Abstract
This chapter covers concepts developed for the directed evolution of enzymes. The principle strategy is given in comparison to rational protein design followed by a description of the most prominent methods for creation of mutant libraries. Screening and selection strategies to identify the best hits in these libraries are presented followed by several assays developed for a range of enzyme classes. Finally, selected examples for the successful application of evolutionary methods to optimize biocatalysts are given.
References
Acker MG, Auld DS (2014) Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Perspect Sci 1:56–73
Arnold FH, Georgiou G (eds) (2003a) Directed enzyme evolution: screening and selection methods. Humana Press, Totawa
Arnold FH, Georgiou G (eds) (2003b) Directed evolution library creation: methods and protocols. Humana Press, Totawa
Bartsch S, Kourist R, Bornscheuer UT (2008) Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase. Angew Chem Int Ed 47:1508–1511
Baumann M, Stürmer R, Bornscheuer UT (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Ed 40:4201–4204
Baxter S, Royer S, Grogan G, Brown F, Holt-Tiffin KE, Taylor IN, Fotheringham IG, Campopiano DJ (2012) An improved racemase/acylase biotransformation for the preparation of enantiomerically pure amino acids. J Am Chem Soc 134:19310–19313
Biles BD, Connolly BA (2004) Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR. Nucleic Acids Res 32:e176
Bornscheuer UT (2016) Protein engineering: beating the odds. Nat Chem Biol 12:54–55
Bornscheuer U, Huisman G, Kazlauskas R, Lutz S, Moore J, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194
Brundiek H, Evitt AS, Kourist R, Bornscheuer UT (2012) Creation of a highly trans fatty acid selective lipase by protein engineering. Angew Chem Int Ed 51:412–414
Caldwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33
Chen B, Lim S, Kannan A, Alford SC, Sunden F, Herschlag D, Dimov IK, Baer TM, Cochran JR (2016) High-throughput analysis and protein engineering using microcapillary arrays. Nat Chem Biol 12:76–81
Colin P-Y, Kintses B, Gielen F, Miton CM, Fischer G, Mohamed MF, Hyvonen M, Morgavi DP, Janssen DB, Hollfelder F (2015) Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics. Nat Commun 6. doi:10.1038/ncomms10008
Currin A, Swainston N, Day PJ, Kell DB (2015) Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44:1172–1239
Dombkowski AA, Sultana KZ, Craig DB (2014) Protein disulfide engineering. FEBS Lett 588:206–212
Dörr M, Fibinger MP, Last D, Schmidt S, Santos-Aberturas J, Böttcher D, Hummel A, Vickers C, Voss M, Bornscheuer UT (2016) Fully automatized high throughput enzyme library screening using a robotic platform. Biotechnol Bioeng 113:1421–1432. doi:10.1002/bit.25925
Engström K, Nyhlen J, Sandström AG, Backväll JE (2010) Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of alpha-substituted esters. J Am Chem Soc 132(20):7038–7042
Enoki J, Meisborn J, Müller A, Kourist R (2016) A multi-enzymatic cascade reaction for the stereoselective production of γ-oxyfunctionalyzed amino acids. Front Microbiol. doi:10.3389/fmicb.2016.00425
Fernández-Álvaro E, Snajdrova R, Jochens H, Davids T, Böttcher D, Bornscheuer UT (2011) A combination of in vivo selection and cell sorting for the identification of enantioselective biocatalysts. Angew Chem Int Ed 50:8584–8587
Fibla J, Gonzalezduarte R (1993) Colorimetric assay to determine alcohol-dehydrogenase activity. J Biochem Biophys Methods 26:87–93
Fox RJ, Davis SC, Mundorff EC, Newman LM, Gavrilovic V, Ma SK, Chung LM, Ching C, Tam S, Muley S, Grate J, Gruber J, Whitman JC, Sheldon RA, Huisman GW (2007) Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 25:338–344
Gassmeyer SK, Yoshikawa H, Enoki J, Hülsemann N, Stoll R, Miyamoto K, Kourist R (2015) STD NMR based protein engineering of the unique arylpropionate racemase AMDase G74C. ChemBioChem 16:1943–1949
Gassmeyer S, Wetzig J, Mügge C, Assmann M, Enoki J, Hilterhaus L, Zuhse R, Miyamoto K, Liese A, Kourist R (2016) Arylmalonate decarboxylase-catalyzed asymmetric synthesis of both enantiomers of optically pure flurbiprofen. ChemCatChem 8:916–921
