title: A flow cytometry-based screening system for directed evolution of proteases.
abstract: Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance.
authors.affiliation: School of Engineering and Science, Jacobs University Bremen (JUB), Bremen, Germany.
authors.forename: Ran
authors.initials: R
authors.lastname: Tu
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanovic
authors.forename: Mathias
authors.initials: M
authors.lastname: Klein
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology; D019020:Directed Molecular Evolution / Q000379:methods*; D004655:Emulsions / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000592:standards; D008958:Models, Molecular; D009154:Mutation; D009821:Oils / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004655:Emulsions; D004791:Enzyme Inhibitors; D009821:Oils; D010447:Peptide Hydrolases; D013381:Subtilisins