WO1999002716A1 - Increased production of secreted proteins by recombinant yeast cells - Google Patents

Abstract

This invention relates to recombinant DNA-technology. Specifically this invention relates to new recombinant yeast cells transformed with SEB1 gene. Yeast cells transformed with several copies of SEB1 gene, or overexpressing the Seb1 protein by some other means, have an increased capacity to produce secreted foreign or endogenous proteins. Further, said new recombinant cells, when transformed with genes expressing suitable hydrolytic enzymes can utilize appropriate macromolecular compounds more efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications.

Description

INCREASED PRODUCTION OF SECRETED PROTEINS BY RECOMBINANT YEAST CELLS

Field of the invention

This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast cells transformed with SEB1 gene or its homologs. A yeast cell transformed with several copies of a SEB1 gene or a gene homologous to SEB1 has an increased capacity to produce secreted foreign or endogenous proteins.

Further, said new recombinant yeast cells, when transformed with genes expressing suitable hydrolytic enzymes can hydrolyze and/or utilize appropriate macro- molecular/polymeric compounds more efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications.

Background of the invention

The development of recombinant DNA methods has made it possible to produce proteins in heterologous host systems. This possibility greatly facilitates production of e.g. proteins of therapeutic importance which normally occur in nature in very low amounts or are otherwise difficult to isolate or purify. Such proteins include growth factors, hormones and other biologically active proteins or peptides which traditionally have been isolated from human or animal tissues or body fluids e.g. blood serum or urine. The increasing danger of the presence of human pathogenic viruses such as HBV, HIV, and oncogenic viruses, prions, or other pathogens in the human or animal tissues or body fluids has greatly speeded up the search for heterologous production systems for these therapeutics. Other proteins of clinical importance are viral or other microbial or human parasite proteins needed for diagnostics and for vaccines especially of such organisms which are difficult to grow in vitro or in tissue culture, or are dangerous human pathogens. These include viruses like HBV, HIV, yellow fever, rubella, FMDV, rabies, and human parasites such as Plasmodium falciparum causing malaria.

A further group of proteins for which heterologous production systems have been or are being developed are secreted enzymes, especially those hydrolyzing plant material, and which are needed in food and fodder production as well as in other industrial processes including textile industry and pulp and paper industry. The possibility of producing proteins in heterologous systems or production of endogenous proteins in genetically engineered cells increases their yields and greatly facilitates their purification and has already by now had a great impact on studies of structure and function of many important enzymes and other proteins. The production and secretion of foreign hydrolytic enzymes in yeast for example, results in improvements in processes based on industrial yeast strains such as distiller's, brewer's or baker's yeasts.

Various production systems have been and are being developed including bacteria, yeasts, filamentous fungi, animal and plant cell cultures and even multicellular organisms like transgenic animals and plants. All of these different systems have their advantages, even if disadvantages, and all of them are needed.

The yeast Saccharomyces cerevisiae is at the moment the best known eukaryote at genetic level. Its whole genome sequence became public in data bases on April 24, 1996. As a eukaryotic microbe it possesses the advantages of a eukaryotic cell like most if not all of the post-translational modifications of eukaryotes, and as a microbe it shares the easy handling and cultivation properties of bacteria. The large scale fermentation systems are well developed for S. cerevisiae which has a long history as a work horse of biotechnology including production of food ingredients and beverages such as beer and wine.

The yeast genetic methods are by far the best developed among eukaryotes based on the vast knowledge obtained by classical genetics. This made it easy to adopt and further develop for yeast the gene technology procedures first described for Escherichia coli. Along other lines the methods for constructing yeast strains producing foreign proteins have been developed to a great extent (Romanos et. al, 1992).

Secretion of the proteins into the culture medium involves transfer of the proteins through the various membrane enclosed compartments constituting the secretory pathway. First the proteins are translocated into the lumen of the endoplasmic reticulum, ER. From there on the proteins are transported in membrane vesicles to the Golgi complex and from Golgi to plasma membrane. The secretory process involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targetted to and fused with the acceptor membrane. At each of these steps function of several different proteins are needed.

The yeast secretory pathway and a great number of genes involved in it have been elucidated by isolation of conditional lethal mutants deficient in certain steps of the secretory process (Novick et al, 1980; 1981). Mutation in a protein, needed for a particular transfer step results in accumulation of the secreted proteins in the preceding membrane compartment. Thus proteins can accumulate in the cyto- plasm, at ER, Golgi or in vesicles between ER and Golgi, or in vesicles between Golgi and plasma membrane.