Grognux J, Reymond JL (2004) Classifying enzymes from selectivity fingerprints. ChemBioChem 5:826–831
Heinze B, Kourist R, Fransson L, Hult K, Bornscheuer UT (2007) Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis. Protein Eng Des Sel 20:125–131
Heitman J, Sun S, James TY (2013) Evolution of fungal sexual reproduction. Mycologia 105:1–27
Henke E, Bornscheuer UT, Schmid RD, Pleiss J (2003) A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases. ChemBioChem 4:485–493
Horsman GP, Liu AMF, Henke E, Bornscheuer UT, Kazlauskas RJ (2003) Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3-bromo-2-methyl propanoate and ethyl 3-phenylbutyrate. Chem Eur J 9:1933–1939
Ljima Y, Matoishi K, Terao Y, Doi N, Yanagawa H, Ohta H (2005) Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism. Chem Commun 21:877–879
Janes LE, Kazlauskas RJ (1997) Quick E. A fast spectroscopic method to measure the enantioselectivity of hydrolases. J Org Chem 62:4560–4561
Kan SJ, Lewis RD, Chen K, Arnold FH (2016) Directed evolution of cytochrome c for carbon–silicon bond formation: bringing silicon to life. Science 354(6315):1048–1051
Köninger K, Gomez-Baraibar A, Mügge C, Paul C, Hollmann F, Nowaczyk M, Kourist R (2016) Recombinant cyanobacteria as tools for asymmetric C = C bond reduction fueled by biocatalytic water oxidation. Angew Chem Int Ed 55:5582–5585. doi:10.1002/anie.201601200
Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, Khabiri M, Plevaka M, Kulik D, Kuta Smatanova I (2013) Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel. Angew Chem Int Ed 52:1959–1963
Kourist R, Bartsch S, Bornscheuer UT (2007) Highly enantioselective synthesis of arylaliphatic tertiary alcohols using mutants of an esterase from Bacillus subtilis. Adv Synth Catal 349:1393–1398
Kourist R, Jochens H, Bartsch S, Kuipers R, Padhi SK, Gall M, Böttcher D, Joosten HJ, Bornscheuer UT (2010) The alpha/beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering. ChemBioChem 11:1635–1643. doi:10.1002/cbic.201000213
Leroy E, Bensel N, Reymond JL (2003) A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone. Bioorg Med Chem Lett 13:2105–2108
Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11–15
Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger KE (2000) Directed evolution of an enantioselective lipase. Chem Biol 7:709–718
Liu AMF, Somers NA, Kazlauskas RJ, Brush TS, Zocher F, Enzelberger MM, Bornscheuer UT, Horsman GP, Mezzetti A, Schmidt-Dannert C, Schmid RD (2001) Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Phiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes. Tetrahedron: Asymmetry 12:545–556
Lutz S, Bornscheuer UT (eds) (2008) Protein engineering handbook. Wiley VCH, Weinheim
Lutz S, Ostermeier M, Benkovic SJ (2001) Rapid generation of incremental truncation libraries for protein engineering using alpha phosphorothioate nucleotides. Nucleic Acids Res 29:e16
Mate DM, Alcalde M (2015) Laccase engineering: from rational design to directed evolution. Biotechnol Adv 33:25–40
Mayer KM, Arnold FH (2002) A colorimetric assay to quantify dehydrogenase activity in crude cell lysates. J Biomol Screen 7:135–140
Meyer MM, Hochrein L, Arnold FH (2006) Structure-guided SCHEMA recombination of distantly related β-lactamases. Prot Eng Des Sel 19:563–570
Miyauchi Y, Kourist R, Uemura D, Miyamoto K (2011) Dramatically improved catalytic activity of an artificial (S)-selective arylmalonate decarboxylase by structure-guided directed evolution. Chem Commun 47:7503–7505. doi:10.1039/c1cc11953b
Molina-Espeja P, Garcia-Ruiz E, Gonzalez-Perez D, Ullrich R, Hofrichter M, Alcalde M (2014) Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Appl Environ Microbiol 80(11):3496–3507
Molina Espeja P, Cañellas M, Plou FJ, Hofrichter M, Lucas F, Guallar V, Alcalde M (2016) Synthesis of 1 naphthol by a natural peroxygenase engineered by directed evolution. ChemBioChem 17:341–349
