We set out to investigate the cyclodipeptide-synthesising capability of two cyclodipeptide synthases which accept histidine as a substrate. Our work uncovered that the use of a collective tRNA pool was sufficient for the CDPSs to yield their expected product in addition to accepting a variety of unnatural amino acids as substrates for CDP formation. This new method can easily be scaled up and has proved useful for generating cyclic dipeptides containing both canonical and non-canonical amino acids.