Biosynthesis of Macrolactam BE-14106 Involves Two Distinct PKS Systems and Amino Acid Processing Enzymes for Generation of the Aminoacyl Starter Unit

Abstract

BE-14106 is a macrocyclic lactam with an acyl side chain previously identified in a marine-derived Streptomyces sp. The gene cluster for BE-14106 biosynthesis was cloned from a Streptomyces strain newly isolated from marine sediments collected in the Trondheimsfjord (Norway). Bioinformatics and experimental analyses of the genes in the cluster suggested an unusual mechanism for assembly of the molecule. Biosynthesis of the aminoacyl starter apparently involves the concerted action of a distinct polyketide synthase (PKS) system and several enzymes that activate and process an amino acid. The resulting starter unit is loaded onto a second PKS complex, which completes the synthesis of the macrolactam ring. Gene inactivation experiments, enzyme assays with heterologously expressed proteins, and feeding studies supported the proposed model for the biosynthesis and provided new insights into the assembly of macrolactams with acyl side chain.

Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

“…Fermentations were performed with Applikon 3-liter fermentors with 1.5 liters of medium. The production cultures were inoculated with 3% (vol/vol) from a 0.5ϫ Trypticase soy broth (TSB)-glucose preculture cultivated as described previously (19). Fermentations were run for 6 days at 25°C with constant aeration (0.25% [vol/vol/ min]) and agitation (1,000 rpm).…”

“…High-performance liquid chromatography (HPLC) fractionation and subsequent liquid chromatography-mass spectrometry-time of flight (LC-MS-TOF) characterization of active fractions were performed as described earlier (19). Molecular masses (10-ppm window) were submitted to the online version of the Dictionary of Natural Products (DNP) in order to search for previously characterized compounds with bioactivity.…”

“…The pH was controlled at 7.3 with 2 M NaOH. Production of ML-449 in shake flasks was performed with 0.3ϫ BPS-3-1 supplemented with glucose as described previously (19). ML-449 was extracted from fermentation broth and purified by preparative HPLC essentially as described previously (19).…”

“…Quantitative and qualitative LC-MS analyses of ML-449 were performed on methanol extracts from culture pellets by using an Agilent 1100 HPLC system connected both to a diode array detector (DAD) and a TOF mass spectrometer. Electrospray ionization was performed in the negative (ESI Ϫ ) mode as described previously (4,19). Quantitative analysis of ML-449 in fermentation broth was performed using BE-14106 purified by preparative HPLC as a standard, assuming that the extinction coefficient of the two compounds is the same.…”

“…We have recently sequenced the biosynthetic gene cluster for the macrolactam BE-14106 ( Fig. 1) and proposed a biosynthetic pathway involving the synthesis of an aminoacyl starter through cooperation between a PKS and NRPS-related enzymes (19). Some of the enzymes presumed to be involved in the biosynthesis of BE-14106 have putative homologs in the other macrolactam gene clusters, suggesting common steps in the biosynthetic pathways.…”