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Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a
Ernest Williams1, Tsvetan Bachvaroff1, Allen Place1
1Institute of Marine and Environmental Technologies, University of Maryland Center for Environmental Science, 701 East Pratt St., Baltimore, MD 21202, USA.
Microorganisms
|April 23, 2022
Summary
Photosynthetic dinoflagellates produce natural products using complex pathways. This study explored protein interactions, finding unexpected flexibility but also technical challenges in identifying specific biosynthetic routes.
Area of Science:
- Biochemistry and Molecular Biology
- Natural Product Synthesis
- Marine Microbiology
Background:
- Photosynthetic dinoflagellates synthesize diverse compounds, including toxins and potential therapeutics, through polyketide/non-ribosomal peptide synthesis.
- Identifying these biosynthetic pathways in dinoflagellates is challenging due to gene complexity and fragmented genomic data.
- Thiolation domains and phosphopantetheinyl transferases (PPTases) are crucial for scaffolding and activating natural product synthesis.
Purpose of the Study:
- To investigate the specificity of interactions between dinoflagellate thiolation domains and PPTases.
- To identify distinct biosynthetic pathways by testing functional pairings of these key enzymes.
- To assess the feasibility of expressing catalytically active dinoflagellate proteins in *E. coli*.
Main Methods:
- Replaced the thiolation domain of the *Streptomyces lavendulae* BpsA gene with domains from three dinoflagellate transcripts.
- Coexpressed these constructs with three different dinoflagellate PPTases in *E. coli*.
- Analyzed the production of indigoidine to assess the functional activity of enzyme pairings.
Main Results:
- All three tested PPTases activated thiolation domains from one dinoflagellate transcript, indicating broad specificity.
- Significant differences in indigoidine production were observed among functional pairings.
- Thiolation domains from other transcripts and a lipid synthesis domain showed limited or no activity/expression.
Conclusions:
- Dinoflagellate PPTases and thiolation domains exhibit a surprising lack of specificity, complicating pathway elucidation.
- Technical hurdles, including inconsistent protein expression, were encountered.
- Expression of active dinoflagellate proteins in *E. coli* represents a valuable tool for future research despite challenges.
