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Fungal Taxol acquired lots of attention in the last few decades mainly because of the hope that fungi could be manipulated more easily than yew trees to scale up the production level of this valuable anticancer drug. Several researchers have studied diverse factors to enhance fungal Taxol production. However, up to date fungal Taxol production has never been enhanced to the commercial level. We have hypothesized that optimization of fungal Taxol production may require clear understanding of the fungal habitat in its original host plant. One major feature shared by all fungal endophytes is that they are located in the internal plant tissues where darkness is prominent; hence here the effect of light on fungal Taxol production was tested. Incubation of Taxol-producing endophytic SSM001 fungus in light prior to inoculation in Taxol production culture media showed dramatic loss of Taxol accumulation, significant reduction in Taxol-containing resin bodies and reduction in the expression of genes known to be involved in Taxol biosynthesis. The loss of Taxol production was accompanied by production of dark green pigments. Pigmentation is a fungal protection mechanism which is photoreceptor mediated and induced by light. Opsin, a known photoreceptor involved in light perception and pigment production, was identified in SSM001 by genome sequencing. SSM001 opsin gene expression was induced by white light. The results from this study indicated that the endophytic fungus SSM001 required the dark habitat of its host plant for Taxol production and hence this biosynthetic pathway shows a negative response to light.
Taxol production has been reported from several endophytic fungi (Zhou et al., 2010); however, the mechanism and the factors affecting its production are rarely investigated. Recently, Soliman et al. (2015) demonstrated that Taxol is sequestered in resin bodies to protect plant cells from the cytotoxic effect of Taxol during transport from the site of biosynthesis to the site of storage. Furthermore, the Taxol-containing bodies are transported through the parenchyma rays to the outer tissues, the bark, in order to block pathogen invasion when plant crack openings occur (Soliman et al., 2015). On the other hand, fungal Taxol production is enhanced when cultured in conditions that mimic the internal plant habitat such as crude plant metabolite extracts and plant defensive substances (Soliman and Raizada, 2013).
Taxol-producing endophytic fungi are unstable and may lose Taxol production activity after certain generations (Soliman et al., 2011; Venugopalan and Srivastava, 2015). Several factors have been studied in order to enhance fungal Taxol production (Metz et al., 2000; Flores-Bustamante et al., 2010; Somjaipeng et al., 2016; Qiao et al., 2017). However, the major factor responsible for the loss of Taxol production activity by fungi has never been investigated. Furthermore, the fungal original habitat as a factor that can affect fungal Taxol production is rarely studied.
In general, the shared characteristic of all fungal endophytes is their prominent dark habitat in the internal host plant tissues. Furthermore, light is considered as a crucial factor in fungal metabolite production since the fungal master regulator, LaeA/VeA (Bok and Keller, 2004; Sarikaya Bayram et al., 2010; Kim et al., 2013), is mainly controlled by light (Bayram et al., 2008). Here in this study, the effect of white light on fungal Taxol production was tested. Isolation of the fungal endophyte followed by immediate light treatment significantly affected later Taxol production.
Previously isolated Taxol-producing endophytic fungus (Paraconiothyrium SSM001 spp.) from Taxus media plants cultivated on the University of Guelph Main Campus and Arboretum (Guelph, ON, Canada) was used in this study. The fungal genotyping was performed using internal transcribed spacer regions (ITS) sequence of 18S rDNA as previously described in Soliman et al. (2011).
Purified fungal tips were transferred onto fresh PDA plates at 25C and incubated for 1 week either in full darkness or full light (white fluorescent light, 300 μmol m-2 s-1 at plate level) (Fett-Neto et al., 1995) prior to inoculation into 500 mL liquid YPD broth in 2 L flasks. In parallel, fungal hyphal tips were inoculated onto microscopic slides covered with a thin film of PDA and allowed to grow in full light for different time periods (3, 5, 6, and 7 days) prior to inoculation for fungal Taxol production and detection. Control fungal cultures fully grown in darkness were employed. Furthermore, a small hyphae tip was inoculated onto PDA plates and completely covered with aluminum foil except a small wedge was exposed to full light for 1 week at 25C prior to inoculation into liquid YPD broth culture.
