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

Karolina Subko,Francisca Vicente,Frederik H Nielsen,Charlotte H Gotfredsen,Thomas Isbrandt,Xinhui Wang,Olga Genilloud,Jens C Frisvad,Thomas O Larsen,Thomas Mackenzie,Maria C Ramos

doi:10.3389/fmicb.2020.618730

Karolina Subko, Francisca Vicente + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2020.618730

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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.

Highlights

Asperphenamate (1) is a linear amino acid (AA) ester, comprised of N-benzoylphenylalanine (2) and N-benzoylphenylalaninol (3)
To investigate the secondary metabolite profile from P. astrolabium, the fungus was inoculated on three media (MEA, Czapek yeast agar (CYA), and yeast extract sucrose agar (YES)) and incubated at 20 and 25◦C for 7, 10, and 14 days
We report that the choice of complex growth medium and/or a simple growth media supplement with selected building blocks, such as proteogenic and non-proteogenic AAs, P. astrolabium and related species can produce a series of novel asperphenamate analogs, which can be readily characterized by HRMS/MS

Summary

Introduction

Asperphenamate (1) is a linear amino acid (AA) ester, comprised of N-benzoylphenylalanine (2) and N-benzoylphenylalaninol (3). The compound has been isolated in trace amounts from a number of unrelated plant species (Wu et al, 2004; Dang et al, 2014; Zhou et al, 2017; Bunteang et al, 2018; Caridade et al, 2018), suggesting endophytic fungi being the actual producers, rather than the plants. A handful of new natural analogs have been isolated, namely Asperphenamates B (4) and C (5) (Liu et al, 2018), and 4-OMe-asperphenamate (Zheng et al, 2013; Ratnaweera et al, 2016) (6) from filamentous fungi. Other analogs containing partial structural similarities include: patriscabratine (7), a N-benzoylphenylalanine phenylalanynol acetate ester, aurantiamide (8) and aurantiamide acetate (9) (Zhou et al, 2017), N-benzoylphenylalanine phenylalanynol and phenylalanynol acetate amides, all isolated from plant material; cordyceamides A (10) and B (11) (Jia et al, 2009), a N-benzoyl-L-tyrosinyl-L-phenylalaninol and N-benzoylL-tyrosinyl-L-p-hydroxyphenylalaninol acetates, from an insect pathogen fungus (Figure 1); along with a number of tentatively identified related metabolites (Kildgaard et al, 2014; Sica et al, 2016)

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