More detailed analysis of the genes and proteins involved in the secretory process has become possible upon cloning the genes and characterization of the function of their encoded proteins. A picture is emerging which indicates that in all steps several interacting proteins are functioning. The number of genes is rapidly increasing that are involved in protein secretion and that were first identified in and isolated from S. cerevisiae and were later found in other organisms including lower and higher eukaryotes. The structural and functional homology has been shown for many of such proteins. We have recently cloned a new yeast gene, SEB1 (Toikkanen et al, 1996) which encodes the β-subunit of the trimeric Secόl complex (hence the name: SEB = SEcόl J?eta subunit) that is likely to represent the protein conducting channel of the ER both in co- and post-translational translocation (Hanein et al, 1996). In the former it functions in close connection with the ribosome and in latter it forms a heptameric membrane protein complex with the tetrameric Sec62/Sec63 complex (Panzner et al, 1995). Genes with sequence similarity to the SEB1 gene are found in plant and mammalian cells indicating that the Secόl translocation complex is conserved in evolution. In fact, similar components function in protein translocati- on also in prokaryotes (discussed in Toikkanen et al, 1996). This further supports the conserved and central role of the SEB1 gene in protein secretion and intracellular transport. However, no reports exist so far on any positive effect of the SEB1 or its homologs in other yeasts, plant or animal cells on secretion when overex- pressed, which effect we are showing in this invention for the yeast SEB1 gene. It should be noticed that Sebl protein is present in a different protein complex and at different location than the Sso proteins which we have previously shown to enhance production of secreted proteins when present in the cells in higher than normal amounts.

Knowledge on the protein secretion process in S. cerevisiae is rapidly increasing. Less is known about the secretory system of other yeasts such as Kluyveromyces, Schizosaccharomyces, Pichia and Hansenula which, however, have proven useful hosts for production of foreign proteins (Buckholz and Gleeson, 1991; Romanos et al, 1992), or Candida and Yarrowia which also are interesting as host systems. The genetics and molecular biology of these yeasts are not as developed as for Saccharomyces but the advantages of these yeasts as production hosts are the same as for Saccharomyces.

Several attempts have been made and published previously to increase foreign protein production in yeast and filamentous fungi as well as in other organisms. Much work has been devoted to various promoter and plasmid constructions to increase the transcription level or plasmid copy number (see e.g. Baldari et al. 1987; Martegani et al. 1992; Irani and Kilgore, 1988). A common approach to try and increase secretion is to use yeast signal sequences (Baldari, et al. 1987, Vanoni et al. 1989). Random mutagenesis and screening for a secreted protein (Smith et al, 1985; Sakai et al, 1988; Shuster et al, 1989; Suzuki et al, 1989; Sleep et al, 1991; Lamsa and Bloebaum, 1990; Dunn-Coleman et al, 1991) or fusion of the foreign protein to an efficiently secreted endogenous protein (Ward et al, 1990; Harkki et al, 1989; Nyyssonen et al 1993; Nyyssonen et al, 1992) have been widely used both for yeast and filamentous fungi in order to make the secretion of foreign proteins more efficient. Both of these methods are of limited use. Overproduction mutants isolated by random mutagenesis and screening are almost exclusively recessive and thus cannot be transferred into industrial yeast strains which are polyploid. Often the overproduction results from changes other than increased secretion and in many cases affects only the protein used for screening. Fusion protein approach requires tailoring of the fusion construction for each foreign protein separately. The fusion protein is often not functional and thus the final product must be released by proteolytic cleavage which complicates the production procedure.

Our approach, increasing the copy number of genes functioning in secretion and thus the amount of components of the secretory machinery is more universal: it is applicable to any protein without specific fusion constructions and applicable to diploid and polyploid strains.

It is not exactly known which steps form the bottle necks in the secretory process, but it can be anticipated that there are more than one stage that may become rate limiting especially under overproduction conditions. The SEB1 gene according to the invention was cloned using a yeast genetic approach and it was shown to interact genetically with the SEC61 gene, encoding the major component of the ER translocation complex. The fact that overexpression of SEB1 gene increases the production of secreted proteins into the culture medium suggests that the Sebl protein is a rate limiting component in the translocation process. This was surprising since Sebl protein is a component of a multiprotein complex and the enhancing effect did not require increased levels of the other components of the complex and since the SEBl gene is not essential for yeast growth. This could mean that there is another gene which can perform the same function as SEBl. We have isolated another gene, SEB2, homologous to SEBl but disruption of both SEBl and SEB2 was not lethal either, indicating that the function of SEBl is not essential for yeast growth.

Summary of the invention

The present invention describes a method for enhanced production of secreted proteins based on overexpression of the previously isolated SEBl gene of Saccharomyces cerevisiae. Specifically, the present invention describes the construction of S. cerevisiae strains overexpressing the SEBl gene either on a multicopy plasmid or when integrated into the yeast genome in single or multiple copies or placed under regulation of a strong promoter. In addition, this invention describes identification of SEBl homologs from other yeasts, and detection of Seblp homologous protein in Kluyveromyces lactis.

This invention thus provides new recombinant yeast cells expressing enhanced levels of Sebl protein of S. cerevisiae.

This invention also provides ρrocess(es) for production of increased amounts of secreted proteins by overexpressing genes interacting with the SEBl gene, such as SEC61.

The yeast cells according to the invention being transformed with the SEBl gene or genes interacting with the SEBl gene have an increased capacity to produce secreted proteins. The new yeast cells according to the invention can also be used for more efficient production of hydrolytic enzymes and hydrolysis of e.g. polymeric substrates which results in improvements in biotechnical processes such as single cell or baker's yeast production due to increased cell mass or in other processes where efficient production of hydrolytic enzymes and/or efficient hydrolysis of plant material is beneficial.

Brief description of the drawings

Fig. 1 shows the S. cerevisiae vector YEpSEBl in which the SEBl gene cDNA is integrated between the ADH1 promoter and CYC1 terminator on the multicopy plasmid pMAC561.

