Elucidating the final step of domoic acid biosynthesis

Pseudo-nitzschia is a cosmopolitan diatom genus that lives in coastal waters off the coast of California and worldwide. Blooms of Pseudo-nitzschia often produce domoic acid (DA), a potent neurotoxin that accumulates in shellfish and can cause serious illnesses in humans and marine mammals. DA biosynthesis was recently decoded by our group (Brunson et al 2018). The group established a cluster of of transcriptionally co-regulated genes (dabA, dabB, dabC, dabD) in several toxic Pseudo-nitzschia species that encode enzymes that perform each step of the biosynthesis of DA. This project aims to determine the final missing step of the biosynthesis in which isodomoic acid is isomerized to domoic acid. Intermediates to domoic acid that are synthesized as labeled with a ¹⁵N and deuterium were fed to live diatom cultures of a high domoic acid producer Pseudo-nitzschia australis, a low domoic acid producer Pseudo-nitzschia pungens, and a non-domoic acid producer Pseudo-nitzschia delicatissima to compare production of labeled intermediates and labeled domoic acid. Results that [15N,D]-L-NGG could be converted into [15N,D]-Domoic Acid were confirmed using LC-MS, achieving higher yields of the labeled domoic acid than reported in previous work (Maeno et al., 2018). A similar feeding experiment will be performed for four other labeled intermediates that have been synthesized by our group: 7’- hydroxy-L-NGG, carboxy-L-NGG, dainic acid A, and isodomoic acid A in the three species described.