The apicoplast ferredoxin (Fd) redox system of Apicomplexa and flavodoxin (fldA) redox system of bacteria serves as electron donors to aid the catalytic activity of enzymes of various metabolic pathways. One of the enzymes that benefit from the redox activity of both proteins in the respective organisms is the 4Fe-4S containing IspH, involved in the last step of the MEP pathway for isoprenoid precursor synthesis, an essential pathway for survival in both, Escherichia coli and apicomplexan parasites harboring an apicoplast. We are interested in knowing whether these two structurally very different redox proteins can replace each other and if it would allow us to use E. coli as a surrogate for the apicoplast for drug screening purposes.

We report the complementation of EcDIspH by Plasmodium falciparum and Toxoplasma gondii IspH (PfIspH and TgIspH). Using homologous recombination, we deleted the essential EcfldA and introduced the mevalonate by-pass pathway to make the E. coli double mutant strain entirely dependent on mevalonate. Removing mevalonate in a simple growth assay allowed us to further use this strain as a model to show that whereas Cyanobacteria (Nostoc) flavodoxin complements EcMP2, Chromera velia flavodoxin does not, raising interesting evolutionary questions. Strain EcMP2 is entirely dependent on the simultaneous expression of plasmid-encoded PfFd, PfFNR, PfIspH, which shows that the three parasite proteins are active and required to replace the respective endogenous E. coli proteins. We have also used the model to test for likely residues at the interaction interface between PfFd and PfIspH based on AlphaFold2 predictions and verified their importance for growth. Metabolomic studies are on the way to unravel the impact of the various complementation results on the MEP pathway. Altogether, we describe an engineered E. coli strain useful for functional and drug screening purposes for inhibitors of the essential MEP pathway. It will be very useful as a first screening platform for functional mutations or drug effects before more laborious and time-consuming assays in the parasite are performed.