• A Comparison of Dinoflagellate Thiolation Domain Binding Proteins Using In Vitro and Molecular Methods.

      Williams, Ernest; Bachvaroff, Tsvetan; Place, Allen (2022-09-18)
      Dinoflagellates play important roles in ecosystems as primary producers and consumers making natural products that can benefit or harm environmental and human health but are also potential therapeutics with unique chemistries. Annotations of dinoflagellate genes have been hampered by large genomes with many gene copies that reduce the reliability of transcriptomics, quantitative PCR, and targeted knockouts. This study aimed to functionally characterize dinoflagellate proteins by testing their interactions through in vitro assays. Specifically, nine Amphidinium carterae thiolation domains that scaffold natural product synthesis were substituted into an indigoidine synthesizing gene from the bacterium Streptomyces lavendulae and exposed to three A. carterae phosphopantetheinyl transferases that activate synthesis. Unsurprisingly, several of the dinoflagellate versions inhibited the ability to synthesize indigoidine despite being successfully phosphopantetheinated. However, all the transferases were able to phosphopantetheinate all the thiolation domains nearly equally, defying the canon that transferases participate in segregated processes via binding specificity. Moreover, two of the transferases were expressed during growth in alternating patterns while the final transferase was only observed as a breakdown product common to all three. The broad substrate recognition and compensatory expression shown here help explain why phosphopantetheinyl transferases are lost throughout dinoflagellate evolution without a loss in a biochemical process.
    • Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter.

      Williams, Ernest; Bachvaroff, Tsvetan; Place, Allen (MDPI AG, 2022-03-23)
      Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward.