Metabolic engineering of arabinose-fermenting yeast cells

Patent metadata

Field	Value
Publication number	US-9303253-B2
Application number	US-201213345146-A
Country	US
Kind code	B2
Filing date	Jan 6, 2012
Priority date	Oct 2, 2006
Publication date	Apr 5, 2016
Grant date	Apr 5, 2016

Abstract

The invention relates to an eukaryotic cell expressing nucleotide sequences encoding the ara A, ara B and ara D enzymes whereby the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol. Optionally, the eukaryotic cell is also able to convert xylose into ethanol.

First claim

The invention claimed is:

1. A eukaryotic cell capable of expressing the following nucleotide sequences, wherein the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product:
(a) a nucleotide sequence encoding an arabinose isomerase (araA), wherein said nucleotide sequence is selected from the group consisting of:
i. nucleotide sequences encoding an araA, said araA comprising an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1,
ii. nucleotide sequences comprising a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:2,
iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii) under moderate conditions;

(b) a nucleotide sequence encoding a L-ribulokinase (araB), wherein said nucleotide sequence is selected from the group consisting of:
i. nucleotide sequences encoding an araB, said araB comprising an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:3,
ii. nucleotide sequences comprising a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:4,
iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii) under moderate conditions;

(c) a nucleotide sequence encoding an L-ribulose-5-P-4-epimerase (araD), wherein said nucleotide sequence is selected from the group consisting of:
i. nucleotide sequences encoding an araD, said araD comprising an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5,
ii. nucleotide sequences comprising a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:6,
iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii) under moderate conditions;

wherein moderate conditions is defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides to hybridize at a temperature of about 45° C. in a solution comprising about 1 M salt and washing at room temperature in a solution comprising about 1 M salt.

2. A cell according to claim 1 , wherein one, two or three of the araA, araB and araD nucleotide sequences originate from a Lactobacillus genus, optionally a Lactobacillus plantarum species.

3. A cell according to claim 1 , wherein the cell is a yeast cell, optionally belonging to one of the genera: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.

4. A cell according to claim 3 , wherein the yeast cell belongs to one of the species: S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis.

5. A cell according to claim 1 , wherein the nucleotide sequences encoding the araA, araB and/or araD are operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in the cell to confer to the cell the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product.

6. A cell according to claim 1 , wherein the cell exhibits the ability to directly isomerise xylose into xylulose.

7. A cell according to claim 6 , wherein the cell comprises a genetic modification that increases the flux of the pentose phosphate pathway.

8. A cell according to claim 6 , wherein the genetic modification comprises overexpression of at least one gene of the non-oxidative part of the pentose phosphate pathway.

9. A cell according to claim 8 , wherein the gene is selected from the group consisting of the genes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase.

10. A cell according to claim 8 , wherein the genetic modification comprises overexpression of at least the genes coding for a transketolase and a transaldolase.

11. A cell according to claim 8 , wherein the cell further comprises a genetic modification that increases the specific xylulose kinase activity.

12. A cell according to claim 11 , wherein the genetic modification comprises overexpression of a gene encoding a xylulose kinase.

13. A cell according to claim 8 , wherein the gene that is overexpressed is endogenous to the cell.

14. A cell according to claim 5 , wherein the cell comprises a genetic modification that reduces unspecific aldose reductase activity in the cell.

15. A cell according to claim 14 , wherein the genetic modification reduces the expression of, or inactivates a gene encoding an unspecific aldose reductase.

16. A cell according to claim 15 , wherein the gene is inactivated by deletion of at least part of the gene or by disruption of the gene.

17. A cell according to claim 14 , wherein the expression of each gene in the cell that encodes an unspecific aldose reductase is reduced or inactivated.

18. A cell according to claim 1 , wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin.

19. A nucleic acid construct comprising a nucleic acid sequence encoding an araA, a nucleic acid sequence encoding an araB and/or a nucleic acid sequence encoding an araD all as defined in claim 1 .

20. A process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin, whereby the process comprises:
(a) fermenting a medium containing a source of arabinose and optionally xylose with a modified cell as defined in claim 1 , whereby the cell ferments arabinose and optionally xylose to the fermentation product; and optionally,
(b) recovering the fermentation product.

21. A process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin, wherein the process comprises:
(a) fermenting a medium containing at least a source of L-arabinose and a source of xylose with a cell as defined in claim 1 and a cell able to use xylose and/or exhibiting the ability to directly isomerise xylose into xylulose, whereby each cell ferments L-arabinose and/or xylose to the fermentation product; and optionally,
(b) recovering the fermentation product.

22. A process according to claim 20 , wherein the medium also contains a source of glucose.

23. A process according to claim 20 , wherein the fermentation product is ethanol.

24. A process according to claim 23 , wherein the volumetric ethanol productivity is at least 0.5 g ethanol per liter per hour.

25. A process accordi

Assignees

• Van Maris Antonius Jeroen Adriaan
• Pronk Jacobus Thomas
• Wisselink Hendrik Wouter
• Van Dijken Johannes Pieter
• Winkler Aaron Adriaan
• Winde Johannes Hendrik
• Dsm Ip Assets Bv

Inventors

• Van Maris Antonius Jeroen Adriaan
• Pronk Jacobus Thomas
• Wisselink Hendrik Wouter
• Van Dijken Johannes Pieter
• Winkler Aaron Adriaan
• Winde Johannes Hendrik

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• Y02E50/17

  Cross-Sectional Technologies · mapped topic

• C12P7/08

  produced as by-product or from waste or cellulosic material substrate · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• C12N9/90 Primary

  Isomerases (5.) · CPC title