# US 11,898,181 B2 · Genetically modified isopropylmalate isomerase enzyme complexes and processes to prepare elongated 2-ketoacids and C5-C10 compounds therewith

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,898,181 B2
- Document ID: US11898181B2
- Status: Granted
- Kind: B2
- Application number: 17/547,689
- Filed: 2021-12-10
- Published: 2022-03-31
- Granted: 2024-02-13
- Earliest claimed: 2017-09-29
- Assignees: Dow Global Technologies LLC
- Applicants: Dow Global Technologies LLC
- Inventors: Paresh Sanghani; Eric C. Shiue; Scott A. Greenwalt
- Web page: https://patentgrep.com/patent/US11898181B2

## CPC

- C12N 9/88: Lyases (4.)
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1025: {Acyltransferases (2.3)}
- C12P 7/04: acyclic
- C12P 7/24: containing a carbonyl group
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12Y 101/01085: 3-Isopropylmalate dehydrogenase (1.1.1.85)
- C12Y 203/03013: 2-Isopropylmalate synthase (2.3.3.13)
- C12Y 402/01033: 3-Isopropylmalate dehydratase (4.2.1.33)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- 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
- 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)

## Abstract

Genetically modified isopropylmalate isomerase enzyme complexes (e.g., LeuCD′ enzyme complexes), microbial organisms including genetically modified isopropylmalate isomerase enzyme complexes (e.g., LeuCD′), and processes for preparing C7-C11 2-ketoacids with genetically modified isopropylmalate isomerase enzyme complexes (e.g., LeuCD′). The genetically modified isopropylmalate isomerase enzyme complexes (e.g., LeuCD′ enzyme complexes), microbial organisms, and processes for preparing C7-C11 2-ketoacids can be used to produce C6-C10 aldehydes, alkanes, alcohols, and carboxylic acids, both in vivo and in vitro.

## Claims

### Claim 1

A genetically modified LeuCD′ enzyme complex comprising:
(a) a genetically modified LeuC′ subunit comprising:
(1) an amino acid sequence with at least 95% homology to SEQ ID NO: 1 having a C-terminus; and
(2) a C-terminal amino acid or peptide coupled to the C-terminus of the amino acid sequence of (a)(1), wherein the C-terminal amino acid is chosen from any naturally occurring amino acid, and wherein the C-terminal peptide comprises an amino acid sequence:
(Xaa)1(Xaa)2(Xaa)3(Xaa)4(Xaa)5(Xaa)6(Xaa)7(Xaa)8(Xaa)9(Xaa)10 (SEQ ID NO: 39) in which:
(Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, and (Xaa)10 are each independently chosen from any naturally occurring amino acid, or are absent, provided that at least two of (Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, or (Xaa)10 are present in the C-terminal peptide; and
(b) a LeuD subunit comprising an amino acid sequence with at least 90% homology to SEQ ID NO: 2, wherein the genetically modified LeuCD′ enzyme complex has activity against 2-hexylmalate (2-HM).

### Claim 2 (depends on 1)

The genetically modified LeuCD′ enzyme complex of claim 1, wherein the genetically modified LeuC′ subunit comprises the C-terminal peptide, and wherein at least six of (Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, or (Xaa)10 are present.

### Claim 3 (depends on 1)

The genetically modified LeuCD′ enzyme complex of claim 1, wherein the genetically modified LeuC′ subunit comprises the C-terminal peptide, and wherein each of (Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, or (Xaa)10 present in the C-terminal peptide is independently chosen from Ser, Thr, His, Lys, and Arg.

### Claim 4 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein:
the genetically modified LeuC′ subunit comprises the C-terminal peptide;
(Xaa)1, (Xaa)2, (Xaa)9, and (Xaa)10 are each independently chosen from Ser and Thr; and
(Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, and (Xaa)8 are each independently chosen from His, Lys, and Arg.

### Claim 5 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the genetically modified LeuC′ subunit comprises the C-terminal peptide (SEQ ID NO: 40).

### Claim 6 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the LeuD subunit consists of the amino acid sequence with at least 90% homology to SEQ ID NO: 2.

### Claim 7 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the valine at position 35 of SEQ ID NO: 1 is substituted with alanine or glycine.

### Claim 8 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the leucine at position 411 of SEQ ID NO: 1 is substituted with valine, alanine, or glycine.

### Claim 9 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the leucine at position 31 of SEQ ID NO: 2 is substituted with valine, alanine, or glycine.

### Claim 10 (depends on 1)

The genetically modified LeuCD′ enzyme complex according to claim 1, wherein the histidine at position 88 of SEQ ID NO: 2 is substituted with alanine or serine.

### Claim 11 (depends on 1)

A process for preparing a C7-C11 2-ketoacid, the process comprising: (I) providing at least one of a C4-C10 2-ketoacid substrate with:
(A) at least one isopropylmalate synthase enzyme having isopropylmalate synthase activity;
(B) at least one isopropylmalate dehydrogenase enzyme having isopropylmalate dehydrogenase activity; and
(C) at least one genetically modified LeuCD′ enzyme complex comprising the genetically modified LeuCD′ enzyme complex according to claim 1;
under conditions that the at least one of the C4-C10 2-ketoacid substrate is converted to the C7-C11 2-ketoacid, wherein:
the at least one genetically modified LeuCD′ enzyme complex has activity against 2-hexylmalate (2-HM), and
conversion of the at least one of the C4-C10 2-ketoacid substrate to the C7-C11 2-ketoacid occurs via one or more biochemical reactions.

### Claim 12 (depends on 11)

The process of claim 11, wherein the genetically modified LeuC′ subunit comprises the C-terminal peptide, and wherein at least six of (Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, or (Xaa)10 are present.

### Claim 13 (depends on 11)

The process according to claim 11, the genetically modified LeuC′ subunit comprises the C-terminal peptide, wherein each of (Xaa)1, (Xaa)2, (Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, (Xaa)8, (Xaa)9, or (Xaa)10 present in the C-terminal peptide is independently chosen from Ser, Thr, His, Lys, and Arg.

### Claim 14 (depends on 11)

The process according to claim 11, wherein:
the genetically modified LeuC′ subunit comprises the C-terminal peptide;
(Xaa)1, (Xaa)2, (Xaa)9, and (Xaa)10 are each independently chosen from Ser and Thr; and
(Xaa)3, (Xaa)4, (Xaa)5, (Xaa)6, (Xaa)7, and (Xaa)8 are each independently chosen from His, Lys, and Arg.

### Claim 15 (depends on 11)

The process according to claim 11, wherein the genetically modified LeuC′ subunit comprises the C-terminal peptide (SEQ ID NO: 40).