PROFICS: A bacterial selection system for directed evolution of proteases

Abstract

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner. Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner. Proteases are important enzymes in various industry sectors and are used for a wide range of applications. Wild-type proteases have evolved to cut a specific group of proteins, while for therapeutic, diagnostic, and biotechnological applications, a more diverse substrate specificity is needed (1Kalwasińska A. Jankiewicz U. Felföldi T. Burkowska-But A. Brzezinska M.S. Alkaline and halophilic protease production by Bacillus luteus H11 and its potential industrial applications.Food Technol. Biotechnol. 2018; 56: 553-561Crossref PubMed Scopus (13) Google Scholar). They often lack the necessary stability (2You L. Arnold F.H. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide.Protein Eng. 1996; 9: 77-83Crossref PubMed Scopus (220) Google Scholar, 3Zhao H. Arnold F.H. Directed evolution converts subtilisin E into a functional equivalent of thermitase.Protein Eng. Des. Sel. 1999; 12: 47-53Crossref Scopus (262) Google Scholar), efficiency (4Sanchez M.I. Ting A.Y. Directed evolution improves the catalytic efficiency of TEV protease.Nat. Methods. 2020; 17: 167-174Crossref PubMed Scopus (31) Google Scholar), specificity (5Packer M.S. Rees H.A. Liu D.R. Phage-assisted continuous evolution of proteases with altered substrate specificity.Nat. Commun. 2017; 8: 956Crossref PubMed Scopus (51) Google Scholar), or are not functional under the desired reaction conditions. Other industrially important characteristics of proteases are the incidence of their recognition site and their ability to create an authentic N-terminus (for a review see (6Tavano O.L. Berenguer-Murcia A. Secundo F. Fernandez-Lafuente R. Biotechnological applications of proteases in food technology.Compr. Rev. Food Sci. Food Saf. 2018; 17: 412-436Crossref PubMed Scopus (106) Google Scholar)). A toolbox with a set of similar proteases fulfilling these criteria and optimized for specific conditions and substrates would allow efficient processing of a wide range of substrates. In order to turn wild-type proteases into optimized, therapeutically, diagnostically, and industrially applicable enzymes, we developed a versatile selection system, which can be applied to many different proteases and demonstrated its functionality with two viral proteases (pestiviral N-terminal autoprotease (Npro) (7Zogg T. Sponring M. Schindler S. Koll M. Schneider R. Brandstetter H. Auer B. Crystal structures of the viral protease Npro imply distinct roles for the catalytic water in catalysis.Structure. 2013; 21: 929-938Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) main protease (Mpro) (8Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. B. X. L. Y. L. Liu F. of from and of its 2020; PubMed Scopus Google and a human that was optimized for tag cleavage M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar). The two viral proteases the of their specific to Npro is to N-terminal of all residues R. F. H. M. F. A. Auer B. to with authentic in Methods. PubMed Scopus Google Scholar), a specificity for a recognition site S. The main protease as drug 2020; PubMed Scopus Google Scholar). for the selection with the autoprotease Npro is a enzyme (7Zogg T. Sponring M. Schindler S. Koll M. Schneider R. Brandstetter H. Auer B. Crystal structures of the viral protease Npro imply distinct roles for the catalytic water in catalysis.Structure. 2013; 21: 929-938Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) that S. activity and substrate specificity of the N-terminal protease PubMed Scopus Google Scholar) and is used in industrial R. F. H. M. F. A. Auer B. to with authentic in Methods. PubMed Scopus Google Scholar, B. Schneider R. T. Sponring M. Koll M. Schindler S. T. Brandstetter H. Npro autoprotease a for industrial production in Biotechnol. Google Scholar, S. B. Sponring M. M. Auer B. Schneider R. A. Npro to efficiency in PubMed Scopus Google Scholar). In we used the main protease of the which is one of the targets for drug (8Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. B. X. L. Y. L. Liu F. of from and of its 2020; PubMed Scopus Google Scholar, S. The main protease as drug 2020; PubMed Scopus Google Scholar, B. the of the main protease of A potential therapeutic Sci. 2020; PubMed Scopus Google Scholar, L. X. U. L. S. R. Crystal of main protease a for of improved 2020; PubMed Scopus Google Scholar). can be enzymes for the cleavage of enzymes in and have a specificity and efficiency their substrates. have for tag cleavage to characteristics B. protein an N-terminal protein and a PubMed Scopus Google Scholar, M.S. S. with cleavage site for of tag PubMed Google Scholar). is are as and have to cleavage and of to functional The protein structures that activity, and PubMed Scopus Google Scholar), which their production and their biotechnological of the cleavage site (the site A. the of the site in Commun. PubMed Scopus Google and are to create native the site residues their activity M. M. substrates the preferences of human and PubMed Scopus