# US 2013/0157332 A1 · Gene inactivation allowing immediate growth on xylose medium by engineered zymomonas

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- Number: US 2013/0157332 A1
- Document ID: US20130157332A1
- Status: Published application
- Kind: A1
- Application number: 13/711,647
- Filed: 2012-12-12
- Published: 2013-06-20
- Earliest claimed: 2011-12-20
- Assignees: E. I. Du Pont de Nemours and Company
- Inventors: WILLIAM D. HITZ; Min Qi; Luan Tao; Paul V. Viitanen; Jianjun Yang
- Web page: https://patentgrep.com/patent/US20130157332A1

## CPC

- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1022: {transferring aldehyde or ketonic groups (2.2)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/92: Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)}
- C12P 7/065: {with microorganisms other than yeasts}
- C12P 7/10: substrate containing cellulosic material
- C12Y 101/01021: Aldehyde reductase (1.1.1.21), i.e. aldose-reductase
- C12Y 202/01001: Transketolase (2.2.1.1)
- C12Y 202/01002: Transaldolase (2.2.1.2)
- C12Y 207/01017: Xylulokinase (2.7.1.17)
- 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

Zymomonas cells that are genetically engineered to have a disrupted aldose reductase gene such that aldose reductase activity for conversion of xylose to xylitol in the presence of NADPH is reduced by greater than 90%, and that are engineered to express a xylose utilization metabolic pathway, were found to have the ability to gro on medium containing xylose as the only sugar without adaptation in media containing xylose.

## Claims

### Claim 1

A method of making a xylose-competent Zymomonas cell comprising:
a) providing a Zymomonas host cell;
b) creating a genetic modification in said cell in at least one endogenous gene encoding an aldose reductase enzyme of EC 1.1.1.21 having the ability to convert xylose to xylitol in the presence of NADPH; and
c) introducing into said cell a xylose utilization metabolic pathway;
wherein the order of steps (b) and (c) is not specified and wherein said xylose-competent Zymomonas cell has aldose reductase activity reduced by greater than 90% as compared with a Zymomonas cell lacking the genetic modification of step (b).

### Claim 2 (depends on 1)

The method of claim 1 further comprising selecting the xylose-competent Zymomonas cell using medium containing xylose as the only sugar.

### Claim 3 (depends on 1)

The method of claim 1 wherein the Zymomonas host cell is a wild type cell.

### Claim 4 (depends on 1)

The method of claim 1 wherein the Zymomonas host cell is a Zymomonas cell that has not previously been exposed to xylose.

### Claim 5 (depends on 1)

The method of claim 1 wherein steps (b) and (c) occur concurrently

### Claim 6 (depends on 1)

The method of claim 1 wherein the endogenous gene encoding aldose reductase encodes a protein having at least about 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO:2.

### Claim 7 (depends on 1)

The method of claim 1 wherein the endogenous gene encoding aldose reductase has a coding region sequence having at least about 95% identity to the nucleotide sequence as set forth in SEQ ID NO:1.

### Claim 8 (depends on 1)

The method of claim 1 wherein the genetic modification of step (b) is selected from the group consisting of mutation, insertion, deletion, and combinations thereof.

### Claim 9 (depends on 1)

The method of claim 1 wherein the xylose utilization metabolic pathway comprises a series of polynucleotides encoding polypeptides, each having xylose isomerase, xylulokinase, transketolase, or transaldolase enzymatic activity.

### Claim 10 (depends on 9)

The method of claim 9 wherein the polypeptide having xylose isomerase activity is a Group I xylose isomerase and is included in the class of enzymes identified by EC 5.3.1.5.

### Claim 11 (depends on 10)

The method of claim 10 wherein the polynucleotide encoding the xylose isomerase polypeptide is isolated from Actinoplanes missouriensis

### Claim 12 (depends on 11)

The method of claim 11 wherein the polynucleotide encoding the xylose isomerase polypeptide is operably linked to a mutant glyceraldehyde-3-phosphate dehydrogenase gene promoter, wherein the mutant promoter has higher activity than the native promoter.

### Claim 13 (depends on 1)

The method of claim 1 wherein the xylose-competent Zymomonas cell further comprises at least one genetic modification which reduces glucose-fructose oxidoreductase activity.

### Claim 14 (depends on 1)

The method of claim 1 wherein the xylose-competent Zymomonas cell further comprises a genetic modification which reduces expression of the endogenous himA gene.

### Claim 15 (depends on 1)

The method of claim 1 wherein the xylose-competent Zymomonas cell further comprises polynucleotides encoding polypeptides for arabinose utilization, each having L-arabinose isomerase activity, L-ribulokinase activity, or L-ribulose-5-phosphate-4-epimerase activity, and optionally a polynucleotide encoding a polypeptide that is an arabinose-proton symporter.

### Claim 16 (depends on 1)

The method of claim 1 wherein the xylose-competent Zymomonas cell further comprises a genetic modification which increases ribose-5-phosphate isomerase activity.

### Claim 17 (depends on 1)

The method of claim 1 further comprising adapting the xylose-competent Zymomonas cell in xylose-containing medium wherein xylose utilization is improved.

### Claim 18

A xylose-competent and ethanol-producing Zymomonas cell having the following characteristics:
a) the cell does not require a xylose-adaptation step for immediate growth on media containing xylose as the only sugar;
b) the cell has aldose reductase activity for conversion of xylose to xylitol in the presence of NADPH reduced by greater than 90%; and
c) the cell comprises a xylose utilization metabolic pathway comprising a series of polynucleotides encoding polypeptides, each having xylose isomerase, xylulokinase, transketolase, or transaldolase enzymatic activity.

### Claim 19 (depends on 18)

A method for producing ethanol comprising growing the xylose-competent and ethanol-producing Zymomonas cell of claim 18 under conditions wherein ethanol is produced.