TITLE OF THE INVENTION

YEAST STRAINS FOR PROTEIN PRODUCTION

BACKGROUND OF THE INVENTION

(1 ) Field of the Invention

The present invention relates to the field of molecular biology, in particular, the invention is concerned with novel selection genes to be used for improved protein production from transformed expression systems. (2) Description of Related Art

In recent years the budding yeast Pichia pastoris has become a popular organism for the expression of heterologous proteins of academic and commercial interest (Cereghino et al, Curr. Opin. Biotechnol. 4: 329-332 (2002); Cereghino and Cregg, FEMS Microbiol. Rev. 24: 45-66 (2000). It was recently shown that it is possible to genetically modify the glycosylation machinery of P. pastoris and express heterologous glycoproteins decorated with complex type human glycans (Choi et al, Proc. Natl. Acad. Sci. 100: 5022-5027 (2003); Hamilton et al, Science 301: 1244-1246 (2003); Bobrowicz et α/., Glycobiology 14: 757-766 (2004); Hamilton, Science, 313: 1441-1443 (2006). However, a need remains for methods and materials to achieve higher cellular productivity in transformed cell lines, such as transformed P. pastoris cell lines. Over the years, numerous auxotrophic and dominant selectable markers have been developed (Higgins et al, Methods MoI. Biol. 103: 41-53 (1998); Lin Cereghino et al, Gene 263: 159-169 (2001); Nett and Gerngross, Yeast 20: 1279-1290 (2003); Nett et al. , Yeast 22: 295-304 (2005) and used to construct protein expression vectors for various applications. Commonly, a gene of interest is integrated into the P. pastoris genome using a plasmid that is either linearized in the marker gene, another homologous region on the plasmid or in the AOXl promoter fragment and transformed into the appropriate auxotrophic mutant. Homologous recombination of the free DNA termini then results in single-crossover type integration into these loci. Most P. pastoris transformants will contain a single copy of the expression vector, but to obtain transformants that express a high level of the protein of interest it is often desirable to screen for multi copy integrants. Using expression vectors that contain drug resistance genes as selection markers like Kan ^R or Zeo ^R it is possible to increase the number of transformants harboring multiple copies of the expression vector by increasing the level of drug used for selection. One significant disadvantage of the single-crossover type integration lies in the fact that the multiple integrated copies can collapse back into a single copy by homologous recombination. This can be especially problematic during scale-up of the expression reaction during fermentation if the protein of interest is toxic to the cells or the eviction of several copies of expression plasmid possesses other growth benefit for the cells.

U.S. Patent No. 5,584,039 relates to a selectable marker gene ADE2 isolated from Pichia methanolica. Piontek et al, Appl Microbiol. Biotechnol. 50:331-338 (1998) relates to novel gene expression systems in Schwanniomyces occidentalis and Pichia stipitis, which systems utilize vectors containing an ADE2 marker and a putative replication sequence. However, no corresponding gene has previously been isolated from Pichia pastor is, and the effects of transformation with ADE2 in P. pastoris have not previously been identified.

Accordingly, a need exists for improved methods of transformation, selection and expression of heterogeneous genes using the Pichia pastoris yeast as the host expression system.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and materials for the use of lower eukaryotic cells such as yeast or filamentous fungi as an expression system for expressing recombinant proteins.

In one aspect, the method is based on constructing slower growing ade2 auxotrophic strains of the lower eukaryote cells and using integration vectors that are capable of integrating into the genome of the ade2 auxotrophic strain and which comprises nucleic acids encoding an ADE2 marker gene or open reading frame (ORF) operably linked to a promoter and a recombinant protein, wherein the integration vector integrates into the genome of the ade2 auxotrophic strain, the ADE2 renders the auxotrophic strain prototrophic for adenine, and the recombinant protein is expressed.

Thus, provided is an expression system comprising (a) a Pichia pastoris host cell in which the endogenous ADE2 gene encoding Ade2p has been removed from the genome of the host cell; and (b) an integration vector comprising (1) a nucleic acid encoding the Ade2p; (2) a nucleic acid having an insertion site for the insertion of one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination.

Also, provided is a method for producing a recombinant Pichia pastoris host cell that expresses a heterologous protein or peptide comprising (a) providing the host cell in which the endogenous ADE2 gene encoding an Ade2p has been removed from the genome of the host cell; and (a) transforming the host cell with an integration vector comprising (1) a nucleic acid encoding the Ade2p; (2) a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination, wherein the transformed host cell produces the recombinant protein.

Further provided is an isolated nucleic acid comprising the ADE2 gene of Pichia pastoris. In particular aspects, the nucleic acid comprises the open reading frame that encodes the Ade2p protein or the nucleic acid has a nucleotide sequence with 95% identity to the nucleic acid sequence shown in SEQ ID NO:60 from nucleotide 127 to nucleotide 1,815. Further provided is an isolated polypeptide comprising an amino acid sequence with 95% identity to the amino acid sequence shown in SEQ ID NO:61.

The applicants further discovered that operably linking an auxotrophic marker gene or ORF to a minimal promoter in the integration vector, that is a promoter that has low transcriptional activity, enabled the production of recombinant host cells that contain a sufficient number of copies of the integration vector integrated into the genome of the auxotrophic host cell to render the cell prototrophic and which render the cells capable of producing amounts of the recombinant protein or functional nucleic acid of interest that are greater than the amounts that would be produced in a cell that contained only one copy of the integration vector integrated into the genome. Therefore, provided is a method in which an auxotrophic strain of a lower eukaryote cell is obtained or constructed and an integration vector is provided that is capable of integrating into the genome of the auxotrophic strain and which comprises nucleic acids encoding a marker gene or ORF that compliments the auxotrophy and is operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, or a truncated endogenous or heterologous promoter and a recombinant protein. Host cells in which a number of the integration vectors have been integrated into the genome to compliment the auxotrophy of the host cell are selected in medium that lacks the metabolite that compliments the auxotrophy and maintained by propagating the host cells in medium that lacks the metabolite that compliments the auxotrophy or in medium that contains the metabolite because in that case, cells that evict the plasmids including the marker will grow more slowly.

In a further embodiment, provided is an expression system comprising (a) a host cell in which the endogenous gene encoding an auxotrophic selectable marker protein has been removed from the genome of the host cell; and (b) an integration vector comprising (1) a nucleic acid comprising an open reading frame (ORF) encoding a function that is complementary to the function of the endogenous gene encoding the auxotrophic selectable marker protein and which is operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter; (2) a nucleic acid having an insertion site for the insertion of one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination.

In a further still embodiment, provided is a method for expression of a recombinant protein in a host cell comprising (a) providing the host cell in which the endogenous gene encoding an auxotrophic selectable marker protein has been removed from the genome of the host cell; and (a) transforming the host cell with an integration vector comprising (1) a nucleic acid comprising an open reading frame (ORF) encoding a function that is complementary to the function of the endogenous gene encoding the auxotrophic selectable marker protein and which is operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter; (2) a nucleic acid having one or more expression cassettes comprising a nucleic acid encoding one or more heterologous peptides, proteins, and/or functional nucleic acids of interest, and (3) a targeting nucleic acid that directs insertion of the integration vector into a particular location of the genome of the host cell by homologous recombination, wherein the transformed host cell produces the recombinant protein.

In further aspects of the above embodiments, the auxotrophic selectable marker protein is encoded by a gene selected from the group consisting of ADE, URA, and LYS. In a further still aspect, the auxotrophic selectable marker protein is encoded by the ADEl gene or the ADE2 gene.

In further still aspects, the integration vector comprises multiple insertion sites for the insertion of one or more expression cassettes encoding the one or more heterologous peptides, proteins and/or functional nucleic acids of interest. In further still aspects, the integration vector comprises more than one expression cassette. In further still aspects, the integration vector comprises little or no homologous DNA sequence between the expression cassettes. In further still aspects, the integration vector comprises a first expression cassette encoding a light chain of a monoclonal antibody and a second expression cassette encoding a heavy chain of a monoclonal antibody.

In further still aspects, the host cell is a lower eukaryote. In further still aspects, the host cell is from a species selected from the group consisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta {Ogataea minuta, Pichia lindnerϊ), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. In further still aspects, the expression system of claim 1 , wherein the host cell is Pichia pastoris or a Pichia pastoris cell that has been modified to be capable of producing glycoproteins having hybrid or complex N- glycans.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, VoI I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, VoI II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999). All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.

The genetic nomenclature for naming chromosomal genes of yeast is used herein. Each gene, allele, or locus is designated by three italicized letters. Dominant alleles are denoted by using uppercase letters for all letters of the gene symbol, for example, ADE2 for the adenine 2 gene, whereas lowercase letters denote the recessive allele, for example, the auxotrophic marker for adenine 2, ade2. Wild-type genes are denoted by superscript "+" and mutants by a "-" superscript. The symbol δ can denote partial or complete deletion. Insertion of genes follow the bacterial nomenclature by using the symbol "::", for example, trp2::ARG2 denotes the insertion of the ARG2 gene at the TRP2 locus, in which ARG2 is dominant (and functional) and trp2 is recessive (and defective). Proteins encoded by a gene are referred to by the relevant gene symbol, non-italicized, with an initial uppercase letter and usually with the suffix 'p", for example, the adenine 2 protein encoded by ADE2 is Ade2p. Phenotypes are designated by a non- italic, three letter abbreviation corresponding to the gene symbol, initial letter in uppercase. Wild-type strains are indicated by a "+" superscript and mutants are designated by a "-" superscript. For example, Ade2 ⁺ is a wild-type phenotype whereas Ade2- is an auxotrophic phenotype (requires adenine).