Google Scholar). The is in caused by the after the cleavage site T. B. A to and substrate PubMed Scopus Google Scholar). caspase-2 is an for cleavage industrial to its recognition site the N-terminal to the site of Y. and enzymatic into caspase-2 protein substrate recognition and Full Text Full Text PDF PubMed Scopus Google Scholar). We have established a caspase-2 which not only a specificity can be and in in Escherichia thus a main in the industrial of M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar). The the activity with the the site residue a great cleavage we that these of caspase-2 can be with PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a for enables the optimization of this and other proteases by mutation and the selection for improved in of activity and P1′ tolerance or with altered Directed evolution is the of mutation and or selection for improved properties (for a review see R. Zhao H. Directed and 2013; PubMed Scopus Google Scholar)). the are used as for a new round of mutation and or selection. can be the desired are can be in the or by R. Zhao H. Directed and 2013; PubMed Scopus Google Scholar). mutation can create for only in a protein are to all their a of all can be that a in in is the efficiency of the which the of the The of can be by which to the protein for characteristics and to mutations and for the evolution of 21: PubMed Scopus Google Scholar). B. A. Schneider R. the of a of the protease by 2020; PubMed Scopus Google Scholar) have used an in to optimize caspase-2. that the P1′ tolerance by of different and in in The of evolution is improved the of that an or a selection with a specific is In variant of the be with properties are and functional of a protein the of a by of the and and of the a different site. The order of the and are in the of the protein Y. S. A different to enzyme and Biotechnol. Full Text Full Text PDF PubMed Scopus Google Scholar). that is not needed for and protein activity U. M. of for protein and PubMed Scopus Google Scholar) and that properties are not by In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google Scholar). is an essential coli enzyme and the in the of the pyrimidine the of which is In the coli all are the the B. of Escherichia coli of the catalytic and and of the PubMed Scopus Google Scholar). a of and catalytic and is The of and two catalytic The are the of the catalytic and of Escherichia coli PubMed Scopus Google Scholar). the catalytic and into an with the properties as the native enzyme for the and catalytic activity of PubMed Scopus Google Scholar). that catalytic of by of residues and of the wild-type and of new can and in with containing catalytic Sci. U. S. A. PubMed Scopus Google Scholar). The the of the its ability to catalytic In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google Scholar, R. of and of the to the catalytic of Sci. U. S. A. 1996; PubMed Scopus Google Scholar). by and to a protease selection as an essential The of the protease selection PROFICS is the that the new of a enzyme are in a with of to We the established by and In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google Scholar) the interest of from as reporter enzyme with the desired to a protease selection The new N-terminus with residue A and of in the wild-type and enzymes this is with wild-type The new and of its are in the a of the protein In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google Scholar) with we that the of or to of the would the of enzyme and its catalytic of the sequence selection system, we thus pyrimidine cells containing by a or the sequence is the into the cells can in with a protease to the of by specific The protease activity, and P1′ tolerance the of the and simple allow only proteases with the desired to the In an overnight process, the are selected from the of with is and enables and new coli knockout strain coli was by of the and its with a The strain is for and can only in containing or the cells are with an expression the for catalytic and the of the for the selection of different the of the catalytic of was by the of an or an variant of the viral autoprotease Npro was of a and of the The cleavage by Npro S. activity and substrate specificity of the N-terminal protease PubMed Scopus Google Scholar) the of containing the or the protease protein in of cells autoprotease not the of cleavage was by a in Npro from the protein R. T. of cleavage site autoprotease and protein of PubMed Google Scholar), which the to after of the that can be by the of Npro to its we to the of the selection from with cleavage activity to with We a of caspase-2 caspase-2 M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google as its selection a by a and a recognition site to the N-terminus of We that the tag of would and cells the enzyme would only be to with a a protease to the tag and the of A of PROFICS by of the two is in and that the cells and and They are for and in their of and The functionality of PROFICS for was in coli cells by different with the caspase-2 that with the caspase-2 recognition site with the caspase-2 the an altered recognition site which is not and by and