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Updated: Feb 9, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Complete Switch of Reaction Specificity of an Aldolase by Directed Evolution In Vitro: Synthesis of Generic Aliphatic
Sebastian Junker1, Raquel Roldan2, Henk-Jan Joosten3
1Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287, Darmstadt, Germany.
Abstract:
A structure-guided engineering of fructose-6-phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, that is, unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26, and N28, which form a binding pocket around the nucleophilic carbon atom. Double-selectivity screening was executed by high-performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. D6 turned out to be the key residue that enabled activity with non-hydroxylated nucleophiles. Altogether 25 single- and double-site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6-phosphate.
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