Abstract
We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process ∼4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.
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References
Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).
Schoemaker, H.E., Mink, D. & Wubbolts, M.G. Dispelling the myths—biocatalysis in industrial synthesis. Science 299, 1694–1697 (2003).
Hibbert, E.G. & Dalby, P.A. Directed evolution strategies for improved enzymatic performance. Microb. Cell Fact. 4, 29 (2005).
Tawfik, D.S. Biochemistry. Loop grafting and the origins of enzyme species. Science 311, 475–476 (2006).
Castle, L.A. et al. Discovery and directed evolution of a glyphosate tolerance gene. Science 304, 1151–1154 (2004).
Crameri, A., Raillard, S.A., Bermudez, E. & Stemmer, W.P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).
Ness, J.E. et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 20, 1251–1255 (2002).
Ness, J.E. et al. DNA shuffling of subgenomic sequences of subtilisin. Nat. Biotechnol. 17, 893–896 (1999).
Stemmer, W.P.C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).
Yuan, L., Kurek, I., English, J. & Keenan, R. Laboratory-directed protein evolution. Microbiol. Mol. Biol. Rev. 69, 373–392 (2005).
Kubinyi, H. QSAR and 3D QSAR in drug design Part1: methodology. Drug Disc. Today 2, 457–467 (1997).
Hellberg, S., Sjostrom, M. & Wold, S. The prediction of bradykinin potentiating potency of pentapeptides. An example of a peptide quantitative structure-activity relationship. Acta Chem. Scand. B 40, 135–140 (1986).
Eroshkin, A.M., Fomin, V.I., Zhilkin, P.A., Ivanisenko, V.A. & Kondrakhin, Y.V. PROANAL version 2: multifunctional program for analysis of multiple protein sequence alignments and studying structure-activity relationships in protein families. Comp. Appl. Biosci. 11, 39–44 (1995).
Eroshkin, A.M., Zhilkin, P.A. & Fomin, V.I. Algorithm and computer program Pro__Anal for analysis of relationship between structure and activity in a family of proteins or peptides. Comput. Appl. Biosci. 9, 491–497 (1993).
Fox, R. Directed molecular evolution by machine learning and the influence of nonlinear interactions. J. Theor. Biol. 234, 187–199 (2005).
Fox, R. et al. Optimizing the search algorithm for protein engineering by directed evolution. Protein Eng. 16, 589–597 (2003).
Nakamura, T., Nagasawa, T., Yu, F., Watanabe, I. & Yamada, H. A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide. Biochem. Biophys. Res. Commun. 180, 124–130 (1991).
van den Wijngaard, A.J., Reuvekamp, P.T. & Janssen, D.B. Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2. J. Bacteriol. 173, 124–129 (1991).
Matsuda, H., Shibata, T., Hashimoto, H. & Kitai, M. Method for producing (R)-4-cyano-3-hydroxybutyric acid lower alkyl ester. US patent 5,908,953 (1999).
Stemmer, W.P. DNA in vitro shuffling by random fragmentation and reassembly in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).
Wells, J.A. Additivity of mutational effects in proteins. Biochemistry 29, 8509–8517 (1990).
Sandberg, W.S. & Terwilliger, T.C. Engineering multiple properties of a protein by combinatorial mutagenesis. Proc. Natl. Acad. Sci. USA 90, 8367–8371 (1993).
de Jong, S. SIMPLS: an alternative approach to partial least squares regression. Chemomet. and Intell. Lab. Sys. 18, 251–263 (1993).
Mee, R.P., Auton, T.R. & Morgan, P.J. Design of active analogues of a 15-residue peptide using D-optimal design, QSAR and a combinatorial search algorithm. J. Pept. Res. 49, 89–102 (1997).
Bennett, K. & Embrechts, M. An Optimization Perspective on Partial Least Squares. Advances in Learning Theory: Methods, Models and Applications, NATO Science Series III: Computer & Systems Sciences vol. 190. (eds. Suykens, J., Horvath, G., Basu, S., Micchelli, J. & Vandewalle, J.) 227–250, (IOS Press, Amsterdam, 2003).
de Jong, R.M. et al. Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site. EMBO J. 22, 4933–4944 (2003).
Li, W.H., Wu, C.I. & Luo, C.C. Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications. J. Mol. Evol. 21, 58–71 (1984).
Hartl, D.L. & Taubes, C.H. Towards a theory of evolutionary adaptation. Genetica 102-103, 525–533 (1998).
Hylckama Vlieg, J.E.T. et al. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases. J. Bacteriol. 183, 5058–5066 (2001).
Kauffman, S. . The Origins of Order (Oxford University Press, New York, 1993).
Thayer, A. Competitors want to get a piece of Lipitor. Chem. Eng. News 84, 26–27 (2006).
Zhang, J.H., Dawes, G. & Stemmer, W.P. Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94, 4504–4509 (1997).
Stutzman-Engwall, K. et al. Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis. Metab. Eng. 7, 27–37 (2005).
Reetz, M.T., Wang, L.W. & Bocola, M. Directed evolution of enantioselective enzymes: Iterative cycles of CASTing for probing protein-sequence space. Angew Chem. Int. Ed. Engl. 45, 1236–1241 (2006).
Acknowledgements
We thank David Gray, Alice Wang, Su Chen, John Peterson, Walter Heath, Tim Brandon, Jon Postlethwaite, Anjali Srivastava and Bill Dewhirst for help with bioprocess development and production; Malissa Jefferson and Susan Louie for additional assay support; Patricia Babbitt, Pim Stemmer, Lynne Gilson, Lori Giver, Birthe Borup, Anke Krebber, Stephen DelCardayre and Jonathan Blanding for careful reading of the manuscript and helpful suggestions; and three anonymous reviewers for insightful commentary and critical feedback.
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Fox, R., Davis, S., Mundorff, E. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 25, 338–344 (2007). https://doi.org/10.1038/nbt1286
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DOI: https://doi.org/10.1038/nbt1286
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