the cells to of used for with into coli cells and ability to pyrimidine of to distinct to to distinct to not to to to distinct to not to to not to to to distinct to not to to in a new of a can create many The of a selection many a can be with and of caspase-2 in and as into coli cells containing a with a distinct showed that all the while cells containing the to The selection efficiency of was by the efficiency of the which was of was used for total reaction as with have a and while a efficiency of coli by PubMed Scopus Google Scholar). The of in the and the efficiency are as the to optimized great was in the of the and the optimization of the in the of caspase-2 generated by and The applied conditions caused in The efficiency is by the of the by of with cells containing the substrate of the into these up to the of for selection in a with or which the efficiency with in coli cells containing with a of caspase-2 and in with an of The with to mutations was with the and the The after to and to was in and was found in the in two one mutation and is to a caspase-2 sequence in the the variant was showed that the cleavage activity of the caspase-2 variant was significantly improved with caspase-2 M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar). The activity is to an of the the reaction the of the new variant for the substrate with the in the P1′ was 25-fold which is the all substrates. was used as P1′ residue the selection. The tolerance for all other P1′ residues was In to the found for the P1′ residues with for substrates with and in the P1′ to with the variant after one round of the tolerance of variant which the for all P1′ substrates. and to the of the mutation the altered the P1′ substrates. The was from the of A. Crystal of of the Full Text Full Text PDF PubMed Scopus Google Scholar), and from the of with and the substrate was in the with a to the catalytic the are of the catalytic of the and a to the in is with its with protein as and as in The caspase-2 with the site of the of the cleavage site the with the site N-terminal of the cleavage site The important and with all residues of residue in a its two and with the of of the and and with the group of a of The of is to the The for residue the for are for the site can of the and cleavage of the by to the site. the P1′ which is in to the catalytic a residues in the P1′ site the and the site of while the The showed that residue caspase-2 an important in the of the is the as in The from to in caspase-2 with the of the The residue from and as in which the in the The of with as from a is in In the the with its in to the not as a The mutation to a of the which is in The the and the of the The is more as the is the site is more which of the with the this the enzyme to be more the P1′ residue the catalytic the tolerance of a protease for a specific P1′ a selection in which this residue can be is to the as P1′ residue of all in of the The was not only to which mutations to the of coli into was that the of is to in the protein In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google Scholar), that of this residue be necessary for its that of the of was a for its activity can be found in the The in this are with the N-terminal and only its by of this was after cleavage by caspase-2 for P1′ tolerance the of the other be by and and of the the N-terminus with after of the are as for only a and to be with the of and can not be used for a selection. In to the autoprotease Npro and we PROFICS with the main protease recognition site (8Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. B. X. L. Y. L. Liu F. of from and of its 2020; PubMed Scopus Google Scholar) and a tag to the N-terminus of the to the The main protease of was is a for drug (8Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. B. X. L. Y. L. Liu F. of from and of its 2020; PubMed Scopus Google Scholar), and we to that PROFICS can be used as a to drug-resistance-conferring that the and specifically the activity of bacterial in the selection selection of a protease in the of the can mutations that as only cells with a and drug protease can and the selection. A is that the can only be used for that are for the and under conditions in the as as in the with the and with a mutation used as was protease as of used for into coli cells with and ability to pyrimidine of to distinct to to distinct to to distinct to not to to not to distinct to to distinct to not to to not in a new was the cells only cells one of the and cells and under conditions and after to PROFICS is a bacterial selection for which the of an essential enzyme by tag cleavage to The deficient coli cells containing a by N-terminal of an autoprotease or a tag a protease recognition site. The is specific cleavage by a protease its The of the cells is the activity and specificity of the protease and the selection of with altered The selection of the in selection that only with off-target activity are A protease with specificity would coli and or have to be The of the protease not have or the the other the protease to be and under the conditions selection in is important that the recognition site of the protease