Complete Switch of Reaction Specificity of an Aldolase by Directed Evolution In Vitro: Synthesis of Generic Aliphatic Aldol Products

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.

nanoDSF as screening tool for enzyme libraries and biotechnology development

“…In the study, commercial purified hexokinase from Saccharomyces cerevisiae (HK) was included as received, ketoreductase from L. brevis (KRED Lb ) was used as CFE from recombinant expression and DERA Tm was additionally heat treated and dialysed. Transketolase variant L397F/D399G/H479Q from G. stearothermophilus (TK) , fructose‐6‐phosphate aldolase from E. coli (FSA) and DERA from E. coli (DERA Ec ) were available from current synthetic projects. These enzymes represent distinct folding patterns and come from different organisms, including mesophilic and thermophilic, bacterial and eukaryotic origins.…”

“…DERA Ec was purified to homogeneity by Ni‐nitrilotriacetic acid chromatography using its His‐tag, lyophilised, stored at −20 °C until use and then the lyophilisate was dissolved to 2 g·L −1 in 50 m m TEA pH 7.5 for use. Transketolase variant L397F/D399G/H479Q from G. stearothermophilus (TK) and fructose‐6‐phosphate aldolase from E. coli (FSA) were heat treated according to published protocols before lyophilisation, dissolved to 1 and 2 g·L −1 , respectively, in 50 m m TEA buffer (pH 7.5). Prozomix provided heat treated, dialysed and lyophilised DERA from Thermotoga maritima (DERA Tm ) and lyophilised ketoreductase from L. brevis (KRED Lb ).…”

Biocatalytic Aldol Addition of Simple Aliphatic Nucleophiles to Hydroxyaldehydes

“…In this work, we show that engineered variants FSA D6X 32 catalyze aldol additions of ethanal ( 1a ), linear ( 1b – d ), and cyclic ( 1e – g ) simple aliphatic ketones to nonphosphorylated hydroxyaldehyde derivatives ( 2a – c ) ( Figure 1 ), expanding the synthetic utility to rare and unusual sugar derivatives as well as related chiral building blocks.…”

Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilised glycerol dehydrogenase

“…As safe path for enzyme optimization, the scientic community exploits, either separately or synergistically, random and extensive mutagenesis campaigns through directed evolution 29 and minimalist mutations through site-directed mutagenesis guided by structural knowledge. 22,30,31 In the particular case of GlyDH, one of the few documented successes is the combination of DNA-shuffling and site-directed mutagenesis to expand the GlyDH substrate scope to other diols. This variant was 2.6 times more active towards 1,3-butanediol than the wild-type.…”