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Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

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Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell—required for the induction of cutinase production—and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.

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  • Bitter GA, Egan KM, Koski RA, Jones MO, Elliott SG, Giffin JC (1987) Expression and secretion vectors for yeast. In: Wu R, Grossman L (eds) Methods in enzymology, vol 153. Academic Press, San Diego, Calif., pp 516–544

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brown R, O'Kennedy RD, Helwigh J, Madden E, Hoare M (2000) Accelerated prediction of recombinant protein production in Saccharomyces cerevisiae by using rapid monitoring techniques. Enzyme Microb Technol 26:801–807

    Article  CAS  PubMed  Google Scholar 

  • Calado CRC, Hamilton GE, Fonseca LP, Cabral JMS, Lyddiatt A (2001) Direct product sequestration of a recombinant cutinase from batch fermentations of S. cerevisiae. Bioseparation 10:87–97

    Article  CAS  PubMed  Google Scholar 

  • Calado CRC, Monteiro SMS, Cabral JMS, Fonseca LP (2002a) Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae. J Biosci Bioeng 93:354–359

    Article  CAS  Google Scholar 

  • Calado CRC, Taipa MA, Cabral JMS, Fonseca LP (2002b) Optimisation of culture conditions and characterization of cutinase produced by recombinant S. cerevisiae. Enzyme Microb Technol 31:161–170

    Article  CAS  Google Scholar 

  • Carvalho CML, Aires-Barros MR, Cabral JMS (1999) Cutinase: from molecular level to bioprocess. Biotechnol Bioeng 66:17–34

    Article  CAS  PubMed  Google Scholar 

  • Cortassa S, Aon MA (1998) Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behaviour in glucose-limited chemostat cultures. Biotechnol Bioeng 59:203–213

    Article  PubMed  Google Scholar 

  • Das RC, Shultz JL (1987) Secretion of heterologous proteins from Saccharomyces cerevisiae. Biotechnol Prog 3:43–48

    CAS  Google Scholar 

  • Ferreira BS, van Keulen F, da Fonseca MMR (1998) Novel calibration method for mass spectrometers for on-line gas analysis. Set-up for the monitoring of a bacterial fermentation. Bioprocess Eng 19:289–296

    Article  CAS  Google Scholar 

  • Gemeren IA van, Musters W, van den Hondel CAMJJ, Verrips CT (1995) Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi. J Biotechnol 40:155–162

    Article  PubMed  Google Scholar 

  • Gimenez JA, Monkovik DD, Dekleva ML (2000) Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 67:245–251

    Article  CAS  PubMed  Google Scholar 

  • 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 cultures. Appl Environ Microbiol 59:52–59

    CAS  PubMed  Google Scholar 

  • Horak J, Wolf DH (1997) Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis and degradation in the vacuole. J Bacteriol 179:1541–1549

    CAS  PubMed  Google Scholar 

  • Horak J, Wolf DH (2001) Glucose-induced monoubiquitination of the Saccharomyces cerevisiae galactose transporter is sufficient to signal its internalization. J Bacteriol 183:3083–3088

    Article  CAS  PubMed  Google Scholar 

  • Kapat A, Jung JK, Park YH (2000) Effect of continuous feeding of galactose on the production of recombinant glucose oxidase using Saccharomyces cerevisiae. Bioprocess Eng 23:37–40

    Article  CAS  Google Scholar 

  • Käppeli O (1986) Regulation of carbon metabolism in Saccharomyces cerevisiae and related yeasts. In: Rose AH, Tempest DW (eds) Advances in microbial physiology, vol 28. Academic Press, London, pp 181–209

  • Lagunas R (1993) Sugar transport in Saccharomyces cerevisiae. FEMS Microbiol Rev 104:229–242

    Article  CAS  Google Scholar 

  • Mathews CK, van Holde KE (1990) Biochemistry. Benjamin/Cummings, Redwood City, Calif.

  • Meijer MMC, Boonstra J, Verkleij AJ, Verrips CT (1998) Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux. J Biol Chem 273:24102–24107

    Article  CAS  PubMed  Google Scholar 

  • Özcan S, Johnston M (1999) Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63:554–569

    CAS  Google Scholar 

  • Park J-B, Kweon Y-E, Rhee S-K, Seo J-H (1995) Production of hirudin by recombinant Saccharomyces cerevisiae in a membrane-recycle fermenter. Biotechnol Lett 17:1031–1036

    CAS  Google Scholar 

  • Rohde JR, Trinh J, Sadowski I (2000) Multiple signals regulate GAL transcription in yeast. Mol Cell Biol 20:3880–3886

    Article  CAS  PubMed  Google Scholar 

  • Sierkstra LN, Nouwen NP, Verbakel JMA, Verrips CT (1992) Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose-limited continuous culture. Yeast 8:1077–1087

    CAS  PubMed  Google Scholar 

  • Smith RA, Duncan MJ, Moir DT (1985) Heterologous protein secretion from yeast. Science 229:1219–1224

    CAS  PubMed  Google Scholar 

  • Stephanopoulos G, Aristidou AA, Nielsen J (1998) Metabolic engineering. Academic Press, San Diego, Calif.

  • Verrips T, Duboc P, Visser C, Sagt C (2000) From gene to product in yeast: production of fungal cutinase. Enzyme Microb Technol 26:812–818

    Article  PubMed  Google Scholar 

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This work was supported by Fundação para a Ciência e a Tecnologia, PRAXIS XXI programme (grant GGP XXI/BD/2936/96 awarded to B.S. Ferreira and grant GGP XXI/BD/18276/98 awarded to C.R.C. Calado). The authors wish to thank Dr. Maarten Egmond, Dr. Maurice Mannessi and Dr. Arthur Fellinger and Unilever Research Laboratory for providing the transformed S. cerevisiae strain.

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Correspondence to B. S. Ferreira.

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Ferreira, B.S., Calado, C.R.C., van Keulen, F. et al. Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects. Appl Microbiol Biotechnol 61, 69–76 (2003).

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