Fig. 2 shows Western analysis demonstrating overexpression of Sebl protein in yeast transformed with YEpSEBl. Lane 1: SEBl overexpression strain transformed with plasmid YEpSEBl, Lane 2: Trp^~ segregant of SEBl overexpression strain, Lane 3: Control strain (with plasmid pMA56).

Fig. 3 shows increased production of secreted Bacillus -amylase by S. cerevisiae transformed with the multicopy plasmid YEpSEBl expressing SEBl and with YEpαaό expressing Bacillus α-amylase gene. Growth (filled symbols) and secretion of α-amylase (open symbols) in the yeast transformant overexpressing the SEBl gene (YEpαaό and YEpSEBl; squares), in control transformant (YEp- αa6 and pMA56; diamonds), in YEpSEBl segregant (YEpαaό; circles) and in α- amylase transformant (YEpαaό; stars).

Fig. 4 shows Western analysis of Bacillus α-amylase secreted by S. cerevisiae with or without the multicopy plasmid expressing SEBl. Lane 1: Culture medium from YEpSEBl transformant, Lane 2: Culture medium from control strain, Lane 3: Bacillus α-amylase standard.

Fig. 5 shows increased production of secreted Bacillus α-amylase by S. cerevisiae strain containing an integrated copy of a Bacillus α-amylase gene and transform- ed with multicopy plasmid, expressing SEBl gene. The secretion of Bacillus α- amylase integrant yeast (open symbols); the transformant overexpressing the SEBl gene (YEpSEBl; squares), control transformant (pMA56, circles), YEpSEBl segregant (diamonds) and pMA56 segregant (stars). The growth of the yeast transformants is shown with filled symbols.

Fig. 6 shows the SEBl expression cassette flanked by ribosomal sequences integrated into BS+, generating the vector YrbSEBl.

Fig. 7 Identification of SEBl homologs in other yeast species by heterologous hybridization under non-stringent conditions. Hindlll digested genomic DNA of Saccharomyces cerevisiae (lane 1), Schizosaccharomyces pombe (lane 2), Kluyve- romyces lactis (lane 3), Pichia pastoris (lane 4), Pichia stipitis (lane 5), Candida utilis (lane 6) and Yarrowia lipolytica (lane 7).

Fig. 8 Detection of the Seblp and its homolog in K. lactis yeast using Seblp specific antibody. S. cerevisiae (lane 1), K. lactis (lane 2).

Detailed description of the invention

For better understanding of the following detailed description of the invention the following definitions of certain terms are given to be used hereinafter.

Homologous genes, homologs: Genes which are related, but not identical, in their DNA sequence and/or perform the same function are homologous with each other and are called each other's homologs.

Overexpression of a gene: A protein encoded by said gene is produced in increased amounts in the cell. This can be achieved by increasing the copy number of the gene by introducing extra copies of the gene into the cell on a plasmid, or integrated into the genome. Overexpression can also be achieved by placing the gene under a promoter stronger than its own promoter. The amount of the protein in the cell can be varied by varying the copy number of the gene and/or the strength of the promoter used for the expression. Secreted proteins: Proteins which inside of the cell are directed to the secretory pathway and transported through it to the exterior of the cell, outside of the plasma membrane, are called secreted proteins. In yeast the proteins may remain associated with the cell wall such as invertase or may be released through the cell wall into the growth medium such as the foreign protein Bacillus α-amylase.

Suppression of a mutation: When the effect of a mutation in a given gene is alleviated or abolished by a mutation in another gene, this second gene is called a suppressor of the first gene. Suppression can occur also by overexpression of the wild type allele of the second gene by the means described above. This is called overexpression suppression. If the overexpression is caused by multiple copies of the suppressing gene the suppression can also be called multicopy suppression. Suppression phenomenon indicates that these two genes interact at genetic level. The interaction may also occur at physical level as direct, physical contact between the two proteins encoded by the interacting genes.

Transformant/segregant: When yeast is transformed with a plasmid it is called transformant, i.e. a transformed strain. When the plasmid is lost, i.e. segregated away from the transformant the strain is called a segregant.

SEBl gene to be used in this invention is isolated from an organism containing this gene, e.g. Saccharomyces cerevisiae or Kluyveromyces lactis. Also other suitable yeasts, such as Schizosaccharomyces pombe, Yarrowia lipolytica, Candida spp., Pichia spp. and Hansenula spp. can be used. It is to be noted that homolo- gous genes from other organisms can also be used.

Furthermore, overexpression of other genes functioning at the same step with the SEBl gene, such as SEC61, in the presence of normal or increased levels of Sebl protein results in increased production of secreted proteins.

Genes functioning at the other steps of the secretory process may well have a similar effect. Thus, release of the secretory vesicles from ER or the Golgi compartment may be facilitated by increasing the copy number of appropriate genes known to function at this step or by searching for and increasing the copy number of genes interacting with the known genes, e.g. suppressors of their mutations. Likewise any other step of the secretory process may be improved by increasing the copy number of genes involved. The new gene SEBl, which we have isolated from S. cerevisiae, represents a conserved gene which suggests that it plays an important role in the cell.

Based on the conserved nature of SEBl and its homologs in other species, as mentioned above, we propose that increase of the SEBl gene or its homolog in any other yeasts would result in increased protein secretion efficiency.

