The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

The thiamine diphosphate (ThDP)‐dependent pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC) catalyzes the carboligation of aldehydes that yields (R)‐2‐hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor‐made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so‐called S‐pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC‐E469G provides access to (S)‐phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP‐dependent enzymes, which now gives access to all stereo‐ and regioisomers of the asymmetric aliphatic–aromatic cross‐benzoin‐like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities.

höhere Produktivität und geringe Beeinflussung der Organismen durch Metabolitrepression. Somit stellt Festphasenfermentation eine alternative Anwendung zur fermentativen Produktion von seltenen Isoprenoiden dar.