Abstract In order to evaluate the potential of Saccharo- myces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae- based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the con- trol of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultiva- tions on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluy- veri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain. Introduction The advantages of using Saccharomyces cerevisiae as host for the production of proteins from higher eukary- otes are numerous, including ease of genetic manipula- tion, ability of performing post-translational modifica- tions, ease of growth and well established industrial pro- cesses. The major disadvantages are the low levels of ex- pression of foreign genes and an inefficient secretion ap- paratus (Romanos et al. 1992). Furthermore, the Crabtree effect exhibited by S. cerevisiae necessitates fed-batch cultivation, which has to be operated at low dilution rates to prevent reduction in biomass yield and build-up of toxic levels of metabolites (Hensing et al. 1995; Pronk et al. 1996). Several other yeasts have been employed for heterologous protein production, e.g. Hansenula poly- morpha, Kluyveromyces lactis, Pichia pastoris, Schizo- saccharomyces pompe, Schwanniomyces occidentalis and Yarrowia lipolytica (Buckholz and Gleeson 1991). Yeasts, which can be employed for heterologous protein production, are heavily patented; and this is why it is of great interest to find new expression systems. Some of the drawbacks of non-conventional yeasts are the limited number of cloned genes, a limited understanding of the regulation of their metabolism and a limited availability of molecular biological tools (Sudbery 1994; Flores et al. 2000). In order to take advantage of the enormous amount of knowledge available for S. cerevisiae, we have searched for new hosts for heterologous protein production among the Saccharomyces yeasts. S. kluyveri is the most distant Saccharomyces relative of S. cerevi- siae (Kurtzman and Robnett 1998); and recently its ge- nome has been extensively analysed (Langkjær et al. 2000; Neuvéglise et al. 2000). S. kluyveri has been shown to be transformable with S. cerevisiae plasmids, using the URA3 marker (Fujimura 1991; Gojkovic et al. 2000). In this study, S. kluyveri is evaluated for its potential for heterologous protein production, using proteinase A (PrA) from S. cerevisiae as a model protein. PrA is a vacuolar aspartate protease, encoded by the PEP4 gene, which is secreted by S. cerevisiae if the gene is overex- pressed (Rothmann et al. 1986). PrA is synthesised as a zymogen, but can be autoactivated extracellulary in strains that secrete the zymogen as a result of overex- pression. Autoactivation of PrA is dependent on pH and ionic strength (van den Hazel et al. 1997). Overexpres- sion of PEP4 has previously been used to study protein secretion in S. cerevisiae (Jochumsen 1995; Carlsen et al. 1997). K. Møller · L.D. Tidemand · L. Olsson · J. Nielsen ( ✉ ) Center for Process Biotechnology, BioCentrum–DTU, Technical University of Denmark, 2800 Lyngby, Denmark e-mail: jens.nielsen@biocentrum.dtu.dk Tel.: +45-45252696, Fax: +45-45884148 J.R. Winther Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Copenhagen Valby, Denmark J. Pis ˇkur Section for Molecular Microbiology, BioCentrum–DTU, Technical University of Denmark, 2800 Lyngby, Denmark Appl Microbiol Biotechnol (2001) 57:216–219 DOI 10.1007/s002530100680 SHORT CONTRIBUTION K. Møller · L.D. Tidemand · J.R. Winther L. Olsson · J. Pis ˇkur · J. Nielsen Production of a heterologous proteinase A by Saccharomyces kluyveri Received: 25 January 2001 / Received revision: 28 February 2001 / Accepted: 28 February 2001 / Published online: 9 June 2001 © Springer-Verlag 2001