Evolution of bond-forming enzymes

Patent metadata

Field	Value
Publication number	US-9267127-B2
Application number	US-201313922812-A
Country	US
Kind code	B2
Filing date	Jun 20, 2013
Priority date	Jun 21, 2012
Publication date	Feb 23, 2016
Grant date	Feb 23, 2016

Abstract

Strategies, systems, methods, reagents, and kits for the directed evolution of bond-forming enzymes are provided herein. Evolution products, for example, evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are also provided herein, as are methods for using such evolved bond-forming enzymes. Kits comprising materials, reagents, and cells for carrying out the directed evolution methods described herein are also provided.

First claim

What is claimed is:

1. A sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of Staphylococcus aureus ( S. aureus ) Sortase A as provided as SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO: 21, wherein the amino acid sequence of the sortase comprises two or more mutations selected from the group consisting of P94S, P94R, E106G, F122Y, K154R, D160N, D165A, G174S, K190E, and K196T.

2. The sortase of claim 1 , wherein the sortase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO: 21.

3. The sortase of claim 1 , wherein the amino acid sequence of the sortase comprises at least three mutations selected from the group consisting of P94S, P94R, E106G, F122Y, K154R, D160N, D165A, G174S, K190E, and K196T.

4. The sortase of claim 1 , wherein the sortase comprises a P94S or P94R mutation, a D160N mutation, a D165A mutation, a K190E mutation, and a K196T mutation.

5. The sortase of claim 1 , wherein the sortase comprises a P94S or P94R mutation, a D160N mutation, and a K196T mutation.

6. The sortase of claim 1 , wherein the sortase comprises a P94S or P94R mutation, a D160N mutation, and a D165A mutation.

7. The sortase of claim 1 , wherein the sortase comprises a P94S or P94R mutation, a D160N mutation, a D165A mutation, and a K196T mutation.

8. The sortase of claim 1 , wherein the sortase exhibits a k cat that is at least 1.5-fold greater than the k cat of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

9. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is at least 2-fold-less than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

10. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence GGG that is not more than 2-fold greater than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

11. The sortase of claim 1 , wherein the sortase exhibits a ratio of K cat /K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is least 2-fold greater than the K cat /K M ratio of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

12. A sortase catalyzing transpeptidation of substrates other than LPETG (SEQ ID NO: 32), the sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO: 21,
wherein the amino acid sequence comprises two or more mutations selected from the group consisting of P86L, P94R, N98S, A104T, A118T, F122S, D124G, N127S, K134R, D160N, D165A K173E, K177E, K190E, and K196T.

13. The sortase of claim 12 , wherein the substrate comprises the amino acid sequence LPXS, LAXT, MPXT, LAXS, NPXT, NAXT, NAXS, LPXP, or LPXTA (SEQ ID NO: 40), wherein each occurrence of X represents independently any amino acid residue.

14. The sortase of claim 12 , wherein the substrate comprises the amino acid sequence LPESG (SEQ ID NO: 38).

15. A method for transpeptidation, the method comprising
contacting the sortase of claim 1 with a substrate comprising an LPETG (SEQ ID NO: 32) sequence and with a substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation.

16. The method of claim 15 , wherein the LPETG (SEQ ID NO: 32) substrate and/or the GGG substrate are on the surface of a cell.

17. The method of claim 16 , wherein the cell expresses a surface marker protein that is C-terminally fused to an LPETG (SEQ ID NO: 32) sequence.

18. The method of claim 16 , wherein the cell expresses a surface marker protein that is N-terminally fused to a GGG sequence.

19. The method of claim 15 , wherein the LPETG (SEQ ID NO: 32) substrate and/or the GGG substrate are polypeptides or proteins, and wherein the method results in the generation of a protein fusion.

20. The method of claim 15 , wherein the LPETG (SEQ ID NO: 32) substrate or the GGG substrate comprises a non-protein structure.

21. The sortase of claim 1 , wherein the sortase comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO: 21.

22. The sortase of claim 1 , wherein the sortase comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 1 or to amino acid residues 1-150 of SEQ ID NO: 21.

23. The sortase of claim 1 , wherein the amino acid sequence of the sortase comprises at least four mutations selected from the group consisting of P94S, P94R, E106G, F122Y, F154R, D160N, D165A, G174S, K190E, and K196T.

24. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is at least 5-fold less than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

25. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is at least 10-fold less than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or of the Sortase A having the amino acid sequence provided by amino acid residues 1-150 of SEQ ID NO: 21.

26. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence GGG that is not more than 5-fold greater than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

27. The sortase of claim 1 , wherein the sortase exhibits a K M for a substrate comprising the amino acid sequence GGG that is not more than 10-fold greater than the K M of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

28. The sortase of claim 1 , wherein the sortase exhibits a ratio of K cat /K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is least 5-fold greater than the K cat /K M ratio of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

29. The sortase of claim 1 , wherein the sortase exhibits a ratio of K cat /K M for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is least 10-fold greater than the K cat /K M ratio of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

30. The sortase of claim 1 , wherein the sortase exhibits a ratio of K cat /K m for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 32) that is least 50-fold greater than the K cat /K M ratio of the Sortase A having the amino acid sequence provided as SEQ ID NO: 1 or amino acid residues 1-150 of SEQ ID NO: 21.

31. The sortase of claim 1 , wherein the sortase has an amino acid sequence provided by a sequence selected from the group consisting of SEQ ID NO

Assignees

• Harvard College

Inventors

• Liu David R
• Chen Irwin
• Dorr Brent M

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/6472

  Cysteine endopeptidases (3.4.22) · CPC title

• C12N15/1058 Primary

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title