Nitric oxide mediates red light-induced perylenequinone production in Shiraia mycelium culture.

Perylenequinones (PQs) from bambusicolous Shiraia fungi serve as excellent photosensitizers for photodynamic therapy. However, the lower yield of PQ production in mycelium cultures is an important bottleneck for their clinical application. Light has long been recognized as a pivotal regulatory signal for fungal secondary metabolite biosynthesis. In this study, we explored the role of nitric oxide (NO) in the growth and PQ biosynthesis in mycelium cultures of Shiraia sp. S9 exposed to red light. The continuous irradiation with red light (627 nm, 200 lx) suppressed fungal conidiation, promoted hyphal branching, and elicited a notable increase in PQ accumulation. Red light exposure induced NO generation, peaking to 81.7 µmol/g FW on day 8 of the culture, with the involvement of nitric oxide synthase (NOS)- or nitrate reductase (NR)-dependent pathways. The application of a NO donor sodium nitroprusside (SNP) restored conidiation of Shiraia sp. S9 under red light and stimulated PQ production, which was mitigated upon the introduction of NO scavenger carboxy-PTIO or soluble guanylate cyclase inhibitor NS-2028. These results showed that red light-induced NO, as a signaling molecule, was involved in the regulation of growth and PQ production in Shiraia sp. S9 through the NO-cGMP-PKG signaling pathway. While mycelial H2O2 content exhibited no significant alternations, a transient increase of intracellular Ca2+ and extracellular ATP (eATP) content was detected upon exposure to red light. The generation of NO was found to be interdependent on cytosolic Ca2+ and eATP concentration. These signal molecules cooperated synergistically to enhance membrane permeability and elevate the transcript levels of PQ biosynthetic genes in Shiraia sp. S9. Notably, the combined treatment of red light with 5 µM SNP yielded a synergistic effect, resulting in a substantially higher level of hypocrellin A (HA, 254 mg/L), about 3.0-fold over the dark control. Our findings provide valuable insights into the regulation of NO on fungal secondary metabolite biosynthesis and present a promising strategy involving the combined elicitation with SNP for enhanced production of photoactive PQs and other valuable secondary metabolites in fungi.

Bamboo polysaccharides elicit hypocrellin A biosynthesis of a bambusicolous fungus Shiraia sp. S9.

Hypocrellin A (HA), a fungal perylenequinone from bambusicolous Shiraia species, is a newly developed photosensitizer for photodynamic therapy in cancer and other infectious diseases. The lower yield of HA is an important bottleneck for its biomedical application. This study is the first report of the enhancement of HA production in mycelium culture of Shiraia sp. S9 by the polysaccharides from its host bamboo which serve as a strong elicitor. A purified bamboo polysaccharide (BPSE) with an average molecular weight of 34.2 kDa was found to be the most effective elicitor to enhance fungal HA production and characterized as a polysaccharide fraction mainly composed of arabinose and galactose (53.7: 36.9). When BPSE was added to the culture at 10 mg/L on day 3, the highest HA production of 422.8 mg/L was achieved on day 8, which was about 4.0-fold of the control. BPSE changed the gene expressions mainly responsible for central carbon metabolism and the cellular oxidative stress. The induced generation of H2O2 and nitric oxide was found to be involved in both the permeabilization of cell membrane and HA biosynthesis, leading to enhancements in both intra- and extracellular HA production. Our results indicated the roles of plant polysaccharides in host-fungal interactions and provided a new elicitation technique to improve fungal perylenequinone production in mycelium cultures.

Volatiles of Shiraia fruiting body-associated Pseudomonas putida No.24 stimulate fungal hypocrellin production.

Hypocrellins are major bioactive perylenequinones from Shiraia fruiting bodies and have been developed as efficient photosensitizers for photodynamic therapy. Pseudomonas is the second dominant genus inside Shiraia fruiting bodies, but with less known actions on the host fungus. In this work, the effects of bacterial volatiles from the Shiraia-associated Pseudomonas on fungal hypocrellin production were investigated. Pseudomonas putida No.24 was the most active to promote significantly accumulation of Shiraia perylenequinones including hypocrellin A (HA), HC, elsinochrome A (EA) and EC. Headspace analysis of the emitted volatiles revealed dimethyl disulfide as one of active compounds to promote fungal hypocrellin production. The bacterial volatiles induced an apoptosis in Shiraia hyphal cell, which was associated with the generation of reactive oxygen species (ROS). ROS generation was proved to mediate the volatile-induced membrane permeability and up-regulation of gene expressions for hypocrellin biosynthesis. In the submerged volatile co-culture, the bacterial volatiles stimulated not only HA content in mycelia, but also HA secretion into the medium, leading to the enhanced HA production to 249.85 mg/L, about 2.07-fold over the control. This is the first report on the regulation of Pseudomonas volatiles on fungal perylenequinone production. These findings could be helpful to understand the roles of bacterial volatiles in fruiting bodies and also provide new elicitation method using bacterial volatiles to stimulate fungal secondary metabolite production.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures.

BACKGROUND: Perylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including L-isoleucine (L-Ile), L-leucine (L-Leu) and L-valine (L-Val) on Shiraia perylenequinone production were investigated. RESULTS: Based on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by L-Val treatment at 1.5 g/L, but inhibited by L-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA-EC) was stimulated significantly by L-Val at 1.5 g/L, but sharply suppressed by L-Leu. After L-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by L-Val, but most of them were down-regulated by L-Leu. CONCLUSIONS: Our transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. L-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.