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). The term "integration vector" refers to a vector that can integrate into a host cell and which carries a selection marker gene or open reading frame (ORF), a targeting nucleic acid, one or more genes or nucleic acids of interest, and a nucleic acid sequence that functions as a microorganism autonomous DNA replication start site, herein after referred to as an origin of DNA replication, such as ORI for bacteria. The integration vector can only be replicated in the host cell if it has been integrated into the host cell genome by a process of DNA recombination such as homologous recombination that integrates a linear piece of DNA into a specific locus of the host cell genome. For example, the targeting nucleic acid targets the integration vector to the corresponding region in the genome where it then by homologous recombination integrates into the genome.

The term "selectable marker gene", "selection marker gene", "selectable marker sequence" or the like refers to a gene or nucleic acid sequence carried on a vector that confers to a transformed host a genetic advantage with respect to a host that does not contain the marker gene. For example, the P. pastoris URA5 gene is a selectable marker gene because its presence can be selected for by the ability of cells containing the gene to grow in the absence of uracil. Its presence can also be selected against by the inability of cells containing the gene to grow in the presence of 5-FOA. Selectable marker genes or sequences do not necessarily need to display both positive and negative selectability. Non-limiting examples of marker sequences or genes from P. pastoris include ADEl, ADE2 ARG4, HIS4, LYS2, URA5, and URA3. In general, a selectable marker gene as used the expression systems disclosed herein encodes a gene product that complements an auxotrophic mutation in the host. An auxotrophic mutation or auxotrophy is the inability of an organism to synthesize a particular organic compound or metabolite required for its growth (as defined by IUPAC). An auxotroph is an organism that displays this characteristic; auxotrophic is the corresponding adjective. Auxotrophy is the opposite of prototrophy.

The term "a targeting nucleic acid" refers to a nucleic acid carried on the vector plasmid that directs the insertion by homologous recombination of the vector integration plasmid into a specific homologous locus in the host called the "target locus".

The term "sequence of interest" or "gene of interest" or "nucleic acid of Interest" refers to a nucleic acid sequence, typically encoding a protein or a functional RNA, that is not normally produced in the host cell. The methods disclosed herein allow efficient expression of one or more sequences of interest or genes of interest stably integrated into a host cell genome. Non- limiting examples of sequences of interest include sequences encoding one or more polypeptides having an enzymatic activity, e.g., an enzyme which affects N-glycan synthesis in a host such as mannosyltransferases, N-acetylglucosaminyltransferases, UDP-N-acetylglucosamine transporters, galactosyltransferases, UDP-N-acetylgalactosyltransferase, sialyltransferases, fucosyltransferases, erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, soluble IgE receptor α-chain, IgG, IgM, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor- 1, and osteoprotegerin.

The term "operatively linked" refers to a linkage in which a expression control sequence is contiguous with the gene or sequence of interest or selectable marker gene or sequence to control expression of the gene or sequence, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term "recombinant host cell" ("expression host cell," "expression host system," "expression system" or simply "host cell"), as used herein, is intended to refer to a cell into

which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

The term "eukaryotic" refers to a nucleated cell or organism, and includes insect cells, plant cells, mammalian cells, animal cells, and lower eukaryotic cells. The term "lower eukaryotic cells" includes yeast, unicellular and multicellular or filamentous fungi. Yeast and fungi include, but are not limited to Pichiapastoris, Pichia fmlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnerϊ), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

The term "peptide" as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs, derivatives, and mimetics that mimic structural and thus, biological function of polypeptides and proteins.

The term "polypeptide" encompasses both naturally-occurring and non-naturally- occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomelic or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions also include larger polypeptides, or even entire proteins, such as the green fluorescent protein (GFP) chromophore-containing proteins having particular utility. Fusion

proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. The term "functional nucleic acid" refers to a nucleic acid molecule that, upon introduction into a host cell or expression in a host cell, specifically interferes with expression of a protein. In general, functional nucleic acid molecules have the capacity to reduce expression of a protein by directly interacting with a transcript that encodes the protein. Ribozymes, antisense nucleic acids, and siRNA molecules, including shRNA molecules, short RNAs (typically less than 400 bases in length), and micro-RNAs (miRNAs) constitute exemplary functional nucleic acids.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an alignment of the P. pastor is Ade2p amino acid sequence (SEQ ID NO:61) to the S. cerevisiae Ade2p amino acid sequence (SEQ ID NO:62).

Figure 2 A shows a map of plasmid pGLY1065. Figure 2B shows a map of plasmid pGLY2057.

Figure 2C shows a map of plasmid pGLY225.

Figure 2D shows a map of plasmid pGLY1083.

Figure 2E shows a map of plasmid pGLY2092.

Figure 2F shows a map of plasmid pGLY2094. Figure 3 shows western blots and Coomassie gels of the protein produced in adel auxotrophic yeast strains transformed with integration vectors expressing glucocerebrosidase, a single-chain anti-HER2 antibody, or human CD40 ectodomain. Panel A shows the single-chain anti-HER2 antibody produced in seven clones of YGLY563 adel- cells transformed with pJ903 encoding a single-chain anti-HER2 antibody operably linked to the GAPDH promoter and ADEl ORF operably linked to its native promoter and the single chain anti-HER2 antibody produced in seven clones of YGLY563 adel- cells transformed with pJ904 encoding single-chain anti-HER2 antibody operably linked to the GAPDH promoter and ADEl ORF not operably linked to a

promoter. Panel B shows the glucocerebrosidase (GBA) produced in produced in seven clones of YGLY564 adel- cells transformed with pGlylO84 encoding GBA operably linked to the GAPDH promoter and ADEl ORF operably linked to its native promoter and the GBA produced in seven clones of YGLY564 adel- cells transformed with pGLY1085 encoding GBA operably linked to the GAPDH promoter and ADEl ORF not operably linked to a promoter. Panel C shows Coomassie gels of the human CD40 ectodomain produced in six clones of YGLY563 adel- cells transformed with pGLY1073 encoding human CD40 ectodomain operably linked to the AOXl promoter and ADEl ORF operably linked to its native promoter and the human CD40 ectodomain produced in six clones of YGLY563 adel- cells transformed with pGLY1074 encoding human CD40 ectodomain operably linked to the GAPDH promoter and ADEl ORF not operably linked to a promoter. Panel D shows the human CD40 ectodomain produced in six clones of YGLY564 ade-1 cells transformed with pGLY1073 encoding human CD40 ectodomain operably linked to the AOXl promoter and ADEl ORF operably linked to its native promoter and the human CD40 ectodomain produced in six clones of YGLY564 a de- cells transformed with pGLY1074 encoding human CD40 ectodomain operably linked to the AOXl promoter and ADEl ORF not operably linked to a promoter.

Figure 4 shows western blots of the protein produced in ade2 auxotrophic yeast strains transformed with integration vectors encoding erythropoietin (εPO). Panel A shows the εPO produced in six clones of YGLY1215 ade2- cells transformed with pGly2663 encoding εPO operably linked to the AOXl promoter and ADEl ORF operably linked to its native promoter and the εPO produced in six clones of YGLYl 215 ade2- cells transformed with pGly2664 encoding εPO operably linked to the AOXl promoter and ADE2 ORF not operably linked to a promoter. Panel B shows the εPO produced in six clones of YGLYl 216 ade2 ^~ cells transformed with pGly2663 encoding εPO operably linked to the AOXl promoter and ADEl ORF operably linked to its native promoter and the εPO produced in six clones of YGLY1216 ade2- cells transformed with pGly2664 encoding εPO operably linked to the AOXl promoter and ADE2 ORF not operably linked to a promoter.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods and materials for the use of lower eukaryotic cells such as yeast or filamentous fungi as an expression system for expressing recombinant peptides, proteins, or functional nucleic acids. In one aspect, the method provides a method for expressing a recombinant protein comprising obtaining or constructing slower growing ade2 auxotrophic strains of the lower eukaryote cells and introducing into the cells integration vectors that are capable of integrating into the genome of the ade2 auxotrophic strain and which comprises a nucleic acid encoding an ADE2 marker gene or open reading frame (ORF) operably linked to a promoter and a nucleic acid expressing a recombinant protein or functional nucleic

acid of interest, wherein the integration vector integrates into the genome of the ade2 auxotrophic strain, the ADE2 renders the auxotrophic strain prototrophic for adenine and the recombinant peptide, protein, or functional nucleic acid is expressed. The recombinant host cells are selected for in medium that lacks the metabolite adenine but can be maintained in medium that lacks the metabolite adenine or in medium that includes the metabolite adenine. In general, those recombinant host cells that might lose the ADE2 marker (revertants) will grow more slowly and will be lost over time as the recombinant cells are grown. The loss of revertants over time will occur whether the recombinant host cells are grown in medium that includes the metabolite adenine or in medium that either lacks the metabolite adenine. In developing the above invention, the applicants discovered that when the integration vector for introducing a recombinant protein into a lower eukaryote host cell that is auxotrophic for a particular marker gene includes in the integration vector a nucleic acid encoding the complimentary marker gene or ORF but wherein the marker gene or ORF is operably linked to a weak, attenuated, cryptic, truncated promoter that reduces the native activity of the promoter to level less than the native promoter, or no promoter, the auxotrophy of the host cell can be complimented provided that more than one copy of the integration vector is integrated into the genome of the host cell. Because the recombinant host cell contains more than one copy of the integration vector and each copy of the integration vector is transcriptionally active, the recombinant host cell is capable of producing a sufficient quantity of the marker gene or ORF to render the host cell prototrophic for the auxotrophic marker and thus capable of growing in medium that lacks the metabolite that can compliment the auxotrophy. The weaker the promoter linked to the complimentary marker gene or ORF, the more copies of the integration vector integrated into the genome of the host cell that are needed to render the host cell prototrophic for the auxotrophic marker. Host cells that lose copies of the integration vector integrated into the host genome during cell growth or passage in medium that lack the metabolite that can compliment the auxotrophy are rendered auxotrophic again for the marker gene. These newly auxotrophic host cells are at a selective disadvantage in the culture medium and in general, are lost as the remaining prototrophic host cells continue to grow and replicate. Importantly, because the integration vector contains an expression cassette that expresses one or more recombinant proteins or functional nucleic acids of interest, host cells containing one or more copies of the integration vector will produce more of the recombinant protein than would be produced in host cells that contained only one copy of the integration vector.

