Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries.

How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.

[1]  Ryota Fujii,et al.  RAISE: a simple and novel method of generating random insertion and deletion mutations , 2006, Nucleic acids research.

[2]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[3]  Frances H. Arnold,et al.  Molecular evolution by staggered extension process (StEP) in vitro recombination , 1998, Nature Biotechnology.

[4]  Andreas Vogel,et al.  Expanding the substrate scope of enzymes: combining mutations obtained by CASTing. , 2006, Chemistry.

[5]  C. Maranas,et al.  IPRO: an iterative computational protein library redesign and optimization procedure. , 2006, Biophysical journal.

[6]  K. Gruber,et al.  Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution. , 2007, Journal of biotechnology.

[7]  L. Young,et al.  TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening. , 2003, Nucleic acids research.

[8]  N. Ben-Tal,et al.  The ConSurf‐HSSP database: The mapping of evolutionary conservation among homologs onto PDB structures , 2004, Proteins.

[9]  Cameron Neylon,et al.  Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. , 2004, Nucleic acids research.

[10]  V. Heinrichs,et al.  Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies , 2004, Expert opinion on biological therapy.

[11]  Tal Pupko,et al.  Structural Genomics , 2005 .

[12]  Itay Mayrose,et al.  Rate4Site: an algorithmic tool for the identification of functional regions in proteins by surface mapping of evolutionary determinants within their homologues , 2002, ISMB.

[13]  T. S. Wong,et al.  Sequence saturation mutagenesis with tunable mutation frequencies. , 2005, Analytical biochemistry.

[14]  B. Robinson,et al.  Approaches to DNA mutagenesis: an overview. , 1997, Analytical biochemistry.

[15]  B. Dahiyat,et al.  Combining computational and experimental screening for rapid optimization of protein properties , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Tal Pupko,et al.  In silico identification of functional regions in proteins , 2005, ISMB.

[17]  P. Højrup,et al.  Characterization of the Oligomer Structure of Recombinant Human Mannan-binding Lectin* , 2005, Journal of Biological Chemistry.

[18]  L. Otten,et al.  Directed evolution: selecting today's biocatalysts. , 2005, Biomolecular engineering.

[19]  Costas D Maranas,et al.  Identifying residue–residue clashes in protein hybrids by using a second-order mean-field approach , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Andreas Vogel,et al.  Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. , 2006, Angewandte Chemie.

[21]  R. Duggleby,et al.  A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step , 2004, BMC biotechnology.

[22]  M. Reetz,et al.  Infrared-thermographic screening of the activity and enantioselectivity of enzymes , 2001, Applied Microbiology and Biotechnology.

[23]  Leighton Pritchard,et al.  A general model of error-prone PCR. , 2005, Journal of theoretical biology.

[24]  Valérie Taly,et al.  A combinatorial approach to substrate discrimination in the P450 CYP1A subfamily. , 2007, Biochimica et biophysica acta.

[25]  Tal Pupko,et al.  A branch-and-bound algorithm for the inference of ancestral amino-acid sequences when the replacement rate varies among sites: Application to the evolution of five gene families , 2002, Bioinform..

[26]  J. Chiu,et al.  Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. , 2004, Nucleic acids research.

[27]  Andreas Seyfang,et al.  Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round. , 2004, Analytical biochemistry.

[28]  T. S. Wong,et al.  Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM‐3 heme domain , 2007, Biotechnology journal.

[29]  Wayne M Patrick,et al.  User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. , 2003, Protein engineering.

[30]  Narendra Maheshri,et al.  Computational and experimental analysis of DNA shuffling , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  U. Baumann,et al.  An efficient one-step site-directed and site-saturation mutagenesis protocol. , 2004, Nucleic acids research.

[32]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Deem,et al.  A hierarchical approach to protein molecular evolution. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Kauffman,et al.  Search strategies for applied molecular evolution. , 1995, Journal of theoretical biology.

[35]  T. Vernet,et al.  Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes. , 2006, Analytical biochemistry.

