Testing the transacylation site through mutations to KS surface residues
To assess the proposed transacylation site, PikKS6 surface residues were mutated within P1 -P6 -P7 , a triketide synthase composed of PikMod1, PikMod6, and PikMod7 (Figure 5b).24, 25 In the top solution for the association of PikACP6 and PikKS6, the PikKS6 surface residues N275, H281, L315, I319, Q322, D382, and D385 make favorable contacts with PikACP6, while Q217, S230, Q286, S300, R314, Q330, E379, S387, E392, and T395 make little or no contact. Each of these residues was individually mutated to alanine withinP1 -P6 -P7 .34 The prominent putative interface residues N275 and L315 were also individually mutated to aspartate and valine, respectively. As a control, a key residue at the condensation site, T140, was individually mutated to alanine.7, 8P1 -P6 -P7 and its 20 point mutants were expressed in Escherichia coli K207-3,35and triketide lactone production was measured by LC/MS after 8 days. The observed decreases in production are consistent with the predicted transacylation site.