Therefore, methods, materials, and systems that are particularly useful for producing recombinant host cells that are capable of producing large quantities of recombinant proteins (including peptides), or functional nucleic acids are provided. Thus, the present invention provides a method in which an auxotrophic strain of a lower eukaryote cell is obtained or constructed and an integration vector is provided that is capable of integrating into the genome of

the auxotrophic strain and which comprises nucleic acids encoding a marker gene or ORF that compliments the auxotrophy and is either operably linked to a weak, cryptic, attenuated, or truncated promoter or no promoter and a recombinant protein. Host cells in which a number of the integration vectors have been integrated into the genome to compliment the auxotrophy of the host cell are selected in medium that lacks the metabolite that compliments the auxotrophy and maintained by propagating the host cells in medium that either lacks the metabolite that compliments the auxotrophy or includes the metabolite that compliments the auxotrophy. In general, those recombinant host cells that might lose the auxotrophic marker (revertants) will grow more slowly and will be lost over time as the recombinant cells are grown. The loss of revertants over time will occur whether the recombinant host cells are grown in medium that includes the metabolite or in medium that either lacks the metabolite. This phenomenon has been observed at least for the auxotrophic markers ADE, URA, or LYS and is currently believed to be due at least in part to poor transport of the metabolite from the medium into the recombinant host cell. In a general aspect, recombinant host cells are rendered auxotrophic for a particular organic compound by removing or deleting the gene or locus encoding the gene product necessary for producing the organic compound or an intermediate for producing the organic compound or metabolite. The auxotrophic host cells are then transformed with an integration vector that comprises (1) a nucleic acid comprising an open reading frame (ORF) encoding a selectable marker gene or other nucleic acid that complements the auxotrophy; (2) a nucleic acid encoding one or more ORFs encoding a heterologous or recombinant protein or peptide or expressing a functional nucleic acid of interest; and (3) nucleic acid comprising a targeting sequence that directs insertion of the integration vector into a particular target location or locus of the genome of the host cell by homologous recombination. The targeting sequence in the plasmid can comprise any sequence within the host cell genome such as a host cell gene, a host cell promoter or terminator sequence, or a sequence of unknown function. For integrating into a host cell promoter or termination sequence, the promoter and/or the terminator sequence in the expression cassette used for regulating expression of the one or more ORFs encoding a heterologous or recombinant protein or peptide or expressing a functional nucleic acid of interest can also function as the targeting sequence for targeting the integration vector to the target location. For example, the nucleic acid of (1) or (2) above can be operably linked to a host cell promoter for a host cell gene adjacent to the promoter to which the integration vector is targeted. Integration of the vector into the promoter via roll-in single crossover homologous recombination results in a duplication of the promoter sequences. Thus, after integration, the expression cassette is still operably linked to the promoter comprising the targeting sequence and the host cell gene adjacent to the promoter that was the targeting sequence is still operable. Thus, in the recombinant host cell, expression of a heterologous

protein, peptide, or functional nucleic is effected without disrupting expression of the host cell gene adjacent to the targeting site.

To integrate the integration vector into the genome of a host cell by roll-in single crossover homologous recombination, the vector is linearized by cleaving the integration vector at a site within the targeting sequence so as to produce a linear nucleic acid molecule in which the targeting sequences are at the ends of the molecule. Single cross-over events lead to a duplication of the genomic locus and generates direct repeats. While these direct repeats display a high recombination rate and can result in the loss of the marker and expression cassette during propagation of the recombinant host cell, the method disclosed herein where the marker is operably linked to a weak, cryptic, attenuated, or truncated promoter or no promoter ensures that only host cells that maintain the copy number of integration vectors sufficient to render the host cell prototrophic for the marker during propagation. In a preferred aspect, the integration vector is linearized at a restriction enzyme site that occurs only once in the targeting sequence. The vector then integrates into the target site by roll-in single crossover homologous recombination. Roll-in single crossover homologous recombination enables integration of the integration vector into the genome without disrupting expression of the gene at the target site. Roll-in single crossover homologous recombination has been described in Nett et al, Yeast 22: 295-304 (2005).

An important feature of the integration vector is that the ORF encoding the selectable marker gene or other nucleic acid is not operably linked to its endogenous full-strength promoter or to a heterologous full-strength promoter but to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, or a truncated endogenous or heterologous promoter in which the truncation renders the promoter with a transcription activity that is less than the native promoter. In particular embodiments, the attenuated or truncated promoter has a transcription activity that is no more than 50% of the activity of the full-strength promoter. In further embodiments, the attenuated or truncated promoter has a transcription activity that is no more than 10% of the activity of the full-strength promoter. In further embodiments, the attenuated or truncated promoter has a transcription activity that is no more than 1% of the activity of the full-strength promoter. While not wishing to be bound by any theory, it is believed that in general, the nucleic acid sequence adjacent to the ORF encoding the selectable marker gene will contain a so-called cryptic promoter that enables a low level of expression of the selectable marker gene. A cryptic promoter will allow a sufficient amount of spurious transcription initiation adjacent to the ORF sufficient to produce a low amount of the selectable marker. Since expression of the selectable marker gene is below the level needed to fully complement the auxotrophy, multiple integrations of the integration vector into the target sequence in the host cell is necessary for full complementation of the auxotrophy. Because multiple copies of the integration vector must be integrated into the genome of the host cell to

complement the auxotrophy, there are multiple copies of the ORF encoding the protein or peptide or functional nucleic acid of interest, all of which are expressed. Thus, the host cell is capable of encoding more of the protein or peptide or functional nucleic acid of interest than a host cell that includes only one copy of the integration vector integrated into its genome. In practicing the method, it is preferable that there not be any of the selectable marker gene sequence in the auxotrophic host cell that could compete with the targeting sequence for integration. Thus, in further embodiments, either the entire gene encoding the marker (including upstream and downstream regions) is deleted or removed from the genome or at least the open reading frame encoding the marker gene is deleted or removed from the genome. Stable recombinant host cells in which the integration vector is integrated into the target locus are selected by cultivating the transformed host cells in a culture medium that lacks the particular organic compound (metabolite). Because the selectable marker gene or ORF is not operably linked to an endogenous or heterologous full-strength promoter but is operably linked to a weak, attenuated, cryptic promoter, or truncated promoter (or in particular aspects, no promoter), the recombinant, transformed host cells containing only one copy of the integration vector inserted into the target locus are not rendered prototrophic for the organic compound or metabolite. For the transformed host cells to be rendered prototrophic for the organic compound or metabolite, multiple copies of the integration vector must be integrated into the target locus for the host cell, hi addition, because multiple copies of the integration vector must be integrated into the target locus, significant quantities of the protein or peptide encoded by the gene or sequence of interest are also produced.

Lower eukaryotes such as yeast are preferred for expression of proteins because they can be economically cultured, give high yields, and when appropriately modified are capable of suitable glycosylation. Yeast particularly offers established genetics allowing for rapid transformations, tested protein localization strategies and facile gene knock-out techniques. Suitable vectors have expression control sequences, such as promoters, including 3- phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha are generally preferred for cell culture because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp, Neurospora crassa and others can be used to produce glycoproteins of the invention at an industrial scale. Other cells useful as host cells in the present invention include prokaryotic cells, such as E. coli, and eukaryotic host cells in cell culture, including lower eukaryotic cells, plant cells, and mammalian cells, such as Chinese Hamster Ovary (CHO).

Lower eukaryotes, particularly yeast, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or supplying exogenous enzymes as described by Gerngross et al., US 2004/0018590, the disclosure of which is hereby incorporated herein by reference. For example, a host cell can be selected or engineered to be depleted in 1,6-mannosyl transferase activities, which would otherwise add mannose residues onto the N-glycan on a glycoprotein.

U.S. Published application No. 20070072262 discloses ARGl, ARG2, ARG3, HISl, HIS2, HIS5, and HIS6 genes and methods of using the genes for stable genetic integration into yeast and U.S. published application No. 20040229306 discloses the Pichia pastoris URA5 gene and its use for genetic stable integration in yeast. Selectable marker genes that are particularly useful in practicing the methods and systems herein include, but are not limited to, URA3, URA5, HIS3, LEU2, TRPl, LYS2, ADEl, and ADE2 loci. Useful are auxotrophic host cells and selectable marker genes in which the particular auxotrophy renders the cell less able to compete with or grow more slowly than the corresponding prototroph. Thus, particularly useful selectable marker genes are the selectable marker genes ADEl, ADE2. LYS2, URA3, and URA5.

The ADEl gene has been cloned from various species of yeast and fungi, including Saccharomyces cerevisiae (Myasnikov et al., Gene,109:143-147 (1991); Kluyveromyces lactis (Zonneveld and van der Zanden, et al., Yeast, 11 :823-827 (1995), Pichia pastoris (Cereghino et al., Gene 263: 159-169 (2001)). ADEl gene encodes N-succinyl-5-aminoimidazole-4- carboxamide ribotide (SAICAR) synthetase, which is required for de novo purine nucleotide biosynthesis. Red pigment accumulates in mutant cells deprived of adenine.

