In situ mass spectrometry monitoring of fungal cultures led to the identification of four peptaibols with a rare threonine residue

Abstract

Peptaibols are an intriguing class of fungal metabolites due both to their wide range of reported bioactivities and to the structural variability that can be generated by the exchange of variable amino acid building blocks. In an effort to streamline the discovery of structurally diverse peptaibols, a mass spectrometry surface sampling technique was applied to screen the chemistry of fungal cultures in situ. Four previously undescribed peptaibols, all containing a rare threonine residue, were identified from a fungal culture (MSX53554), which was identified as Nectriopsis Maire (Bionectriaceae, Hypocreales, Ascomycota). These compounds not only increased the known threonine-containing peptaibols by nearly 20%, but also, the threonine residue was situated in a unique place compared to the other reported threonine-containing peptaibols. After the initial in situ detection and characterization, a large-scale solid fermentation culture was grown. The four peptaibols were isolated and characterized by mass spectrometry. In addition, one of the peptaibols was fully characterized by NMR and amino acid analysis using Marfey’s reagent and exhibited moderate in vitro anticancer activity.

Media studies to enhance the production of verticillins facilitated by in situ chemical analysis

“…The use of the droplet probe with fungal cultures has been detailed previously [ 67 ] and several examples have been published [ 57 , 65 , 66 , 68 , 69 ]. A CTC/LEAP HTC Pal auto-sampler was converted to a droplet probe with assistance from colleagues at Oak Ridge National Laboratories [ 38 , 39 ].…”

Prealamethicin F50 and related peptaibols from Trichoderma arundinaceum: validation of their authenticity via in situ chemical analysis

“…10, 19, 22 Briefly, approximately 0.2 mg of each amino acid standard was weighed into 2 mL reaction vials. To each standard was added 50 μl of H 2 O, 20 μl of 1 M NaHCO 3 , and 100 μl 1% Marfey’s reagent ( Nα -(2,4-dinitro-5-fluorophenyl)-L-alaninamide, Acros Organics) in acetone.…”

“…The droplet-LMJ-SSP is a manually controlled surface sampling tool that performs a microextraction on the culture surface. The droplet-LMJ-SSP has been used in various types of analysis of fungal cultures, including the in situ scouting and identification of peptaibols, 19 dereplication 18 and mapping of the spatial and temporal distribution of fungal metabolites, 20, 21 and to monitor the biosynthesis of targeted non-natural natural products. 22 …”

Drug Leads from Endophytic Fungi: Lessons Learned via Scaled Production

“…The droplet probe has been used to monitor the production of secondary metabolites in fungal cultures [25][26][27][28][29][30][31][32][33][34][35], with several notable examples highlighted in a recent review [16]. One benefit of this technique for this application was the ability to generate heat maps, showing in a relative manner where secondary metabolites are concentrated.…”

“…Monitoring the biosynthesis of secondary metabolites in agarbased fungal cultures in situ was performed using the droplet probe coupled with a Waters Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation) to a Thermo QExactive Plus (Thermo Fisher Scientific) via procedures described previously [16,[26][27][28][29] and reviewed recently [16]. This technique probes the chemistry of the cultures via a mini-extraction of about 2 µL of 1 : 1 MeOH : H 2 O, permitting the mapping of secondary metabolites in live fungal cultures [26][27][28]41].…”