title: Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase.
abstract: Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.
authors.affiliation: DSM Anti-Infectives, Delft, The Netherlands.
authors.forename: Stefan
authors.initials: S
authors.lastname: Jennewein
authors.forename: Martin
authors.initials: M
authors.lastname: Schürmann
authors.forename: Michael
authors.initials: M
authors.lastname: Wolberg
authors.forename: Iris
authors.initials: I
authors.lastname: Hilker
authors.forename: Ruud
authors.initials: R
authors.lastname: Luiten
authors.forename: Marcel
authors.initials: M
authors.lastname: Wubbolts
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics*; D002617:Chemical Industry / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C020019:deoxyribose-phosphate aldolase