A Multidisciplinary Approach Toward the Rapid and Preparative-Scale Biocatalytic Synthesis of Chiral Amino Alcohols: A Concise Transketolase-/ ω-Transaminase-Mediated Synthesis of (2S,3S)-2-Aminopentane-1,3-diol Mark E. B. Smith, † Bing H. Chen, ‡,¶ Edward G. Hibbert, ‡,0 Ursula Kaulmann, §,9 Kirsty Smithies, † James L. Galman, † Frank Baganz, ‡ Paul A. Dalby, ‡ Helen C. Hailes, † Gary J. Lye, ‡ John M. Ward, § John M. Woodley, ⊥ and Martina Micheletti* ,‡ Department of Chemistry, UniVersity College London, 20 Gordon Street, London WC1H 0AJ, U.K., Department of Biochemical Engineering, UniVersity College London, Torrington Place, London WC1E 7JE, U.K., Institute of Structural and Molecular Biology, UniVersity College London, Gower Street, London WC1E 6BT, U.K., and Department of Chemical and Biochemical Engineering, Technical UniVersity of Denmark, 2800 Lyngby, Denmark Abstract: Chiral amino alcohols represent an important class of value-added biochemicals and pharmaceutical intermediates. Chemical routes to such compounds are generally step intensive, requiring envi- ronmentally unfriendly catalysts and solvents. This work describes a multidisciplinary approach to the rapid establishment of bio- catalytic routes to chiral aminodiols taking the original synthesis of (2S,3S)-2-aminopentane-1,3-diol as a specific example. An engineered variant of Escherichi coli transketolase (D469T) was used for the initial asymmetric ynthesis of (3S)-1,3-dihydroxypen- tan-2-one from the achiral substrates propanal and hydroxypyru- vate. A bioinformatics led strategy was then used to identify and clone an ω-transaminase from Chromobacterium Wiolaceum (DSM30191) capable of converting the product of the transketo- lase-catalysed step to the required (2S,3S)-2-aminopentane-1,3-diol using isopropylamine as an inexpensive amine donor. Experiments to characterize, optimize and model the kinetics of each reaction step were performed at the 1 mL scale using previously established automated microwell processing techniques. The microwell results provided excellent predictions of the reaction kinetics when the bioconversions were subsequently scaled up to preparative scales in batch stirred-tank reactors. The microwell methods thus provide process chemists and engineers with a valuable tool for the rapid and early evaluation of potential synthetic strategies. Overall, this work describes a concise and efficient biocatalytic route to chiral amino alcohols and illustrates an integrated multidisciplinary approach to bioconversion process design and scale-up. 1. Introduction The pharmaceutical industry today faces significant chal- lenges in bringing new drugs to the market. A key area of interest is in harnessing new technologies to progress from initial drug discovery to the final manufacturing process as rapidly and cost-effectively as possible. Rising costs related to the development of increasingly complex pharmaceuticals (with multiple chiral centers and functional groups) are compounded by increasingly stringent environmental legislation and the drive toward sustainable processes. Working together with industry these are the technologies and challenges that the multidisci- plinary UCL Bioconversion - Chemistry - Engineering interface (BiCE) programme aims to address. While there are extensive synthetic transformations for which chemical (catalytic) conversions are most appropriate, biocata- lytic (enzyme and microbial) strategies have been increasingly adopted where mild conditions and high regio- or stereoselec- tivity are desired. 1,2 The majority of biocatalytic processes reported to date have involved the use of a single isolated enzyme. However, recent developments in metabolic engineer- ing and synthetic biology have highlighted the possibility of using multienzyme synthetic pathways to perform more com- plex syntheses. 3,4 Furthermore, many of the enzymes that could be used in series do not necessarily exist naturally together in known metabolic pathways. This raises the possibility of creating de noVo pathways in engineered microorganisms using existing powerful tools such as rDNA technology. As a demonstration of such an approach we have previously devised a synthetic scheme using a transketolase (TK) and a transami- nase (TAm) to create an optically enriched 2-amino-1,3-diol using an engineered Escherichia coli strain. 5 Chiral 2-amino-1,3-diols are an important class of pharma- ceutically relevant compounds, and their motif is present in antibiotics, 6-10 antiviral glycosidase inhibitors, 11,12 and sphingo- * Corresponding author. E-mail: m.micheletti@ucl.ac.uk. † Department of Chemistry, University College London. ‡ Department of Biochemical Engineering, University College London. § Institute of Structural and Molecular Biology, University College London. ⊥ Department of Chemical and Biochemical Engineering, Technical University of Denmark. ¶ Current address: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. 0 Current address: Illumina Cambridge Ltd., Chesterford Research Park, Cambridge CB10 1XL, UK. 9 Current address: Biotica Ltd., Chesterford Research Park, Cambridge CB10 1XL, UK. (1) Pollard, D. J.; Woodley, J. M. Trends Biotechnol. 2007, 25, 66. (2) Woodley, J. M. Trends Biotechnol. 2008, 26, 321. (3) Ro, D. K.; Paradise, E. M.; Ouellet, M.; Fisher, K. J.; Newman, K. L.; Ndungu, J. M.; Ho, K. A.; Eachus, R. A.; Ham, T. S.; Kirby, J.; Chang, M. C.; Withers, S. T.; Shiba, Y.; Sarpong, R.; Keasling, J. D. 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