Previous research by us and others has shown that mutations in active site residues seriously reduce product formation in CDPS enzymes. However, attempts at changing the substrate scope of a CDPS by mutating select residues have been largely unsuccessful until now. Inspired by HisRS and histidine recognition more generally (Supplementary Figure ) we systematically altered three unique residues in ParcuCDPS P1, which we hypothesised to be participating in crucial interactions with the polar side chain of histidine. We first designed Generation 1 containing seven P1 mutants (Y55F; Y55V; E174A; E174H; E174L; Y189F and Y189L), and these variants were cloned, expressed in E.coli, purified and used for activity assays. The presence of the mutation was confirmed using intact protein mass spectrometry (Supplementary Figure ) and the enzymatic activity was confirmed using LC-MS as previously described. The active site mutants -S26A; S26C; D58A; D58N; Y167A; Y167F; E171A; and E171Q -were also generated to confirm the loss of activity upon removing catalytic residues. (Fig. ).