Optimizing production and evaluating biosynthesis in situ of a herbicidal compound, mevalocidin, from Coniolariella sp.

Abstract

Mevalocidin is a fungal secondary metabolite produced by Coniolariella sp. It is a unique phytotoxin that demonstrates broad spectrum post-emergent herbicidal properties. With limited options for weed control, the commercialization of a natural product pesticide would be beneficial to organic farming. In this study, two mevalocidin-producing fungal strains, coded MSX56446 and MSX92917, were explored under a variety of growth conditions, including time, temperature, and media. The concentration of mevalocidin was quantitatively measured via LC-MS to determine the optimal setting for each condition. Maximum production was achieved for each condition at 20 days, at 30 °C, with YESD + agar, and with a media containing 2.5 % dextrose. Furthermore, an advanced surface sampling technique was incorporated to gain a better understanding of the fungal culture's natural ability to biosynthesize and distribute this herbicide into its environment. It was shown that both fungi actively exude mevalocidin into their environment via liquid droplet formations known as guttates.

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

“…3). Guttates, which we and others hypothesize concentrate secondary metabolites [25, 34, 66, 75], were noted when these were grown on Oat-A, YESDA, and PD-mushroom agar (Fig. S6), but not found on the fungi grown on the other nutrient media.…”

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.…”