In principle, wild-type nucleoside phosphorylases are relatively promiscuous, enabling the preparation of nucleoside analogs that are otherwise challenging to access with chemical methods. Indeed, the literature harbors a range of examples, including the pharmaceutically relevant nucleosides cladribine, ribavirine, 48,49 vidarabine 37 and nelarabine. This inherent promiscuity may explain why comparably few studies exist which describe the engineering of nucleoside phosphorylases to broaden their substrate spectrum. In fact, structure-function studies on the human nucleoside phosphorylases (predominantly showing decreases in activity) currently account for the majority of literature examples. However, the limits of this natural promiscuity have thus far prevented these enzymes from becoming the go-to option for nucleoside synthesis. As such, accessing nucleoside targets outside the natural substrate spectrum currently requires either an extensive engineering campaign (as pursued by Merck in their synthesis of islatravir) or the serendipitous discovery of active homologs by screening (as exemplified by our work on the diversification of 4ꞌ-methylated nucleosides). Since the three-dimensional structure of nucleoside phosphorylases as well as all active site residues are highly conserved, it is typically very challenging to rationalize which amino acid exchanges could yield improved activities. As a result, engineering efforts of nucleoside phosphorylases currently rely on random mutations introduced by error-prone PCR or extensive screening of single-site mutation libraries. For instance, Nannemann et al.'s engineering of the human purine nucleoside phosphorylase for the synthesis of dideoxy nucleosides provided a more than ten-fold improved variant over four rounds of semi-rational engineering and evolution. In the impressive examples mentioned above, Merck achieved an 80-fold improvement of activity of E. coli uridine phosphorylase for the production of 5-isobutyryl uridine en route to molnupiravir 21 and a approx. 1000-fold improvement of activity of E. coli purine nucleoside phosphorylase for the synthesis of islatravir. In both studies, extensive screening of single-site mutation libraries and their combination provided highly active enzyme variants over four to five rounds of evolution. While it is unclear if similar engineering campaigns could expand the promiscuity of nucleoside phosphorylases to provide true "generalists", we expect that recent advances in sequencing technology and the advent of reliable computational methods for enzyme design will accelerate the expansion of the substrate scope of these enzymes.