Production of fungal hypocrellin photosensitizers: Exploiting bambusicolous fungi and elicitation strategies in mycelium cultures

Abstract

Hypocrellins, a group of naturally occurring perylenequinone pigments produced by Shiraia bambusicola, are notable for their potential use in photodynamic therapy (PDT) for treating cancers and viruses. Traditionally, hypocrellins have been extracted from the fruiting bodies of S. bambusicola, a parasitic fungus on bamboo. However, the yield from wild Shiraia fruiting bodies is often insufficient, prompting a shift towards seeking other fungi with higher yields of hypocrellins as alternative sources. This review comprehensively examines the current research on the isolation, identification, and bioactivity of fungal perylenequinones from Shiraia isolates from ascostromata or fruiting bodies, Shiraia-like endophytes, and other endophytes from bamboos. Additionally, the review discusses the culture methods and conditions for solid-state and submerged fermentation of hypocrellin-producing fungi, including medium components, culture conditions, and optimisation of fermentation factors, as mycelium cultures have emerged as a promising alternative for the production of hypocrellins. Furthermore, novel elicitation strategies are presented to address the bottleneck of lower production of hypocrellins in mycelium cultures, focusing on the preparation, characterisation, and application of biotic and abiotic elicitors. This review aims to facilitate further exploration and utilisation of fungal resources and elicitation strategies for enhanced production of hypocrellins in mycelium cultures.

Keywords: Shiraia; elicitation; endophytes; hypocrellins; mycelium culture.

Graphical abstract

Figure 1.

The chemical structure of hypocrellin and its derivatives.

Figure 2.

Shiraia bambusicola and the color reaction of hypocrellin pigments. (A) The pinkish Shiraia ascostromata on living bamboo branches. (B) The pigment acetone extract with addition of sodium hydroxide solution (1), hydrochloric acid solution (2), and FeCl₃ solution at 1 mol/L (3), respectively. (C, D) Top view of non-hypocrellin producing strain (C) and hypocrellin-producing strain of S. bambusicola (D).

Figure 3.

Maximum likelihood phylogenetic tree for hypocrellin-producing fungi generated from MEGA11, based on ITS sequences data. Confidence values above 50% obtained from a 1,000-replicate bootstrap analysis are indicated at the branch nodes. The scale bar indicates the number of estimated substitutions per site. Pleospora herbarum (CBS 191.86) was used as outgroup for rooting the tree. GenBank accession numbers are given parentheses.

Figure 4.

The fungal pellet formation and perylenequinone production in liquid culture of Shiraia bambusicola. (A) The culture was maintained in 150-mL flask containing 50 mL of liquid medium at 150 r/min and 28 °C. (B) Morphology (100 ×) of the pellet during the cultivation. (C) The chromatogram of individual perylenequinone in the mycelium. The figure was redrawn based on findings from our previous study (Wang et al. 2024).

Figure 5.

The effects of live Pseudomonas fulva SB1 on the growth and hypocrellins production of Shiraia sp. S9. (A) Scheme of the in vitro confrontation assay. A small piece (5 mm × 5 mm) of the fungal strain was placed in the center of 10-cm PDA plate at 28 °C for 4 d. The bacterial suspension (10 μL) was streaked in two parallel straight lines on PDA, approximately 7 cm apart from each other. (B) The liquid submerged culture of Shiraia sp. S9 with or without live SB1 treatment at 400 cells/mL on day 6. (C) Time profiles of HA content in mycelium and the released HA in cultural broth in the submerged culture. Values are mean ± SD from three independent experiments (**p < 0.01 versus control). The figure was redrawn based on findings from our previous study (Ma et al. 2019a, 2019b).

Figure 6.

The proposed structure of EPS-1 (n ≈ 37) (A) and the repeating unit of OPS (n ≈ 1,600; R = t-Rhap, t-Galp) (B). The figure was redrawn based on findings from our previous study (Zhou et al. ; Li et al. 2024).

Figure 7.

Effects of bacterial volatiles of Bacillus cereus No.1 on Shiraia HA production in the submerged volatile co-cultures. The mode diagrams (A) and the “donut” plate (B) for the bacterial volatiles and the fungus Shiraia sp. S9. The mode diagrams for the submerged volatile co-cultures (C). Two culture flasks were connected through sealed glass tube. The culture was maintained in 250-mL flask containing 100 mL of the liquid medium at 150 r/min and 28 °C for 8 d. An equal volume of sterile LB broth instead of bacterial suspension added to flask was used as control group. Effect of addition time for the bacteria on fungal HA content in mycelium or in medium during the submerged volatile co-cultures (D). Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (**, ^##p < 0.01 versus control, *, ^#p < 0.05). The figure was redrawn based on findings from our previous study (Xu et al. 2022).

Figure 8.

Distribution of hypocrellins in cloud point system. (A) Phase separation of Triton X-100 micelle aqueous solution. (B) Microscopic observation of the cloud point system stained with the oil soluble dye Sudan black B; a) dilute phase, oil-in-water emulsion (40×); b) coacervate phase, water-in-oil emulsion (40×). (C) The chromatogram of HA in cloud point system. The figure was redrawn based on findings from our previous study (Li et al. 2020).

Figure 9.

Effect of different wavelengths of light on fungal hypocrellin a (HA) production of Shiraia bambusicola S8 in solid-state cultures. (A) Fungus in PDA plate was kept at 28 °C for 8 d under different light treatments with LED lamps at 100 lx. (B) Fungal colony morphology in solid-state cultures under different light treatments. (C) HA content in solid state culture. Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (p < 0.05). The figure was redrawn based on findings from our previous study (Ma et al. 2019).