Polypeptides with permease activity
The invention relates to a polypeptide having one or more substitution at a position corresponding to position 339 or 376 of SEQ ID NO: 59, wherein the polypeptide is a member of the Major Facilitator Superfamily (MFS). In an embodiment, the substitution is at position corresponding to 376 and wherein the amino acid at that position is replaced by an amino acid that has a van der Waals volume of 80 to 138 Å 3 and a side chain hydrophobicity of 10 to 100 Δt R .
1. A Saccharomyces cell transformed with a polynucleotide encoding a polypeptide having at least 90% sequence identity with SEQ ID NO:59, comprising:
a) one or more substitution at a position corresponding to position 376 of SEQ ID NO:59, selected from the group consisting of N376M, N376T, N376C, N376L, N376I, N376F, and N376V; and
b) one or more of the following amino acid motifs:
i) G-R-x(3)-G-x(3)-G-x(11)-E-x(5)-[LIVM]-R-G-x(12)-[GA], corresponding to residues 179-221 of SEQ ID NO:59;
ii) R-x(14)-G-x(2)-Y-x(2)-[YF]-[YF]-[GSAL], corresponding to residues 330-353 of SEQ ID NO:59; and
iii) V-x(15)-[GNR]-[RH]-R-x(2)-[LM]-x(2)-[GA], corresponding to residues 375-399 of SEQ ID NO:59;
wherein the polypeptide has sugar transporter activity.
2. The Saccharomyces cell according to claim 1 , wherein the polypeptide encodes amino acid motifs i), ii), and iii).
3. The Saccharomyces cell according to claim 2 , wherein the polypeptide is a mutant of a polypeptide that is native in an untransformed Saccharomyces cell, selected from the group consisting of Gal2, Hxt1, Hxt2, Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, Hxt11, Hxt12, Hxt13, Hxt14, Hxt15, Hxt16, and Hxt17.
4. The Saccharomyces cell according to claim 1 , wherein the polypeptide has reduced glucose transport activity compared to the polypeptide of SEQ ID NO:59.
5. The Saccharomyces cell according to claim 1 , wherein the polypeptide has improved xylose transport activity compared to the polypeptide of SEQ ID NO:59.
6. The Saccharomyces cell according to claim 1 , wherein the Saccharomyces cell has decreased glucose transport activity and improved xylose transport activity compared to a Saccharomyces cell expressing SEQ ID NO:59.
7. The Saccharomyces cell polypeptide according to claim 2 , wherein the polypeptide is a mutant of a polypeptide that is native in an untransformed Saccharomyces cell, selected from the group consisting of SEQ ID NOs: 59 and 150 through 167.
8. A nucleic acid construct encoding a polypeptide having at least 90% sequence identity with SEQ ID NO:59, comprising:
a) one or more substitution at a position corresponding to position 376 of SEQ ID NO:59, selected from the group consisting of N376M, N376T, N376C, N376L, N376I, N376F, and N376V; and
b) one or more of the following amino acid motifs:
i) G-R-x(3)-G-x(3)-G-x(11)-E-x(5)-[LIVM]-R-G-x(12)-[GA], corresponding to residues 179-221 of SEQ ID NO:59;
ii) R-x(14)-G-x(2)-Y-x(2)-[YF]-[YF]-[GSAL], corresponding to residues 330-353 of SEQ ID NO:59; and
iii) V-x(15)-[GNR]-[RH]-R-x(2)-[LM]-x(2)-[GA], corresponding to residues 375-399 of SEQ ID NO:59;
wherein the polypeptide has sugar transporter activity.
9. The transformed Saccharomyces cell according to claim 1 , which belongs to the species Saccharomyces cerevisiae.
10. The transformed Saccharomyces cell according to claim 1 , wherein the polynucleotide encodes a polypeptide that is a mutant of a polypeptide that is native in an untransformed Saccharomyces cell.
11. The transformed Saccharomyces cell according to claim 10 , wherein the polypeptide that is native in the untransformed Saccharomyces cell has sugar transporter activity.
