# US 11,078,467 B2 · Highly active self-sufficient nitration biocatalysts

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- Number: US 11,078,467 B2
- Document ID: US11078467B2
- Status: Granted
- Kind: B2
- Application number: 16/345,224
- Filed: 2017-10-26
- Published: 2019-09-26
- Granted: 2021-08-03
- Earliest claimed: 2016-10-26
- Assignees: University of Florida Research Foundation, Inc.
- Applicants: University of Florida Research Foundation, Incorporated
- Inventors: Yousong Ding; Ran Zuo
- Web page: https://patentgrep.com/patent/US11078467B2

## CPC

- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C07K 14/80: Cytochromes
- C12N 9/0042: {NADPH-cytochrome P450 reductase (1.6.2.4)}
- C12P 17/10: Nitrogen as only ring hetero atom
- C12P 17/16: containing two or more hetero rings {(thiamine open chain analogs C12P17/167, i.e. not condensed among themselves or through a common carbocyclic ring system)}
- C12P 17/165: {Heterorings having nitrogen atoms as the only ring heteroatoms}
- C12P 17/182: {Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00)}
- C12Y 106/02004: NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
- C12Y 114/14001: Unspecific monooxygenase (1.14.14.1)
- C07K 2319/00: Fusion polypeptide
- 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)
- 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

## Abstract

The disclosure relates to the field of fusion proteins. In some aspects, the disclosure relates to artificial fusion proteins comprising cytochrome P450 enzymes linked to reductase enzymes and uses thereof. In some aspects, the disclosure relates to corn-pounds produced by artificial cytochrome P450 enzymes.

## Claims

### Claim 1

A fusion protein comprising:
(i) a TxtE enzyme comprising the amino acid sequence set forth in SEQ ID NO: 56 or 58;
(ii) an amino acid linker that is between 14 and 16 amino acids in length; and,
(iii) a catalytic domain of a CYP102A1 (P450BM3) reductase enzyme;
wherein the linker joins (iii) to a terminus of (i); wherein the fusion protein comprises increased nitration activity compare to cytochrome p450 enzyme.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the terminus is a C-terminus.

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein the TxtE enzyme comprises the amino acid sequence set forth in SEQ ID NO: 56.

### Claim 4 (depends on 1)

The fusion protein of claim 1, wherein the catalytic domain comprises the amino acid sequence set forth in SEQ ID NO: 57.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the amino acid linker is 16 amino acids in length.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein the amino acid linker is 15 amino acids in length.

### Claim 7 (depends on 1)

The fusion protein of claim 1, wherein the amino acid linker is 14 amino acids in length.

### Claim 8 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is not produced by overlap PCR.

### Claim 9 (depends on 1)

The fusion protein of claim 1, wherein the amino acid linker comprises or consists of the sequence set forth in any one of SEQ ID NO: 36, 38, or 40.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the amino acid linker is selected from the group consisting of flexible amino acid linker, rigid amino acid linker and cleavable amino acid linker.