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.

Keywords: Epipolythiodioxopiperazine alkaloids; Fermentation; Filamentous fungi; In situ extraction; Verticillin.

Fig. 1

Structures of verticillin A and related analogues. Compounds 1–5 were the target for the biosynthetic optimization studies

Fig. 2

Relative production of various verticillin analogues grown on rice for 4 weeks across ten different fungal strains as measured by LC-HRMS in three replicates. Strains MSX59553 and MSX79542 demonstrated the highest relative biosynthesis of the targeted verticillin analogues (compounds 1–5). Interestingly, specific strains (such as MSX75296 and MSX75281) showed the biosynthesis of higher amounts of verticillin D (8), corresponding to low to minimal production of the other analogues

Fig. 3

Top: Photos of strains MSX59553 (top) and MSX79542 (bottom). These fungal cultures were grown on SDA, PD-mushroom, Oat-A, SNA and PDA(A)/MEA(B)/YESDA(C) (from left to right, respectively) for 4 weeks. Fungal growths were morphologically distinct based on media, and guttates were observed only on cultures grown on PD-mushroom and Oat-A. Bottom: Relative biosynthesis of verticillin analogues via droplet probe directly from the surface of the strains MSX59553 (bottom left) and MSX79542 (bottom right)

Fig. 4

Relative production of verticillin analogues via droplet probe directly from the surface of strains MSX59553 (a) and MSX79542 (b) grown for 4 weeks. Oatmeal agar showed a higher amount of analogues, likely due to the formation of guttates on the surface of the culture. The same cultures grown on the Petri dishes, and used previously for the droplet probe analysis, were fully extracted and then analyzed via UPLC-HRMS to generate graphs (c) and (d), which represent the relative total production of verticillin analogues by strains MSX59553 and MSX79542, respectively. The relative percentages were normalized by multiplying the peak areas by the weight of their corresponding organic extracts (Fig S2 Supplementary information). The results from c and d demonstrate that oatmeal was the best media for total production of verticillin analogues

Fig. 5

The amounts of the secondary metabolites of interest in mg present in each culture of strains MSX59553 and MSX79542 grown on rice or oatmeal media for 4 weeks in three biological replicates (a). The quantity of Sch52901 (2), the verticillin analogue produced in the highest amount by the fungal strains, was calculated per flask. The data plotted are mean ± SD of three biological replicates per medium and per strain. The quantity of 2 produced by strain MSX59553 grown on oatmeal showed a significant difference relative to the same strain grown on rice and also compared to strain MSX79542 grown on oatmeal and rice, respectively (**P < 0.004, ***P < 0.0003) (b). Similarly, the sum of all verticillin analogues showed a significant difference between the oatmeal culture of strain MSX59553 and the other growth conditions (**P < 0.005, ***P < 0.0003) (c)

Fig. 6

Amount of verticillin A (mg) produced by MSX59553 per flask (with 10 g of oatmeal) during a period of 35 days. The cultures were extracted and then analyzed using UPLC-HRMS. The fungus showed the most production from the 7th to 22nd days of culture before the start of a decline. These data are mean ± SD of two biological replicates, each analyzed in triplicate (n = 6)