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

Abstract

Verticillins are a group of epipolythiodioxopiperazine alkaloids that have displayed potent cytotoxicity. To evaluate their potential further, a larger supply of these compounds was needed for both in vivo studies and analogue development via semisynthesis. To optimize the biosynthesis of these secondary metabolites, their production was analyzed in two different fungal strains (MSX59553 and MSX79542) under a suite of fermentation conditions. These studies were facilitated by the use of the droplet-liquid microjunction-surface sampling probe (droplet probe), which enables chemical analysis in situ directly from the surface of the cultures. These experiments showed that the production of verticillins was greatly affected by growth conditions; a significantly higher quantity of these alkaloids was noted when the fungal strains were grown on an oatmeal-based medium. Using these technologies to select the best among the tested growth conditions, the production of the verticillin analogues was increased while concomitantly decreasing the time required for fermentations from 5 weeks to about 11 days. Importantly, where we could previously supply 5–10 mg every 6 weeks, we are now able to supply 50–150 mg quantities of key analogues per month via laboratory scale fermentation.

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.…”

“…Solid state-fermentation was used for scaling-up production of 1 in the laboratory because it is less expensive than pure liquid broth medium and often yields higher amounts of the fungal extract [36]. Previous studies on the scaled production of fungal secondary metabolites have also successfully used solid-state fermentation [29,37].…”

“…We also designed a new method of extraction and purification that was superior to the methodology employed when 1 was isolated previously [2], taking advantage of the economies of scale. Modern instrumentation was used to evaluate the biosynthesis of secondary metabolites, specifically the droplet probe [16], so as to rapidly evaluate in situ different solid media to determine the most appropriate nutrient media for seed culture, thus optimizing fermentation conditions for laboratory-scale production of the target compound; this is the second example of the value of this tool for this purpose [29]. The benefits obtained from using the improved methods are summarized (▶ Figs.…”

“…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].…”

Droplet probe: coupling chromatography to the in situ evaluation of the chemistry of nature

“…One of the more prominent examples of using the droplet probe to scout for optimized fermentation conditions comes via our studies on verticillins. 87 This class of compounds (epipolythiodioxopiperazine alkaloids) has been in the literature since the early 1970s. 88 While they have been studied by prominent organic chemists, 89–91 a scalable total synthesis has yet to be reported.…”