Skip to main content
SpringerLink
Log in

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium

  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Adams MR, Bryan JJ, Thurston PJ (1989) A medium designed for monitoring pitching yeast contamination in beer using a conductimetric technique. Lett Appl Microbiol 8:55–58

    CAS  Google Scholar 

  • Albers E, Larsson C, Lidén G, Niklasson C, Gustafsson L (1996) Influence of nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl Environ Microbiol 62:3187–3195

    CAS  PubMed  Google Scholar 

  • Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270

    Article  CAS  Google Scholar 

  • Barkholt V, Jensen AL (1989) Amino acid analysis determination of cysteine plus half-cysteine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal Biochem 177:318–322

    CAS  PubMed  Google Scholar 

  • Blechl AE, Thrasher KS, Vensel WH, Greene FC (1992) Purification and characterization of wheat α-gliadin synthesized in the yeast Saccharomyces cerevisiae. Gene 116:119–127

    Article  CAS  PubMed  Google Scholar 

  • Boze H, Celine L, Patrick C, Fabien R, Christine V, Yves C, Guy M (2001) High-level secretory production of recombinant porcine follicle-stimulating hormone by Pichia pastoris. Process Biochem 36:907–913

    Article  CAS  Google Scholar 

  • Cha HJ, Kim M-H, Kim SH, Yeo JS, Chae HJ, Yoo YJ (1998) Enhancement, by succinate addition, of the production of cloned glucoamylase from recombinant yeast using a SUC2 promoter. Process Biochem 33:257–261

    Article  CAS  Google Scholar 

  • Chen Y, Kirk N, Piper PW (1993) Effects of medium composition on MFα1 promoter-directed secretion of a small protease inhibitor in Saccharomyces cerevisiae batch fermentation. Biotechnol Lett 15:223–228

    Article  CAS  Google Scholar 

  • Choi W-A, Oh GH, Kang HA, Chung BH (2000) Improvement of intact human lipocortin-I production in Saccharomyces cerevisiae by inhibiting proteolysis. J Biosci Bioeng 89:77–80

    Article  CAS  Google Scholar 

  • Chung BH, Park KS (1998) Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae. Biotechnol Bioeng 57:245–249

    Article  CAS  PubMed  Google Scholar 

  • Cooper TG (1982) Nitrogen metabolism in Saccharomyces cerevisiae. In Strathern JN, Jones EW, Broach JR (eds), The molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbour Laboratory, pp 39–100

  • Coppella SJ, Dhurjati P (1989) α-Factor directed expression of the human epidermal growth factor in Saccharomyces cerevisiae. Biotechnol Bioeng 33:976–983

    CAS  Google Scholar 

  • Donald KAG, Carle A, Gibbs MD, Bergquist PL (1994) Production of a bacterial thermophilic xylanase in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 42:309–312

    Article  CAS  Google Scholar 

  • Dubois E, Messenguy F (1997) Integration of the multiple controls regulating the expression of the arginase gene CAR1 of Saccharomyces cerevisiae in response to different nitrogen signals: role of Gln3p, ArgRp-Mcm1p, and Ume6p. Mol Gen Genet 253:568–580

    Article  CAS  PubMed  Google Scholar 

  • Du Preez JC, Mare JE, Albertyn J, Kilian SG (2001) Transcriptional repression of ADH2-regulated β-xylanase production by ethanol in recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res 1:233–240

    Article  PubMed  Google Scholar 

  • Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H, Wan NC (2001) High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system. Biotechnol Bioeng 74:492–497

    Article  CAS  PubMed  Google Scholar 

  • Görgens JF, Van Zyl WH, Knoetze JH, Hahn-Hägerdal B (2001) The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium. Biotechnol Bioeng 73:238–245

    Article  PubMed  Google Scholar 

  • Görgens JF, Planas J, van Zyl WH, Knoetze JH, Hahn-Hägerdal B (2004) Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium. Yeast 21:1205–1217

    Article  PubMed  Google Scholar 

  • Greasham RL, Herber WK (1997) Design and optimization of growth media. In: Rhodes, PM, Stanbury PF (eds) Applied microbial physiology—a practical approach. Oxford University Press, Oxford, pp 53–74

    Google Scholar 

  • Grenson M (1992) Amino acid transporters in yeast: structure, function and regulation. In: Molecular aspects of transport proteins. Elsevier, Amsterdam, pp 219–245

  • Gu MB, Park MH, Kim D-I (1991) Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg). Appl Microbiol Biotechnol 35:46–50

    Article  CAS  PubMed  Google Scholar 

  • Herraiz T, Ough CS (1993) Formation of ethyl esters of amino acids by yeasts during the alcoholic fermentation of grape juice. Am J Enol Viticul 44:41–48

    CAS  Google Scholar 

  • Horák J (1997) Yeast nutrient transporters. Biochim Biophys Acta 1331:41–79

    PubMed  Google Scholar 

  • Jin S, Ye K, Shimizu K (1997) Metabolic flux distributions in recombinant Saccharomyces cerevisiae during foreign protein production. J Biotechnol 54:161–174

    Article  CAS  PubMed  Google Scholar 

  • Jiranek V, Langridge P, Henschke PA (1995) Amino acid and ammonium utilization by Saccharomyces cerevisiae wine yeasts from a chemically defined medium. Am J Enol Vitic 46:75–83

