Cytochrome C protein variants for catalyzing carbon-silicon bond formation

Patent metadata

Field	Value
Publication number	US-10093906-B2
Application number	US-201715422360-A
Country	US
Kind code	B2
Filing date	Feb 1, 2017
Priority date	Feb 2, 2016
Publication date	Oct 9, 2018
Grant date	Oct 9, 2018

Abstract

The present invention provides compositions and methods for catalyzing the formation of carbon-silicon bonds using heme proteins. In certain aspects, the present invention provides heme proteins, including variants and fragments thereof, that are capable of carrying out in vitro and in vivo carbene insertion reactions for the formation of carbon-silicon bonds. In other aspects, the present invention provides methods for producing an organosilicon product, the method comprising providing a silicon-containing reagent, a carbene precursor, and a heme protein; and combining the components under conditions sufficient to produce an organosilicon product. Host cells expressing the heme proteins are also provided by the present invention.

First claim

What is claimed is:

1. A cytochrome c protein variant comprising the amino acid sequence set forth in SEQ ID NO:1 that enantioselectively catalyzes the formation of a carbon-silicon bond and comprises one or more mutations selected from the group consisting of V75, M100, and M103 relative to the amino acid sequence set forth in SEQ ID NO:1.

2. The cytochrome c protein variant of claim 1 , wherein the one or more mutations comprise a V75 mutation and an M100 mutation relative to the amino acid sequence set forth in SEQ ID NO:1.

3. The cytochrome c protein variant of claim 1 , wherein the one or more mutations comprise an M100 mutation and an M103 mutation relative to the amino acid sequence set forth in SEQ ID NO:1.

4. The cytochrome c protein variant of claim 1 , wherein the one or more mutations comprise a V75 mutation, an M100 mutation, and an M103 mutation relative to the amino acid sequence set forth in SEQ ID NO:1.

5. The cytochrome c protein variant of claim 1 , wherein the V75 mutation is a V75T mutation.

6. The cytochrome c protein variant of claim 1 , wherein the M100 mutation is an M100D or M100E mutation.

7. The cytochrome c protein variant of claim 1 , wherein the M103 mutation is an M103E mutation.

8. The cytochrome c protein variant of claim 1 , wherein the protein variant comprises a heme cofactor that is a non-native cofactor.

9. The cytochrome c protein variant of claim 1 , wherein the protein variant has a higher total turnover number (TTN) compared to a wild-type cytochrome c protein.

10. The cytochrome c protein variant of claim 9 , wherein the protein variant has a TTN greater than about 70.

11. The cytochrome c protein variant of claim 10 , wherein the protein variant has a TTN greater than about 1,800.

12. The cytochrome c protein variant of claim 1 , wherein the protein variant has a higher turnover frequency (TOF) compared to a wild-type cytochrome c protein.

13. The cytochrome c protein variant of claim 12 , wherein the TOF is at least about 2-fold greater than the wild-type protein.

14. The cytochrome c protein variant of claim 13 , wherein the TOF is at least about 7-fold greater than the wild-type protein.

15. The cytochrome c protein variant of claim 12 , wherein the TOF is at least about 10 min −1 .

16. The cytochrome c protein variant of claim 15 , wherein the TOF is at least about 45 min −1 .

17. The cytochrome c protein variant of claim 1 , wherein the protein variant produces an organosilicon product with a % ee of at least about 75%.

18. The cytochrome c protein variant of claim 17 , wherein the protein variant produces an organosilicon product with a % ee of at least about 95%.

19. The cytochrome c protein variant of claim 18 , wherein the protein variant produces an organosilicon product with a % ee of at least about 99%.

Assignees

• California Inst Of Techn

Inventors

• Kan Sek Bik Jennifer
• Lewis Russell D
• Chen Kai
• Arnold Frances H

Classifications

• C07F7/0896

  Compounds with a Si-H linkage · CPC title

• C07F7/0818

  Chemistry & Metallurgy · mapped topic

• C12N9/0042 Primary

  NADPH-cytochrome P450 reductase (1.6.2.4) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y114/14001

  Unspecific monooxygenase (1.14.14.1) · CPC title