The host to be transformed with the genes of the invention can be any yeast cell suitable for foreign or endogenous protein production, e.g. any S. cerevisiae yeast strain, (e.g. DBY746, AH22, S150-2B, GPY55-15Ba, VTT-A-63015) any

Kluyveromyces lactis yeast (e.g. MW270-7B, MW179-1D), Schizosaccharomyces pombe, Hansenula polymorpha, Candida, Pichia or Yarrowia spp. Transfer of the genes into these cells can be achieved, for instance, by using the conventional methods described for these organisms.

The DNA sequence containing SEBl is isolated from S. cerevisiae by conventional methods. In a preferred embodiment the known DNA sequence of the SEBl gene of S. cerevisiae (Toikkanen et al, 1996) and SEBl-like genes is used to design probes for heterologous hybridization or PCR primers for cloning the SEBl gene. In another approach antibodies to the proteins encoded by the known SEBl and SEBl -like genes are used for cloning the gene by standard methods.

The DNA sequence of K. lactis containing the K. lactis SEBl gene is isolated from the chromosomal DNA or from a cDNA or a chromosomal gene bank prepared from K. lactis by heterologous hybridization in non-stringent conditions as described in Example 5, and characterized by conventional methods, and its function can be shown as described above. Similar approach is suitable for all organisms which have shown to possess chromosomal sequences homologous to the yeast SEBl gene as analyzed for instance by Southern hybridization of total DNA. It is also possible to isolate the gene from an expression library with antibodies prepared against the yeast Sebl protein.

Alternatively, oligonucleotide primers can be designed based on the homologies found between the sequences of the corresponding genes isolated from several organisms. These primers are used to amplify the K. lactis gene in a PCR reaction.

To construct a plasmid suitable for transformation into a yeast, the SEBl gene is cloned into a suitable yeast expression vector, such as pAAH5 (Ammerer, 1983) or vectors derived from it (Ruohonen et al, 1991; Ruohonen et al, 1995) comprising the appropriate yeast regulatory regions. These regulatory regions can be obtained from yeast genes such as the ADH1, GAL1/GAL10, PGK1, CUP1, GAP, CYC1, PH05, TPI1 or asparagine synthetase gene, for instance. Alternatively, also the regulatory regions of SEBl can be used to express the genes in S. cerevisiae. The plasmid carrying the SEBl gene is capable of replicating autonomously when transformed into the recipient yeast strain. The gene SEBl together with the appropriate yeast regulatory regions can also be cloned into a single copy yeast vector such as ρHR70 of Hans Ronne or pRS313, pRS314, pRS315 or pRS316 (Sikorski and Hieter, 1989).

Alternatively, extra copies of SEBl gene can also be integrated into the yeast chromosome, into the ribosomal RNA locus, for instance. For this purpose the ribosomal sequences of a suitable plasmid, e.g. plasmid pIRL9 (Hallborn et al, 1991) are released, and cloned appropriately into BS+ vector, as shown in Fig. 6. The gene SEBl coupled in between suitable yeast promoter and terminator regions, is released from the hybrid vector comprising the gene and cloned into the plasmid obtained at the previous stage. From this resulting plasmid the expression cassette, flanked by ribosomal sequences can be released. This fragment is cotransformed into a yeast with an autonomously replicating plasmid carrying a suitable marker for transformation. The plasmid can be later on removed from the cells containing the extra copies of SEBl gene integrated in the chromosome by cultivating the cells in non-selective conditions. Using this procedure recombinant strains can be obtained which carry no extra foreign DNA such as bacterial vector sequences. If a polyploid yeast strain, such as VTT-A- 63015, is used the gene can be integrated also to an essential locus such as the ADH1 or the PGK1 locus.

To express the SEBl gene in K. lactis the SEBl gene between the ADH1 promoter and CYCl terminator is transformed into a K. lactis strain either on a multicopy plasmid or integrated in the genome using methods known in the art. Suitable promoters in addition to the ADH1 promoter or promoter of the SEBl gene itself are for instance the other S. cerevisiae promoters, as listed hereinbefore.

An object of this invention is thus to provide yeast strains overexpressing the

SEBl gene of S. cerevisiae as well as homologous gene(s) of K. lactis and other yeasts. The sequence of the genes can be determined from the plasmids carrying them by using e.g. the double stranded dideoxy nucleotide sequencing method (Zagursky et al, 1986). The nucleotide sequence of the open reading frame of SEBl gene of S. cerevisiae is given as the SEQ ID NO:l.

Another object of this invention is to provide specific vectors comprising the SEBl genes. For yeast such a vector is either an autonomously replicating multicopy or a single copy plasmid or a vector capable of integrating into the chromosome, as described above.

Still another object of this invention is to provide yeast strains containing extra copies of SEBl gene either on replicating plasmid(s) or integrated into the chromosome, which results in increased production of secreted proteins, such as Bacillus α-amylase, yeast invertase or Trichoderma cellulases or other hydrolases. Thus a method for constructing new yeast cells capable of expressing enhanced levels of Sebl protein comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding Sebl protein; and (b) transforming at least one such vector to a yeast host cell.

Still another object of this invention is to provide yeast cells which in addition to extra copies of SEBl gene comprise a DNA molecule encoding a secreted foreign or endogenous protein, such as α-amylase, cellulase, or an antibody, and are capable of expressing this protein.

