Spatial and Temporal Profiling of Griseofulvin Production in Xylaria cubensis Using Mass Spectrometry Mapping

Abstract

A large portion of natural products research revolves around the discovery of new, bioactive chemical entities; however, studies to probe the biological purpose of such secondary metabolites for the host organism are often limited. Mass spectrometry mapping of secondary metabolite biosynthesis in situ can be used to probe a series of ecological questions about fungi that may be lost through traditional natural products chemistry extraction protocols. A griseofulvin-producing fungal culture of the Xylariaceae family, isolated as an endophyte of the tree Asimina triloba, was analyzed through a series of spatial and temporal mapping experiments. This fungus produced unique fungal characteristics, such as guttates and stroma, both of which were explored spatially. The distribution of griseofulvin on this culture in isolation was compared to its dispersal when grown in co-culture with a competing Penicillium species via a droplet–based surface sampling system. The fungistatic properties of griseofulvin were visualized, including the consequences for biosynthesis of polyhydroxyanthraquinones in a rival culture.

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 autosampler was converted to a droplet probe with assistance from colleagues at Oak Ridge National Laboratories [38, 39].…”

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

“…D or L). Given the ability to study peptaibols by mass spectrometry, coupled with their interesting biological activities (Figueroa et al, 2013; He et al, 2006; Schiell et al, 2001; Thirumalachar, 1968), we sought to study fungal cultures in situ toward the discovery of peptaibols by using the droplet–LMJ–SSP, (Kertesz and Van Berkel, 2010; Paguigan et al, 2016; Sica et al, 2016b; Sica et al, 2015; Sica et al, 2016c). The fungal culture, coded MSX53554, which was identified as Nectriopsis Maire (Bionectriaceae), was readily noted as a peptaibol producer based on the mass spectral data (Figure 1).…”

“…Mass spectrometry monitoring of fungal secondary metabolites in situ can address important ecological information that could be lost via traditional extraction process (Sica et al, 2016b; Sica et al, 2015; Sica et al, 2016c). To better understand the fungal metabolites biosynthesized in a living axenic culture, we analyzed a culture of strain MSX53554 by the droplet–LMJ–SSP system.…”

“…These small peptides have been reported to possess a wide variety of bioactivities, such as antimicrobial (Figueroa et al, 2013), anthelmintic (Schiell et al, 2001; Thirumalachar, 1968), and cytotoxic (He et al, 2006). As part of a multidisciplinary program to identify new anticancer leads from nature (Ayers et al, 2012; Figueroa et al, 2013; Kinghorn et al, 2016), a protocol was implemented to monitor the chemistry of fungal cultures in situ using a recently described droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) coupled to an ultraperformance liquid chromatography–high resolution mass spectrometry (UPLC–HRMS) system (Kertesz and Van Berkel, 2010; Paguigan et al, 2016; Sica et al, 2016b; Sica et al, 2015; Sica et al, 2016c). …”

“…This system is designed to perform microextractions on the surface of a sample and directly inject the extract into LC-MS (Kertesz and Van Berkel, 2010, 2013, 2014). Recently, this technique has been applied towards in situ analyses of fungal and plant metabolites with various goals, such as dereplication of fungal cultures (Sica et al, 2015), spatial and temporal mapping of the biosynthesis of targeted leads in fungal cultures (Paguigan et al, 2016; Sica et al, 2016b; Sica et al, 2016c), and the characterization of complex mixtures in a suite of plant tissues (Sica et al, 2016a). A major benefit of this technique is the ability to monitor the biosynthesis of fungal secondary metabolites, both with respect to space and time, without having to extract the entire culture.…”