Xylose Isomerases that confer efficient xylose fermentation capability to yeast

Patent metadata

Field	Value
Publication number	US-2019316111-A1
Application number	US-201716472514-A
Country	US
Kind code	A1
Filing date	Dec 21, 2017
Priority date	Dec 21, 2016
Publication date	Oct 17, 2019
Grant date	—

Abstract

The present invention relates to novel nucleic acid sequences encoding bacterial xylose isomerases that upon transformation of a eukaryotic microbial host cell, such as yeast, to confer to the host cell the ability of isomerising xylose to xylulose. The nucleic acid sequences encode xylose isomerases that originate from bacteria such as Eubacterium sp., Clostridium cellulosi and others. The invention further relates to fermentation processes wherein the transformed host cells ferment a xylose-containing medium to produce ethanol or other fermentation products.

First claim

1 . A eukaryotic microbial cell comprising a nucleotide sequence, the expression of which confers to, or increases in the cell the ability to directly isomerise xylose into xylulose, wherein the nucleotide sequence encodes a polypeptide with xylose isomerase activity, which polypeptide comprises an amino acid sequence that has at least 68% sequence identity with the amino acid sequence of SEQ ID NO. 7, and wherein preferably the nucleotide sequence encodes an amino acid sequence that is obtainable from a bacterium of the genus Eubacterium , more preferably a bacterium of the species Eubacterium sp. CAG_180.

2 . The cell according to claim 1 , wherein the cell further comprises a nucleotide sequence, the expression of which confers to, or increases in the cell the ability to directly isomerise xylose into xylulose, wherein the nucleotide sequence encodes a polypeptide with xylose isomerase activity, which polypeptide comprises an amino acid sequence that has at least 71% sequence identity with the amino acid sequence of SEQ ID NO. 10, and wherein preferably the nucleotide sequence encodes an amino acid sequence that is obtainable from a bacterium of the genus Clostridium , more preferably a bacterium of the species Clostridium cellulosi.

3 . The cell according to claim 1 , wherein the cell is a yeast or a filamentous fungus of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Naumovia, Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium , and Penicillium.

4 . The cell according to claim 3 , wherein the cell is a yeast is capable of anaerobic alcoholic fermentation.

5 . The cell according to claim 4 , wherein the yeast belongs to a Saccharomyces species selected from the group consisting of S. cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus, S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis and S. unisporus.

6 . The cell according to claim 1 , wherein the nucleotide sequence encoding the polypeptide with xylose isomerase activity is operably linked to a promoter that is insensitive to catabolite repression and that does not require xylose for induction.

7 . The cell according to claim 1 , whereby the cell comprises at least one genetic modification selected from:
a) a genetic modification that increases the specific xylulose kinase activity; b) a genetic modification that increases the flux of the pentose phosphate pathway; and, c) a genetic modification that reduces unspecific aldose reductase activity in the cell.

8 . The cell according to claim 1 , wherein the cell further comprises at least one genetic modification that results in a characteristic selected from the group consisting of:
a) increased tolerance to ethanol; b) increased tolerance to acetic acid; c) reduced production of glycerol; d) increased xylose to ethanol fermentation rate; and, e) increased thermotolerance.

9 . The cell according to claim 8 , wherein, in:
a) the genetic modification is a modification that introduces an allele of one or more of the ADE1, KIN3, MKT1, VPS70, SWS2 and APJ1 genes that confers increased tolerance to ethanol as described in WO 2012/175552 and WO 2014/170330; b) the genetic modification is a modification that introduces an allele of one or more of the GLO1, DOT5, CUP2 and HAA1 genes that confers increased tolerance to acetic acid as described in WO 2015/181169 and WO 2016/083397; c) the genetic modification is a modification that introduces a mutant SSK1 gene encoding a truncated ssk1 protein as described in WO 2014/048863; d) the genetic modification is a modification that introduces an allele of the NNK1 gene that confers an increased xylose to ethanol fermentation rate as described in WO 2015/086805; and, e) the genetic modification is overexpression of at least one of a gene encoding the Prp42 protein and a gene encoding the Smd2 protein.

10 . The cell according to claim 1 , wherein the nucleotide sequence encoding the polypeptide with xylose isomerase activity is integrated into the genome of the cell.

11 . The cell according to claim 1 , wherein the cell is a cell of an industrial yeast strain or derived from an industrial yeast strain.

12 . The cell according to claim 1 , wherein the cell is a diploid, aneuploid or polyploid cell.

13 . The cell according to claim 1 , wherein the cell is improved in at least one industrially relevant phenotype by evolutionary engineering, wherein preferably the industrially relevant phenotype is xylose utilisation rate.

14 . The cell according to claim 1 , wherein the cell has the ability to produce at least one fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins.

15 . 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, amino acids, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins, whereby the process comprises the steps of: (a) fermenting a medium containing a source of xylose, and optionally a source of glucose, with a cell as defined in claim 1 , whereby the cell ferments the xylose, and optionally the glucose, to the fermentation product, and optionally, (b) recovery of the fermentation product.

16 . The cell according to claim 2 , wherein the cell belongs to a Saccharomyces species selected from the group consisting of S. cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus, S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis and S. unisporus.

17 . The cell according to claim 2 , wherein the nucleotide sequence encoding the polypeptide with xylose isomerase activity is operably linked to a promoter that is insensitive to catabolite repression and that does not require xylose for induction.

18 . The cell according to claim 2 , whereby the cell comprises at least one genetic modification selected from:
a) a genetic modification that increases the specific xylulose kinase activity; b) a genetic modification that increases the flux of the pentose phosphate pathway; and, c) a genetic modification that reduces unspecific aldose reductase activity in the cell.

19 . The cell according to claim 2 , wherein the cell further comprises at least one genetic modification that results in a characteristic selected from the group consisting of:
a) increased tolerance to ethanol; b) increased tolerance to acetic acid; c) reduced production of glycerol; d) increased xylose to ethanol fermentation rate; and, e) increased thermotolerance.

20 . The cell according to claim 19 , wherein, in:
a) the genetic modification is a modification that introduces an allele of one or more of the ADE1, KIN3, MKT1, VPS70, SWS2 and APJ1 genes that confers increased tolerance to ethanol as described in WO 2012/175552 and WO 2014/170330; b) the genetic modification is a modification that introduces an allele

Assignees

• Vib Vzw
• Univ Leuven Kath
• Globalyeast N V

Inventors

• Thevelein Johan
• Demeke Mekonnen
• Foulquié Moreno Maria Remedios
• De Graeve Stijn
• Valdomiro Charles Belo Edgard

Classifications

• C12P7/16

  Butanols · CPC title

• C12N9/92 Primary

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

• C12P7/6409

  Fatty acids · CPC title

• C12N1/22 Primary

  Processes using, or culture media containing, cellulose or hydrolysates thereof · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title