Genome as Resources for Biocatalyst
Genomes as Resources for Biocatalysis
JON D. STEWART
Department of Chemistry, University of Florida Gainesville, Florida 32611
I. II. III. IV.
Introduction Yeast Dehydrogenase Gene Identification Expression and Isolation of Yeast Dehydrogenases Characterization of Yeast Dehydrogenases A. Results from Ethyl Acetoacetates B. Results from Higher Homologs C. Synthetic Applications V. Conclusions and Future Directions References
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I. Introduction How well biocatalysis enables novel routes to target molecules ultimately rests on the properties of the available enzymes. In most applications, proteins are called on to catalyze reactions for which they did not specifically evolve. This means one must first identify the best enzyme ‘‘tool’’ for a particular synthetic task, and this phase of bioprocess development has often consumed a significant fraction of the total effort expended. Biocatalyst identification was originally conducted by screening whole organisms (usually bacteria and fungi) to uncover those that catalyzed the desired transformation. This strategy has proven especially successful with steroid biotransformations in which site‐selective hydroxylation or carbonyl reductions are the typical goals (Mahato and Majumdar, 1993; Warhurst and Fewson, 1994). Many organizations have accumulated large microbial culture collections, and these have provided the ‘‘hits’’ that have been—and continue to be—developed into commercial bioprocesses. Despite the many success stories derived from whole organism screening, this approach suffers from several drawbacks. First, the strategy is purely empirical, and the screening process must be repeated for each new substrate/reaction pair. While accumulated experience can help narrow the search to those organisms known to mediate related reactions, the exploration remains labor‐intensive since cells of each organism must be grown anew for every screening. Another problem with using whole cells is that
JON D. STEWART
Department of Chemistry, University of Florida Gainesville, Florida 32611
I. II. III. IV.
Introduction Yeast Dehydrogenase Gene Identification Expression and Isolation of Yeast Dehydrogenases Characterization of Yeast Dehydrogenases A. Results from Ethyl Acetoacetates B. Results from Higher Homologs C. Synthetic Applications V. Conclusions and Future Directions References
31 33 34 35 37 40 43 46 49
I. Introduction How well biocatalysis enables novel routes to target molecules ultimately rests on the properties of the available enzymes. In most applications, proteins are called on to catalyze reactions for which they did not specifically evolve. This means one must first identify the best enzyme ‘‘tool’’ for a particular synthetic task, and this phase of bioprocess development has often consumed a significant fraction of the total effort expended. Biocatalyst identification was originally conducted by screening whole organisms (usually bacteria and fungi) to uncover those that catalyzed the desired transformation. This strategy has proven especially successful with steroid biotransformations in which site‐selective hydroxylation or carbonyl reductions are the typical goals (Mahato and Majumdar, 1993; Warhurst and Fewson, 1994). Many organizations have accumulated large microbial culture collections, and these have provided the ‘‘hits’’ that have been—and continue to be—developed into commercial bioprocesses. Despite the many success stories derived from whole organism screening, this approach suffers from several drawbacks. First, the strategy is purely empirical, and the screening process must be repeated for each new substrate/reaction pair. While accumulated experience can help narrow the search to those organisms known to mediate related reactions, the exploration remains labor‐intensive since cells of each organism must be grown anew for every screening. Another problem with using whole cells is that
References: 1 Genes upregulated ! twofold by saline osmotic stress: YJR096w, YBR149w, YOR120w, YHR104w, YDR368w, YNL274c, YGL157w, and YOL151w (Causton et al., 2001). HIGHER HOMOLOGS In addition to ethyl acetoacetates, we also examined the interaction of higher b‐keto ester homologs with the collection of yeast dehydrogenases (Kaluzna et al., 2004, 2005; Stowe and Stewart, 2005)