Thus a process for producing increased amounts of a secreted foreign or endogenous protein by overexpressing the SEBl gene is provided. This process comprises: (a) obtaining a vector comprising an isolated DNA molecule encoding said protein;

(bl) transforming the vector obtained into a suitable yeast host expressing enhanced levels of Sebl protein to obtain recombinant host cells; or

(b2) transforming the vector obtained into a suitable yeast host and retransforming this transformant with SEBl or a gene homologous to SEBl;

(c) screening for cells with enhanced production of said protein; and

(d) cultivating said recombinant host cells under conditions permitting expression of said protein.

A further object of this invention is to improve secretion by optimizing the Sebl protein level using different promoters and different copy numbers of the gene and combining the SEBl gene with other genes involved in secretion, such as SEC61.

Thus the invention provides a process for producing increased amounts of a secreted foreign or endogenous protein, by overexpressing a gene interacting with the SEBl gene, e.g. SEC61, in the presence of normal or increased amounts of the Sebl protein, which process comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding said protein; (bl) transforming the vector obtained into a suitable yeast host expressing normal or enhanced levels of Sebl protein and overexpressing another gene interacting with SEBl gene, e.g. SEC61, to obtain recombinant host cells; or,

(b2) transforming the vector into a suitable yeast host and retransforming this transformant with SEBl or a gene homologous to SEBl and by the gene interacting with SEBl gene;

(c) screening for cells with enhanced production of said protein; and

(d) cultivating said recombinant host cells under conditions permitting expression of said protein.

Still another object of this invention is to provide a process for increased production of an endogenous secreted protein, the process comprising:

(a) transforming cells producing said protein with a SEBl gene or a gene homologous to SEBl, alone or together with a gene interacting with the SEBl gene, such as SEC61, (b) screening for transformants producing enhanced level of said protein thus obtaining recombinant cells for enhanced protein production, and

(c) cultivating said recombinant cells in conditions permitting expression of said protein.

Still another object of this invention is to provide yeast strains which in addition to extra copies of SEBl gene or its homolog comprise a DNA sequence coding for a hydrolytic enzyme such as α-amylase and/or glucoamylase or lignocellulose hydrolyzing enzymes such as cellulases, hemicellulases or ligninases, which render the yeast capable of increased hydrolysis of, and/or enhanced growth on polymeric compounds such as starch or lignocellulose. Thus an efficient biomass production on said raw material or efficient hydrolysis of said raw material is provided. This process comprises:

(a) obtaining a yeast vector comprising an isolated DNA molecule encoding an endogenous or foreign hydrolytic enzyme;

(bl) transforming the vector obtained into a suitable yeast host expressing enhanced levels of Sebl protein to obtain recombinant yeast host cells; or

(b2) transforming the vector to a suitable yeast host and retransforming this transformant with SEBl or a gene homologous to SEBl; (c) screening for cells with enhanced production of said enzyme; and

(d) cultivating said recombinant host cells under conditions permitting expression of said hydrolytic enzyme.

A process is also provided for efficient biomass production on a raw material or efficient hydrolysis of a raw material, by overexpressing genes interacting with the

SEBl gene, e.g. SEC61, in the presence of normal or increased amounts of the

Sebl protein. This process comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding an endogenous or foreign hydrolytic enzyme; (bl) transforming the vector obtained to a suitable yeast host expressing enhanced levels of a protein interacting with the Sebl protein in the presence of normal or increased amounts of the Sebl protein to obtain recombinant yeast host cells; or,

(b2) transforming the vector to a suitable yeast host and retransforming this transformant with SEBl gene or a gene homologous to SEBl and with a gene interacting with SEBl gene, such as SEC61;

(c) screening for cells with enhanced production of said enzyme; and

(d) cultivating said recombinant yeast host cells under conditions permitting expression of said hydrolytic enzyme. Possible applications of said recombinant cells are e.g. in single cell production, in improved alcohol production or in processes where efficient hydrolysis of raw material is desired.

EXPERIMENTAL

The S. cerevisiae strain used in all experiments was DBY746 (a his3Dl leu2-3 leu2-112 ura3-52 trpl-289 Cyh^R) (obtained from David Botstein, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA)

Example 1: Overexpression of the Sebl protein in yeast transformed with YEpSEBl

The S. cerevisiae strains transformed (Ito et al, 1983) with the control plasmid pMA56 (1) (Ammerer, 1983) or with YEpSEBl (3) (Toikkanen et al, 1996) were grown in synthetic complete medium (Sherman et al, 1983) lacking Trp, and a strain from which the YEpSEBl plasmid was segregated away (2) was grown in synthetic complete medium. Yeast cell lysates were prepared in the presence of SDS as described by Keranen (1986). Thirty μg of total yeast protein present in the lysates were separated by SDS-PAGE (Schagger and von Jagow, 1987) and detected by Western blotting using polyclonal antibodies made in rabbit against an 18 amino acids long N-terminal peptide of the Sebl protein (Toikkanen et al, 1996) and alkaline phosphatase conjugated goat anti-rabbit IgG for detection. As shown in Fig. 2, greatly increased amount of Sebl protein was seen in the YEpSEBl transformant. When the YEpSEBl plasmid was segregated away from the yeast, the Sebl protein level was reduced to the level of the control strain transformed with the vector plasmid not containing the SEBl gene. Example 2: Enhanced production of a secreted foreign protein, Bacillus a- amylase and an endogenous protein, invertase in a yeast strain overexpressing SEBl

