# US 9,752,171 B2 · Pentose fermentation by a recombinant microorganism

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- Number: US 9,752,171 B2
- Document ID: US9752171B2
- Status: Granted
- Kind: B2
- Application number: 14/871,483
- Filed: 2015-09-30
- Published: 2016-01-14
- Granted: 2017-09-05
- Earliest claimed: 2010-05-28
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Ezhilkani Subbian; Xiyun Zhang; Benjamin N. Mijts; Catherine M. Cho
- Web page: https://patentgrep.com/patent/US9752171B2

## CPC

- C12P 19/24: produced by the action of an isomerase, e.g. fructose
- C12N 9/90: Isomerases (5.)
- C12N 9/92: Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)}
- C12N 15/81: for yeasts
- C12P 7/04: acyclic
- C12P 7/10: substrate containing cellulosic material
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12Y 503/01005: Xylose isomerase (5.3.1.5)
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention provides recombinant nucleic acid constructs comprising a xylose isomerase polynucleotide, a recombinant fungal host cell comprising a recombinant xylose isomerase polynucleotide, and related methods.

## Claims

### Claim 1

A recombinant fungal host cell comprising a polynucleotide sequence that encodes a polypeptide that is capable of catalyzing the isomerization of D-xylose directly to D-xylulose, wherein the polynucleotide is a recombinant polynucleotide encoding a xylose isomerase variant— polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2, wherein said polypeptide further comprises the substitutions T64Q/K233C/F250/E372G, relative to SEQ ID NO:2, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:2.

### Claim 2 (depends on 1)

The recombinant fungal host cell of claim 1, wherein said isolated xylose isomerase variant is a mature form having xylose isomerase activity further comprising a substitution at one or more positions selected from E2, N6, Q13, K16, T18, E29, G62, T67, Q70, S71, A74, A75, K78, V81, L91, S106, K111, Q116, K127, Q128, A139, S156, A164, Y182, M199, K201, M206, K211, K223, , T236, K237, T244, V247, L248, H274, Q277, R281, R284, A325, F328, T329, N330, A339, G342, G356, F360, I371, D373, R375, K378, V380, D382, S386, T389, G390, I391, A393, A397, G398, K399, A400, S404, K407, E414, R423, Q424, M426, V431, N433, V434, L435, and/or F436, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2.

### Claim 3 (depends on 2)

The recombinant fungal host cell of claim 2, wherein said variant is a mature form having xylose isomerase activity further comprising a substitution at one or more positions selected from E2S, N6G, N6H, Q13K, K16E, T18C, T18K, T18L, T18M, E29N, G62F, T67S, Q70E, S71L, A74G, A75T, K78R, V81I, L91M, S109D, K111A, K111L, Q116C, K127I, K127R, Q128A, A139G, S156T, A164V, Y182C, M199A, M199V, L201H, M206T, K211H, K223T, T236A, T236L, K237A, T244S, V247A, L248S, H274R, Q277R, R281L, R284H, A325R, A325S, F328H, T329S, N330G, N330H, N330L, N330W, N330Y, A339R, G342P, G342V, G356A, F360M, I371G, I371L, I371Q, I371R, I371T, D373G, R375Q, R375T, R375V, K378A, K378D, V380W, D382G, D382N, S386K, T389H, G390M, I391A, I391L, A93T, A397L, A397S, G398E, K399E, K399T, K399V, A400G, S404Y, K407E, K407L, K407R, E414A,R423G, Q424H, M426R, V431E, N433A, N433H, N433R, V434Q, V434S, L435S, and/or F436G, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2.

### Claim 4 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the polynucleotide is integrated into the host cell genome.

### Claim 5 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the host cell is a yeast cell.

### Claim 6 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the host cell has had one or more native genes deleted from its genome.

### Claim 7 (depends on 6)

The recombinant fungal host cell of claim 6, wherein the deletion results in one or more phenotypes including increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and/or reduced production of by products, wherein comparison is made with respect to the corresponding host cell without the deletion(s).

### Claim 8 (depends on 1)

The recombinant fungal host cell of claim 1, wherein said host cell is altered to overexpress one or more polynucleotides.

### Claim 9 (depends on 8)

The recombinant fungal host cell of claim 8, wherein overexpression results in one or more phenotypes, including increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and/or reduced product of by products, wherein comparison is made to the corresponding unaltered host cell.

### Claim 10 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the host cell is capable of growth in a xylose-based culture medium.

### Claim 11 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the host cell is capable of fermentation in a xylose-based culture medium.

### Claim 12 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the host cell is capable of faster growth in a xylose-based culture medium as compared to wild-type Saccharomyces cerevisiae.

### Claim 13 (depends on 1)

The recombinant fungal host cell of claim 1, wherein the xylose-based culture medium is a product from a cellulosic saccharification process and/or a hemicellulosic feedstock.