Having hindered the capacity of ParcuCDPS to recognize histidine, we then focussed on Generation 3 composed of three double mutants (Y55V+E174L; Y55V+Y189L; E174L+Y189L) and a triple mutant (Y55V+E174L+Y189L), aimed at switching the substrate specificity to a less polar amino acid. This was because Generation 1 and 2 variants essentially incorporate hydrophobic and non-polar amino acids potentially altering the overall environment and electrostatics of P1. Therefore, we hypothesised that whilst these enzymes were incapable of cHP production, they would tolerate a different, less charged substrate in P1. Previously we showed that CDPSs can use both tRNA and DBE substrates to yield a CDP and so using a combination of these, the production of cLP and cFP by ParcuCDPS mutants was investigated (Fig. and). Interestingly, reactions using Pro tRNAPro and Leu-DBE/Phe-DBE gave a higher yield of product compared to Pro tRNAPro and Leu tRNALeu/ Phe tRNAPhe. This indicates that the enzyme is still able to recognise and reject the tRNA body, which can be circumvented by using a smaller DBE substrate. It is evident that whilst wild-type ParcuCDPS is unable to use either leucine or phenylalanine as substrates, the new mutants can accept these amino acids. All double mutants were capable of producing cLP and cFP albeit with varying yields: 84% of the total products formed by Y55V+E174L was cLP, whilst for E174L+Y189L, 70% of products formed was cFP. These mutants, however, did not accept other small hydrophobic amino acids such as valine and isoleucine thus demonstrating that the enzyme is still actively selecting its substrates.