# US 2023/0119263 A1 · Pseudomonas mutant strains with enhanced xylose and galactose utilization

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- Number: US 2023/0119263 A1
- Document ID: US20230119263A1
- Status: Published application
- Kind: A1
- Application number: 17/709,332
- Filed: 2022-03-30
- Published: 2023-04-20
- Earliest claimed: 2021-03-31
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Hyungyu Lim; Thomas T. Eng; Adam M. Feist
- Web page: https://patentgrep.com/patent/US20230119263A1

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C07K 14/21: from Pseudomonadaceae (F)
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/88: Lyases (4.)
- C12N 9/90: Isomerases (5.)
- C12N 9/92: Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Y 207/01006: Galactokinase (2.7.1.6)
- C12Y 207/07012: UDP-glucose--hexose-1-phosphate uridylyltransferase (2.7.7.12)
- C12Y 402/01082: Xylonate dehydratase (4.2.1.82)
- C12Y 501/03002: UDP-glucose 4-epimerase (5.1.3.2), i.e. UDP-galactose 4-epimerase
- C12N 2500/34: Sugars
- C12R 2001/38: Pseudomonas
- C12R 2001/385: Pseudomonas aeruginosa
- C12R 2001/39: Pseudomonas fluorescens
- C12R 2001/40: Pseudomonas putida
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention provides for a Pseudomonas cell is able to grow in a medium with xylose or galactose as a sole carbon source with a growth rate of equal to or higher than 0.10 h−1. The present invention provides for methods and compositions relating to an engineered Pseudomonas putida KT2440 utilizing a non-native carbon source, such as galactose or xylose or both.

## Claims

### Claim 1

A Pseudomonas cell is able to grow in a medium with xylose or galactose as a sole carbon source with a growth rate of equal to or higher than 0.10 h−1.

### Claim 2 (depends on 1)

The Pseudomonas cell of claim 1, wherein the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P, oryzihabitans, P. parafluva, or P. plecoglossicida.

### Claim 3 (depends on 2)

The Pseudomonas cell of claim 2, wherein the Pseudomonas cell is a P. putida cell.

### Claim 4 (depends on 3)

The Pseudomonas cell of claim 3, wherein the P. putida cell is strain KT2440.

### Claim 5 (depends on 1)

The Pseudomonas cell of claim 1, wherein the Pseudomonas cell comprises the following genes: (A) a gene encoding a heterologous xylonate dehydratase, and mutations in native ptxS and/or kguT genes encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, respectively; and/or, (B) genes encoding heterologous UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase, and mutation(s) in a native gtsABCD gene encoding an ATP-binding cassette (ABC) sugar transporting system.

### Claim 6 (depends on 5)

The Pseudomonas cell of claim 5, wherein the heterologous xylonate dehydratase is Caulobacter crescentus xylD gene.

### Claim 7 (depends on 5)

The Pseudomonas cell of claim 5, wherein the genes encoding heterologous UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-l-epimerase are Escherichia coli galETKM genes.

### Claim 8 (depends on 5)

The Pseudomonas cell of claim 5, wherein the Pseudomonas cell is capable of utilizing xylose as a sole carbon source, and the Pseudomonas cell comprises the following genes: a gene encoding xylonate dehydratase, and mutations in ptxS and/or kguT genes encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, respectively.

### Claim 9 (depends on 8)

The Pseudomonas cell of claim 8, wherein the gene encoding xylonate dehydratase is Caulobacter crescentus xylD gene.

### Claim 10 (depends on 8)

The Pseudomonas cell of claim 8, wherein the Pseudomonas cell growing in a medium with xylose as a sole carbon source has a growth rate of equal to or higher than 0.10 h−1, 0.15 h−1, 0.20 h−1, or 0.25 h−1.

### Claim 11 (depends on 5)

The Pseudomonas cell of claim 5, wherein the Pseudomonas cell capable of utilizing galactose as a sole carbon source, and the Pseudomonas cell comprises the following genes: genes encoding UDP-glucose 4-epimerase, galactose-l-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase, and mutation(s) in gtsABCD genes encoding an ATP-binding cassette (ABC) sugar transporting system.

### Claim 12 (depends on 11)

The Pseudomonas cell of claim 11, wherein the genes encoding UDP-glucose 4-epimerase, galactose-l-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase are Escherichia coli galETKM genes.

### Claim 13 (depends on 11)

The Pseudomonas cell of claim 11, wherein the Pseudomonas cell growing in a medium with galactose as a sole carbon source has a growth rate of equal to or higher than 0.10 h−1, 0.15 h−1, 0.20 h−1, 0.25 h−1, 0.30 h−1, 0.35 h−1, 0.40 h−1, 0.45 h−1, 0.50 h−1, or 0.52 h−1.

### Claim 14 (depends on 5)

The Pseudomonas cell of claim 5, wherein the native ptxS, kguT, and/or gtsABCD genes are knocked out or deleted from the chromosome of the Pseudomonas cell.

### Claim 15 (depends on 5)

The Pseudomonas cell of claim 5, wherein the xylD gene and/or the galETKM genes are each independently capable of expression from the chromosome under a constitutive promoter and 5′-untranslated region.