New facets in the biosynthesis of the protein-transport inhibitor Brefeldin A

Brefeldin A is a natural product from the filamentous fungi Eupenicillium brefeldianum with a wide-range of bioactivities, including antiviral, anti fungal and cytotoxic against several cancer cell lines. However, it is most important for its inhibition of protein transport among eukaryotes, which makes it a useful tool in biological labs. This compound is a 16-membered macrolactone that contains an unusual cyclopentane ring. By sequencing the producing strain and performing bioinformatic analysis on the assembled genome, we have identified a candidate cluster containing the main biosynthetic enzymes, including a highly-reducing polyketide synthase (HR-PKS, herein referred to as BrefA), a hydrolase (BrefB) and several P450 monooxygenases. We investigated the activity of the megasynthase BrefA and the releasing enzyme BrefB both in vitro and in vivo (in a Saccharomyces cerevisiae host), and found that they produce four linear octaketide molecules differing in the degree of reduction of the polyketide scaffold, which substantiates the involvement of the PKS in creating the C-16 backbone of the molecule. We believe that these compounds are not precursors but are shunt products of the enzyme. These results illustrate that contrary to previous hypotheses, the cyclopentane ring of BFA needs to form first in order to bring the terminal hydroxyl closer to the ACP tether to facilitate the intramolecular cyclization. From these studies, we propose a pathway for the production of this important molecule that involves a P450-mediated ring cyclization and a lactonizing hydrolase, demonstrating the utility of biosynthetic studies in discovering enzymes with new functions.