The ADE2 gene has been cloned from various species of yeast and fungi, including Saccharomyces cerevisiae (Jones and Fink, "Regulation of amino acid and nucleotide biosynthesis in yeast" pp.181 -299 in The Molecular Biology of the Yeast Saccharomyces:

Metabolism and Gene Expression, Strathern et al. (Eds.) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press); Candida albicans (Kurtz et al, MoI. Cell. Biol., 6:142-149 (1986)); Aspergillus oryzae, (Jin et al., Biosci Biotechnol Biochem. 68:656-62(2004), and Pichia pastoris (herein). The ADE2 gene encodes phosphoribosyl-aminoimidazole carboxylase, which catalyzes a step in the de novo purine nucleotide biosynthetic pathway. Red pigment accumulates in mutant cells deprived of adenine.

In further embodiments, a cell line can be transformed with a vector that will displace or knock out the function of one or more auxotrophic genes, for example, knocking out or displacing the ADEl or ADE2 genes to render the cells auxotrophic for adenine, for example, cells with an Adel- or Ade2- phenotype. Thus, the present invention includes methods for genetically engineering cell lines such that they contain auxotrophic mutations which impede the growth of the cells. These cell lines containing auxotrophic mutations can then serve as the host

cells for selection as taught herein, in which the host cells are transformed with integration vectors encoding one or more desired glycoproteins and genes which complement the auxotrophic mutations such that the cells expressing the desired protein(s) will also carry the gene(s) which complement the auxotrophic mutations and provide a phenotype which is readily identifiable and selectable.

The method disclosed herein using the ADEl oτADE2 markers and adel or ade2 auxotrophic host cells is particularly useful for making recombinant Pichia pastoris host cells because it addresses the scarcity of suitable markers for Pichia pastoris that can be used for multicopy selection. To date, primarily dominant markers, like Zeocin, are used for this purpose. However, dominant markers possess significant disadvantages. For example, during fermentation, it is frequently not feasible to add the antibiotic, in order to make sure all integrated copies of the heterologous gene stay integrated. However, if the constructs disclosed herein are evicted, the cells will become unable to produce adenine and will exhibit the selectable phenotype of slower growth and pinkish color. Therefore, heterologous constructs which are evicted during fermentation are easily selected against by virtue of this slower growth.

The advantages of the disclosed system are the ability to select transformants with multiple copies of the marker and desired gene for expression in the host cell integrated in the genome by color, and the stable retention of the transformants with one or more copies integrated into the genome because of the slow growth of adel or ade2 cells. In one aspect, the system utilizes the differential phenotypes of pink/white color selection oϊade/ADE strains coupled with the slow growth of strains having an Adel- or Ade2- phenotype and the integration of a plasmid comprising a copy of the ADEl or ADE2 open reading frame (ORF) operably linked to a promoter and a desired gene for expression in the host cell in order to in order to provide a system that is an improvement over the current system for making recombinant host cells that relies upon dominant Zeocin selection. In another aspect, the system utilizes the differential phenotypes of pink/white color selection of ade/ADE strains coupled with the slow growth of strains having an Adel- or Ade2- phenotype and the forced multiple integration of a plasmid comprising a copy of the ADEl or ADE2 ORF operably linked to a weak, attenuated, or cryptic promoter and a desired gene for expression in the host cell in order to provide a system that is an improvement over the current system for making recombinant host cells that relies upon dominant Zeocin selection. Thus, the methods and materials are useful for stable high level expression of heterologous proteins.

Thus, in particular embodiments, the method and system comprises recombinant host cells, non-human eukaryotic host cells, in particular lower eukaryotic host cells such as yeast and filamentous fungal host cells, with improved productivity for the production of recombinant proteins, including glycoproteins when using host cells capable of making glycoproteins having hybrid or complex N-glycans. The recombinant host cells are modified by the reduction or

elimination of the function of at least one endogenous gene encoding an auxotrophic marker gene, such as ADEl or ADE2. Cells with a mutation leading to adenine deficiency grow quite slowly, and accumulate a reddish pigment, which results in production of pink colonies (that is, cells with an Adel- or Ade2- phenotype). When these cells with an Adel- or Ade2- phenotype are transformed with a plasmid comprising an ADEl or ADE2 ORF, respectively, operably linked to a promoter for expressing the ADEl or ADE2 ORF, the Adel or Ade2 mutation is complemented and the cell is rendered prototrophic for adenine, that is, the cells are rendered to have an Adel ⁺ or Ade2 ⁺ phenotype. These complemented recombinant cells exhibit a white color and large colony size, which facilitates identification and selection of the recombinant cells. Alternatively, when these cells with an Adel- or Ade2- phenotype are transformed with a plasmid comprising the ADEl or ADE2 ORF, respectively, not operably linked to a promoter for expressing the ADEl or ADE2 ORF, the Adel or Ade2 mutation is complemented only in recombinant cells that contain more than one copy of the ADEl or ADE2 gene integrated into the genome. In other embodiments, the integration vectors are provided for the selectable expression of heterologous genes in an expression system employing host cells, which exhibit an Adel- or Ade2- phenotype, such as the host cells described above. The integration vectors comprise a nucleic acid comprising a promoter sequence and a transcription termination sequence separated by and operably linked to a cloning site. A nucleic acid sequence encoding one or more desired heterologous proteins or peptides or functional nucleic acid of interest is inserted into the cloning site using standard ligation techniques in the proper orientation to be expressed via the promoter. The integration vector preferably comprises at least one promoter, which is functional in the host cell, followed by at least one restriction site, preferably a multiple cloning site, followed by a transcription terminator sequence which is functional in the host cell. Using appropriate known techniques, a nucleotide fragment encoding the desired protein or polypeptide can be ligated into the restriction sites of cloning site of the integration vector. The integration vectors also comprises at least one copy of a selectable marker ORF selected from the group consisting of ADEl and ADE2, which may be under the control of appropriate transcription termination terminator sequences, which are functional in the host cell. In some embodiments, the ORF is operably linked to a full-strength homologous or heterologous promoter and in other embodiments, the ORF is operably linked to a cryptic promoter, weak promoter, attenuated promoter, or a truncated homologous or heterologous promoter with reduced transcriptional activity compared to the full-strength promoter.

In a further embodiment, provided are methods, materials, and systems for the construction of recombinant host cells for expressing heterologous or recombinant proteins and peptides wherein the ADEl gene has been removed or deleted to render the host cells auxotrophic for adenine, for example, render the cells adel . The adel host cells are then

transformed with an integration vector comprising (1) a nucleic acid encoding the Adelp or Adelp activity operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter; (2) one or more nucleic acids encoding a gene or functional nucleic acid of interest to produce a heterologous or recombinant protein or peptide or functional nucleic acid ectopically; and (3) a targeting nucleic acid sequence that directs insertion of the integration vector into a particular target location or locus of the genome of the host cell by homologous recombination. Stable recombinant host cells in which the integration vector is integrated into the target locus are selected by cultivating the transformed host cells in a culture medium that lacks adenine. Because the nucleic acid encoding the Adelp activity is operably linked to a weak, attenuated, cryptic promoter, or truncated promoter, the recombinant, transformed host cells containing only one copy of the integration vector inserted into the target locus are not rendered prototrophic for adenine. For the transformed host cells to be rendered prototrophic, multiple copies of the integration vector must be integrated into the target locus for the host cell. In addition, because multiple copies of the integration vector must be integrated into the target locus, significant quantities of the protein or peptide encoded by the gene or sequence of interest are produced.

In another embodiment, provided are methods, materials, and systems for the construction of recombinant host cells for expressing heterologous or recombinant proteins and peptides wherein the ADE2 gene has been removed or deleted to render the host cells auxotrophic for adenine. The ade2 host cells are then transformed with an integration vector comprising (1) a nucleic acid encoding the Ade2p or Ade2p activity operably linked to a weak promoter, an attenuated endogenous or heterologous promoter, a cryptic promoter, a truncated endogenous or heterologous promoter, or no promoter; (2) one or more nucleic acids encoding a gene or functional nucleic acid of interest to produce a heterologous or recombinant protein or peptide or functional nucleic acid ectopically; and (3) a targeting nucleic acid sequence that directs insertion of the integration vector into a particular target location or locus of the genome of the host cell by homologous recombination. Stable recombinant host cells in which the integration vector is integrated into the target locus are selected by cultivating the transformed host cells in a culture medium that lacks adenine. Because the nucleic acid encoding the Ade2p activity is operably linked to a weak, attenuated, or cryptic promoter, the recombinant, transformed host cells containing only one copy of the integration vector inserted into the target locus are not rendered prototrophic for adenine. For the transformed host cells to be rendered prototrophic for adenine, more than one copy of the integration vector must be integrated into the target locus for the host cell. In addition, because multiple copies of the integration vector must be integrated into the target locus, significant quantities of the protein or peptide encoded by the gene or sequence of interest are produced.

In both of the above embodiments, adel or ade2 auxotrophs grow more slowly than prototrophs (e.g., ADEl or ADE2, respectively) or cells rendered prototrophic by integration of multiple copies of the integration vector into the genome. In culture, revertants and transformed cells that lose multiple copies of the integration vector inserted into the target locus will grow more slowly and be out-competed by those cells that maintain the multiple copies of the integration vector integrated into the target locus. In addition, in particular organisms such as yeast, adel or ade2 auxotrophs are red or pink in color whereas prototrophs or cells rendered prototrophic by integration of more than one copy of the integration vector into the genome are white. Thus, selection of recombinant cells containing multiple copies of the integration vector inserted into the target sequence can be based upon selecting white colonies.

