The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemocatalysis and biocatalysis. Recent research has focused on the enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases), and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, because of the enzymes’ generally low turnover numbers (k cat < 0.1 s–1) and variable stability under reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former exhibiting an increased thermostability (melting temperature, T m) of 83.5 °C, compared to 56.5 °C for the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (c = 96%, ee (R) < 99%) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs, compared to their PheDH counterparts.

Imine reductases (IREDs) have shown great potential as catalysts for the asymmetric synthesis of industrially relevant chiral amines, but a limited understanding of sequence activity relationships makes rational engineering challenging. Here, we describe the characterization of 80 putative and 15 previously described IREDs across 10 different transformations and confirm that reductive amination catalysis is not limited to any particular subgroup or sequence motif. Furthermore, we have identified another dehydrogenase subgroup with chemoselectivity for imine reduction. Enantioselectivities were determined for the reduction of the model substrate 2-phenylpiperideine, and the effect of changing the reaction conditions was also studied for the reductive aminations of 1-indanone, acetophenone, and 4-methoxyphenylacetone. We have performed sequence-structure analysis to help explain clusters in activity across a phylogenetic tree and to inform rational engineering, which, in one case, has conferred a change in chemoselectivity that had not been previously observed.

The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets.

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein a number of guidelines and considerations toward the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resourceefficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch, it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L −1 h −1 and TON above 48 000 were achieved.

Successful screening of enzyme libraries in functional metagenomics and directed evolution becomes more likely after uniform cell growth in droplets amplifies genotype and phenotype.