# US 2013/0157343 A1 · Biological methods for preparing adipic acid

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- Number: US 2013/0157343 A1
- Document ID: US20130157343A1
- Status: Published application
- Kind: A1
- Application number: 13/549,076
- Filed: 2012-07-13
- Published: 2013-06-20
- Earliest claimed: 2009-07-02
- Assignees: Verdezyne, Inc.
- Inventors: Stephen PICATAGGIO; Tom Beardslee
- Web page: https://patentgrep.com/patent/US20130157343A1

## CPC

- C12N 15/815: {for yeasts other than Saccharomyces}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/44: Polycarboxylic acids
- 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)}

## Abstract

The technology relates in part to biological methods for producing adipic acid and engineered microorganisms capable of such production.

## Claims

### Claim 1

A genetically modified Candida yeast, comprising:
a genetic modification that reduces or eliminates a POX4 activity, thereby partially blocking beta oxidation activity;
a genetic modification that adds or increases a CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity; and
a genetic modification that adds or increases a monooxygenase reductase activity.

### Claim 2 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the Candida yeast is a genetically modified ATCC20336 yeast.

### Claim 3 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A15 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A15 monooxygenase coding sequence.

### Claim 4 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises: (i) an increased copy number of a CYP52A16 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A16 monooxygenase coding sequence.

### Claim 5 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A19 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A19 monooxygenase coding sequence.

### Claim 6 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRA monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRA monooxygenase reductase coding sequence.

### Claim 7 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRB monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRB monooxygenase reductase coding sequence.

### Claim 8 (depends on 1)

The genetically modified Candida yeast of claim 1, wherein the genetic modification that reduces or eliminates the POX4 activity comprises (i) a disrupted, deleted or knocked out polynucleotide that encodes a POX4 polypeptide, or (ii) a disrupted promoter and/or 5′UTR in functional connection with a polynucleotide that encodes a POX4 polypeptide.

### Claim 9 (depends on 1)

The genetically modified Candida yeast of claim 1, which is capable of producing adipic acid, at a level of at least 0.4 grams per liter of culture medium, from a feedstock comprising a mixture of fatty acids from a vegetable oil.

### Claim 10 (depends on 9)

The genetically modified Candida yeast of claim 9, wherein the vegetable oil is a palm oil, soybean oil or fatty acid soap stock.

### Claim 11

A genetically modified Candida yeast, comprising:
a genetic modification that reduces or eliminates a POX5 activity, thereby partially blocking beta oxidation activity,
a genetic modification that adds or increase a CYP52A15, CYP52A16, or CYP52A19 monooxygenase activity, and
a genetic modification that adds or increases a monooxygenase reductase activity.

### Claim 12 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the Candida yeast is a genetically modified ATCC20336 yeast.

### Claim 13 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A15 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A15 monooxygenase coding sequence.

### Claim 14 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises: (i) an increased copy number of a CYP52A16 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A16 monooxygenase coding sequence.

### Claim 15 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A19 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A19 monooxygenase coding sequence.

### Claim 16 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRA monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRA monooxygenase reductase coding sequence.

### Claim 17 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRB monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRB monooxygenase reductase coding sequence.

### Claim 18 (depends on 11)

The genetically modified Candida yeast of claim 11, wherein the genetic modification that reduces or eliminates the POX5 activity comprises (i) a disrupted, deleted or knocked out polynucleotide that encodes a POX5 polypeptide, or (ii) a disrupted, deleted or knocked out promoter and/or 5′UTR in functional connection with a polynucleotide that encodes a POX5 polypeptide.

### Claim 19 (depends on 11)

The genetically modified Candida yeast of claim 11, which is capable of producing adipic acid, at a level of at least 0.4 grams per liter of culture medium, from a feedstock comprising a mixture of fatty acids from a vegetable oil.

### Claim 20 (depends on 19)

The genetically modified Candida yeast of claim 19, wherein the vegetable oil is a palm oil, soybean oil or fatty acid soap stock.

### Claim 21

A genetically modified Candida yeast, comprising:
a genetic modification that reduces or eliminates a POX4 activity and a POX5 activity, thereby blocking beta oxidation activity,
a genetic modification that adds or increase a CYP52A15, CYP52A16, or CYP52A19 monooxygenase activity, and
a genetic modification that adds or increases a monooxygenase reductase activity.

### Claim 22 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the Candida yeast is a genetically modified ATCC20336 yeast.

### Claim 23 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A15 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A15 monooxygenase coding sequence.

### Claim 24 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises: (i) an increased copy number of a CYP52A16 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A16 monooxygenase coding sequence.

### Claim 25 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A19 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A19 monooxygenase coding sequence.

### Claim 26 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRA monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRA monooxygenase reductase coding sequence.

### Claim 27 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRB monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRB monooxygenase reductase coding sequence.

### Claim 28 (depends on 21)

The genetically modified Candida yeast of claim 21, wherein the genetic modification that reduces or eliminates the POX4 activity and the POX5 activity comprises (i) a disrupted, deleted or knocked out polynucleotide that encodes a POX4 polypeptide and/or a POX5 polypeptide, or (ii) a disrupted, deleted or knocked out promoter and/or 5′UTR in functional connection with a polynucleotide that encodes a POX4 polypeptide and/or a POX5 polypeptide.

### Claim 29 (depends on 21)

The genetically modified Candida yeast of claim 21, which is capable of producing adipic acid, at a level of at least 0.4 grams per liter of culture medium, from a feedstock comprising a mixture of fatty acids from a vegetable oil.

### Claim 30 (depends on 29)

The genetically modified Candida yeast of claim 29, wherein the vegetable oil is a palm oil, soybean oil or fatty acid soap stock.