The methods and systems herein can be practiced in any organism in which auxotrophic mutations can be made such as the adel or ade2 and complementation thereof results in the selectable phenotype described herein. The methods involve transforming host cells which exhibit adel or ade2 minus phenotype with integration vectors which include nucleotide sequences encoding the complementary Adelp or Ade2p proteins, such that when the host cells are transformed with the integration vector encoding a desired secreted glycoprotein, the complementation of the Adel- and/or Ade2- phenotype leads to stable integration of the genes encoding the desired glycoprotein, and contributes to improved quality of the transformed recombinant host cells, particularly, increased yield of the desired recombinant glycoprotein. hi further embodiments, the host cells of the present invention carry other genetic manipulations in their genome, such that the host cells, and/or the proteins or peptides produced therefrom, exhibit desired properties. For example, the host cell may be manipulated in accordance with the methods described in for example, U.S. Patent No. 7,029,872, U.S. Published Application No. 2004/0018590, and Hamilton et al, Science, 313: 1441-1443 (2006); such that the host cells are capable of producing recombinant glycoproteins with highly homogeneous levels of one or more desired glycoforms. In other embodiments, the host cells may be modified by deleting one or more endogenous genes encoding molecular chaperone proteins and/or transforming the host cell with one or more heterologous genes encoding molecular chaperone genes originating from the species of the heterologous protein or polypeptide to be produced. For example, a host cell of the species Pichia may be modified by elimination of the endogenous protein PDI and/or BiP, and transformed with one or ore plasmids encoding mammalian PDI, BiP and/or GRP94 genes. See, Choi et al. supra, the disclosure of which is hereby incorporated herein by reference.

In further still embodiments, methods are provided for increasing the productivity of recombinant human or mammalian glycoproteins in a non-human eukaryotic host cell, lower eukaryotic host cell, or a yeast or filamentous fungal host cell. The methods comprise the step of transforming a host cell, which is adel or ade2 and capable of producing glycoproteins having

hybrid or complex N-glycans, with a vector comprising a nucleic acid encoding ADEl or ADE2 ORF and a nucleic acid encoding a glycoprotein of interest.

The following examples are intended to promote a further understanding of the present invention.

EXAMPLE 1

Cloning of P 'ichia pastoris ADEl and ADE2 genes was performed as follows. The cloning of the P. pastoris ADEl gene has been published before (Cereghino et al., supra). Additional 5'- and 3'- sequence was obtained using a partial P. pastoris genomic sequence obtained from Integrated Genomics, Chicago, IL. The nucleotide sequence of the P. pastoris ADEl open reading frame (ORF), including promoter and transcription termination sequences, is shown in SEQ ID NO: 56. The amino acid sequence of the P. pastoris ADEl is shown in SEQ ID NO:57. Querying the same genomic sequence with the S. cerevisiae ADE2 ORF, the P. pastoris ADE2 homologue (563 amino acids with 69% identity) was identified using the program BLAST (Altschul et al, J. MoI. Biol. 215: 403-410 (1990)). The nucleotide sequence encoding the P. pastoris ADE2 ORF, including promoter and transcription termination sequences, is shown in SEQ ID NO:60. The ADE2 ORF is encoded by nucleotides 127 to 1,815 of the nucleotide sequence shown in SEQ ID NO:60 and has the amino acid sequence shown in SEQ ID N0:61. Alignment of the P. pastoris ADE2 amino acid sequence (SEQ ID N0:61) to the S. cerevisiae ADE2 amino acid sequence (SEQ ID NO:62) is shown in Figure 1.

EXAMPLE 2

Construction of ADEl and ADE2 Knock-out vectors and strains was as follows. In the first step of plasmid construction, we created a universal knock-out plasmid containing DNA regions of: (a) the ARG3 gene of P. pastoris (Nett et al. 2005, supra) as space holders for the 5' and 3' regions of the gene to be knocked out; (b) the P. pastoris C/iL4J-blaster(Nett and Gerngross, Yeast 20: 1279-1290 (2003) as auxotrophic marker; and (c) an expression cassette with a multiple cloning site for insertion of a foreign gene.

To create a WL45-blaster cassette compatible with the architecture of the universal knock-out plasmid the Sacl-Pvύll fragment of lacZ was cloned into the Sacl-Smal sites of pUC19. The resulting plasmid was digested with Hindi and the Sacϊ-Pvull fragment of lacZ that had been blunt-ended using T4DNA polymerase was inserted into this plasmid in a head to tail orientation to yield pGLY8. A 1.0 kb DNA fragment of the P. pastoris URA5 gene was amplified using primers Ura5comp5 (SEQ ID NO: 1) and Ura5comp3 (SEQ ID NO: 2) and yeast strain NRRL Y-11430 genomic DNA as template and cloned into the Bamlil-Xbaϊ sites of pGLY8 to generate pGLYlO. In order to remove the internal Sad and Xhol sites three overlapping fragments of URA5 were amplified using pGLYlO as template and primer pairs

URA5MUT1 (SEQ ID NO:3) and URA5MUT2 (SEQ ID NO:4), URA5MUT3 (SEQ ID NO:5,) and URA5MUT4 (SEQ ID NO:6), and URA5MUT5 (SEQ ID NO:7) and URA5MUT6 (SEQ ID NO: 8) respectively. The resulting PCR products were gel purified, mixed and served as template in a fusion PCR using URA5MUT1 (SEQ ID NO:3) and URA5MUT6 (SEQ ID NO:8) as primers. The resulting PCR product was then cloned into vector pCR2.1 TOPO ^® , removed again using Clal and 55-5HII and cloned into pGLYlO that also had been digested with Clal and BssHll to yield pGLY12. To remove the Sad and BamHl sites, pGLY12 was first cut with Sad, blunt- ended using T4DNA polymerase and religated creating pGLY13a and then cut with BamHl, blunt-ended and religated to yield pGLY13b. In both cases, the lacZ-URA5 cassette can be released by digestion with EcoKL and Sphl.

A 1.1 kb DNA fragment of the ARG3-5 ¹ region was amplified by PCR using primers ARG355DIS (SEQ ID NO:9) and ARG353-2 (SEQ ID NO: 10) with P. pastoris genomic DNA as a template and cloned into the Sad— SaR sites of pUC19. The resulting plasmid was cut with BamHl and Sail and a 0.7 kb DNA fragment of the ARG3-3' region that had been amplified using primers ARG335-2 (SEQ ID NO:11) and ARG333 (SEQ ID NO:12) was cloned into the open sites creating pGLY21. The plasmid was cut with BamRI blunt-ended with T4DNA polymerase and the EcoRl and Sphl cut and blunted /αcZ-URA5 cassette from pGLY 13a or pGLY13b were inserted resulting in plasmids pGLY22b and pGLY23 respectively. Plasmid pGLY22b constitutes the universal knock-out plasmid without additional expression cassette, whereas pGLY23 was further modified to also contain a cassette for the additional expression of a heterologous gene.

To create an expression cassette with Notl and Pad as cloning sites, a 0.5 kb DNA fragment containing the GAPDH promoter of P. pastoris was amplified using primers GAP5CLEAN (SEQ ID NO: 13) and GAP3CLEAN (SEQ ID NO: 14) and P. pastoris genomic DNA as template and cloned into the BamHl - Sphl sites of pUC 19. The resulting plasmid was cut with Spel and Sphl and a 0.3 kb fragment containing the S. cerevisiae CYCl transcriptional terminator region that had been amplified using primers CYC5CLEAN (SEQ ID NO: 15) and CYC3CLEAN (SEQ ID NO: 16) and S. cerevisiae genomic DNA as template and had been cut with Nhel and Sphl was cloned into the open sites creating pGLY17. The expression cassette was released by BamHl digestion and cloned into pGLY23 to yield pGLY24.

The ADEl knock-out plasmid was constructed from pGLY22b in the following way. A 1.8 kb fragment of the ADEl -5' region that had been amplified using primers ADEl 55L (SEQ ID NO: 17) and ADEl 53L (SEQ ID NO: 18) was cut with Sad and Pmel and cloned into pGLY22b to yield pGLY1064. Then a 1.5 kb fragment of the ADEl-V region that had been amplified using primers ADE1KO35 (SEQ ID NO:19) and ADE133L (SEQ ID NO:20) was cut with Swal and Sphl and cloned into pGLY1064 creating the ADEl knock-out plasmid pGLY1065. (Figure IA)

The ADE2 knock-out plasmid was constructed from pGLY24 in the following way. The P. pastoris ALG3 transcriptional terminator was PCR amplified using primers RCD534 (SEQ ID NO:21) and RCD535 (SEQ ID NO:22) and P. pastoris genomic DNA as template, cut with EcoRW and Aβll and cloned into the Pmel-Aflll sites of pGLY24 to create pGLY566. This modification is irrelevant for the following ADE2 knock out plasmid, but served to construct a plasmid used for a different project. A 1.7 kb fragment of the ADE2-3' region that had been amplified using primers ADE235 (SEQ ID NO:25) and ADE233 (SEQ ID NO:26) was cut with Swaϊ and Sail and cloned into pGLY566 to yield pGLY1079. Then a 1.0 kb fragment of the ADE2-5' region that had been amplified using primers ADE255KO (SEQ ID NO:23) and ADE253KO (SEQ ID NO :24) was cut with Sad and Fsel and cloned into pGLYl 079 to yield the ADE2 knock-out plasmid pGLY2057. (Figure IB)

ADEl and ADE2 knock-out strains were constructed the following way. The strain YGLY24-3 ochl A:\lacZ, bmt2A::lacZ/KMNN2-2, mnn4LlA::lacZ/MmSLC35A3,pnolAmnn4A::lacZ, that had been constructed using methods described earlier (Nett and Gerngross, Yeast 20: 1279-1290 (2003); Choi et al., Proc. Natl. Acad. Sci. 100: 5022-5027 (2003); Hamilton et al., Science 301 : 1244-1246 (2003) was transformed with pGLY1065 and two pink transformants were designated YGLY563 and YGLY564. Their Adel phenotype was confirmed by their inability to grow on media lacking Adenine. These strains are capable of producing glycoproteins having predominantly Man5GlcNAc2 N-glycans.