[36]  Costas D Maranas,et al.  eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments. , 2002, Nucleic acids research.

[37]  C D Maranas,et al.  Creating multiple-crossover DNA libraries independent of sequence identity , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Martin,et al.  Altering protein specificity: techniques and applications. , 2005, Bioorganic & medicinal chemistry.

[39]  Andrew E. Firth,et al.  Statistics of protein library construction , 2005, Bioinform..

[40]  S. L. Mayo,et al.  Protein design automation , 1996, Protein science : a publication of the Protein Society.

[41]  J. Salerno,et al.  INSULT: a novel mutagenesis method generating high yields of closed circular mutant DNA with one primer per mutant. , 2005, Molecular biotechnology.

[42]  Martin Zacharias,et al.  A statistical analysis of random mutagenesis methods used for directed protein evolution. , 2006, Journal of molecular biology.

[43]  Peter T. Lansbury,et al.  A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries , 2005, Nucleic acids research.

[44]  Stephen J Benkovic,et al.  FamClash: A method for ranking the activity of engineered enzymes , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Delcourt,et al.  High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips. , 2005, BioTechniques.

[46]  W. Wang,et al.  Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. , 1999, BioTechniques.

[47]  C D Maranas,et al.  Modeling DNA mutation and recombination for directed evolution experiments. , 2000, Journal of theoretical biology.

[48]  Motowo Nakajima,et al.  Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution , 2006, Applied Microbiology and Biotechnology.

[49]  Andreas Vogel,et al.  Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. , 2005, Angewandte Chemie.

[50]  Claes Gustafsson,et al.  Optimizing the search algorithm for protein engineering by directed evolution. , 2003, Protein engineering.

[51]  Motowo Nakajima,et al.  Directed evolution by accumulating tailored mutations: thermostabilization of lactate oxidase with less trade-off with catalytic activity. , 2006, Protein engineering, design & selection : PEDS.

[52]  Christopher A. Voigt,et al.  Protein building blocks preserved by recombination , 2002, Nature Structural Biology.

[53]  J. Punnonen,et al.  DNA shuffling and screening strategies for improving vaccine efficacy. , 2005, DNA and cell biology.

[54]  Costas D Maranas,et al.  Using multiple sequence correlation analysis to characterize functionally important protein regions. , 2003, Protein engineering.

[55]  Jeffrey B. Endelman,et al.  Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450 , 2006, PLoS biology.

[56]  Frances H Arnold,et al.  To whom correspondence should be addressed. , 2022 .

[57]  Piero Fariselli,et al.  ConSeq: the identification of functionally and structurally important residues in protein sequences , 2004, Bioinform..

[58]  W. P. Russ,et al.  Evolutionary information for specifying a protein fold , 2005, Nature.

[59]  J. Sayers,et al.  Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach. , 1992, BioTechniques.

[60]  Cheng Zhao,et al.  Estimation of the Mutation Rate During Error-prone Polymerase Chain Reaction , 2000, J. Comput. Biol..

[61]  Costas D Maranas,et al.  Predicting out-of-sequence reassembly in DNA shuffling. , 2002, Journal of theoretical biology.

[62]  Hiroshi Murakami,et al.  Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs , 2002, Nature Biotechnology.

[63]  P. Acharya,et al.  Multiplex‐PCR‐Based Recombination as a Novel High‐Fidelity Method for Directed Evolution , 2005, Chembiochem : a European journal of chemical biology.

[64]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[65]  Ling Yuan,et al.  Laboratory-Directed Protein Evolution , 2005, Microbiology and Molecular Biology Reviews.

[66]  W. Patrick,et al.  Natural history as a predictor of protein evolvability. , 2006, Protein engineering, design & selection : PEDS.

[67]  J. Punnonen,et al.  Development of novel vaccines using DNA shuffling and screening strategies. , 2004, Current opinion in molecular therapeutics.

[68]  Frances H Arnold,et al.  General method for sequence-independent site-directed chimeragenesis. , 2003, Journal of molecular biology.

[69]  Hideo Nakano,et al.  Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression. , 2003, Journal of molecular biology.

