Appl Microbiol Biotechnol (2005) 67: 684–691 DOI 10.1007/s00253-004-1803-3 APPLIED MICROBIAL AND CELL PHYSIOLOGY Johann F. Görgens . Willem H. van Zyl . Johannes H. Knoetze . Bärbel Hahn-Hägerdal Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium Received: 23 August 2004 / Revised: 22 September 2004 / Accepted: 19 October 2004 / Published online: 4 January 2005 # Springer-Verlag 2005 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 correspond- ing xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylan- ase 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 re- combinant 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. Introduction Cultivation of recombinant yeast in defined media for heterologous protein production is advantageous due to easier quantification of growth requirements, simpler pu- rification of the product and the propensity towards rapid scale-up (Greasham and Herber 1997). In the present study, the possible limitation of heterologous protein pro- duction in recombinant Saccharomyces cerevisiae by an insufficient availability of amino acids or succinate during growth in defined medium was investigated. A prototro- phic recombinant S. cerevisiae strain producing a fungal xylanase under the transcriptional control of the ADH2 promoter was used as a model system. Compared to pro- duction levels in complex medium, very low production levels of heterologous xylanases by auxotrophic recombi- nant S. cerevisiae in defined media have been reported (Donald et al. 1994; Pérez-González et al. 1996; La Grange et al. 1996; Nuyens et al. 2001; our unpublished results). In an auxotrophic recombinant S. cerevisiae strain, heterologous xylanase production improved dra- matically by supplementation of the defined medium with an excess of amino acids (Görgens et al. 2004). An ap- parent limitation in the availability of amino acid building blocks for heterologous xylanase production by auxotro- phic yeasts thus existed. In the present study, a proto- trophic strain was used to investigate the presence of a similar limitation for amino acids or succinate for hete- rologous xylanase production. The defined medium was supplemented during growth on ethanol in batch culture or during glucose-limited growth in chemostats, when the native ADH2 promoter, used to regulate xylanase expres- sion, was derepressed. Supplementation of defined media with exogenous nitrogen sources has previously improved biomass forma- tion by S. cerevisiae, especially during fully respiratory growth on ethanol (Chen et al. 1993; Gu et al. 1991; Thomas and Ingledew 1990, 1992). Nitrogen sources most strongly preferred by S. cerevisiae include glutamine, asparagine and ammonium. These compounds are utilised first from mixtures of nitrogen sources and support higher growth rates than less preferred nitrogen sources (Ter Schure et al. 2000; Dubois and Messenguy 1997; Wiame et al. 1985; Cooper 1982). Exogenous amino acids can be incorporated directly into biomass during consumption (Albers et al. 1996), whilst amino acids have been known to improve the production of heterologous proteins by S. cerevisiae in defined media (Mendoza-Vega et al. 1994; J. F. Görgens . B. Hahn-Hägerdal (*) Department of Applied Microbiology, Lund University, Box 124, 221 00 Lund, Sweden e-mail: Barbel.hahn-hagerdal@tmb.lth.se Fax: +46 46 222 4203 J. F. Görgens . J. H. Knoetze Department of Process Engineering, University of Stellenbosch, South Africa W. H. van Zyl Department of Microbiology, University of Stellenbosch, South Africa