Strains YGLY227 and YGLY228 (direct descendants of YGLY24-3 that had been transformed with a URA5 marked Trichoderma reesei 1 ,2 mannosidase expressing plasmid and counterselected on 5-FOA in an unrelated experiment) were transformed with pGLY2057 and for each strain one pink transformant was isolated generating strains YGLYl 215 and YGLYl 216 respectively. Their ade2 phenotype was also confirmed by their inability to grow on media lacking Adenine (results not shown). As expected (Cereghino et al., supra), both the adel and ade2 strains exhibited a slow growth phenotype even on media supplemented with Adenine. These strains are capable of producing glycoproteins having predominantly Man5GlcNAc2 N- glycans.

EXAMPLE 3

Construction of ADEl and ADE2 Marked Integration Vectors was as follows. A vector with a more suitable multiple cloning site containing sites for BgHl, EcoRl, Kpnl, Swal, BamHl, Notl, Pad, Asd and Sβl was constructed by cutting pUC19 with EcoRI and Hindlll and inserting annealed oligos EXMCSl (SEQ ID NO:27) and EXMCS2 (SEQ ID NO:28), creating pGLY192. A 0.3 kb DNA fragment containing the S. cerevisiae CYCl transcriptional terminator region that had been amplified using primers CYCTT5 (SEQ ID

NO:29) and CYCTT3 (SEQ ID NO:30) and S. cerevisiae genomic DNA as template was cut with BamHl and Swaϊ and cloned into pGLY192 yielding pGLY213. Then the P. pastor is AOXl promoter was amplified from genomic DNA using oligos AOX1P-5 (SEQ ID NO:31) and AOX1P-3 (SEQ ID NO:32), cut with Bgϊil and EcoRl and ligated into pGLY213 to create pGLY214. Since both ade knock-out plasmids had been designed to remove the complete ORF, a region for integration of the plasmid as an alternative to the promoter region was added. To this end a 1.8 kb fragment containing the P. pastoris TRP2 gene was amplified from genomic DNA using oligos TRP2-5 (SEQ ID NO:33) and TRP2-3revised (SEQ ID NO:34), cut with Sfλ and cloned into pGLY214 to yield pGLY215. This plasmid contains an EcoRl, Kpnl, Swal site for addition of the gene of interest and a Barriαl, No/I, Pad, Ascl, site for addition of the truncated ADE markers.

ADEl marker cassettes containing the ADEl ORF operably linked to its native promoter or to various truncations of the native promoter were constructed as follows. The ADEl markers with full-length or truncated promoters were PCR amplified using oligo ADEl ^' -3 (SEQ ID νO:35) as 3'-oligo and ADE1-5C-BAM (SEQ ID NO:36), ADE1-5-100 (SEQ ID NO:37),

ADE1-5-186 (SEQ ID NO:38), ADE1-5-295 (SEQ ID NO:39), ADE1-5-325 (SEQ ID NO:40), and ADE1-50RF (SEQ ID NO:41) as 5'-oligos, yielding fragments with 370, 276, 191, 82, 50, and 0 nucleotides of promoter region respectively. The first five fragments were cut with Nøtl and Ascl and the last fragment was cut with Pad and Asd and all fragments were cloned into pGLY215 to generate the ADEl marked integration plasmids pGLY220 to pGLY225 respectively (See Figure 1C for a plasmid map of pGLY225). To create plasmids for constitutive protein expression, the AOXl promoter in pGLY220 and pGLY225 was removed and replaced by a GAPDH promoter that had been amplified using primers GAPDHP-5 (SEQ ID νO:42) and GAPDHP-3 (SEQ ID NO:43), yielding plasmids pGLYl 082 and pGLY1083 respectively (See Figure ID for a plasmid map of pGLY1083).

ADE2 marker cassettes containing the ADE2 ORF operably linked to its native promoter or to various truncations of the native promoter were constructed as follows. An unmarked integration plasmid equivalent to pGLY215 for the ADE2 marker cassettes was constructed essentially the same way as above. The main difference between this plasmid, called pGFI4, and pGLY215 was that it contained the GAPDH promoter that had been amplified as above and the multiple cloning site for addition of the gene of interest had been expanded using oligos 5oligoERSSKFS (SEQ ID NO:44) and 3oligoSFSSRF (SEQ ID NO:45) to contain the restriction sites EcoRl, Rsrll, Sphl, Stul, Kprή, Fsel and Swal. The truncated ADE2 markers were amplified using oligo ADE23 (SEQ ID NO:46) as 3'-oligo and oligos ADE25NotI-l (SEQ ID NO:47), ADE25NotI-2 (SEQ ID NO:48), ADE25NotI-3 (SEQ ID NO:49), ADE25NotI-4 (SEQ ID

NO:50), and ADE25'PacInew (SEQ ID NO:51) as 5'-oligos, yielding fragments with 126, 82, 51, 13, and 0 nucleotides of promoter region respectively. The first four DNA fragments were cut

with Notl and Ascl and the last DNA fragment was cut with Pad and Ascl and all fragments were cloned into pGFI4 to generate the ADE2 marked integration plasmids pGLY2077 to pGLY2081 respectively. In addition to the EcøRI site in the multiple cloning site these plasmids also contain an EcoRl site in the ADE2 ORF. In pGLY2077 and pGLY2081 the EcoKl site in the ORF was therefore removed by site directed mutagenesis creating pGLY2091 and pGLY2092 respectively. (See Figure IE for a plasmid map of pGLY2092). To create plasmids for inducible protein expression, the GAPDH promoter in these two last constructs was removed and replaced by an AOXl promoter that had been amplified using primers AOX1P-5 (SEQ ID NO:52) and AOX1P-3 (SEQ ID NO:53) as above, yielding plasmids pGLY2093 and pGLY2094 respectively (See Figure IF for a plasmid map of pGLY2094).

In order to test the effect of the truncated markers on protein expression, several vectors expressing various proteins of interest were constructed.

Human glucocerebrosidase (GBA) was fused to the human serum albumin (HSA) signal sequence and cloned into the EcoRI / Kpnl sites of pGLYl 082 and pGLYl 083 to create GAPDH driven and ADEl marked integration vectors pGLY1084 and pGLY1085 respectively. A single- chain version of the anti-HER2 monoclonal antibody Herceptin® (U.S. Patent Application No. 20060252096) fused to the S. cerevisiae alpha mating factor pre-sequence and cloned into the EcoRUSwal sites of pGLY1082 and pGLY1083 to yield GAPDH driven and ADEl marked integration vectors pJN904 and pJN905 respectively. The human CD40 ectodomain (amino acids 20 to 192, a gift of R.J. Noelle; Lu, L. et ai, J. Biol. Chem. 278: 45414-45418 (2003) was fused to the S. cerevisiae alpha mating factor prepro-sequence and cloned into the EcoKl/Kpnl sites of pGLY 220 and pGLY225 to create AOXl driven and ADEl marked integration vectors pGLY1073 and pGLY1074 respectively. Human EPO was fused to the S. cerevisiae alpha mating factor pre-sequence and cloned into the EcoRUKpnl sites of pGLY2093 and pGLY2094 to yield AOXl driven and ADE2 marked integration vectors pGLY2663 and pGLY2664 respectively.

EXAMPLE 4 Effect of ADE marker promoter length on copy number and protein expression was determined.

To test the effect of the various ADE marker promoter truncations on copy number and protein expression, we considered the following assumptions: 1) Since all integration plasmids are integrated into the same genomic locus (TRP2), it is not expected that a reduction of marker promoter strength will lead to an increased copy number of plasmid integrants per se; 2) If the marker promoter strength drops below a certain threshold it is expected that clones integrating only a single copy of the plasmid will grow at a slower rate than clones integrating multiple

copies of the plasmid due to the slow growth phenotype of the ade minus phenotype. This should also be concomitant with the appearance of pink color in the low copy clones; 3) A gradual drop in marker promoter strength should therefore lead to decreasing numbers of fast growing white clones and on a relative basis increasing numbers of slow growing pink clones; and, 4) In order to eliminate any effect that the expression of a heterologous protein might exert on the growth of transformants, the empty expression plasmids should be tested initially.

Auxotrophic adel strains YGLY563 and YGLY564 were therefore transformed with equal amounts (0.2 μg) of integration plasmids pGLY220 to pGLY225 that had been linearized in the TRP2 integration region using BspEl and spread on minimal media plates. After five days of incubation at 23 °C the transformation plates were assessed for colony number. Surprisingly, integration plasmids pGLY220 to pGLY224 all yielded approximately the same number of colonies. Both yeast strains that had been transformed with pGLY225 however yielded less than 10% of the number of white transformants with a significant number of barely visible, pink transformants in the background (See Table 1). It had been anticipated that the plasmids with the promoter truncations would give rise to smaller number of colonies as the length of the promoter decreased, with the shortest one, only containing the ORF, yielding none. The results however suggest that the CYCl terminator region and the multiple cloning site in front of the marker contain a cryptic promoter activity that allows for a background level of transcription, thereby resulting in levels of ADEl gene product that in some cases are enough to complement the adel auxotrophic phenotype.

