Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella

Abstract

Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron spp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium.

Fig. 1.

Peptides and symbionts from L. patella. (Upper) Single cell of P. didemni (Right) isolated from the ascidian L. patella (Left) (photograph by Chris Ireland, University of Utah). The green pockets near the surface of L. patella are monocultures of P. didemni. (Lower) Patellamides A and C.

Fig. 2.

PatE sequence. Italic type, the conserved leader sequence; bold type, the proposed start and stop cyclization sequences; underlined type, product CDSs. Sequences corresponding to patellamide C (Upper) and A (Lower) are aligned for clarity.

Fig. 3.

The pat gene cluster (A) and GC skew (B). Colored genes represent those that can have a function assigned. White genes are those that have no significant homolog; blue genes contains protease activity. The G+C% skew below is altered where a coding region is present, as is common in many species and suggests that the gene predictions are correct (53). Additionally, the increase of the G+C% in this area suggests that this region may have been transferred into this species by means of horizontal gene transfer.

Fig. 4.

Proof of function of the pat cluster. (A) Standard from 25 ml of culture broth containing 20 μg of patellamides under SRM conditions observing m/z = 725 (patellamide A daughter ion). (B) Two-liter sample of pCR2.1-pat no. 9 under SRM conditions for m/z = 725. (C) Blind control: SRM using a sample identical to that shown in B except that empty pCR2.1 vector was used. y-axis scales are in units of relative abundance (0-100%).

Fig. 5.

Proposed pathway to patellamides showing the route to patellamide A. Timing of epimerization of alanine remains unclear; it may take place in tandem with thiazole formation (PatD2-catalyzed), at another biosynthetic step, or nonenzymatically (see Results and Discussion).