Regulatory region for heterologous gene expression in yeast

Claim 2

A DNA fragment comprising a regulatory region; wherein said regulatory region is responsive to at least one of the conditions selected from the group (i) the presence of methanol in the culture medium with which a host organism for said DNA fragment is in contact, (ii) the presence of a non-catabolite repressing carbon source other than methanol in the culture medium with which a host organism for said DNA fragment is in contact, and (iii) carbon source starvation in the culture medium with which a host organism for said DNA fragment is in contact after growth of the host organism on a . catabolite repressing carbon and energy source; wherein said regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said messenger RNA, and mutants of said DNA fragment.

Claim 3

The DNA fragment of claim 1 characterized in that said regulatory region is derived from yeast; in particular wherein said yeast is selected from the genus Pichia; in particular wherein said yeast is a member of the species Pichia pastoris; in particular wherein said yeast is Pichia pastoris NRRL Y-11430.

Claim 4

. The DNA fragment of claim 2 characterized in that said regulatory region controls the transcription of messenger RNA which codes for the production of alcohol oxidase; in particular said DNA fragment further comprising the 3' sequence of DNA downstream of the DNA which codes for the production of alcohol oxidase; in particular wherein said fragment is characterized as shown by the restriction map in Fig. 5 of the drawings; in particular said DNA fragment further comprising an additional fragment selected from the group characterized as shown by the restriction maps in Fig. 8 of the drawings; in particular wherein said fragment has the nucleotide sequence: in particular said DNA fragment of said nucleotide sequence further comprising an additional fragment positioned at the 3' end of the DNA which codes for the production of messenger RNA; wherein said additional fragment is selected from the group characterized by the restriction maps in Fig. 8 of the drawings; in particular wherein said fragment has the nucleotide sequence: in particular wherein said fragment has the nucleotide sequence:

Claim 5

The DNA fragment of claim 2 characterized in that said regulatory region controls the transcription of messenger RNA which codes for the production of the polypeptide p76; in particular said DNA fragment further comprising the 3' sequence of DNA downstream of the DNA which codes for the production of polypeptide p76; in particular wherein said fragment is selected from the group characterized as shown by the restriction maps in Fig. 4 of the drawings; in particular said DNA fragment further comprising the additional fragment characterized as shown by the restriction map in Fig. 7 of the drawings; in particular wherein said fragment has the nucleotide sequence:

Claim 8

The DNA fragment of claim 2 characterized in that said regulatory region controls the transcription of messenger RNA which codes for the production of the polypeptide p40; in particular said DNA fragment further comprising the 3' sequence of DNA downstream of the DNA which codes for the production of polypeptide p40; in particular wherein said fragment is characterized as shown by the restriction map in Fig. 6 of the drawings; in particular said DNA fragment further comprising the additional fragment characterized as shown by the restriction map in Fig. 9 of the drawings.

Claim 9

A DNA fragment in accordance with claim 1 wherein said messenger RNA codes for the production of a heterologous polypeptide.

Claim 10

The DNA fragment of claim 1 further comprising: a polypeptide coding region, wherein said regulatory region is positioned at the 5' end of said polypeptide coding region; in particular said DNA fragment further comprising a 3' sequence of DNA downstream of the polypeptide coding region, wherein said 3' sequence of DNA is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA coded for by said polypeptide coding region; in particular wherein said DNA fragment further comprises one or more additional DNA sequences derived from the group bacterial plasmid DNA, bacteriophage DNA, yeast plasmid DNA, and yeast chromosomal DNA; in particular wherein said yeast chromosomal DNA comprises an autonomously replicating DNA sequence and a marker gene.

Claim 11

The DNA fragment of claim 7 characterized in that said polypeptide coding region codes for the production of alcohol oxidase; in particular wherein said DNA fragment is characterized by the restriction map in Fig. 2 of the drawings; in particular wherein said fragment has the nucleotide sequence:

Claim 12

The DNA fragment of claim 7 characterized in that said polypeptide coding region codes for the production of polypeptide p76, in particular wherein said DNA fragment is characterized by the restriction map in Fig. 10 of the drawings; or wherein said polypeptide coding region codes for the production of polypeptide p40, in particular wherein said DNA fragment is characterized by the restriction map in Fig. 11 of the drawings; or wherein said polypeptide coding region codes for the production of a heterologous polypeptide, in particular wherein said heterologous polypeptide is β-galactosidase, in particular wherein said DNA fragment is selected from the group characterized by the restriction maps in Fig. 15 or Fig. 16 of the drawings.

Claim 13

A DNA fragment comprising a regulatory region, wherein said regulatory region is capable of controlling the polyadenylation, termination of transcription and ter-. mination of translation of messenger RNA when positioned at the 3' end of the polypeptide coding region which codes for the production of said messenger RNA, wherein the transcription and translation of said messenger RNA is controlled by a second regulatory region, wherein said second regulatory region is responsive to at least one of the conditions selected from the group (i), (ii) and (iii) as identified in claim 1; in particular wherein said DNA fragment is characterized by the restriction map in Fig. 7 of the drawings; in particular wherein said DNA fragment is selected from the group characterized by the restriction map in Fig. 8 of the drawings; in particular wherein said DNA fragment is characterized by the restriction map in Fig. 9 of the drawings.