When ade2 auxotrophic strains YGLY1215 and YGLYl 216 were transformed with integration plasmids pGLY2077 to pGLY2081, a somewhat similar picture was obtained. In the case of the truncated ADE2 markers however, a gradual reduction in colony number concomitant with a shorter promoter was observed. As was the case for ADEl, the vector only containing the ADE2 ORF with no native promoter sequence at all yielded less than 10% of the number of white transformants than the construct with the full promoter sequence (See Table 1).

In order to test how this anticipated multicopy integration affected protein expression levels, plasmids expressing GBA, single-chain anti-HER2 antibody, human CD40 ectodomain or human EPO were transformed into adel or ade2 auxotrophic yeast strains (See Table 2). Transformants were grown in 96 well deep well plates, expression was induced using the appropriate carbon source and protein levels were assessed by Western Blot or Coomassie gel (See Figures 3 and 4). For most transformations using the promoterless ADE2 ORF as marker, as expected, a very low number of white transformants (5 to 20) were observed. However the expression level of those clones was generally significantly higher than clones obtained from transformations using the complete ADE2 gene as marker, which usually gave rise to hundreds of transformants (See Figures 3 and 4). Especially striking is the amount of protein produced from the clone shown in the lane marked in Figure 3D with an asterisk.

Materials and Methods

Escherichia coli strain DH5α was used for recombinant DNA work. Wild type P. pastoris strain NRRL-Y 11430 was used for construction of yeast strains (ATCC #76273). PCR reactions were performed according to supplier recommendations using EXTAQ (TaKaRa), Taq Poly (Promega) or Pfu Turbo ^® (Stratagene, La Jolla, CA). Restriction and modification enzymes were from New England Biolabs (Beverly, MA). Yeast strains were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose and 1.5% agar) or synthetic defined medium (1.4% yeast nitrogen base, 2% dextrose, 4xlO ^"5 % biotin and 1.5% agar) supplemented as appropriate. Yeast transformations were performed by electroporation as described in (Nett et al., 2005).

Coomassie gels and Western Blots were performed using 4-20% precast TRIS-SDS gels and the Mini PROTEAN 3 cell from Biorad according to the manufacturer's instructions. Primary antibodies for detection were: Goat Anti -Human IgG (Fc) #31413 from Pierce at 1:10000 dilution for Herceptin; Anti human EPO #sc7956 from Santa Cruz Biotechnology at 1 :500 dilution; Anti-GBA rabbit polyclonal (custom made) from Rockland Immunochemicals, Inc. at 1 :500 dilution.

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

BRJEF DESCRIPTION OF THE SEQUENCES

CCAAGTTTGACTCTTGCTCAATATCCAGATCTATGG

AATCTTGAGCAGGTCTTTTGGAATAAAATGCGACT

AAAAACCCAGAAAGTAGCCCAATTATATGCAGTCT

GAACATGAGTGGTACTTTGGTGAGTGACCTCCATA

TCCATGACATGGATGGATTTCGCCCTTTTCTTGTGT

AATATGACATCAACAACGACGTGGATGACACAGTA

ACAACAGTCAAGGAGAGTTTGAGACTTTCTTTTAC

GCTTTTTATGACTATCTGTTTGTAATACTTCCATTT

GCTAGCCGCTTTCAGCTGTTCCAATTCTTCCGTGCT

AAGTCTCAAGTTCATAAAGAAGAAAAATGGAAAG

AGGTATTCAAGGACTACCGTGTATTTTCTTGGCAA

ATATCGCAACAGAAAGTTTCTCAGATCAAATGCAA

ATCGATTTTTCATGCTATTCTTACCAATTATGCTTT

CCAGTTCATAGAAAGATTTGACCATATCACCAGAT

GAAACCATGCGAGAAGTTCCTCTTTTGACTAATAG

GCCTTCACCCATAAAGTTTAAGATGTTCCTGAAAT

ATACTGGACAGTTCTCGTAATCCATGATAAACGAC

TTGAAAATCTGCGAGTAACATAATGGGAATAGATA

CCATGAACGTAAGAGTTTGTCTCTCTTTGGAACACT

TTTTAGCGCTTTGAGCCTACGAATGAAACAACTATT

TTCTGGTTGATCTTCGAATTCAGCGTTGTCTGTGTC

TTTCATATCAGAATCCTTGATAACGTATATAGAGG

ATGTCTCTTTGGAAAATTGGTCGGGGTAAACCTGT

TCCAAGAACTTATAGCCATACTCTACCATTAATACC

GTAAAATATATTGATGCATAATTCTTTTGGTAATAT

ATTTTACTGGGATACAGGGCAAATGACACCACTGA

TGTGAATAGACTGGAAACGACTGAATTGAAAAGAA

ACTTTTGCTTCTCAGTGACTTTTAAATAGCTCTCTG

CGAAAATGTCAAGAATCTTGTTGAACAATGGTTTA

ACTGAAAATAAGAGACCCAGTGATGTAGAAAATTT

TAGCAAATTCACCCGATCATTGAACATTAAATTTCT

TCTAGAATTTGCAATATTCAACTTTCTTAAGATCTT

AAATATTACGCCCAACGATCCAAACAACAATAGAA

ACCATCTGTTGAAGTTTCTAGCTGCCTTTATGGTGA

CTTTTAGTATTCCTGTTGTGTCGTTCTCATAAAATG

ACTGTTCTACAGTCGATAATAAGCCACTCATCTTCC

ACAACTTCAACTGCACTTCCTCCAATGCAACTAGA

TCATGCTTTTCAAGCTGCTTGAGATTGATCTTCAGT

AATTCTTTAACTTCATCGTGTGATGTGAGCAAGAC

GAGTAAATACTTGAGTTTTGTCAAGTTATTACTGCC

CTTGTTTGACATGGATTGCTGTATTTGAGAAGAAA

AATGAACGTAAACTTGAATCTCCCCAGGTGAACTT

GGCGTGTATCTTATCTACCCCAGCTCTAAAGTTTAC

CCGATGAGGTAATTCTTAGGGATAATTTGGTGTAT

GGATTTGACTAAATTGCCGGAGTTGATTCAATGAC

AGAGAAGCTTACATGCAAGGAACATGATTCGTTGA

TCAACATTAGATGGGTGGCATGGAG

ADE 1 3" seq, ATGATTAGTACCCTCCTCGCCTTTTTCAGACATCTG for KO AAATTTCCCTTATTCTTCCAATTCCATATAAAATCC

TATTTAGGTAATTAGTAAACAATGATCATAAAGTG

AAATCATTCAAGTAACCATTCCGTTTATCGTTGATT

TAAAATCAATAACGAATGAATGTCGGTCTGAGTAG

TCAATTTGTTGCCTTGGAGCTCATTGGCAGGGGGTC

TTTTGGCTCAGTATGGAAGGTTGAAAGGAAAACAG

ATGGAAAGTGGTTCGTCAGAAAAGAGGTATCCTAC

ATGAAGATGAATGCCAAAGAGATATCTCAAGTGAT

AGCTGAGTTCAGAATTCTTAGTGAGTTAAGCCATC

CCAACATTGTGAAGTACCTTCATCACGAACATATTT

CTGAGAATAAAACTGTCAATTTATACATGGAATAC

TGTGATGGTGGAGATCTCTCCAAGCTGATTCGAAC

ACATAGAAGGAACAAAGAGTACATTTCAGAAGAA

AAAATATGGAGTATTTTTACGCAGGTTTTATTAGC

ATTGTATCGTTGTCATTATGGAACTGATTTCACGGC

TTCAAAGGAGTTTGAATCGCTCAATAAAGGTAATA

GACGAACCCAGAATCCTTCGTGGGTAGACTCGACA

AGAGTTATTATTCACAGGGATATAAAACCCGACAA

CATCTTTCTGATGAACAATTCAAACCTTGTCAAACT

GGGAGATTTTGGATTAGCAAAAATTCTGGACCAAG

AAAACGATTTTGCCAAAACATACGTCGGTACGCCG

TATTACATGTCTCCTGAAGTGCTGTTGGACCAACCC

TACTCACCATTATGTGATATATGGTCTCTTGGGTGC

GTCATGTATGAGCTATGTGCATTGAGGCCTCCTTTT

CAAGCCACTACACATTTACAATTACAACAAAAGAT

CCAAGAAGGGACATTCCCTCCACTTCCGGACGTAT

TTTCACCCCGGTTAAGATCTCTGATCAATGCTTGCA

TAACCATAGACCTGAACCAACGACCATCTACTCAC

GAACTTCTTCAGGAAAGTTGCTTCAATGTGTATATC

AAGGAGGTTAATTTAGAGATAAGGGAGGACAGATT

GAATGAGCGTGAACGCAAACTGAAAATACGAGAG

AACAAGTTAATCTTGAGCGAAGAGGGAATAGTGAA

ACAACTGAATGAAGAACTGGAATTTCAAAGAAAGT

TGCTTGAACAAGAAGTAGAGGAAATAAGGAAGTC

ATACAAGAACGAATTTCAGTTCGTACTGGAACAAC

AGGTGCAACAGGCATTGAGCAAAATTCTAGGTCCC

CAATACAATCAAAAGCCATTGAACAGGAATCAGCA

ACAAAAACAAATACAACAAATTTACAGCAGACAG

GATCCGCAATTATCAAGCCCAAAGTCACAACAAGC

TCAGATCCAAGGACCAAGAGAGTTGCTTAAAAGAG

G

ADEl gene GGGTGCTATCGTTTTGTGCAATTTGGTTTGCTGGAG (including AGTCGACCAAGAGATGATAACTGTTACTAAGCTTC promoter and TCCGTAATTAGTGGTATTTTGTAACTTTTACCAATA terminator) ATCGTTTATGAATACGGATATTTTTCGACCTTATCC

