Fig. 5: Overview of workflow and results of engineering PfDHFR for pyrimethamine resistance.

a Schematic of overall strategy for engineering pyrimethamine resistance in yeast. Modifications to YHM7 (top left) target TRIDENT mutations to the PfDHFR gene expression cassette. Replicates of YHM7 were grown in a 96-well plate containing pyrimethamine and inducer. Diversity generation and enrichment of beneficial mutations occur simultaneously as the cells grow to saturation. After reaching saturation, replicates are back-diluted into an increased amount of pyrimethamine until full resistance to the drug has been achieved. Pyrimethamine resistant samples were sequenced and results are shown in the table (bottom left). This schematic was created using Servier Medical Art images, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com. b PfDHFR (green) binding pocket crystal structure with bound pyrimethamine (light blue). The D54 residue on PfDHFR is shown in detail. The potential interactions between D54 and pyrimethamine are highlighted by distance measurements given in angstroms. c Distribution of all mutations found in PfDHFR gene when 177 experimental replicates were sequenced after five passages in pyrimethamine. Mutations of all types possible in the TRIDENT system were found. The distribution of base mutation types is different from other experiments using TRIDENT on other genes with different selection pressures (Figs. 3d and 4c) and reflects the dominance of the D54N mutation.