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.

Transketolase variants were engineered to utilize arylalkanals and benzaldehyde as substrates with up to 28-fold rate acceleration for C–C bond formation with good yields (50–73%) and virtually complete (3S)-stereoselectivity (>99% ee).

Naturally rare l-erythro (3S,4S)-ketoses were prepared at high temperatures through a simultaneous two-step enzymatic cascade synthesis with excellent stereoselectivity.

The transketolase from Geobacillus stearothermophilus (TK ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK for various applications in asymmetric carboligation.

We propose an ecofriendly, efficient, stereoselective procedure for the two-carbon elongation of nonphosphorylated aldoses (C4–C6) to the corresponding C n+2 ketoses (C6–C8) in one step, using hydroxypyruvate (HPA) as a ketol donor substrate and an evolved thermostable transketolase from Geobacillus stearothermophilus (TKgst) as a biocatalyst. Simultaneous site saturation mutagenesis (SSM) at two or three key positions in the TKgst active site yielded efficient variants, L382F/F435Y, R521Y/S385/H462N, and R521V/S385D/H462S, with increased activity compared to wild-type TKgst for conversion of two tetroses (d-threose, l-erythrose), two pentoses (d-xylose, d-ribose), and two hexoses (d-allose, d-glucose), respectively. These six C n aldoses as acceptor and HPA as donor substrates were transformed by the TKgst variants at 60 °C with practically complete conversion. The corresponding C n+2 ketoses, including two hexuloses (d-tagatose, l-psicose), two heptuloses (d-altro-heptulose, d-ido-heptulose), and two octuloses (d-glycero- d-ido-octulose, d-glycero-d-altro-octulose) are naturally rare compounds with important biological functions, which were obtained with high diastereoselectivity.

Transketolase (TK) from various origins (including Escherichia coli and yeast) has been described to be fully enantiomer specific for (2R)-hydroxyaldehyde substrates. A thermostable TK from Geobacillus stearothermophilus (TK gst ) was found to display a minor reactivity for (2S)-hydroxylated aldehydes. To improve this activity by directed protein evolution, we have built a library of TK gst variants by site saturation mutagenesis on two key positions L382 and D470. The best TK gst double mutant L382D/D470S shows up to 4-and 5-fold higher activities towards l-lactaldehyde and l-glyceraldehyde as acceptor substrates, respectively. Preparative utility of this mutant was demonstrated by the one-step synthesis of valuable l-ribulose and its 5-deoxy analogue with the l-erythro (3S,4S) configuration, which were previously inaccessible by using common TK sources.

We described an efficient in situ generation of hydroxypyruvate from d-serine catalyzed by a damino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l-serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbonÀ carbon bond. The enzymatic cascade reaction was performed in one-pot in the presence of d-serine and appropriate aldehydes for the synthesis of valuable (3S)-hydroxyketones, which were obtained with high enantio-and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d-serine and aldehydes. Scheme 1. In situ generation of HPA from TA or dAAOcatalyzed reaction coupled with TK-catalyzed reaction.Scheme 2. Reaction of DAAO Rg coupled with l-lactate dehydrogenase (LDH) in the presence of NADH.

We describe an efficient three-enzyme, sequential one-pot cascade reaction where both transketolase substrates are generated in situ in a convergent fashion. The nucleophilic donor substrate hydroxypyruvate was obtained from l-serine and pyruvate by a transaminase-catalyzed reaction. In parallel, three different (2S)-α-hydroxylated aldehydes, l-glyceraldehyde, d-threose, and l-erythrose, were generated as electrophilic acceptors from simple achiral compounds glycolaldehyde and formaldehyde by d-fructose-6-phosphate aldolase catalysis. The compatibility of the three enzymes was studied in terms of temperature, enzyme ratio and substrate concentration. The efficiency of the process relied on the irreversibility of the transketolase reaction, driving a shift of the reversible transamination reaction and securing the complete conversion of all substrates. Three valuable (3S,4S)-ketoses, l-ribulose, d-tagatose, and l-psicose were obtained in good yields with high diastereoselectivity.