Fungal tips from 1-week old pure PDA plate fungal cultures were used for production and extraction of fungal taxanes as described previously (Stierle et al., 1993; Soliman et al., 2011). Briefly, 1 mg of 1-week-old fungal tips was inoculated into 500 mL YPD broth in 2 L Erlenmeyer flasks and incubated in darkness at room temperature for 21 days. The culture was filtered and the filtrate was extracted with chloroform: methanol (9:1 v/v). The organic layer was separated, washed, and evaporated until dried. The residue was dissolved in 30 μL methanol, and 10 μl was spotted onto TLC silica gel plates (10 cm 20 cm, Fisher Scientific #4861-320) alongside Taxol standard at a concentration of 10 μg/mL. TLC plates were then developed in chloroform/methanol (5.0:0.5) and visualized with 0.5% vanillin/sulfuric acid reagent. Fungal Taxol in the extracted liquid media was identified as previously described by Soliman et al. (2011). For Taxol quantification, 10 μL of the total extract was injected into HPLC according to Soliman et al. (2011, 2013). The peak area of each sample injected was measured by a UV detector at 233 nm and then factored against a calibration curve generated from injecting different Taxol concentrations.
Fungal hyphal tips were transferred onto microscopic slides containing a thin film of PDA media under aseptic conditions and were left to grow for 1 week prior to microscopic examination. Fungal hyphae on the microscopic slides were stained for 1 h with Sudan IV in 50% ethanol followed by light microscopy.
Culturing purified SSM001fungus onto PDA plates under light exposure for 1 week caused production of dark green pigments covering the fungal hyphae compared to pigment-free fungus when grown in darkness (Figure 1A). Post-inoculation of hyphae tips from each fungal growth source into YPD broth showed production of Taxol only from the fungus previously grown in darkness (D) compared to undetectable Taxol from the culture grown in light (L). Fungal Taxol detection was performed on TLC plates (Figure 1B) and by HPLC (Figure 1C).
Light pre-incubation inhibited fungal Taxol production. (A) Growth of Taxol-producing endophyte SSM001 fungus in light (L) and darkness (D) on PDA at 25C for 1 week. (B) Detection of extracted fungal Taxol after inoculation for 3 weeks in liquid YPD broth on TLC-silica plates (10 μL sample, developing system chloroform: methanol; 5:0.5 and visualized using 0.5% vanillin/sulfuric acid reagent). (C) Detection and quantification of fungal Taxol by HPLC-UV when fortified with 5 ng standard Taxol. The peak area of each sample (10 μL injection volume) was measured at 233 nm. The quantification data display the mean of three replicates. The asterisk is the diagnostic peak of Taxol.
Growth of fungal hyphae at different light durations was accompanied by the production of different levels of dark green pigments based on the duration of light exposure (Figure 2A). On the other hand, duration of light exposure was inversely correlated with accumulation of resin bodies (Figure 2B) and fungal Taxol production (Figure 3). Fungal Taxol is known to be localized to resin bodies to protect plant cells from the cytotoxic effects of Taxol (Soliman et al., 2015).
Fungus exposed to different durations of light affects resin body production. (A) Incubation of fungal hyphae tips on microscopic slides covered with a thin film of PDA exposed to different light durations compared to dark incubation for 1 week at 25C. (B) Fungal growth from A stained with Sudan IV for 1 h prior to detection of resin bodies (known as sequestering organelles for fungal Taxol) visualized using a light microscope.
Dark-incubated versus light-incubated fungal hyphae tips. (A) Incubation of a fungal tip under both light and darkness caused production of dark pigments only from the place exposed to white light. (B,C) Detection of fungal Taxol inoculated from different locations of the same fungal hyphae on (B) a TLC-silica plate and by (C) HPLC. HPLC quantification of fungal Taxol is represented in a column graph showing the mean standard error of the mean. (D) Gene expression of opsin, HMGR and DHQ genes in both complete light (location # 4) and complete darkness (location #1). The asteriks denote a significant change in mean expression (at P < 0.05).
The results from this study demonstrate that light exposure of Taxol-producing endophytic SSM001 fungus is accompanied by the production of dark green pigments; and causes complete loss of Taxol production. Previously, it was reported that light stimulates the fungal production of specific plant-associated biomolecules in particular pigments and chlorophyll (Fang and Bidochka, 2006). Furthermore, light-grown Taxus suspension cultures showed significant reductions in Taxol production compared to those grown in dark (Fett-Neto et al., 1995). In another report, light inhibited nicotine production from tobacco plant tissues, and the inhibitory effect increased upon increased intensity and length of exposure (Ohta and Yatazawa, 1978).
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