Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.

Application of aldolases for the asymmetric synthesis of multifunctional chiral products is hampered by their reputed strict nucleophile (=aldol donor) specificity owing to a mechanistic requirement for creating a carbanion nucleophile in aqueous medium. Here we report that a minimalist engineering can extensively broaden the substrate scope of native D-fructose-6-phosphate aldolase (FSA) from Escherichia coli, for which hydroxyacetone is the most proficient substrate, to accept an unprecedented wide variety of alternative nucleophiles. By single-or double-space-generating mutations using simple conservative Leu to Ala replacement of active site residues, we found enzyme variants to efficiently convert larger ketols and bioisosteric ether components with up to seven skeletal atoms, including linear and branched-chain structures. All reactions occurred with full retention of the natural D-threo diastereospecificity. These FSA variants open new avenues toward the synthesis of novel product families that hitherto were inaccessible by biological catalysis.

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.

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.

Enzyme,d ie asymmetrische C-C-Verknüpfungen katalysieren, sind typischerweise hochs pezifisch füri hre nukleophilen Donorsubstrate.M ittels strukturbasierter gerichteter Evolution konnten neue Enzymvarianten der thermostabilen Transketolase aus Geobacillus stearothermophilus erzeugt werden, die anstelle ihrer natürlichen Substrate,w ie polyhydroxylierte Ketosephosphate oder Hydroxypyruvat, auch Pyruvat und dessen hçhere aliphatische Homologe als Nukleophile fürd en Acyltransfer verwenden kçnnen. Die Einfachvariante H102T war am besten geeignet, 3-Methyl-2-oxobutyrat als Donor zu verwerten, während die Doppelvariante H102L/H474S die hçchste katalytischeE ffizienz fürP yruvat als Donorsubstrat aufwies.D ie letztgenannte Variante war in der Lage,den auxotrophen Defekt eines Deletionsstamms von Escherichia coli zu komplementieren, dem das dxs-Gen für den ersten Schritt in die Terpenoid-Biosynthese fehlt. Dies erçffnet die Mçglichkeit zur weiteren Enzymoptimierung mithilfe eines Wachstumsselektionstests.