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Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering

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Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering Ronnie Machielsen Promotoren: Prof. dr. J. van der Oost Hoogleraar Microbiologie en Biochemie Wageningen Universiteit Prof. dr. W.M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Leden van de Promotiecommissie: Prof. dr. P.C. Wright University of Sheffield Prof. dr. R. Wever Universiteit van Amsterdam Prof. dr. M.A. Huynen Radboud Universiteit Nijmegen Prof. dr. H. Gruppen Wageningen Universiteit Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering Ronnie Machielsen Proefschrift Ter verkrijging van de graad van doctor Op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op dinsdag 16 oktober 2007 des namiddags te half twee in de Aula Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering Ronnie Machielsen Ph.D. thesis Wageningen University, Wageningen, The Netherlands (2007) 136 p., with summary in Dutch ISBN 978 90 8504 710 0 Abstract Machielsen, R. (2007). Microbial Alcohol Dehydrogenases: Identification, Characterization and Engineering. PhD thesis. Laboratory of Microbiology, Wageningen University, The Netherlands. Alcohol dehydrogeases (ADHs) catalyze the interconversion of alcohols, aldehydes and ketones. They display a wide variety of substrate specificities and are involved in an astonishingly wide range of metabolic processes, in all living organisms. Besides the scientific interest in ADHs, they are also attractive biocatalysts for industrial applications. Especially, their capability to catalyze chemo-, stereo-, and region-selective reactions endows them with considerable potential for a range of applications in food, pharmaceutical and fine chemicals industries. Natural enzymes are, however, optimized and often highly specialized for certain biological functions; hence, in many instances natural enzymes do not perform optimally in an industrial process. Laboratory evolution (random methods) and/or rationale- based protein engineering (design methods) can be applied to obtain enzyme variants, which are suitable for a particular industrial application. This study describes the identification, characterization and engineering of microbial ADHs. Because protein stability is an important issue for in vitro applications, thermophiles are considered a potential source of thermozymes. The genes coding for sixteen putative ADHs were identified in the genome of the hyperthermophilic archaeon Pyrococcus furiosus and a more detailed level of function prediction was attempted for all identified genes. Subsequently, fifteen of the putative ADHs were heterologously produced and an initial screening for activities was performed in which two of the putative ADHs showed activities. Next, these two ADHs, AdhD and Pf-TDH, were purified and characterized. AdhD is a member of the aldo-keto reductase superfamily and Pf- TDH is a L-threonine dehydrogenase. In addition, either laboratory evolution or rationale protein engineering was applied to improve the properties of two selected ADHs to extend their potential for industrial applications. AdhA, a thermostable ADH of P. furiosus, was engineered by laboratory evolution to improve the production of (2S,5S)-hexanediol at moderate temperatures. High-resolution 3D structures are required to perform the directed engineering of proteins. The ADH of Lactobacillus brevis (Lb-ADH) was selected because a 1.0 Å resolution structure was available, and because of its potential for industrial application. Structure-based computational protein engineering was applied to adjust its cofactor specificity. Keywords: alcohol dehydrogenase, laboratory evolution, rational protein engineering, Pyrococcus furiosus, biocatalysis, characterization, computational design, thermostability. Table of contents Chapter 1 Aim and outline of the thesis 9 Chapter 2 General introduction 13 Chapter 3 Identification and heterologous production of NAD(P)-dependent 43 alcohol dehydrogenases from the hyperthermophilic archaeon Pyrococcus furiosus Chapter 4 Production and characterization of a thermostable alcohol 57 dehydrogenase that belongs to the aldo-keto reductase superfamily Chapter 5 Production and characterization of a thermostable L-threonine 71 dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus Chapter 6 Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to 85 improve the production of (2S,5S)-hexanediol at moderate temperatures Chapter 7 Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase 101 by computational design Chapter 8 Summary and concluding remarks 115 Samenvatting en conclusies 121 Dankwoord / Acknowledgements 129 Curriculum vitae 131 List of publications 133 Activities in the frame of VLAG research school 135 Chapter 1 Aim and outline of the thesis Chapter 1 Alcohol dehydrogenases (ADHs) are present in virtually all organisms. They catalyze the interconversion of alcohols, aldehydes and ketones. ADHs require a cofactor, display a wide variety of substrate specificities, and play an important role in a broad range of physiological processes (e.g. alcohol and alkane metabolism, and cell defense towards exogenous alcohols and aldehydes). These features make them very interesting from a scientific perspective. Stable and enantioselective ADHs (e.g. Pyrococcus furiosus AdhA and Lactobacillus brevis ADH, mentioned further on) have also considerable potential for a range of applications in food, pharmaceutical and fine chemicals industries. The production of enantio- and diastereomerically pure diols is particularly desired because these are important chemical building blocks. Biocatalysis is gradually taking over from chemical catalysis in many industrial applications. Enzymes are environmentally friendly, biodegradable, efficient, and low cost in terms of resource requirements; as such they provide benefits compared to traditional chemical approaches in various industrial processes. In many instances, however, natural enzymes do not perform optimally in a particular unnatural process, and as such can be unsuitable for large-scale industrial applications. Industry needs enzymes that have (i) a high activity over longer periods of time, (ii) a high stability under harsh physical (high temperature) and chemical (non-aqueous solvents) conditions, and (iii) a high specificity and selectivity that does allow the enzyme to efficiently generate specific products that are not necessarily present in nature. There are three major and principally different routes to obtain enzyme variants with improved features: (1) isolating enzymes from organisms living in environments that correspond as much as possible to actual process conditions, (2) rationale- based protein engineering, and (3) laboratory evolution (random methods). The aim of the research described in this thesis was to identify and characterize novel and stable alcohol dehydrogenases for the production of fine chemicals, and to improve the properties of selected alcohol dehydrogenases to extend their potential for industrial applications. The latter has been done by laboratory evolution (random methods) and rationale protein engineering. In search for (thermo)stable ADHs for the production of aldehydes, ketones and (chiral) alcohols the hyperthermophilic archaeon Pyrococcus furiosus was chosen. Enzymes from hyperthermophiles, i.e., microorganisms that grow optimally above 80°C, generally display an extreme stability at high temperature, high pressure, as well as high concentrations of chemical denaturants; this feature generally makes hyperthermophiles a very good source of robust enzymes. In addition, the genome sequence of P. furiosus and of several closely related micro-organisms were available. Furthermore, the research described in this thesis was part of the EU project PYRED, which aimed to exploit the very high reducing power of P. furiosus (including possibly engineered strains) for the production of aldehydes, ketones and (chiral) alcohols. Therefore, identification, production and characterization of ADHs from P. furiosus, as described in this thesis, were important for the possible in vivo and in vitro production of valuable fine chemicals. 10 Aim and outline To extend their potential for industrial applications AdhA, an alcohol dehydrogenase of P. furiosus, and the alcohol dehydrogenase of Lactobacillus brevis (Lb-ADH) were selected to be improved. AdhA was selected to improve its low-temperature activity for the production of (2S,5S)-hexanediol, while retaining the stability of the enzyme. Lb-ADH is R- specific, relatively stable and has a broad substrate specificity. These features have already made the enzyme very attractive for industrial applications. However, a drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). As the atomic resolution (1.0 Å) structure of Lb-ADH is available, it was possible to perform structure-based computational protein engineering. In both projects a clear engineering challenge, either improving the low-temperature activity or adjusting the cofactor specificity, could be dealt with. In addition, both laboratory evolution and rationale protein engineering could be utilized. Chapter 2 gives a general introduction and a review on improving enzyme performance by the random approach of laboratory evolution as well as by rational and computational design. The
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