INSIGHTS INTO DINOFLAGELLATE NATURAL PRODUCT SYNTHESIS VIA CATALYTIC DOMAIN INTERACTIONS

Abstract

Dinoflagellates are protists that can be split into two evolutionary groups, the parasitic syndinians and the largely photosynthetic “core” dinoflagellates. They represent a major portion of aquatic biomass which means that they are responsible for large portions of carbon that are both fixed and released. Other than biomass, the fixed carbon can be made into natural products such as polyunsaturated fatty acids that support the biota of many ecosystems or toxins that are harmful to aquatic life and humans. DNA and RNA analyses have been used to discover the putative genes that may make these compounds, but their non-colinear arrangement in the genome is very different from model organisms and their gene copy number is very high, making it nearly impossible to determine the exact biosynthetic pathways. The goal of my studies was to develop methods to differentiate biosynthetic pathways such as lipid and toxin synthesis by comparing the ability of domains to interact with each other with the assumption that domains that preferentially interact are more likely to participate in the same pathway. Initially, a survey was performed on available dinoflagellate transcriptomes to enumerate domains potentially involved in natural product synthesis and bin them based on sequence similarity to identify genes that could be used in biochemical assays. An interesting integration of analogous genes involved in lipid synthesis with those involved in natural product synthesis was observed as well as trends in domain expansion and contraction during core dinoflagellate evolution. Ultimately, the domain that scaffolds natural product synthesis, the thiolation domain, was chosen for further study because it exhibited two clear functional bins and is acted on directly by another enzyme, a phosphopantetheinyl transferase (PPTase). The PPTase activates the thiolation domain by transferring the phosphopantetheinate group from Coenzyme A to the thiolation domain, creating a free thiol group upon which the natural products are synthesized. These PPTases were then enumerated in dinoflagellates and characterized by looking for sequence motifs and observing expression patterns over a diel cycle as well as during growth in the model species Amphidinium carterae, a basal toxic dinoflagellate. Amphidinium carterae appears to have three PPTases, two of which (PPTase 1 and 2) are very similar, except that PPTase 2 does not appear to have a stop codon and has never been observed as a full-size protein. The remaining two PPTases (PPTase 1 and 3) had alternating expression patterns that did not appear to directly correlate to the acyl carrier protein, the thiolation domain required specifically for lipid biosynthesis. This carrier protein, like other enzymes for natural product synthesis in dinoflagellates, had a chloroplast targeting sequence while the three PPTases did not. To investigate the ability of these three PPTases to activate various thiolation domains, a total of 8 domains from A. carterae were substituted into the blue pigment synthesizing gene BpsA from Streptomyces lavendulae. These recombinant constructs were used for coexpression in E. coli as well as in vitro to reduce as many artifacts as possible and assess the interactions of each PPTase with the thiolation domains. Some of the recombinant BpsA genes were able to make blue dye with all three PPTases, while others never made blue dye both in E. coli as well as in vitro. In vitro quantification of free thiol added by the PPTase showed that all the thiolation domains, as well as the acyl carrier protein could be phosphopantetheinated by all the PPTases. This generalist substrate recognition, along with the alternating expression patterns and lack of chloroplast signaling peptide, indicate that the two active PPTases are performing the same function on all available thiolation domains, probably before export to the chloroplast. This lack of pathway segregation by PPTases is a completely novel way of synthesizing natural products compared to bacteria and fungi, likely due to the acquisition of both photosynthesis and natural product/lipid biosynthesis during dinoflagellate evolution that was not present in the common ancestor. Additionally, the techniques to identify genes of interest and perform biochemical characterization developed here are useful for future experiments annotating the function of dinoflagellate genes.