The S. cerevisiae strain harboring the plasmid YEpαaό containing Bacillus α- amylase gene ligated between the ADH1 promoter and terminator (Ruohonen et al, 1987), modified for more efficient expression by deleting predicted inhibitory sequences 5' to the promoter element (Ruohonen et al, 1991; 1995) was transfor- med either with YEpSEBl or with the control plasmid pMA56 (Ammerer, 1983). The yeast strains obtained containing YEpSEBl and YEpαaό or pMA56 and YEpαaό were grown in selective medium at 30°C and secretion of α-amylase into the culture medium was monitored by measuring the α-amylase activity using the Phadebas amylase test (Pharmacia Diagnostics AB, Sweden). As shown in Fig. 3, increased α-amylase activity was obtained in the strain which carried

SEBl on the multicopy plasmid compared with the control strain transformed with the control plasmid without SEBl gene. No difference was observed in the yeast growth between the control transformant and SEBl transformant. Secretion of the endogenous protein, invertase, was also enhanced by SEBl overexpression measured at late logarithmic to early stationary growth phase. The secreted invertase activity in the YEpSEBl transformant was 1.4 fold compared to that of the control transformant containing pMA56.

Removal of the predicted inhibitory sequences on the ADH1 promoter (see above) used for expression of the SEBl in YEpSEBl results in prolonged expression of SEBl and prolongs existence of increased level of the Sebl protein and consequently even higher final levels of the Bacillus α-amylase are secreted into the medium. Example 3: Enhanced production of secreted foreign protein, Bacillus α- amylase in a yeast strain in which the α-amylase gene is integrated in the genome at HISS locus and the SEBl gene is over- expressed

The cassette expressing α-amylase of Bacillus amyloliquefaciens was released from the plasmid YEpαaό (Ruohonen et al, 1995) as 3.35 kb fragment by digesting with BamHI and Sail and it was cloned into the yeast integrating vector pRS403 (Sikorski and Hieter, 1989) between the BamHI and Sail sites to obtain plasmid YIpαal. The plasmid was linearized by Pstl digestion inside the HIS3 gene and used for transformation of yeast. The transformants were selected for histidine prototrophy. Integration of the α-amylase expression cassette at the HIS3 locus was confirmed by Southern analysis. The integrant strain thus obtained was transformed with YEpSEBl or with the control plasmid pMA56. The transfor- mants were grown in selective medium at 30°C and secretion of α-amylase into the culture medium was monitored by measuring the α-amylase activity as described in Example 2. Clearly enhanced levels of secreted α-amylase were detected in the YEpSEBl transformant, in which the α-amylase level in the culture medium was 4.2 fold compared to the control strain.

Example 4: Enhanced production of secreted foreign protein, Bacillus α- amylase in a yeast strain in which the α-amylase gene is integrated in the genome at URA3 locus and the SEBl gene is over- expressed

An integration cassette for integration of the α-amylase gene in the URA3 locus was construted as follows. Two URA3 fragments were made by PCR and cloned into the multiple cloning site of pBluescript SK(-). The first fragment comprising base pairs (bp) 71-450 of the 1135 bp long URA3 was cloned as a Sacl-Xbal fragment. The second fragment (781-1135 bp) was cloned as a Xhol-Kpnl fragment. The resultant plasmid is pBUF. The 3.35 kb long α-amylase expression cassette was cut as a BamHl-Sall fragment from the plasmid YEpαaό (Ruohonen et al, 1995) and cloned into the pBluescript vector yielding plasmid pBαaό. The expression cassette was then released again as a BamHl-Sall fragment and inserted into pBUF between the URA3 fagments. This construct is pUIαal. The S. cerevisiae strain which is ura3-52 was first converted to wt URA3 by transfor- ming with a fragment containing the entire URA3 gene. This strain was then transformed with the α-amylase integration cassette released as a Sacl-Nsil fragment from pUIαal and the transformants were selected in the presence of 5- FOA which selects for strains in which the URA3 gene is inactivated by integration of the α-amylase cassette. Integration at the URA3 locus was confirmed by Southern analysis. The strain thus obtained was transformed with YEpSEBl or with the control plasmid pMA56. The YEpSEBl transformant obtained was named VTT C-97280 and was deposited according to the Budapest Treaty at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) on 16 June 1997 with the accession number DSM 11615.

The transformants were grown in selective medium at 30°C and secretion of α- amylase into the culture medium was monitored by measuring the α-amylase activity as described in Example 2. As shown in Fig. 4 and Fig. 5 clearly enhanced levels of secreted α-amylase were detected in the YEpSEBl transformant both by Western blotting and by measuring the α-amylase activity.

Example 5: Identification of SEBl homologs in other yeasts by heterologous hybridization

Genomic DNA from the fungal species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Pichia stipitis, Candida utilis, and Yarrowia lipolytica were isolated, digested with the Hindlll restriction enzyme, separated electrophoretically in an 0.8% agarose gel and blotted on a nylon filter. Southern hybridization of the filter was carried out at different stringencies using the Saccharomyces SEBl gene coding region as a probe.