Claim 14

A gene coding for the production of alcohol oxidase, or a sub-unit thereof or an equivalent of such a gene or sub-unit; in particular wherein said gene is characterized by the restriction map in Fig. 13 of the drawings; in particular wherein said gene has the nucleotide sequence identified in claim 8; in particular said gene further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 2a of the drawings.

Claim 15

A gene coding for the production of polypeptide p76, or a sub-unit thereof or an equivalent of such a gene or sub-unit; in particular wherein said gene is characterized by the restriction map in Fig. 12 of the drawings; in particular said gene further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 1a of the drawings.

Claim 16

A gene coding for the production of polypeptide p76, or a sub-unit thereof or an equivalent of such a gene or sub-unit; in particular wherein said gene is characterized by the restriction map in Fig. 12 of the drawings; in particular said gene further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 1a of the drawings.

Claim 17

A gene coding for the production of polypeptide p40, or a sub-unit thereof or an equivalent of such a gene or sub-unit; in particular wherein said gene is characterized by the restriction map in Fig. 14 of the drawings; in particular said gene further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 3a of the drawings.

Claim 18

A gene coding for the production of polypeptide p40, or a sub-unit thereof or an equivalent of such a gene or sub-unit; in particular wherein said gene is characterized by the restriction map in Fig. 14 of the drawings; in particular said gene further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 3a of the drawings.

Claim 19

The DNA fragment of claim 7 characterized in that said fragment is in the form of a closed circular hybrid plasmid; in particular wherein the non-bacterial insert is selected from the restriction maps of the drawings shown in Fig. 1a (pPG 6.0), Fig. 2a (pPG 4.0), Fig. 3a (pPG 4.8), Fig. 1b (pPC 15.0)., Fig. 2b (pPC 8.3), Fig. 2c (pPC 8.0) and Fig. 3b (pPC 6.7); or wherein said hybrid plasmid has a restriction map selected from those shown in Fig. 17 (pSAOH 1), Fig. 18 (pSAOH 5), Fig. 19 (pSAOH 10), Fig. 20 I (pTAFH 85), Fig. 21 (pT76H 1), Fig. 22 (pT76 H2), Fig. 22a (pT76H3), Fig. 22b (pT76H4), Fig. 30 (pTA013) and Fig. 30a (pT76Ul).

Claim 20

A transformed yeast strain, wherein said transformed yeast strain is a host for recombinant DNA material; wherein said recombinant DNA material comprises: (1) a DNA fragment, and (2) a polypeptide coding region; wherein said DNA fragment is responsive to at least one of the conditions selected from the group consisting of: (i) the presence of methanol in the culture medium with which a host organism for said DNA fragment is in contact, and (ii) the presence of a non-catabolite repressing carbon source other than methanol in the culture medium with which a host organism for said DNA fragment is in contact, and (iii) carbon source starvation in the culture medium with which a host organism for said DNA fragment is in contact after growth of the host organism on a carbon and energy source other than methanol, wherein said regulatory region is positioned at the 5' end of said polypeptide coding region, and, wherein said transformed yeast strain is capable of expressing the polypeptide coded for by said polypeptide coding region; in particular wherein said transformed yeast strain is capable of growth on methanol as carbon and energy source; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis and Pichia; in particular wherein said transformed yeast strain is selected from the genus Pichia; in particular wherein said recombinant DNA material further comprises a second DNA fragment, wherein said second DNA fragment is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA when positioned at the 3' end of the polypeptide coding region which codes for the production of said messenger RNA; in particular wherein said transformed yeast is capable of growth on at least one carbon and energy source selected from the group consisting of methanol, glucose, acetate, ethanol, glycerol, sucrose, lactose, fructose and galactose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces and Kluyveromyces; in particular wherein said transformed yeast strain is selected from the genus Saccharomyces; in particular wherein said yeast strain is selected from Pichia pastoris NRRL Y-15852 (GS115-pSAOH 1), Pichia pastoris NRRL Y-15853 (GS115-pSAOH 5), Pichia pastoris NRRL Y-15854 (GS115-pSAOH10), Pichia pastoris NRRL Y-15855 (GS115- pTAFH 85), Pichia pastoris NRRL Y-15856 (GS115-pT76Hl), Pichia pastoris NRRL Y-15857 (GS115-pT76H2) and Saccharomyces cerevisiae NRRL Y-15858 (SEY 2102-pTAP13).

