Method for the production of heterologous polypeptides in transformed yeast cells

#The present invention is related to a method for the production and secretion of heterologous proteins or polypeptides in Crabtree negative Saccharomyces yeast species, and DNA-sequences, vectors and transformed cell lines for use in such method.

#Saccharomyces cerevisiae is an attractive organism for heterologous protein production and many suitable expression systems have been developed to produce high level of proteins with this organism. This yeast is also the best studied eukaryotic organism and many molecular tools have been developed. However, S. cerevisiae has some limitations in its commercial application, due to its relatively poor secretion efficiency of proteins, the need to use fed-batch fermentation techniques to attain high cell densities and hence improve the protein synthesis, and secretion of O- and N-glycosylated proteins which are often hyperglycosylated. Although, S.cerevisiae is regarded as a very proteolysis weak host organism, a further problem encountered in production of some heterologous proteins in S. cerevisiae is low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane cf. Gabrielsen et al. Gene, 90:255-262, 1990, (secretion of human parathyroid hormone by S. cerevisiae), Rokkones et al., J. Biotechnol. 33:293-306 (secretion of human parathyroid hormone by S. cerevisiae), and Bitter et al. Proc. Natl. Acad. Sci. USA, 81:5330-5334, 1984 (secretion of β-endorphine by S. cerevisiae).

#According to Waldron and Lacroute, J. Bacteriol., 122:855-865,1975, the protein synthesis rate in yeast is dependent on the specific growth rate. They demonstrated that the net rate of protein synthesis in yeast lowered with decreasing specific growth rate. This was due to the decrease in average ribosomal efficiency i.e. the rate of protein synthesis per ribosome.

#The present invention will be described in further details by reference to the drawings in which

#FIG. 1a and 1b show batch fermentation profile of S. kluyveri GRY1183 strain along with the exit gas profile during fermentation;

#FIG. 2a and 2b show a batch fermentation profile of S. kluyveri GRY1175 strain along with the exit gas profile during fermentation;

#This invention describes the use of yeast strains within the Saccharomyces genus being a Crabtree negative yeast for expression of heterologous proteins in high yields. As an example proteinase A, a S. cerevisiae protein expressed by the PEP4 gene, was successfully expressed and secreted in Saccharomyces kluyveri. The recombinant strains were obtained by transformation with the 2μ based plasmid derived from S. cerevisiae. Stable maintenance of plasmid bearing cell population was ensured through a selection strategy mechanism, that could depend on the completion of an auxotrophic marker such as the URA3 or TPI1 marker or the introduction of a suitable antibiotic resistance gene such as kanamycin G418 from E. coli or neomycin. Thereby stable recombinant S. kluyveri strains for production and secretion of biologically active heterologous protein were developed. Furthermore, the efficient performance of these recombinant strains in batch fermentations was demonstrated.

#The present invention also discloses an expression vector for expression and secretion of an insulin precursor MI3 with the structure B(1-29)-Ala-Ala-Lys-A(1-21) where B(1-29) is the B chain of human insulin lacking the amino acid residue in position B(30) and A(1-21) is the A chain of human insulin. The vector contains the MI3 encoding DNA linked in proper reading frame with the signal/leader sequence from the MFαS.k. gene under transcriptional control of the MFαS.k promoter.

#S. kluyveri was found as a Crabtree negative yeast having the advantage of biosynthesizing more biomass/proteins than other Saccharomyces yeasts. It is also capable of secreting its own and heterologous proteins. Although, S. kluyveri being different from S. cerevisiae in its genomic structure, it can stably accommodate S. cerevisiae based (YEp24) plasmids. The strain is also capable of secreting heterologous proteins with the same immunological and enzyme activity as that observed in S. cerevisiae. The present invention thus demonstrates that Crabtree negative Saccharomyces yeast species, such as S. kluyveri, are interesting alternatives to the Crabtree positive S. cerevisiae for expressing heterologous peptides or proteins.