We describe a facile strategy for cyclic dipeptide production with superior yield and decreased cost/labour. Using canonical and non-canonical amino acids as substrates we generated a diverse library of unprecedented compounds (Supplementary Table ). We then used small molecule substrates as chemical probes to characterise substrate binding pockets P1, which is more stringent and deeper, and P2, which is shallower and more solvent exposed. To investigate histidine recognition on P1, we solved crystal structures of the cyclo(His-Pro)-producing enzyme, as well as of several mutants. We rationally engineered P1 to become more hydrophobic and deeper, steering the substrate specificity a, Enzymes characterised here, both use aminoacylated tRNAs as substrates to produce cyclic dipeptide. ParaCDPS from Parabacteroides sp. 20_3 (GenBank: EFK64745.1) produces cyclo(His-Phe), and ParcuCDPS from Parcubacteria bacterium RAAC4_OD1_1 (GenBank: ETB63777.1) can synthesise both cyclo(His-Glu) -not shown -and cyclo(His-Pro). b, strategy envisioned here, using a single enzyme catalyst to produce novel cyclodipeptide products; c, representative LC-HRMS spectra showing production of new molecules. Spectra for all cyclodipeptides described here are on Supplementary Figure and Supplementary Figure . away from histidine and towards more hydrophobic amino acids. These mutants displayed a remarkable shift in substrate specificity from the previously accepted histidine to two new substrates -leucine and phenylalanine. Therefore, our CDPS variants highlighted residues in P1 which are key for the recognition of histidine, unveiling important characteristics of how CDPSs select polar substrates. This has wide implications in our capacity to predict function as well as engineer CDPS enzymes to produce molecules of our choosing.