    CAS  Google Scholar 

  • Jones EW (1991) Tackling the protease problem in Saccharomyces cerevisiae. In: Guthrie C, Fink GR (eds) Methods in enzymology, guide to yeast genetics and molecular biology, vol 194. Academic Press, San Diego, pp 428–452

    Google Scholar 

  • Kaclikova E, Lachowicz TM, Gbelska Y, Subik J (1992) Fumaric acid overproduction in yeast mutants deficient in fumarase. FEMS Microbiol Lett 91:101–106

    Article  CAS  Google Scholar 

  • Kang HA, Choi E-S, Hong W-K, Kim J-Y, Ko S-M, Sohn J-H, Rhee SK (2000) Proteolytic stability of recombinant humans serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:575–582

    Article  CAS  PubMed  Google Scholar 

  • La Grange DC, Pretorius IS, Van Zyl WH (1996) Expression of a Trichoderma reesei β-xylanase gene (XYN2) in Saccharomyces cerevisiae. Appl Environ Microbiol 62:1036–1044

    PubMed  Google Scholar 

  • Mendoza-Vega O, Sabatie J, Brown SW (1994) Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 15:369–410

    Article  CAS  PubMed  Google Scholar 

  • Messenguy F, Colin D, Ten Have J-P (1980) Regulation of compartmentalisation of amino acid pools in Saccharomyces cerevisiae and its effects on metabolic control. Eur J Biochem 108:439–447

    CAS  PubMed  Google Scholar 

  • Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethylcellulase activity. Anal Biochem 2:127–132

    Article  Google Scholar 

  • Nuyens F, Van Zyl WH, Iserentant D, Verachtert H, Michiels C (2001) Heterologous expression of the Bacillus pumilus endo-β-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 56:431–434

    Article  CAS  PubMed  Google Scholar 

  • Ogrydziak DM (1993) Yeast extracellular proteases. Crit Rev Biotechnol 13: 1–55

    Google Scholar 

  • Pérez-González JA, De Graaff LH, Visser J, Ramón D (1996) Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes. Appl Environ Microbiol 62:2179–2182

    PubMed  Google Scholar 

  • Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. In: Guthrie C, Fink GR (eds) Methods in enzymology, guide to yeast genetics and molecular biology, vol 194. Academic Press, San Diego, pp 302–318

    Google Scholar 

  • Slaughter JC, McKernan G, Saita M (1990) Intracellular asparagine pool as a factor in control of ammonium uptake by Saccharomyces cerevisiae. Mycol Res 94:1009–1012

    CAS  Google Scholar 

  • Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay J-T, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in methylotropic yeast Pichia pastoris. Gene 190:55–62

    Article  CAS  PubMed  Google Scholar 

  • Ter Schure EG, Van Riel NAW, Verrips CT (2000) The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae. FEMS Microbiol Rev 24:67–83

    Article  CAS  PubMed  Google Scholar 

  • Thomas KC, Ingledew WM (1990) Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes. Appl Environ Microbiol 56:2046–2050

    CAS  PubMed  Google Scholar 

  • Thomas KC, Ingledew WM (1992) Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mashes. Can J Microbiol 38:626–634

    CAS  PubMed  Google Scholar 

  • Toman PD, Chisholm G, McMullin H, Giere LM, Olsen DR, Kovach RJ, Leigh SD, Fong BE, Chang R, Daniels GA, Berg RA, Hitzeman RA (2000) Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae. J Biol Chem 275:23303–23309

    Article  CAS  PubMed  Google Scholar 

  • Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid metabolism on metabolic fluxes in yeast: a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–507

    CAS  PubMed  Google Scholar 

  • Werten MWT, Van den Bosch TJ, Wind RD, Mooibroek H, De Wolf FA (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1087–1096

    Article  CAS  PubMed  Google Scholar 

  • Wiame J-M, Grenson M, Arst HN (1985) Nitrogen catabolite repression in yeasts and filamentous fungi. Adv Microb Phys 26:1–88

    Google Scholar 

  • Wittrup KD, Benig V (1994) Optimisation of amino acid supplements for heterologous protein secretion in Saccharomyces cerevisiae. Biotechnol Tech 8:161–166

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Ms Jenny Ågren (Lund University) and Mr Henk Blignault (Stellenbosch University) are gratefully acknowledged for technical assistance during screening of the medium components.

Author information

Authors and Affiliations

  1. Department of Applied Microbiology, Lund University, Box 124, 221 00, Lund, Sweden

    Johann F. Görgens & Bärbel Hahn-Hägerdal

  2. Department of Process Engineering, University of Stellenbosch, South Africa

    Johann F. Görgens & Johannes H. Knoetze

  3. Department of Microbiology, University of Stellenbosch, South Africa

    Willem H. van Zyl

Authors
  1. Johann F. Görgens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Willem H. van Zyl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johannes H. Knoetze
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bärbel Hahn-Hägerdal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bärbel Hahn-Hägerdal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Görgens, J.F., van Zyl, W.H., Knoetze, J.H. et al. Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium. Appl Microbiol Biotechnol 67, 684–691 (2005). https://doi.org/10.1007/s00253-004-1803-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-004-1803-3

Keywords

Search

Navigation