Natural products containing vinylogous amino acids are rarely found in nature and often possess significant biological activity. Barnesin A was the first NP reported from an anaerobic bacterium (Sulfurospirillum barnesii) postulated to be biosynthesized by a nonribosomal peptide synthetase (NRPS) polyketide synthases (PKS) hybrid. Containing a vinylogous arginine moiety, the lipodipeptide exhibited nanomolar inhibitory activity against cysteine proteases. While a putative NRPS-PKS hybrid biosynthetic gene cluster (brn) was identified and a trans-acting acyltransferase (trans-AT) domain was postulated, experimental validation remained an open question. Here, we report the production of barnesin A by heterologous expression of the trans-AT domain-dependent NRPS-PKS gene cluster in Escherichia coli. Our findings indicate that the native primary metabolism-derived malonyl CoA-acyl carrier protein transacylase homolog (FabD) functions as a trans-AT in the biosynthesis pathway, while the NRPS-PKS megaenzyme exhibited strict selectivity toward its native phosphopantetheinyl transferase. Metabolome mining further allowed for the description of previously unreported barnesin congeners. The results of this study enabled the establishment of a biosynthetic platform for the generation of novel lipopeptidic vinylogous protease inhibitors.
Balluff et al. (Tue,) studied this question.