Origin and variation of tunicate secondary metabolites

Abstract

Ascidians (tunicates) are rich sources of structurally elegant, pharmaceutically potent secondary metabolites and, more recently, potential biofuels. It has been demonstrated that some of these compounds are made by symbiotic bacteria and not by the animals themselves, and for a few other compounds evidence exists supporting a symbiotic origin. In didemnid ascidians, compounds are highly variable even in apparently identical animals. Recently, we have explained this variation at the genomic and metagenomic levels and have applied the basic scientific findings to drug discovery and development. This review discusses what is currently known about the origin and variation of symbiotically derived metabolites in ascidians, focusing on the family Didemnidae, where most research has occurred. Applications of our basic studies are also described.

Figure 1

Bioactive compounds isolated from didemnid ascidians. The natural products shown here have unique mechanisms of action against diverse targets, and some are potent to ~100 pM against cancer-relevant cell lines. (Note: trunkamide has been reported to be both the L- and D-Phe isomer from the natural source. We have found that the pathway naturally produces the L-Phe isomer, which slowly isomerizes to the D-form.)

Figure 2

Didemnid ascidians and their symbiotic cyanobacteria, P. didemni. (A) L. patella. (B) P. didemni.

Figure 3

Didemnid “ascidian” compounds made by symbiotic bacteria. (A) Cyclic peptides were demonstrated to originate in P. didemni. (B) Other compounds from P. didemni. (C) Compounds from other types of bacteria.

Figure 4

Genome level changes in P. didemni lead to a natural combinatorial library of bioactive natural products. (A) A map showing some of the samples examined, with each individual animal represented by a circle. Colors indicate some of the biosynthetic pathways identified in P. didemni. Pathways are basically identical when they are found, but the pathways are not universal. They are present sporadically, leading to mixtures of natural products. (B) Compounds made by the pathways shown in panel A. Chemical products have been identified for gaz, pat, and tru, but they have not yet been found and remain hypothetical for pyr and nkd.

Figure 5

Modifications in ribosomal peptide pathways. Natural genetic variations reveal that (A) identical cyanobactin enzymes accept extremely sequence-diverse substrates and (B) post-translational modifications can be moved between pathway types and are portable.

Figure 6

Proof-of-concept for biotechnological applications. Using cyanobactin biosynthetic pathways, we have demonstrated that direct metagenomic methods can be used (A) to supply bioactive natural products, (B) to discover new compounds in genomes and in tiny coral reef animals, and (C) to synthesize single analogues and combinatorial analogue libraries.

Figure 7

Enzymatic synthesis. Enzymes from cyanobactin pathways have been overexpressed and used in vitro for synthesis of (A) heterocycles, (B) macrocycles, and (C) prenylated amino acids.