Hybridization in a mixture containing 30 % formamide, όxSSC, 10 x Denhardt's, 0.5% SDS, 100 mg/ml herring sperm DNA and 10 mg/ml polyA at 35°C and washing 15 minutes in όxSSC, 0.1% SDS at 42°C and 2 x 30 minutes in 2xSSC, 0.1% SDS at 42°C revealed clear hybridizing bands in DNA derived from S. cerevisiae, S. pombe, K. lactis, P. stipitis and Y. lipolytica, and a much weaker band in DNA of C. utilis (Fig. 7).

Example 6: Detection of Seblp homologous protein in Kluyveromyces lactis yeast

The plasmid YEpSEBl (S. cerevisiae vector containing SEBl gene) and the vector plasmid were converted to shuttle vectors that are able to replicate in K. lactis. The K lactis replication origin (Chen et al, 1986) was released from the KEpό plasmid as about 1000 bp fragment (digestion with Aatll and Clal, fill-in by T4 DNA polymerase) and purified and ligated in the Pvull site of YEpSEBl and the vector control, pMA56 to obtain plasmids KEpSEBl and KpMA56, respectively. The plasmids were transformed into K. lactis and the transformants were selected for growth on SC-Trp plates. SDS lysates prepared from the transformants were analyzed by Western blotting using Seblp specific antibody as described in Example 1. The antibody detected a band with slightly slower migration than that of Seblp of S. cerevisiae (Fig. 8).

Example 7: Enhanced production of secreted foreign protein, Bacillus a- amylase in yeast overexpressing SEC61 in combination with normal or increased levels of functional Sebl protein.

The S. cerevisiae strain in which the Bacillus α-amylase expression cassette is integrated at the URA3 locus was transformed either with a multicopy plasmid YEpSECόl expressing the SEC61 gene or with the control plasmid pRS425 (Christianson et al, 1992). The transformants were grown in selective medium at 30°C and secretion of α-amylase into the culture medium was monitored by measuring the α-amylase activity using the Phadebas amylase test (Pharmacia

Diagnostics AB, Sweden). Increased α-amylase activity (1.3 fold) was obtained in the strain which carries SEC61 on a multicopy plasmid compared with the strain transformed with the vector without SEC61 gene.

Simultaneous overexpression of both Secόlp and Seblp from two different plasmids enhanced production of secreted α-amylase even further (3.3 fold). Under these 2-plasmid conditions enhancement by SEBl alone was 2.4 fold. The plasmid expressing the SEC61 gene is available at VTT, Biotechnical Laboratory, Espoo, Finland.

Deposited microorganism

The following microorganism was deposited according to the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, Germany.

Strain Accession number Deposit date

Saccharomyces cerevisiae DSM 11615 16.06.1997

VTT C-97280 carrying the integrated α-amylase gene and plasmid YEpSEBl

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(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Valtion teknillinen tutkimuskeskus

(B) STREET: Vuorimiehentie 5

(C) CITY: Espoo

(E) COUNTRY: Finland

(F) POSTAL CODE (ZIP): FIN-02150

(ii) TITLE OF INVENTION: Increased production of secreted proteins by recombinant yeast cells

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 249 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Saccharomyces cerevisiae

( ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..249

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG TCA AGC CCA ACT CCT CCA GGT GGT CAA CGT ACT TTG CAA AAG AGA 48 Met Ser Ser Pro Thr Pro Pro Gly Gly Gin Arg Thr Leu Gin Lys Arg 1 5 10 15

AAA CAG GGA AGT TCA CAA AAA GTT GCG GCA TCC GCT CCA AAG AAA AAC 96 Lys Gin Gly Ser Ser Gin Lys Val Ala Ala Ser Ala Pro Lys Lys Asn 20 25 30

ACG AAC AGC AAT AAT TCG ATT TTG AAG ATT TAT TCT GAT GAG GCT ACG 144 Thr Asn Ser Asn Asn Ser lie Leu Lys He Tyr Ser Asp Glu Ala Thr 35 40 45

GGA CTA AGA GTA GAT CCC TTA GTT GTG TTG TTT CTA GCG GTC GGT TTC 192 Gly Leu Arg Val Asp Pro Leu Val Val Leu Phe Leu Ala Val Gly Phe 50 55 60

ATC TTT TCT GTT GTT GCA TTA CAT GTT ATT TCT AAA GTT GCC GGT AAG 240 He Phe Ser Val Val Ala Leu His Val He Ser Lys Val Ala Gly Lys 65 70 75 80

TTA TTT TA 249

Leu Phe (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 82 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Ser Pro Thr Pro Pro Gly Gly Gin Arg Thr Leu Gin Lys Arg 1 5 10 15

Lys Gin Gly Ser Ser Gin Lys Val Ala Ala Ser Ala Pro Lys Lys Asn 20 25 30

Thr Asn Ser Asn Asn Ser He Leu Lys He Tyr Ser Asp Glu Ala Thr 35 40 45

Gly Leu Arg Val Asp Pro Leu Val Val Leu Phe Leu Ala Val Gly Phe 50 55 60

He Phe Ser Val Val Ala Leu His Val He Ser Lys Val Ala Gly Lys 65 70 75 80

Leu Phe

Claims

WE CLAIM:

1. A yeast expression vector comprising a DNA molecule encoding Sebl protein having an amino acid sequence depicted in SEQ ID NO:2, or a functional fragment thereof.

