Review Directed evolution of biocatalytic processes Edward G. Hibbert a , Frank Baganz a , Helen C. Hailes b , John M. Ward c , Gary J. Lye a , John M. Woodley a , Paul A. Dalby a, * a Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK b Department of Chemistry, University College London, Gordon Street, London WC1H 0AJ, UK c Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Received 2 August 2004; received in revised form 28 September 2004; accepted 28 September 2004 Abstract The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts. # 2005 Elsevier B.V. All rights reserved. Keywords: Directed evolution; Biocatalysis; Process; Metabolic engineering; Recombineering; Microscale 1. Directed enzyme evolution and biocatalytic processes While enzymes are potentially a very useful complement to the increasing range of chemical catalysts for the synthesis of complex chiral molecules, the process condi- tions in an industrial catalytic reactor are rarely ideally suited to maintaining a highly active and long lasting enzyme. Natural enzymes have evolved over millions of years to operate in a specific environment and despite their wide diversity, natural, and industrial environments are usually significantly different (Table 1). In a recent paper, some of the ideal properties that would be required of an enzyme for an industrial process have been discussed [1] and it is clear that environments appropriate to deliver such properties are not easily found in nature. In addition, enzyme chemistry in nature is rarely the most useful to assist in the synthesis of complex industrial products. These factors make the application of enzymes for industrial synthesis difficult, in particular, where multiple (rather than singular) traits need to be satisfied to create the ideal industrial enzyme catalyst. Nevertheless, there are very strong reasons for attempting to overcome these barriers, such as the exquisite chemo-, regio-, and stereo-selective and specific properties of enzymes and their ability for effective catalysis in water, under mild conditions. One method of overcoming some of the barriers to implementing enzymes in industry is the application of directed evolution, in which the amino acid sequence of an enzyme is iteratively altered until the enzyme functions in the desired manner. Changing a protein by directed evolution opens the possibilities for moving towards a variety of required properties. The most popular targets for directed enzyme evolution to date have been activity [2–4], substrate specificity [5–10], thermal and oxidative stability www.elsevier.com/locate/geneanabioeng Biomolecular Engineering 22 (2005) 11–19 * Corresponding author. Tel.: +44 20 7679 2962; fax: +44 20 7383 2348. E-mail address: p.dalby@ucl.ac.uk (P.A. Dalby). 1389-0344/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2004.09.003