AGTGCCAAATCACGTAACTTAATCATGGTTTAAAT

ACTCCACTTGAACGATTCATTATTCAGAAAAAAGT

CAGGTTGGCAGAAACACTTGGGCGCTTTGAAGAGT

ATAAGAGTATTAAGCATTAAACATCTGAACTTTCA

CCGCCCCAATATACTACTCTAGGAAACTCGAAAAA

TTCCTTTCCATGTGTCATCGCTTCCAACACACTTTG

CTGTATCCTTCCAAGTATGTCCATTGTGAACACTGA

TCTGGACGGAATCCTACCTTTAATCGCCAAAGGAA

AGGTTAGAGACATTTATGCAGTCGATGAGAACAAC

TTGCTGTTCGTCGCAACTGACCGTATCTCCGCTTAC

GATGTGATTATGACAAACGGTATTCCTGATAAGGG

AAAGATTTTGACTCAGCTCTCAGTTTTCTGGTTTGA

TTTTTTGGCACCCTACATAAAGAATCATTTGGTTGC

TTCTAATGACAAGGAAGTCTTTGCTTTACTACCATC

AAAACTGTCTGAAGAAAAATACAAATCTCAATTAG

GACAAATGAAAGCCGACGAGCCAGCACTTTATAGT

AAGTGCAGGTGAGTCAATAAGAATAAATGTATGGC

TTGCTGTCCCTATCGCGTAAGAAGCTTACTAAGATC

GCCTAAATTGAAAAGTTGAACAAATCAGTTCTAGC

TGGCCTCCATCAGCATTTCGTTCTCCTCTGATCATC

TTTGCCAATCGCTAGCATGCCCTCAGCGTGCAAGG

AAAAGCACGCTTCTTTCTTATCGACGTATTTTCAAC

TATGGCAGAGCCAGGTTAGCAAGTC

ADE 2 3' seq, ATTTAGTATTGTTTTTTAATAGATGTATATATAATA for KO GTACACGTAACTTATCTATTCCATTCATAATTTTAT

TTTAAAGGTTCGGTAGAAATTTGTCCTCCAAAAAG

TTGGTTAGAGCCTGGCAGTTTTGATAGGCATTATTA

TAGATTGGGTAATATTTACCCTGCACCTGGAGGAA

CTTTGCAAAGAGCCTCATGTGCTCTAAAAGGATGT

CAGAATTCCAACATTTCAAAATTATATCTGCATGC

GTCTGTAATACTGGAACTGTTATTTTTCTGGTCAGG

ATTTCACCGCTCTTGTCGTCATGTTTCTCGTCGTCT

GAAAGTAAACTGACTTTCCTCTTTCCATAAACACA

AAAATCGATTGCAACTTGGTTATTCTTGAGATTGA

AATTTGCTGTGTCTTCAGTGCTTAGCTGAATATCAA

CAAACTTACTTAGTACTAATAACGAAGCACTATGG

TAAGTGGCATAACATAGTGGTATTGAAGCGAACAG

TGGATATTGAACCCAAGCATTGGCAACATCTGGCT

CTGTTGATACTGATCCGGATCGTTTGGCACCAATTC

CTGAAACGGCGTAGTGCCACCAAGGTTTCGATTTG

AGAACAGGTTCATCATCAGAGTCAACCACCCCAAT

GTCAATGGCAGGCTCCAACGAAGTAGGTCCAACAA

CAACAGGAAGTATTTGACCTTGAAGATCTGTTCCTT

TATGATCCACCACACCTTGCCCCAATTCCAATAACT

TTACCAGTCCCGATGCAGACATGATAACTGGTACT

AATGATCTCCATTGATTTTCGTCGGCACTACGTAAA

GCCTCCAAAAATGAATTCAGAATATCTTCTGAAAC

TAGATTCTGCTTCTGTGATTCAAGCATTGCTTTATG

TAGACATCTCTTGAATAAAAGCAATTCTCCACATA

TTGGTGTGTGTAAGATAGATCTGGAAAGATGTATC

TGGAATAGTCCAGTCAACGTTGTGCAATTGATTAG

CATTACCTTACTGTGAACATCTCTATCTACAACAAC

AGACTCAATTCGATAGACGTTCCGGGAAAGTTTTT

CAAGCGCATTCAGTTTGCTGTTGAACAAAGTGACT

TTGCTTTCCAATGTGCAAATACCCCTGTATATCAAG

TCCATCACATCACTCAAGACCTTGGTGGAAAAGAA

TGAAACAGCTGGAGCATAATTTTCGAATGAATTAG

GTAAGGTCACTTCATCCTTATCTGTTGTAATGCTAT

AATCAATAGCGGAACTAACATCTTCCCATGTAACA

GGTTTCTTGATCTCTGAATCTGAATCTTTATTTGAA

AAAGAATTGAAAAAAGACTCATCACTCATTGGGAA

TTCAAGGTCATTAGGGTATTCCATTGTTAGTTCTGG

TCTAGGTTTAAAGGGATCACCTTCGTTAAGACGAT

GGAAAATAGCTAATCTGTACAATAACCAGATACTT

CTAACGAAGCTCTCTCTATCCATCAGTTGACGTGTT

GAGGATATCTGAACTAGCTCTTTCCACTGCGAATC

AGGCATGCTCGTATAGCTGGCAAGCATGTTATTCA

GCTTTACCAAGTTAGAAGCCCTTTGGAAACCATCT

ATAGATTCCCGAAAAAACTTATACCCACTGAGGGT

TTCACTGAGCATAGTCAGTGACATCAAAGAGCATT TCAAATCCATCTCA

ADE2 gene GTCAAAGCCGTATACTCGGTAGTGTGCTCGCCAAA (including AATAAATTTGACTTGACTCTTCACTAGCCTATGCAA promoter and ATAAGGTTACCTTTTCCAAGAATCGTAGAAACGAT terminator) TAAAAAACTTCCAAACTCTCATGGATTCTCAGGTA

ATAGGTATTCTAGGAGGAGGCCAGCTAGGCCGAAT

GATTGTTGAGGCCGCTAGCAGGCTCAATATCAAGA

CCGTGATTCTTGATGATGGTTTTTCACCTGCTAAGC

ACATTAATGCTGCGCAAGACCACATCGACGGATCA

TTCAAAGATGAGGAGGCTATCGCCAAGTTAGCTGC

CAAATGTGATGTTCTCACTGTAGAGATTGAGCATG

TCAACACAGATGCTCTAAAGAGAGTTCAAGACAGA

ACTGGAATCAAGATATATCCTTTACCAGAGACAAT

CGAACTAATCAAGGATAAGTACTTGCAAAAGGAAC

ATTTGATCAAGCACAACATTTCGGTGACAAAGTCT

CAGGGTATAGAATCTAATGAAAAGGCGCTGCTTTT

GTTTGGAGAAGAGAATGGATTTCCATATCTGTTGA

AGTCCCGGACTATGGCTTATGATGGAAGAGGCAAT

TTTGTAGTGGAGTCTAAAGAGGACATCAGTAAGGC

ATTAGAATTCTTGAAAGATCGTCCATTGTATGCCG

AGAAGTTTGCTCCTTTTGTTAAAGAATTAGCGGTA

ATGGTTGTGAGATCACTGGAAGGCGAAGTATTCTC

CTACCCAACCGTAGAAACTGTGCACAAGGACAATA

TCTGTCATATTGTGTATGCTCCGGCCAGAGTTAATG

ACACCATCCAAAAGAAAGCTCAAATATTAGCTGAA

AACACTGTGAAGACTTTCCCAGGCGCTGGAATCTT

CGGAGTTGAGATGTTCCTATTGTCTGATGGAGAAC

TTCTTGTAAATGAGATTGCTCCAAGGCCCCACAATT

CTGGTCACTATACAATCGATGCATGTGTAACATCTC

AGTTCGAAGCACATGTAAGAGCCATAACTGGTCTG

CCAATGCCACTAGATTTCACCAAACTATCTACTTCC

AACACCAACGCTATTATGCTCAATGTTTTGGGTGCT

GAAAAATCTCACGGGGAATTAGAGTTTTGTAGAAG

AGCCTTAGAAACACCCGGTGCTTCTGTATATCTGTA

CGGAAAGACCACCCGATTGGCTCGTAAGATGGGTC

ATATCAACATAATAGGATCTTCCATGTTGGAAGCA

GAACAAAAGTTAGAGTACATTCTAGAAGAATCAAC

CCACTTACCATCCAGTACTGTATCAGCTGACACTA

AACCGTTGGTTGGAGTTATCATGGGTTCAGACTCT

GATCTACCTGTGATTTCGAAAGGTTGCGATATTTTA

AAACAGTTTGGTGTTCCATTCGAAGTTACTATTGTC

TCTGCTCATAGAACACCACAGAGAATGACCAGATA

TGCCTTTGAAGCCGCTAGTAGAGGTATCAAGGCTA

TCATTGCAGGTGCTGGTGGTGCTGCTCATCTTCCAG

GAATGGTTGCTGCCATGACTCCGTTGCCAGTCATTG

GTGTTCCTGTCAAGGGCTCTACGTTGGATGGTGTA

GACTCGCTACACTCGATTGTCCAAATGCCTAGAGG

TGTTCCTGTGGCTACGGTTGCTATCAACAACGCCAC

CAATGCCGCTCTGTTGGCCATCAGGATTTTAGGTAC

AATTGACCACAAATGGCAAAAGGAAATGTCCAAGT

ATATGAATGCAATGGAGACCGAAGTGTTGGGGAAG

GCATCCAACTTGGAATCTGAAGGGTATGAATCCTA

TTTGAAGAATCGTCTTTGAATTTAGTATTGTTTTTT