We have uncovered a single transcript (>14 Kb) with three consecutive KS modules, that are conserved across three species of dinoflagellates (Karlodinium veneficum and Amphidinium carterae, have known toxins, and Akashiwo sanguinea, which is non-toxic), as well as many single module KS subunits using RNA sequencing analysis. When comparing the three species, the acyl transferase (AT) subunit in the triple module KS was present only in the non-toxic species and missing in the toxin producing species. To test the functionality of PKS in these species, we added cerulenin, an inhibitor that covalently binds to the KS subunits (Funabashi et al, 1989), to an exponential phase culture of A. carterae and A. sanguinea. Using 14C labeled acetate and liquid chromatography mass spectrometry (LC/MS), we found that cerulenin inhibition resulted in the reduction of both fatty acid and toxin production. This shows KS plays a role in both fat and toxin synthesis leading us to our hypothesis: The triple module KS acts as a scaffold for both toxin and fatty acid production where the final product is mediated by accessory trans-AT subunits, which exist in parallel with the KS. To substantiate that the triple KS transcript produces a protein, we have generated an antibody to an epitope in the TE domain. We clearly have evidence that a partial multi-module protein (~285,000 daltons) is expressed and is predicted to catalyze two carbon additions to a growing fatty acid/polyketide chain. However, an antibody to KS1 domain found a discrete protein containing the KS1 and KS2 domains but not the KS3 domain indicating post-translational processing. We are currently investigating the interacting partners with these two protein scaffolds.
Place et al. (Fri,) studied this question.