Automated screening of enzyme variants

Abstract

Disclosed are methods for identifying bio-molecules with desired properties (or which are most suitable for a round of directed evolution) from complex bio-molecule libraries or sets of such libraries. Some embodiments of the present disclosure provide methods for virtually screening proteins for beneficial properties. Some embodiments of the present disclosure provide methods for virtually screening enzymes for desired activity and/or selectivity for catalytic reactions involving particular substrates. Some embodiments combine screening and directed evolution to design and develop proteins and enzymes having desired properties. Systems and computer program products implementing the methods are also provided.

1 . A method, implemented using a computer system that includes one or more processors and system memory, for screening a plurality of different enzyme variants for activity with a substrate, the method comprising:
 (a) for each enzyme variant, docking, by the computer system, a computational representation of the substrate to a computational representation of an active site of the enzyme variant, wherein docking (i) generates a plurality of poses of the substrate in the active site, and (ii) identifies energetically favorable poses of the substrate in the active site;   (b) for each energetically favorable pose, determining whether the pose is active, wherein an active pose meets one or more constraints for the substrate to undergo catalysis in the active site; and   (c) selecting at least one of the enzyme variants determined to have one or more active poses.   
     
     
         2 . The method of  claim 1 , further comprising: screening the at least one enzyme variant selected in (c) against the substrate by producing a chemical reaction. 
     
     
         3 . The method of  claim 1 , wherein the computational representation of the substrate represents a species along the reaction coordinate for the enzyme activity, the species being selected from the substrate, a reaction intermediate of the substrate, or a transition state of the substrate. 
     
     
         4 . The method of  claim 1 , wherein the plurality of enzyme variants comprise a panel of enzymes that can turn over multiple substrates and wherein the members of the panel possess at least one mutation relative to a reference sequence. 
     
     
         5 . The method of  claim 4 , wherein the at least one mutation is a single-residue mutation in the active site of the enzyme. 
     
     
         6 . The method of  claim 1 , wherein the plurality of variants comprise one or more enzymes that can catalyze a chemical reaction selected from oxidoreduction, transferation, hydrolysis, isomerization, ligation, and chemical bond breaking by a reaction other than hydrolysis, oxidation, or reduction. 
     
     
         7 . The method of  claim 6 , wherein the enzyme is selected from oxidoreductase, transferase, hydrolase, isomerase, ligase, and lyase. 
     
     
         8 . The method of  claim 6 , wherein the plurality of variants comprise one or more enzymes that can catalyze a chemical reaction selected from ketone reduction, transamination, oxidation, nitrile hydrolysis, imine reduction, enone reduction, acyl hydrolysis, and halohydrin dehalogenation. 
     
     
         9 . The method of  claim 8 , wherein the enzyme is selected from ketone reductase, transaminase, cytochrome P450, Baeyer-Villiger monooxygenase, monoamine oxidase, nitrilase, imine reductase, enone reductase, acylase, and halohydrin dehalogenase. 
     
     
         10 . The method of  claim 1 , wherein the plurality of variants comprises members of library produced by one or more rounds of directed evolution in vitro and/or in silico. 
     
     
         11 . The method of  claim 1 , wherein the plurality of variants comprises at least about ten different variants. 
     
     
         12 . The method of  claim 1 , wherein the plurality of variants comprises at least about a thousand different variants. 
     
     
         13 . The method of  claim 1 , wherein the computational representations of active sites are provided from 3-D homology models for the plurality of variants. 
     
     
         14 . The method of  claim 13 , further comprising producing said 3-D homology models for the plurality of variants. 
     
     
         15 . The method of  claim 1 , wherein the computational representation of the substrate is a 3-D model of the substrate. 
     
     
         16 . The method of  claim 1 , wherein the method is applied to screen a plurality of substrates. 
     
     
         17 . The method of  claim 1 , further comprising identifying the constraints for the substrate to undergo the catalyzed chemical transformation by identifying one or more poses of a native substrate, a reaction intermediate of the native substrate, or a transition state of the native substrate when the native substrate undergoes the catalyzed chemical transformation by a wild-type enzyme. 
     
     
         18 . The method of  claim 1 , wherein the constraints comprise one or more of the following: position, distance, angle, and torsion constraints. 
     
     
         19 - 36 . (canceled) 
     
     
         37 . A system, comprising:
 one or more processors;   system memory; and   wherein the one or more processors and memory are configured to implement a method for virtually screening enzyme variants for activity with a substrate, the method comprising:
 (a) for each enzyme variant, docking a computational representation of the substrate to a computational representation of an active site of the enzyme variant, wherein docking (i) generates a plurality of poses of the substrate in the active site, and (ii) identifies energetically favorable poses of the substrate in the active site; 
 (b) for each energetically favorable pose, determining whether the pose is active, wherein an active pose meets one or more constraints for the substrate to undergo catalysis in the active site; and 
 (c) selecting at least one of the enzyme variants determined to have one or more active poses. 
   
     
     
         38 . (canceled) 
     
     
         39 . A method, implemented using a computer system that includes one or more processors and system memory, for screening a plurality of protein variants for interaction with a ligand, the method comprising:
 (a) for each protein variant, docking, by the computer system, a computational representation of the ligand to a computational representation of an active site of the protein variant, wherein docking (i) generates a plurality of poses of the ligand in the active site, and (ii) identifies energetically favorable poses of the ligand in the active site;   (b) for each energetically favorable pose, determining whether the pose is active, wherein an active pose meets one or more constraints for the ligand to undergo a particular interaction with protein variant; and   (c) selecting at least one of the protein variants determined to have one or more active poses.   
     
     
         40 . (canceled)