Evolution of bond-forming enzymes

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.

What is claimed is: 
     
         1 . A method for directed evolution of bond-forming enzymes, the method comprising
 (a) providing a cell population in which
 (i) a first cell surface protein or a cell in the cell population is conjugated to a candidate bond-forming enzyme, wherein different cells within the population of cells comprise different candidate bond-forming enzymes conjugated to the cell surface protein; 
 (ii) a second cell surface protein is conjugated to a substrate A; 
   (b) contacting the cell population with a substrate B conjugated to a detectable label under conditions suitable for the bond-forming enzyme to form a bond between substrate A and substrate B; and   (c) identifying and/or isolating a cell that is conjugated to substrate B.   
     
     
         2 . A method for directed evolution of bond-forming enzymes, the method comprising
 (a) providing a yeast cell population in which
 (i) a library of candidate bond-forming enzymes is expressed as a fusion to an Aga2p cell surface mating factor, wherein different cells within the cell population express different candidate bond-forming enzymes; 
 (ii) an Aga1p cell surface mating factor is covalently bound to the Aga2p cell surface mating factor, wherein the Aga2p cell surface mating factor is conjugated to a substrate A; 
   (b) contacting the cell population with a substrate B conjugated to a detectable label under conditions suitable for the bond-forming enzyme to form a bond between substrate A and substrate B; and   (c) identifying and/or isolating a cell that is conjugated to substrate B.   
     
     
         3 . The method of  claim 1 , further comprising
 (d) identifying and/or isolating the bond-forming enzyme(s) expressed in the cells isolated in (c).   
     
     
         4 . The method of  2 , further comprising
 (d) identifying and/or isolating the bond-forming enzyme(s) expressed in the cells isolated in (c).   
     
     
         5 . The method of  claim 1 , further comprising
 (e) subjecting the bond-forming enzyme(s) expressed in the cells isolated in (c) to a diversification procedure, thus creating a diversified library of candidate bond-forming enzymes, expressing the diversified candidate bond-forming enzyme library as a fusion to an Aga2p cell surface mating factor in a population of yeast cells, and repeating steps (a)-(c).   
     
     
         6 . The method of  claim 5 , wherein the diversification procedure comprises random mutagenesis and/or recombination. 
     
     
         7 . The method of  claim 1 , wherein substrate A is conjugated to the cell surface protein or the Agap2 cell surface mating factor via a reactive handle. 
     
     
         8 . The method of  claim 1 , wherein the candidate enzyme is fused to the cell surface protein or the Agap1 cell surface mating factor via a cleavable linker. 
     
     
         9 . The method of  claim 8 , wherein the cleavable linker comprises a protease cleavage site. 
     
     
         10 . The method of  claim 3 , wherein the method comprises multiple rounds of performing steps (a)-(d) and a final round of performing steps (a)-(c). 
     
     
         11 . The method of  claim 10 , wherein the method comprises decreasing the concentration of substrate B in subsequent rounds of performing steps (a)-(d) or steps (a)-(c). 
     
     
         12 . The method of  claim 3 , wherein the method comprises using a modified substrate A or a modified substrate B in subsequent rounds of performing steps (a)-(d) or steps (a)-(c). 
     
     
         13 . The method of  claim 1 , wherein the method further comprises comparing the bond-forming properties of at least one enzyme identified or isolated in (c) with the corresponding wild type enzyme, wherein if the enzyme isolated in (c) exhibits an improved bond-forming characteristic, it is identified as an enhanced, evolved bond-forming enzyme. 
     
     
         14 . The method of  claim 1 , wherein the bond-forming enzymes are transpeptidases. 
     
     
         15 . The method of  claim 14 , wherein the transpeptidases are sortases. 
     
     
         16 . The method of  claim 1 , wherein the bond-forming enzymes are ligases, polymerases, kinases, transferases, aldolases, diels alderases, and transferases.