UC Santa Cruz

Natural products are structurally optimized by evolution and are often complex and have a wide range of bioactivity. The organisms which produce these compounds use enzymatic machinery which can perform reactions beyond the scope of what is currently possible by purely synthetic routes. Studying these organisms and their natural products can inform scientists of novel biosynthetic mechanisms and potentially novel biocatalysts. The erythrazoles are produced by SNB-035, a Gram negative alphaproteobacterial species, and harbor unique biosynthetic assembly. These structural features include being derived from an unusual hybrid of the methylerythritol phosphate pathway and shikimate pathway, in addition to containing two amino acids. Additionally, erythrazole A and B contain a benzothiazole moiety and show a modified terpene. Their terpene chain ends in a carboxylic acid and erythrazole B has an unusual chain length of 22 carbons. How these molecules arise is of great interest to us as they have negligible biosynthetic precedent in literature. Additionally, these were some of the first metabolites ever discovered from this genus of bacteria and some of the very few known naturally occurring benzothiazoles.

We used isotopic labeling to label the amino acids of the erythrazoles which determined that the benzothiazole of erythrazoles A-B is derived from a ?-carbon cleavage of cysteine, showcasing the first known example of this mechanism. Stable isotope labeling studies also led us to discover that interconversion does not occur between erythrazoles A and B. This indicates that they arise separately and their terminal carboxylic acid is formed through oxidative cleavage rather than an acetate extension of erythrazole A to form erythrazole B, which we originally hypothesized was the source of the unusual C22 terpene of erythrazole B. We also used various methods to propose four putative biosynthetic gene clusters, which were investigated through transcriptional repression using CRISPRi. Though no gene cluster was identified, these experiments provided further insight into erythrazole biosynthesis and showed evidence for the relationship between the erythrazoles and ubiquinone. The work described in this dissertation shines light not only on the erythrazoles but on naturally occurring benzothiazoles.