2. A vector according to claim 1, which vector is capable of replicating autonomously when transformed into yeast cells.

3. A vector according to claim 1, which vector is capable of integrating into the chromosome when transformed into yeast cells.

4. A vector according to claim 1 being a yeast expression vector wherein said DNA sequence is expressed under yeast gene regulatory regions.

5. A vector according to claim 4, wherein said yeast gene regulatory regions are selected from the group consisting of the promoter regions of the SEBl gene, GAL1/GAL10 genes, the alcohol dehydrogenase gene ADH1, asparagine synthetase gene, and functional parts thereof.

6. A vector according to claim 1 which is a yeast vector YEpSEBl, the structure of which is given in Fig. 1.

7. Recombinant yeast cells carrying a vector according to any one of claims 1 to 6 and expressing enhanced levels of Sebl protein.

8. Recombinant yeast cells according to claim 7 being yeast cells belonging to a species selected from the group consisting of Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces pombe, Pichia spp., Hansenula spp., Candida spp. and Yarrowia spp.

9. Recombinant yeast cells according to claim 8 being yeast cells belonging to Saccharomyces species.

10. Recombinant yeast cells according to claim 9 being yeast cells of Saccha- romyces cerevisiae strain VTT C-97280 having the deposit accession number

DSM 11615.

11. A method for constructing new yeast cells capable of expressing enhanced levels of Sebl protein, which method comprises: (a) obtaining a vector comprising an isolated DNA molecule encoding

Sebl protein having an amino acid sequence depicted in SEQ ID NO: 2, and

(b) transforming the vector obtained to suitable yeast host cells.

12. A method according to claim 11, wherein the yeast host to be transformed is selected from the group consisting of Saccharomyces spp., Kluyveromyces spp.,

Schizosaccharomyces pombe, Candida spp., Pichia spp., Hansenula spp. and Yarrowia spp.

13. A method according to claim 12, wherein said yeast host belongs to a species selected from Saccharomyces and Kluyveromyces.

14. A process for producing increased amounts of a secreted foreign or endogenous protein, by overexpressing the SEBl gene, which process comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding said protein;

(bl) transforming the vector obtained into a suitable yeast host comprising a DNA molecule encoding the Sebl protein having an amino acid sequence depicted in SEQ ID NO:2, and expressing enhanced levels of Sebl protein, to obtain recombinant yeast host cells; or, (b2) transforming the vector to a suitable yeast host and retransforming this transformant with a vector according to claim 1;

(c) screening for cells with enhanced production of said protein; and (d) cultivating said recombinant yeast host cells under conditions permitting expression of said protein.

15. A process for producing increased amounts of a secreted foreign or endo- genous protein, by overexpressing a gene interacting with the SEBl gene, in the presence of normal or increased amounts of the Sebl protein, which process comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding said protein; (bl) transforming the vector obtained into a suitable yeast host expressing normal or enhanced levels of Sebl protein and overexpressing another gene interacting with SEBl gene, to obtain recombinant yeast host cells; or,

(b2) transforming the vector to a suitable yeast host and retransforming this transformant with a vector according to claim 1 and by the gene interacting with SEBl gene;

(c) screening for cells with enhanced production of said protein; and

(d) cultivating said recombinant yeast host cells under conditions permitting expression of said protein.

16. A process for increased production of an endogenous secreted protein, the process comprising:

(a) transforming cells producing said protein with a vector according to claim 1, alone or together with a gene interacting with SEBl gene;

(b) screening for transformants producing enhanced levels of said protein thus obtaining recombinant cells for enhanced protein production; and

(c) cultivating said recombinant cells in conditions permitting expression of said protein.

17. A process for efficient biomass production on a raw material or efficient hydrolysis of a raw material, which process comprises:

(a) obtaining a vector comprising an isolated DNA molecule encoding an endogenous or a foreign hydrolytic enzyme; (bl) transforming the vector obtained to a suitable yeast host comprising a DNA molecule encoding the Sebl protein having an amino acid sequence depicted in SEQ ID NO:2, and expressing enhanced levels of Sebl protein, to obtain recombinant yeast host cells; or, (b2) transforming said vector to a suitable yeast host and retransforming this transformant with a vector according to claim 1, and

(c) screening for cells with enhanced production of said enzyme; and

(d) cultivating said recombinant yeast host cells under conditions permitting expression of said hydrolytic enzyme.

18. A process for efficient biomass production on a raw material or efficient hydrolysis of a raw material, by overexpressing genes interacting with the SEBl gene, in the presence of normal or increased amounts of the Sebl protein, which process comprises: (a) obtaining a vector comprising an isolated DNA molecule encoding an endogenous or foreign hydrolytic enzyme;

(bl) transforming the vector obtained to a suitable yeast host expressing enhanced levels of proteins interacting with the Sebl protein in the presence of normal or increased amounts of the Sebl protein to obtain recombinant yeast host cells; or,

(b2) transforming the vector to a suitable yeast host and retransforming this transformant with a vector according to claim 1 and with a gene interacting with SEBl gene, and

(c) screening for cells with enhanced production of said enzyme; and (d) cultivating said recombinant yeast host cells under conditions permitting expression of said hydrolytic enzyme.

19. A process according to any one of claims 15, 16 and 18, characterized in that the gene interacting with the SEBl gene is SEC61 gene.