld HW (1987) Microbial energetics should be considered in manipulating metabolism for biotechnological purposes. Trends Biotechnol 5:149–155
Van den Berg JA, Van der Laken KJ, Van Ooyen AJJ, Renniers TCHM, Rietveld K, Schaap A, Brake AJ, Bishop RJ, Schultz K, Moyer D, Richman M, Shuster JR (1990) Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Biotechnol 8:135–139
Van der Aar PC, Lopes TS, Klootwijk J, Groeneveld P, Van Verseveld HW, Stouthamer AH (1990) Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 32:577–587
Veale RA, Giuseppin MLF, Eijk HMJ van, Sudbery PE, Verrips CT (1992) Development of a strain of Hansenula polymorpha for the efficient expression of guar α-galactosidase. Yeast 8:361–372
Verbakel JMA (1991) Heterologous gene expression in the yeast Saccharomyces cerevisiae. PhD thesis 1991, University of Utrecht, The Netherlands
Verduyn C (1991) Physiology of yeasts in relation to growth yields. Antonie van Leeuwenhoek 60:325–353
Verduyn C, Postma E, Scheffers WA, Dijken JP van (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517
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Hensing, M.C.M., Bangma, K.A., Raamsdonk, L.M. et al. Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis . Appl Microbiol Biotechnol 43, 58–64 (1995). https://doi.org/10.1007/BF00170623
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DOI: https://doi.org/10.1007/BF00170623