Excelzyme: A Swiss University-Industry Collaboration for Accelerated Biocatalyst Development.

Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.

Inverting the Stereoselectivity of an NADH-Dependent Imine-Reductase Variant.

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)-selective IRED from Myxococcus stipitatus (NADH-IRED-Ms) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the (S)-amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (R)-IRED-Ms wild-type enzyme and the NADH-dependent NADH-IRED-Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.

Engineering of Thermostable ß-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

The ß-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-ßHAD), which catalyzes the NADP+ -dependent oxidation of ß-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-ßHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.

Systematic Evaluation of Imine-Reducing Enzymes: Common Principles in Imine Reductases, ß-Hydroxy Acid Dehydrogenases, and Short-Chain Dehydrogenases/ Reductases.

The enzymatic, asymmetric reduction of imines is catalyzed by imine reductases (IREDs), members of the short-chain dehydrogenase/reductase (SDR) family, and ß-hydroxy acid dehydrogenase (ßHAD) variants. Systematic evaluation of the structures and substrate-binding sites of the three enzyme families has revealed four common principles for imine reduction: structurally conserved cofactor-binding domains; tyrosine, aspartate, or glutamate as proton donor; at least four characteristic flanking residues that adapt the donor's pKa and polarize the substrate; and a negative electrostatic potential in the substrate-binding site to stabilize the transition state. As additional catalytically relevant positions, we propose alternative proton donors in IREDs and ßHADs as well as proton relays in IREDs, ßHADs, and SDRs. The functional role of flanking residues was experimentally confirmed by alanine scanning of the imine-reducing SDR from Zephyranthes treatiae. Mutating the "gatekeeping" phenylalanine at standard position 200 resulted in a tenfold increase in imine-reducing activity.

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the "classical" SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.

The Short-chain Dehydrogenase/Reductase Engineering Database (SDRED): A classification and analysis system for a highly diverse enzyme family.

The Short-chain Dehydrogenases/Reductases Engineering Database (SDRED) covers one of the largest known protein families (168 150 proteins). Assignment to the superfamilies of Classical and Extended SDRs was achieved by global sequence similarity and by identification of family-specific sequence motifs. Two standard numbering schemes were established for Classical and Extended SDRs that allow for the determination of conserved amino acid residues, such as cofactor specificity determining positions or superfamily specific sequence motifs. The comprehensive sequence dataset of the SDRED facilitates the refinement of family-specific sequence motifs. The glycine-rich motifs for Classical and Extended SDRs were refined to improve the precision of superfamily classification. In each superfamily, the majority of sequences formed a tightly connected sequence network and belonged to a large homologous family. Despite their different sequence motifs and their different sequence length, the two sequence networks of Classical and Extended SDRs are not separate, but connected by edges at a threshold of 40% sequence similarity, indicating that all SDRs belong to a large, connected network. The SDRED is accessible at https://sdred.biocatnet.de/.