is not by enzymes, as activity be efficient expression of the Proteases that these criteria can be or in from in a simple overnight process. is a simple which not of or In improved can outgrow as in a was the selection the that be can this The and of the as as the efficiency of the selection are for the of a bacterial selection They many can be and the to an improved We generated a with cells that one expression to The and with selection create a for the of and the selection of with an and a PROFICS enables the evolution of proteases by the of mutation and selection. The efficiency and the of coli allow of the characteristics of a protease in a the N-terminus of we the for evolution of a protease and used to the of a caspase-2 for an industrial production M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar). A to be applicable to of the N-terminus of the protein of selection toolbox protease substrates with all P1′ all have and the or of The of the enzyme are a and have that is as the and are the the The is by a by residues not into this and by the of the P1′ with one of the are mutation and selection can be applied to with residues their to that these We found that processing of the by coli to be necessary for A the N-terminus of the catalytic of its activity, enables the of the for the selection of optimized such as or be selected for altered P1′ tolerance or the a with residues its N-terminus be The selection of PROFICS can be by the P1′ residue of the the preferences of a The of PROFICS was demonstrated by the selection of an efficient one round of mutation and we a in enzymatic activity and the P1′ residue used in the selection. the variant an other in the and of the an unbiased mutation or to and for bacterial selection a variant with a that was found to improved catalytic properties with substrate and P1′ tolerance to 25-fold In that the of the mutation from a substrate recognition and more of the The mutation a residue to a The two have different properties and would not be selected in the protein all are S. B. Schneider R. Efficient in of a of the protease PubMed Scopus Google Scholar). The of such which are in to have is to only mutation is to in the as for the to mutation in the L. Rev. 2018; PubMed Scopus Google Scholar). are to or residues such as in as these residues have more similar properties to the properties and of M.S. for Scopus Google Scholar). The two caspase-2 from in B. A. Schneider R. the of a of the protease by 2020; PubMed Scopus Google Scholar) showed cleavage for substrates with and P1′ the mutation found with PROFICS is in a different of the the P1′ tolerance of all is the is in the variant found with be to the mutations found with the different methods and to their are A of in and improved The of PROFICS was with different with is applicable for the selection of various proteases with and cleavage was demonstrated by a variant with significantly activity and production optimization for by of expression or even usage, is by selection criteria that of for by P1′ residues such as in the reporter of and and of all and can be found in the from and the was was by coli cells used for of all the selection system, an strain was generated coli The was in coli cells the coli and to a coli cells containing a for with protease cleavage site to efficiency of coli by PubMed Scopus Google Scholar, of Escherichia coli with PubMed Scopus Google Scholar). The sequence of coli was by from and The was into the site of the bacterial expression with and A cleavage site in the protein was by to of the two the of wild-type sequence as by In of containing catalytic for protein and Sci. 1996; PubMed Scopus Google was and with and into the of the containing the The sequence of the N-terminal protease of the of was N-terminal of the of the A by a for and a caspase-2 recognition site the N-terminus of the by A was with the recognition site the recognition site was to and to with and The caspase-2 M. A. F. M. Schneider R. A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar, B. A. Schneider R. the of a of the protease by 2020; PubMed Scopus Google Scholar) was into the of a with and The of main protease of was optimized for coli with the and into the of a with and The was from and by Scopus Google Scholar). 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A. of caspase-2 for expression in Escherichia and 2020; Scopus Google Scholar). In A substrate with the sequence was used is the is the caspase-2 recognition is the P1′ site all A is as is and is as the The was in with substrate of and and a enzyme of caspase-2. the of the was and was used to the to the the M. and of the for Commun. Scopus Google Scholar). A was the catalytic and the substrate the of the catalytic process. In of this that the of mutations be and the set interaction for in the 8: PubMed Scopus Google Scholar), water was and by of the simple H. for water in to protein B. The and Google Scholar). conditions of the The potential and 9: Scholar), the of the of of a with of Scopus Google Scholar) was applied for for of a range range a reaction R. 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