# US 2019/0316111 A1 · Xylose Isomerases that confer efficient xylose fermentation capability to yeast

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- Number: US 2019/0316111 A1
- Document ID: US20190316111A1
- Status: Published application
- Kind: A1
- Application number: 16/472,514
- Filed: 2017-12-21
- Published: 2019-10-17
- Earliest claimed: 2016-12-21
- Assignees: Vib Vzw; Katholieke Universiteit Leuven, KU LEUVEN R&D; GlobalYeast N.V.
- Applicants: VIB vzw; Katholieke Universiteit Leuven, K.U. Leuven R&D; GlobalYeast N.V.
- Inventors: Johan Thevelein; Mekonnen Demeke; Maria Remedios Foulquié Moreno; Stijn De Graeve; Edgard Valdomiro Charles Belo
- Web page: https://patentgrep.com/patent/US20190316111A1

## CPC

- C12N 1/22: Processes using, or culture media containing, cellulose or hydrolysates thereof
- C12N 9/92: Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 15/905: {in yeast}
- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C12P 7/10: substrate containing cellulosic material
- C12P 7/16: Butanols
- C12P 7/18: polyhydric
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/46: Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
- C12P 7/48: Tricarboxylic acids, e.g. citric acid
- C12P 7/6409: Fatty acids
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 35/00: Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12Y 503/01005: Xylose isomerase (5.3.1.5)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## 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.

## Claims

### 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

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.

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 2)

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.

### Claim 17 (depends on 2)

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.

### Claim 18 (depends on 2)

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.

### Claim 19 (depends on 2)

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.

### Claim 20 (depends on 19)

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 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.