[70]  J. Wells,et al.  Additivity of mutational effects in proteins. , 1990, Biochemistry.

[71]  Frances H. Arnold,et al.  Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine , 2000, Nature Biotechnology.

[72]  Christopher A. Voigt,et al.  Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach. , 2004, Chemistry & biology.

[73]  T. S. Wong,et al.  The diversity challenge in directed protein evolution. , 2006, Combinatorial chemistry & high throughput screening.

[74]  H. Brunner,et al.  Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template , 2003, Journal of biomedicine & biotechnology.

[75]  Frances H. Arnold,et al.  Computational method to reduce the search space for directed protein evolution , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[76]  Frances H. Arnold,et al.  Structure-guided SCHEMA recombination of distantly related β-lactamases , 2006 .

[77]  Costas D Maranas,et al.  Design of combinatorial protein libraries of optimal size , 2005, Proteins.

[78]  Wayne M Patrick,et al.  Novel methods for directed evolution of enzymes: quality, not quantity. , 2004, Current opinion in biotechnology.

[79]  Y. Husimi,et al.  Theory of evolutionary molecular engineering through simultaneous accumulation of advantageous mutations. , 2000, Journal of theoretical biology.

[80]  G. Feijoo,et al.  Strategies for the design and operation of enzymatic reactors for the degradation of highly and poorly soluble recalcitrant compounds , 2007 .

[81]  Directed enzyme evolution guided by multidimensional analysis of substrate-activity space. , 2004, Protein engineering, design & selection : PEDS.

[82]  Motowo Nakajima,et al.  Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation. , 2005, Protein engineering, design & selection : PEDS.

[83]  Marc Ostermeier,et al.  A combinatorial approach to hybrid enzymes independent of DNA homology , 1999, Nature Biotechnology.

[84]  U. Schwaneberg,et al.  Directed evolution of oxygenases: screening systems, success stories and challenges. , 2007, Combinatorial chemistry & high throughput screening.

[85]  K A Dill,et al.  Additivity Principles in Biochemistry* , 1997, The Journal of Biological Chemistry.

[86]  H. Hogrefe,et al.  Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit. , 2002, BioTechniques.

[87]  Adi Doron-Faigenboim,et al.  Selecton: a server for detecting evolutionary forces at a single amino-acid site , 2005, Bioinform..

[88]  Marc Ostermeier,et al.  Finding Cinderella's slipper—proteins that fit , 1999, Nature Biotechnology.

[89]  Gregory Stephanopoulos,et al.  Identifying Functionally Important Mutations from Phenotypically Diverse Sequence Data , 2006, Applied and Environmental Microbiology.

[90]  Markus Wiederstein,et al.  Protein sequence randomization: efficient estimation of protein stability using knowledge-based potentials. , 2005, Journal of molecular biology.

[91]  C D Maranas,et al.  Predicting crossover generation in DNA shuffling , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[92]  Costas D Maranas,et al.  Using a residue clash map to functionally characterize protein recombination hybrids. , 2003, Protein engineering.

[93]  Manfred T Reetz,et al.  Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. , 2006, Angewandte Chemie.

[94]  K. Dellagi,et al.  A novel simple and rapid PCR-based site-directed mutagenesis method , 2004, Molecular biotechnology.

[95]  Z. Jia,et al.  A novel PCR strategy for high-efficiency, automated site-directed mutagenesis , 2005, Nucleic acids research.

[96]  Yu Xue,et al.  An efficient site-directed mutagenesis method for ColE1-type ori plasmid. , 2007, Analytical Biochemistry.

[97]  Tuck Seng Wong,et al.  Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution. , 2004, Nucleic acids research.

[98]  Frances H Arnold,et al.  Library analysis of SCHEMA‐guided protein recombination , 2003, Protein science : a publication of the Protein Society.

[99]  Itay Mayrose,et al.  ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures , 2005, Nucleic Acids Res..

[100]  G. Gilardi,et al.  Directed evolution of enzymes for product chemistry. , 2004, Natural product reports.