# US 2020/0399662 A1 · Alkane oxidation by modified hydroxylases

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- Number: US 2020/0399662 A1
- Document ID: US20200399662A1
- Status: Published application
- Kind: A1
- Application number: 16/872,275
- Filed: 2020-05-11
- Published: 2020-12-24
- Earliest claimed: 2005-03-28
- Assignees: California Institute of Technology
- Applicants: The California Institute of Technology
- Inventors: Frances Arnold; Peter Meinhold; Matthew W. Peters; Rudi Fasan; Mike M.Y. Chen
- Web page: https://patentgrep.com/patent/US20200399662A1

## CPC

- C12P 7/04: acyclic
- C07K 14/32: from Bacillus (G)
- C12N 9/0042: {NADPH-cytochrome P450 reductase (1.6.2.4)}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/0077: {with a reduced iron-sulfur protein as one donor (1.14.15)}
- C12P 7/06: Ethanol, i.e. non-beverage
- 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)
- C12Y 114/15: with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y 114/15003: Alkane 1-monooxygenase (1.14.15.3)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

This invention relates to modified hydroxylases. The invention further relates to cells expressing such modified hydroxylases and methods of producing hydroxylated alkanes by contacting a suitable substrate with such cells.

## Claims

### Claim 1

A polynucleotide that encodes a polypeptide that catalyzes the conversion of a (C1-C12)alkane to an alcohol,
wherein the polypeptide encoded by the polynucleotide has one or more selective amino acid substitutions at residues 47, 52, 74, 78, 82, 94, 142, 175, 184, 188, 205, 226, 236, 252, 255, 290, 328, 353, 366, 443, 464, and 710 in comparison to the amino acid sequence set forth in SEQ ID NO:1, and wherein the one or more selective amino acid substitutions are as follows:
(i) at positions selected from the group consisting of 47, 52, 82, 142, 205, 236, 252, 255, and 464 and any combination thereof, an amino acid residue selected from glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C);
(ii) at positions selected from the group consisting of 94, 175, 184, 188, 290, 353, a and 366 or any combination thereof, an amino acid residue selected from alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), and methionine (M);
(iii) at position 226, an amino acid selected from lysine (K) and arginine (R);
(iv) at positions 78 and 328, an amino acid selected from alanine (A), tyrosine (Y), phenylalanine (F), tryptophan (W) and histidine (H);
(v) at position 74, an amino acid selected from alanine (A), serine (S), and glutamic acid (E);
(vi) at position 443 an alanine (A) residue; and
(vii) at position 710 a threonine (T).

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide further comprises up to 50 conservative amino acid substitutions in comparison to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 3 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide further comprises up to 25 conservative amino acid substitutions in comparison to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 3 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide further comprises up to 10 conservative amino acid substitutions in comparison to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 4 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide further comprises up to 5 conservative amino acid substitutions in comparison to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide comprises at position 52 a leucine (L), at position 74 an alanine (A), at position 188 a leucine (L), and at position 366 an isoleucine (I).

### Claim 6 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide comprises at position 47 a cysteine (C), at position 78 a phenylalanine (F), at position 82 a serine (S), at position 94 an isoleucine (I), at position 142 a serine (S), at position 175 an isoleucine (I), at position 184 a valine (V), at position 205 a cysteine (C), at position 226 an arginine (R), at position 236 a glutamine (Q), at position 252 a glycine (G), at position 255 a serine (S), at position 290 a valine (V), at position 328 a phenylalanine (F), and at position 353 a valine (V).

### Claim 7 (depends on 5)

The polynucleotide of claim 5, wherein the polypeptide encoded by the polynucleotide comprises at position 47 a cysteine (C), at position 78 a phenylalanine (F), at position 82 a serine (S), at position 94 an isoleucine (I), at position 142 a serine (S), at position 175 an isoleucine (I), at position 184 a valine (V), at position 205 a cysteine (C), at position 226 an arginine (R), at position 236 a glutamine (Q), at position 252 a glycine (G), at position 255 a serine (S), at position 290 a valine (V), at position 328 a phenylalanine (F), and at position 353 a valine (V).

### Claim 8 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide encoded by the polynucleotide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

### Claim 9 (depends on 6)

The polynucleotide of claim 6, wherein the polypeptide encoded by the polynucleotide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

### Claim 10 (depends on 7)

The polynucleotide of claim 7, wherein the polypeptide encoded by the polynucleotide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

### Claim 11 (depends on 1)

The polynucleotide of claim 1, wherein the polynucleotide has at least 90% sequence identity to the sequence of SEQ ID NO:15 or SEQ ID NO:17.

### Claim 12 (depends on 11)

The polynucleotide of claim 11, wherein the polynucleotide has at least 95% sequence identity to the sequence of SEQ ID NO:15 or SEQ ID NO:17.

### Claim 13 (depends on 1)

The polynucleotide of claim 1, wherein the (C1-C12)alkane is selected from the group consisting of methane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane.

### Claim 14 (depends on 1)

The polynucleotide of claim 1, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol.

### Claim 15 (depends on 1)

A vector comprising the polynucleotide of claim 1.

### Claim 16 (depends on 15)

The vector of claim 15, wherein the vector is a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC).

### Claim 17 (depends on 15)

The vector of claim 15, wherein the vector is derived from pCWori.

### Claim 18 (depends on 1)

A cell comprising the polynucleotide of claim 1.

### Claim 19 (depends on 15)

A cell comprising the vector of claim 15.

### Claim 20 (depends on 19)

The cell of claim 19, wherein the cell is a prokaryote.