Mass Spectrometry Guided Discovery and Design of Novel Asperphenamate Analogs From Penicillium astrolabium Reveals an Extraordinary NRPS Flexibility

Abstract

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners

“…We therefore reason that the nicotinic acid containing myxochelin congeners 1 – 3 reported here are common byproducts of myxochelin A synthesis, wherein their formation is enabled by substrate promiscuity of the A domain MxcE and biogenic precursor availability. A similar production mode depending on availability of nicotinic acid as precursor has also been assumed for asperphenamate biosynthesis in Penicillium astrolabium [ 32 ].…”

“…Among the few known exceptions are a fungal polyketide synthase (PKS) that incorporates nicotinyl-CoA for meroterpenoid biosynthesis [ 33 ], a PKS from a marine mollusk that uses nicotinic acid as a starter unit to synthesize 3-alkylpyridine alkaloids [ 34 ], an NRPS-unit from Micromonospora sp. that incorporates nicotinic acid to produce kosinostatin [ 35 ], and a fungal NRPS that uses either benzoic acid or nicotinic acid for the biosynthesis of asperphenamates [ 32 ]. Furthermore, also in the biosynthesis of bacillibactins, the incorporation of nicotinic or benzoic acid instead of DHBA has been reported and assumed to happen due to a promiscuous DhbE [ 36 ], which has a pairwise identity of 67.7% (coverage 99.1%) to MxcE as well as the identical A domain Stachelhaus specificity-conferring code of PLPAQGVVNK ( Table S2 ).…”

Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity

“…The MS/MS spectra for each metabolite were compared to several databases (MarinLit, Dictionary of Natural Products (DNP), Natural Products Atlas (NPA), Scifinder and Chemspider) and to published data [ 46 , 47 , 48 , 49 ]. The main compounds produced by the two isolates affiliated to P. olsonii (UBOCC-A-118169 and UBOCC-A-119011) were assigned to xanthoepocin and asperphenamate.…”

“…Among the constellation of nodes, the putative subcluster of the main compound, assigned by comparing the obtained MS/MS data with that of the pure metabolite, contains 61 nodes and 161 edges ( Figure 4 ). In order to assign analogues, MS/MS spectra were compared to published data [ 49 ]. We used the asperphenamate analogue naming system proposed by Subko et al (2021).…”

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners

“…Among the few known exceptions are a fungal polyketide synthase (PKS) that incorporates nicotinyl-CoA for meroterpenoid biosynthesis [33], a PKS from a marine mollusk that uses nicotinic acid as a starter unit to synthesize 3-alkylpyridine alkaloids [34], an NRPS-unit from Micromonospora sp. that incorporates nicotinic acid to produce kosinostatin [35], and a fungal NRPS that uses either benzoic acid or nicotinic acid for the biosynthesis of asperphenamates [32]. Furthermore, also in the biosynthesis of bacillibactins, the incorporation of nicotinic or benzoic acid instead of DHBA has been reported and assumed to happen due to a promiscuous DhbE [36], which has a pairwise identity of 67.7% (coverage 99.1%) to MxcE as well as the identical A domain Stachelhaus specificityconferring code of PLPAQGVVNK (Table S2).…”