Claims
- 1. A recombinant non-yeast DNA encoding a plant protein of interest, wherein an unmodified DNA corresponding to said recombinant non-yeast DNA contains a region having a high content of codons that are poorly suited to for expression in yeasts, wherein at least 75% of the codons that are poorly suited for expression in yeasts are replaced in said region of said recombinant non-yeast DNA with synonymous codons coding for the same amino acid that are well-suited to for expression in yeasts, and wherein the number of replaced codons is sufficient to permit expression of the non-yeast DNA in yeasts.
- 2. The recombinant non-yeast DNA of claim 1, wherein 100% of the codons that are poorly suited for expression in yeast are replaced in said region of said recombinant non-yeast expression in yeasts, wherein the number of replaced codons is sufficient to permit expression of the non-yeast DNA in yeasts, and wherein the plant protein of interest is produced.
- 3. The recombinant non-yeast DNA according to claims 1 or 2, wherein the poorly suited codons are selected from the group consisting of codons whose frequency of use by yeasts is less than about 13 per 1000 codons.
- 4. The recombinant non-yeast DNA according to claim 3, wherein the protein of interest is an enzyme.
- 5. The recombinant non-yeast DNA according to claim 2, wherein the poorly suited codons are selected from the group consisting of CTC, CTG and CTT, which encode leucine, CGG, CGC, CGA, CGT and AGG, which encode arginine, GCG and GCC, which encode alanine, GGG, GGC and GGA, which encode glycine, and CCG and CCC, which encode proline.
- 6. The recombinant non-yeast DNA according to claim 5, wherein the poorly suited codons are selected from the group consisting of CTC and CTG, which encode leucine, CGG, CGC, CGA, CGT and AGG, which encode arginine, GCG and GCC, which encode alanine, GGG and GGC, which encode glycine, and CCG and CCC, which encode proline.
- 7. The recombinant non-yeast DNA according to claim 3, wherein the codons that are well-suited for expression in yeasts are selected from the group consisting of codons whose frequency of use by yeasts is greater than 15 per 1000 codons.
- 8. The recombinant non-yeast DNA according to claim 7, wherein the well-suited codons are selected from the group consisting of TTG and TTA, which encode leucine, AGA, which encodes arginine, GCT and GCA, preferably GCT, which encode alanine, GGT, which encodes glycine, and CCA, which encodes proline.
- 9. The recombinant non-yeast DNA according to claim 3, wherein the region having a high content of codons that are poorly suited for expression in yeasts contains at least 2 poorly suited codons among 10 consecutive codons, wherein the poorly suited codons are adjacent or nonadjacent to each other.
- 10. The recombinant non-yeast DNA according to claim 9, wherein the region having a high content of poorly suited codons contains 2, 3, 4, 5 or 6 poorly suited codons per 10 consecutive codons, or contain at least 2 or 3 adjacent poorly suited codons.
- 11. The recombinant non-yeast DNA according to claim 3, wherein the corresponding unmodified DNA is selected from the group consisting of a dicotyledonous plant DNA and a monocotyledonous plant DNA.
- 12. The recombinant non-yeast DNA according to claim 11, wherein the corresponding unmodified DNA is selected from the group consisting of a wheat DNA, a barley DNA, an oat DNA, a rice DNA, a maize DNA, a sorghum DNA, and a cane sugar DNA.
- 13. The recombinant non-yeast DNA of claim 3, wherein an unmodified DNA corresponding to said recombinant non-yeast DNA contains a region of high CTC codon or high CTC+CTG codon content, wherein a number of said CTC codons and/or CTG codons are replaced in said recombinant non-yeast DNA with TTG and/or TTA codons, and wherein the number of replaced codons is sufficient to permit expression in yeasts.
- 14. The recombinant non-yeast cDNA according to claim 13, wherein the CTC codon(s) and/or the CTG codon(s) are replaced with TTG codon(s).
- 15. The recombinant non-yeast cDNA according to claim 13, wherein the region having a high CTC codon or high CTC+CTG codon content encodes a polypeptide that contains 2, 3, 4, 5 or 6 leucines per 10 consecutive amino acids, or contain at least 2 or 3 adjacent leucines.
- 16. A chimeric gene which comprises a recombinant non-yeast DNA sequence according to claim 3 operably linked to heterologous 5′ and 3′ regulatory elements which are able to function in a yeast.
- 17. A yeast transformation vector comprising at least one chimeric gene according to claim 16.
- 18. A process for transforming a yeast cell using a vector according to claim 17 comprising contacting a yeast cell with said vector under conditions that permit said yeast cell to take up said vector.
- 19. A transformed yeast for expressing a protein of interest, comprising the chimeric gene according to claim 16.
- 20. A The yeast according to claim 19, which is selected from the group consisting of Saccharomyces, Kluyveromyces, Hansenula, Pichia and Yarrowia.
- 21. A process for producing a heterologous protein of interest in a transformed yeast, comprising:a) transforming a yeast with a vector which contains a recombinant non-yeast DNA according to claim 3 operably linked to heterologous 5′ and 3′ regulatory elements which are able to function in a yeast; b) culturing the transformed yeast; and c) extracting the protein of interest from the yeast culture.
- 22. A process for transforming a substrate by enzymatic catalysis with an enzyme which is expressed in a yeast comprising:a) culturing, in the presence of the substrate to be transformed, a transformed yeast that expresses a protein of interest, said transformed yeast comprising a chimeric gene, said chimeric gene comprising a recombinant non-yeast DNA encoding a plant protein of interest, wherein an unmodified DNA corresponding to said recombinant non-yeast DNA contains a region having a high content of codons that are poorly suited for expression in yeasts, wherein a number of the codons that are poorly suited to yeasts are replaced in said region of said recombinant non-yeast DNA with synonymous codons coding for the same amino acid that are well-suited to yeasts, and wherein the number of replaced codons is sufficient to permit expression of the non-yeast DNA in yeasts, said chimeric gene being operable linked to heterologous 5′ and 3′ regulators elements which are able to function in a yeast; and b) recovering the transformed substrate from the yeast culture.
- 23. A recombinant non-yeast DNA encoding a plant protein of interest, wherein an unmodified DNA corresponding to said recombinant non-yeast DNA contains a region having a high content of codons that are poorly suited for expression in yeasts, wherein a number of the codons in the 5′ region of the DNA that are poorly suited for expression in yeasts are replaced in said 5′ region of said recombinant non-yeast DNA with synonymous codons coding for the same amino acid that are well-suited for expression in yeasts, wherein the number of replaced codons is sufficient to permit expression in yeasts, and wherein replaced codons are only in the 5′ region.
- 24. A recombinant non-yeast DNA encoding a plant cytochrome P450, in which the non-yeast DNA encoding the plant cytochrome, P450 contains a region having a high content of codons that are poorly suited for expression in yeasts, wherein a number of the codons that are poorly suited for expression in yeasts are replaced in said region of said recombinant non-yeast DNA with synonymous codons coding for the same amino acid that are well-suited for expression in yeasts, such that the number of replaced codons is sufficient to permit expression of the plant cytochrome P450 DNA in yeasts.
- 25. The recombinant non-yeast DNA according to claim 24, wherein the corresponding unmodified DNA has a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:10.
- 26. The recombinant non-yeast DNA according to claim 24 having a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:14.
Priority Claims (1)
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97 12094 |
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Parent Case Info
This application is continuation of U.S. patent application Ser. No. 09/158,767, filed Sep. 23, 1998, now U.S. Pat. No. 6,180,363, issued Jan. 30, 2001.
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Continuations (1)
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09/158767 |
Sep 1998 |
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09/713794 |
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