12. The transformed Saccharomyces cell according to claim 10 , wherein the polypeptide that is native in the untransformed Saccharomyces cell is a hexose transporter polypeptide.
13. The transformed Saccharomyces cell according to claim 11 , wherein the polypeptide that is native in the untransformed Saccharomyces cell is a hexose transporter polypeptide.
14. The transformed Saccharomyces cell according to claim 13 , wherein the polypeptide that is native in the untransformed host cell is a polypeptide selected from the group consisting of Gal2, Hxt1, Hxt2, Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, Hxt11, Hxt12, Hxt13, Hxt14, Hxt15, Hxt16, and Hxt17.
15. The transformed Saccharomyces cell of claim 1 that, when subjected to the Glucose Transport Activity Counter Screen (GTAC) protocol, consumes xylose from a medium comprising xylose and glucose, while glucose is still present in the medium.
16. The transformed Saccharomyces cell according to claim 15 that, when subjected to the Glucose Transport Activity Counter Screen (GTAC) protocol, consumes xylose faster than said transformed Saccharomyces cell consumes glucose.
17. A polypeptide having at least 90% sequence identity with SEQ ID NO:59, comprising:
a) a substitution corresponding to N376T of SEQ ID NO:59; and
b) one or more of the following amino acid motifs:
i) G-R-x(3)-G-x(3)-G-x(11)-E-x(5)-[LIVM]-R-G-x(12)-[GA], corresponding to residues 179-221 of SEQ ID NO:59;
ii) R-x(14)-G-x(2)-Y-x(2)-[YF]-[YF]-[GSAL], corresponding to residues 330-353 of SEQ ID NO:59; and
iii) V-x(15)-[GNR]-[RH]-R-x(2)-[LM]-x(2)-[GA], corresponding to residues 375-399 of SEQ ID NO:59;
wherein the polypeptide has xylose transporter activity.
18. The polypeptide according to claim 17 , wherein the polypeptide is expressed in an eukaryotic cell and the eukaryotic cell is used to ferment xylose in the presence of glucose.
19. A process for degradation of ligno-cellulosic or hemi-cellulosic material, wherein ligno-cellulosic or hemi-cellulosic material is contacted with an enzyme composition, wherein one or more sugar is produced, and wherein produced sugar is fermented to give ethanol as a fermentation product, wherein the fermentation is conducted with the transformed Saccharomyces cell of claim 1 .
20. The process according to claim 19 , wherein produced sugar comprises xylose and glucose and wherein the Saccharomyces cell co-ferments xylose and glucose.
21. A process for the degradation of ligno-cellulosic or hemi-cellulosic material, wherein ligno-cellulosic or hemi-cellulosic material is contacted with an enzyme composition, wherein one or more sugar is produced, and wherein the produced sugar is fermented to give ethanol as a fermentation product, wherein the fermentation is conducted with the transformed Saccharomyces cell of claim 1 .
22. The Saccharomyces cell of claim 1 , wherein the polynucleotide encodes a polypeptide having at least 95% sequence identity with SEQ ID NO:59.
23. The Saccharomyces cell of claim 1 , wherein the polynucleotide encodes a polypeptide having at least 98% sequence identity with SEQ ID NO:59.
24. The Saccharomyces cell of claim 1 , wherein the polynucleotide encodes a polypeptide having at least 99% sequence identity with SEQ ID NO:59.
25. The nucleic acid construct of claim 8 , encoding a polypeptide having at least 95% sequence identity with SEQ ID NO:59.
26. The nucleic acid construct of claim 8 , encoding a polypeptide having at least 98% sequence identity with SEQ ID NO:59.
27. The nucleic acid construct of claim 8 , encoding a polypeptide having at least 99% sequence identity with SEQ ID NO:59.
28. The polypeptide of claim 17 , having at least 95% sequence identity with SEQ ID NO:59.
29. The polypeptide of claim 17 , having at least 98% sequence identity with SEQ ID NO:59.
30. The polypeptide of claim 17 , having at least 99% sequence identity with SEQ ID NO:59.