Alkane oxidation by modified hydroxylases

Patent metadata

Field	Value
Publication number	US-10648006-B2
Application number	US-201815942001-A
Country	US
Kind code	B2
Filing date	Mar 30, 2018
Priority date	Mar 28, 2005
Publication date	May 12, 2020
Grant date	May 12, 2020

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.

First claim

What is claimed is:

1. An isolated or recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 with up to 50 conservative amino acid substitutions and wherein if residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, and 353 and optionally further residues 464, and 710 are substituted these residues have selective substitutions as follows:
(i) at positions selected from the group consisting of 47, 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, 290 and 353, and 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); and
(v) at position 710 a threonine (T),
wherein the polypeptide catalyzes the conversion of a (C 1 -C 12 )alkane to an alcohol.

2. The isolated or recombinant polypeptide of claim 1 , wherein the polypeptide has up to 25 conservative amino acid substitutions.

3. The isolated or recombinant polypeptide of claim 2 , wherein the polypeptide has up to 10 conservative amino acid substitutions.

4. The isolated or recombinant polypeptide of claim 3 , wherein the polypeptide has up to 5 conservative amino acid substitutions.

5. The isolated or recombinant polypeptide of claim 1 , wherein the isolated or recombinant polypeptide 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).

6. The isolated or recombinant polypeptide of claim 1 , wherein the polypeptide 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).

7. The isolated or recombinant polypeptide of claim 5 , wherein the polypeptide 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).

8. The isolated or recombinant polypeptide of claim 1 , wherein the polypeptide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

9. The isolated or recombinant polypeptide of claim 6 , wherein the polypeptide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

10. The isolated or recombinant polypeptide of claim 7 , wherein the polypeptide comprises at position 464 a glycine (G), and at position 710 a threonine (T).

11. A method of converting a C1 to C12 alkane to an alcohol comprising contacting the C1 to C12 alkane with an isolated or recombinant polypeptide of claim 1 under conditions to produce the alcohol.

12. The method of claim 11 , wherein the C1 to C12 alkane is selected from the group consisting of methane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane.

13. The method of claim 11 , wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol.

14. The method of claim 11 , wherein the contacting is in a cell-free system.

15. A method to convert methane to methanol comprising contacting methane with an isolated or recombinant polypeptide of claim 7 under conditions to produce methanol.

16. A method to convert methane to methanol comprising contacting methane with an isolated or recombinant polypeptide of claim 10 under conditions to produce methanol.

17. A method to convert ethane to ethanol comprising contacting ethane with an isolated or recombinant polypeptide of claim 10 under conditions to produce ethanol.

18. A method to convert octane to 2-octanol comprising contacting octane with an isolated or recombinant polypeptide of claim 7 under conditions to produce 2-octanol.

19. A method to convert octane to 2-octanol comprising contacting octane with an isolated or recombinant polypeptide of claim 10 under conditions to produce 2-octanol.

Assignees

• The California Institute Of Tech
• California Inst Of Techn

Inventors

• Arnold Frances
• Meinhold Peter
• Peters Matthew W
• Fasan Rudi
• Chen Mike M Y

Classifications

• C07K14/32

  from Bacillus (G) · CPC title

• C12Y106/02004

  NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase · CPC title

• C12N9/0077

  with a reduced iron-sulfur protein as one donor (1.14.15) · CPC title

• C12Y114/15003

  Alkane 1-monooxygenase (1.14.15.3) · CPC title

• C12Y114/14001

  Unspecific monooxygenase (1.14.14.1) · CPC title