Cyclodipeptide synthases (CDPSs) use aminoacylated tRNA (aatRNA) substrates to form peptide bonds between two amino acids yielding a cyclic dipeptide product (CDP). CDPs contain a diketopiperazine ring, a scaffold that has been coined as privileged due to its remarkable properties such as proteolytic resistance, blood-brain barrier permeability and the ability to mimic functional pharmacophores. CDPSs act in combination with tailoring enzymes, adding significant complexity to the types of natural products that can be produced. Because computational prediction of the specificity of CDPSs is challenging , the determination of substrates and products of each enzyme requires experimental testing, in a time consuming and low throughput process. Prior to this work there were no reports of successfully engineering CDPS enzymes to direct substrate selection.