Moore JC, Arnold FH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 14:458–467
Neylon C (2004) Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res 32:1448–1459
Obata R, Nakasako M (2010) Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cys188Ser mutant in the liganded form. Biochemistry 49:1963–1969. doi:10.1021/bi9015605
Ostermeier M, Lutz S (2003) The creation of ITCHY hybrid protein libraries. In: Arnold FH, Georgiou G (eds) Directed evolution library creation: methods and protocols, Methods in molecular biology. Humana Press, Totowa, pp 129–141
Ostermeier M, Shim JH, Benkovic SJ (1999) A combinatorial approach to hybrid enzymes independent of DNA homology. Nat Biotechnol 17:1205–1209
Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Gen 16:379–394
Patel PH, Kawate H, Adman E, Ashbach M, Loeb LA (2001) A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity. J Biol Chem 276:5044–5051
Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Biotechnol 2:891–903
Reetz MT, Wilensek S, Zha D, Jaeger KE (2001) Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew Chem Int Ed 40:3589–3591
Reetz MT, Carballeira JD, Vogel A (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Ed 45:7745–7751
Reetz MT, Soni P, Fernandez L (2009) Knowledge-guided laboratory evolution of protein thermolability. Biotechnol Bioeng 102:1712–1717
Reymond JL (ed) (2005) Enzyme assays. Wiley-VCH, Weinheim
Sandström AG, Engström K, Jyhlén J, Kasrayan A, Bäckvall J-E (2009) Directed evolution of Candida antarctica lipase A using an episomally replicating yeast plasmid. Protein Eng Des Sel 22(7):413–420
Sandström AG, Wikmark Y, Engström K, Nyhlén J, Bäckvall J-e (2012) Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library. Proc Natl Acad Sci U S A 109:78–83. doi:10.1073/pnas.1111537108
Schmidt M, Hasenpusch D, Kahler M, Kirchner U, Wiggenhorn K, Lange W, Bornscheuer UT (2006) Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block. ChemBioChem 7:805–809
Schmidt S, Scherkus C, Muschiol J, Menyes U, Winkler T, Hummel W, Gröger H, Liese A, Herz HG, Bornscheuer UT (2015) An enzyme cascade synthesis of ε-caprolactone and its oligomers. Angew Chem Int Ed 54:2784–2787
Schrewe M, Ladkau N, Bühler B, Schmid A (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv Synth Catal 355:1693–1697
Stemmer WPC (1994) Rapid evolution of a protein by in vitro DNA shuffling. Nat Biotechnol 370:389–391
Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31:1707–1721
Terao Y, Miyamoto K, Ohta H (2006) Improvement of the activity of arylmalonate decarboxylase by random mutagenesis. Appl Microbiol Biotechnol 73:647–653
Udit AK, Silberg JJ, Sieber V (2003) Sequence homology-independent protein recombination, SHIPREC. In: Arnold FH, Georgiou G (eds) Directed evolution library creation: methods and protocols, Methods in molecular biology. Humana Press, Totowa, pp 153–164
Wahler D, Reymond JL (2002) The adrenaline test for enzymes. Angew Chem Int Ed 41:1229–1232
Wahler D, Boujard O, Lefevre F, Reymond JL (2004) Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates. Tetrahedron 60:703–710
Yang GY, Shamsuddin AM (1996) Gal-GalNAc: a biomarker of colon carcinogenesis. Histol Histopathol 11:801–806
Yoshida S, Enoki J, Kourist R, Miyamoto K (2015) Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance. Biosci Biotechnol Biochem 79:1965–1971
Zhao H (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer International Publishing AG
About this entry
Cite this entry
Bornscheuer, U.T., Kourist, R. (2016). Evolving Enzymes for Biocatalysis. In: Lee, S. (eds) Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals. Handbook of Hydrocarbon and Lipid Microbiology . Springer, Cham. https://doi.org/10.1007/978-3-319-31421-1_217-1
Download citation
DOI: https://doi.org/10.1007/978-3-319-31421-1_217-1
Received:
Accepted:
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-31421-1
Online ISBN: 978-3-319-31421-1
eBook Packages: Springer Reference Biomedicine and Life SciencesReference Module Biomedical and Life Sciences