Premium
Directed evolution of an industrial biocatalyst: 2‐deoxy‐ D ‐ribose 5‐phosphate aldolase
Author(s) -
Jennewein Stefan,
Schürmann Martin,
Wolberg Michael,
Hilker Iris,
Luiten Ruud,
Wubbolts Marcel,
Mink Daniel
Publication year - 2006
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200600020
Subject(s) - aldolase a , biocatalysis , directed evolution , biochemistry , chemistry , ribose , phosphate , fructose bisphosphate aldolase , enzyme , catalysis , reaction mechanism , gene , mutant
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 (3 R ,5 S )‐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 (3 R ,5 S )‐6‐chloro‐2,4,6‐trideoxyhexapyranoside synthesis, under industrially relevant conditions.