Claim 21

A transformed yeast strain, wherein said transformed yeast strain is a host for recombinant DNA material; wherein said recombinant DNA material comprises: (1) a DNA fragment, and (2) a polypeptide coding region; wherein said DNA fragment is responsive to at least one of the conditions selected from the group consisting of: (i) the presence of methanol in the culture medium with which a host organism for said DNA fragment is in contact, and (ii) the presence of a non-catabolite repressing carbon source other than methanol in the culture medium with which a host organism for said DNA fragment is in contact, and (iii) carbon source starvation in the culture medium with which a host organism for said DNA fragment is in contact after growth of the host organism on a carbon and energy source other than methanol, wherein said regulatory region is positioned at the 5' end of said polypeptide coding region, and, wherein said transformed yeast strain is capable of expressing the polypeptide coded for by said polypeptide coding region; in particular wherein said transformed yeast strain is capable of growth on methanol as carbon and energy source; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis and Pichia; in particular wherein said transformed yeast strain is selected from the genus Pichia; in particular wherein said recombinant DNA material further comprises a second DNA fragment, wherein said second DNA fragment is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA when positioned at the 3' end of the polypeptide coding region which codes for the production of said messenger RNA; in particular wherein said transformed yeast is capable of growth on at least one carbon and energy source selected from the group consisting of methanol, glucose, acetate, ethanol, glycerol, sucrose, lactose, fructose and galactose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces and Kluyveromyces; in particular wherein said transformed yeast strain is selected from the genus Saccharomyces; in particular wherein said yeast strain is selected from Pichia pastoris NRRL Y-15852 (GS115-pSAOH 1), Pichia pastoris NRRL Y-15853 (GS115-pSAOH 5), Pichia pastoris NRRL Y-15854 (GS115-pSAOH10), Pichia pastoris NRRL Y-15855 (GS115- pTAFH 85), Pichia pastoris NRRL Y-15856 (GS115-pT76Hl), Pichia pastoris NRRL Y-15857 (GS115-pT76H2) and Saccharomyces cerevisiae NRRL Y-15858 (SEY 2102-pTAP13).

Claim 22

An essentially pure culture of E. coli bearing any of the hybrid plasmids of claim 14

Claim 23

A process for preparing polypeptides comprising cultivating a transformed yeast strain in a nutrient medium containing methanol wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises (1) a methanol responsive DNA fragment, and (2) a polypeptide coding region, wherein said methanol responsive DNA fragment is positioned at the 5' end of said polypeptide coding region; in particular further comprising isolating and purifying said polypeptide; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, and Pichia.

Claim 24

A process for preparing polypeptides comprising cultivating a transformed yeast strain in a nutrient medium containing methanol wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises (1) a methanol responsive DNA fragment, and (2) a polypeptide coding region, wherein said methanol responsive DNA fragment is positioned at the 5' end of said polypeptide coding region; in particular further comprising isolating and purifying said polypeptide; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, and Pichia.

Claim 25

A process for preparing polypeptides comprising: cultivating a transformed yeast strain in a nutrient medium, wherein said nutrient medium comprises at least one catabolite non-repressing carbon source, wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises: (1) a DNA fragment responsive to the presence of a catabolite non-repressing carbon source in the culture medium with which the transformed yeast strain is in contact, and (2) a polypeptide coding region, wherein said DNA fragment is positioned at the 5' end of said polypeptide coding region; in particular wherein said catabolite non-repressing carbon source is selected from glycerol and galactose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces and Kluyveromyces.

Claim 26

A process for preparing polypeptides comprising: cultivating a transformed yeast strain in a nutrient medium, wherein said nutrient medium comprises at least one catabolite non-repressing carbon source, wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises: (1) a DNA fragment responsive to the presence of a catabolite non-repressing carbon source in the culture medium with which the transformed yeast strain is in contact, and (2) a polypeptide coding region, wherein said DNA fragment is positioned at the 5' end of said polypeptide coding region; in particular wherein said catabolite non-repressing carbon source is selected from glycerol and galactose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces and Kluyveromyces.

Claim 27

A process for preparing polypeptides comprising: (a) cultivating a transformed yeast strain in a nutrient medium, wherein said nutrient medium comprises at least one carbon and energy source, wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises: (1) regulatory region, and (2) a polypeptide coding region, wherein said regulatory region is responsive to carbon source starvation in the culture medium with which the transformed yeast strain is in contact after growth of the transformed yeast strain on said at least one catabolite repressing carbon and energy source, and wherein said regulatory region is positioned at the 5' end of said polypeptide coding region, and (b) subjecting the product of step (a) to conditions of carbon source starvation; in particular wherein said at least one catabolite repressing carbon and energy source is selected from glucose, ethanol, and fructose; in particular wherein said catabolite repressing carbon source is glucose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosacharomyces and Kluyveromyces.

Claim 28

A process for preparing polypeptides comprising: (a) cultivating a transformed yeast strain in a nutrient medium, wherein said nutrient medium comprises at least one carbon and energy source, wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises: (1) regulatory region, and (2) a polypeptide coding region, wherein said regulatory region is responsive to carbon source starvation in the culture medium with which the transformed yeast strain is in contact after growth of the transformed yeast strain on said at least one catabolite repressing carbon and energy source, and wherein said regulatory region is positioned at the 5' end of said polypeptide coding region, and (b) subjecting the product of step (a) to conditions of carbon source starvation; in particular wherein said at least one catabolite repressing carbon and energy source is selected from glucose, ethanol, and fructose; in particular wherein said catabolite repressing carbon source is glucose; in particular wherein said transformed yeast strain is selected from members of the genera Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosacharomyces and Kluyveromyces.