The last decade has seen a dramatic increase in the utilization of enzymes as green and sustainable (bio)catalysts in pharmaceutical and industrial applications. This trend has to a significant degree been fueled by advances in scientists' and engineers' ability to customize native enzymes by protein engineering. A review of the literature quickly reveals the tremendous success of this approach; protein engineering has generated enzyme variants with improved catalytic activity, broadened or altered substrate specificity, as well as raised or reversed stereoselectivity. Enzymes have been tailored to retain activity at elevated temperatures and to function in the presence of organic solvents, salts and pH values far from physiological conditions. However, readers unfamiliar with the field will soon encounter the confusingly large number of experimental techniques that have been employed to accomplish these engineering feats. Herein, we use history to guide a brief overview of the major strategies for protein engineering-past, present, and future.

The preparation of flavin mononucleotide (FMN) and FMN analogs from their corresponding riboflavin precursors is traditionally performed in a two-step procedure. After initial enzymatic conversion of riboflavin to flavin adenine dinucleotide (FAD) by a bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. To simplify the protocol, we have engineered the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain. The newly created biocatalyst is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogs to their corresponding FMN cofactors at preparative-scale.

The rapid advancement of sequencing technology has created an immense reservoir of protein sequence-function information that has yet to be fully utilized for fundamental or biocatalytic applications. For example, ene reductases from the "old yellow enzyme" (OYE) family catalyze the asymmetric hydrogenation of activated alkenes with enhanced stereoselectivity - key transformations for sustainable production of pharmaceutical and industrial synthons. Despite the proven biocatalytic application, the OYE family remains relatively underexplored with only 0.1% of identified members having any experimental characterization. Here, a platform of integrated bioinformatics and synthetic biology techniques was employed to systematically organize and screen the natural diversity of the OYE family. Using protein similarity networks, the known and unknown regions of the >115,000 members of the OYE family were broadly explored to identify phylogenetic and sequence-based trends. From this analysis, 118 novel enzymes were characterized across the family to broadly explore and expand the biocatalytic performance and substrate scope of known OYEs. Over a dozen novel enzymes were identified exhibiting enhanced catalytic activity or altered stereospecificity. Beyond well-established ene reduction, we detected widespread occurrence of oxidative chemistry amongst OYE family members at ambient conditions. Crystallography studies of selected OYEs yielded structures for two enzymes, contributing to a better understanding of their unique performance. Their structures revealed an unusual loop conformation within a novel OYE subclass. Overall, our study significantly expands the known functional and chemical diversity of OYEs while identifying superior biocatalysts for asymmetric reduction and oxidation.