Variants of GAL2 transporter and their uses

Patent metadata

Field	Value
Publication number	US-10308692-B2
Application number	US-201515520814-A
Country	US
Kind code	B2
Filing date	Oct 22, 2015
Priority date	Oct 22, 2014
Publication date	Jun 4, 2019
Grant date	Jun 4, 2019

Abstract

The present invention relates to polypeptides which are Gal2 variants comprising at least one amino acid substitution at a position corresponding to M435, and optionally further amino acid substitution(s). The present invention further relates to nucleic acid molecules encoding the polypeptides and to host cells containing said nucleic acid molecules. The present invention further relates to a method for the production of bioethanol and/or other bio-based compounds, comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention also relates to the use of the polypeptides, nucleic acids molecule or host cells for the production of bioethanol and/or other bio-based compounds, and/or for the recombinant fermentation of biomaterial containing pentose(s), preferably D-xylose and/or L-arabinose.

First claim

The invention claimed is:

1. A polypeptide, comprising at least one amino acid substitution at a position corresponding to M435 and at a position corresponding to N376 of the amino acid sequence of SEQ ID NO: 1,
wherein the polypeptide has at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide has either or both of an in vitro and an in vivo pentose transport function.

2. The polypeptide of claim 1 , wherein the polypeptide is Gal2 of Saccharomyces cerevisiae.

3. The polypeptide of claim 1 , comprising the amino acid substitution M435I.

4. The polypeptide of claim 1 , comprising an amino acid substitution that is selected from N376Y and N376F.

5. The polypeptide of claim 1 , wherein the amino acid substitution at the position corresponding to M435 increases activity of either or both of the in vitro and the in vivo pentose transport function compared to said activity of the polypeptide without the amino acid substitution.

6. The polypeptide of claim 1 , wherein the amino acid substitution at the position corresponding to M435 increases affinity of the polypeptide for a pentose compared to said affinity of the polypeptide without the amino acid substitution.

7. The polypeptide of claim 1 , wherein the pentose for which the polypeptide has pentose transport function is either one or both of D-xylose and L-arabinose.

8. A nucleic acid molecule, comprising a nucleic acid sequence encoding the polypeptide of claim 1 .

9. The nucleic acid molecule of claim 8 , further comprising one or more of:
(i) a vector nucleic acid sequence,
(ii) an expression vector sequence,
(iii) a promoter nucleic acid sequence,
(iv) a terminator nucleic acid sequence,
(v) a regulatory nucleic acid sequence,
wherein the nucleic acid molecule comprises dsDNA, ssDNA, PNA, CNA, RNA, or mRNA, or combinations thereof.

10. A host cell containing the nucleic acid molecule of claim 8 , wherein one or more of:
(a) the host cell expresses said nucleic acid molecule,
(b) the host cell is a fungus cell,
(c) the host cell is a yeast cell, or
(d) the host cell is selected from the group consisting of a Saccharomyces species, a Kluyveromyces sp., a Hansenula sp., a Pichia sp., and a Yarrowia sp.

11. The host cell of claim 10 , which belongs to the species Saccharomyces cerevisiae.

12. The host cell of claim 10 , which has an increased uptake rate for either or both of D-xylose and L-arabinose, compared to a cell that does not contain the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

13. The host cell of claim 10 which further comprises either or both of:
(a) nucleic acid molecules which code for proteins of a xylose metabolic pathway, and
(b) nucleic acid molecules which code for proteins of an arabinose metabolic pathway.

14. The host cell of claim 13 , which has at least one of:
(a) an increased rate of consumption of either or both of D-xylose and L-arabinose, or
(b) a faster growth rate with either or both of D-xylose and L-arabinose present,
compared to a cell that does not contain the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

15. The host cell of claim 13 wherein:
(i) the proteins of the xylose metabolic pathway comprise at least xylose isomerase and xylulokinase, and
(ii) the proteins of the arabinose metabolic pathway comprise at least rabinose isomerase, ribulokinase, and ribulose-5-P 4-epimerase.

16. A method for producing ethanol and/or other fermentation products, comprising expressing, in the host cell of claim 10 , the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

17. The method of claim 16 which comprises recombinant fermentation by the host cell of biomaterial containing pentoses, wherein the pentoses comprise either or both of D-xylose and L-arabinose.

18. The method of claim 16 wherein the other fermentation products are selected from:
1-butanol, isobutanol, 2-butanol, other alcohols,
lactic acid, acetic acid, succinic acid, malic acid, other organic acids,
amino acids, alkanes, terpenes, isoprenoids, solvents, pharmaceutical compounds, and vitamins.

Assignees

• Johann Wolfgang Goethe Univ Frankfurt
• Lallemand Hungary Liquidity Man Llc

Inventors

• Boles Eckhard
• Farwick Alexander
• Kirchner Ferdinand
• Schadeweg Virginia
• Oreb Mislav

Classifications

• C12N9/92

  Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)} · CPC title

• C12Y207/01016

  Ribulokinase (2.7.1.16) · CPC title

• C12Y501/03004

  L-Ribulose-5-phosphate 4-epimerase (5.1.3.4) · CPC title

• C12N1/18

  Baker's yeast; Brewer's yeast · CPC title

• C12P7/12

  substrate containing sulfite waste liquor or citrus waste · CPC title