Promotion of the Hypocrellin Yield by a Co-Culture of Shiraia bambusicola (GDMCC 60438) with Arthrinium sp. AF-5 Fungus

Hypocrellin is a natural 3,10-xylene-4,9-anthracene derivative compound that originates from the stroma of Shiraia bambusicola (S. bambusicola) and Hypocrella bambusae with excellent photobiological activities. Submerged fermentation with the mycelia of S. bambusicola is generally regarded as an ideal technology for hypocrellin production. This study developed a co-cultivation strategy for an obvious promotion of the hypocrellin yield by incubating S. bambusicola (GDMCC 60438) with the endophyte fungus Arthrinium sp. AF-5 isolated from the bamboo tissue. The results indicated that the yield of hypocrellin A (HA) reached a 66.75 mg/g carbon source after an 84-h co-cultivation of the two strains, which was a four-time increase of that by the fermentation only with the S. bambusicola. The microscope observation found that the mycelia of the two strains were intertwined with each other to form the mycelium pellets during the co-cultivation. Moreover, the mycelium pellets of the co-culture showed a contracted and slightly damaged morphology. The addition of H2O2 in the fermentation media could further increase the HA production by 18.31%.

The signaling role of extracellular ATP in co-culture of Shiraia sp. S9 and Pseudomonas fulva SB1 for enhancing hypocrellin A production

Background Adenosine 5′-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. Results The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10–300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. Conclusions Our results indicate that eATP release is an early event during the intimate bacterial–fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal–bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites. Graphic abstract

Nitric oxide regulates perylenequinones biosynthesis in Shiraia bambusicola S4201 induced by hydrogen peroxide

Shiraia bambusicola has been used as a traditional Chinese medicine for a long history. Its major medicinal active metabolites are perylenequinones, including hypocrellin A, elsinochrome A and so on. At present, the fermentation yield of perylenequinones is low, and its complex biosynthesis and regulatory pathways are still unclear. In this study, nitric oxide, as a downstream signal molecule of hydrogen peroxide, regulates the biosynthesis of perylenequinones. Exogenous addition of 0.01 mM sodium nitroprusside (nitric oxide donor) can promote perylenequinones production by 156% compared with the control. Further research found that hydrogen peroxide and nitric oxide increased the transcriptional level of the biosynthetic genes of hypocrellin A. The results showed that nitric oxide is involved in the biosynthesis and regulation of perylenequinones in Shiraia bambusicola as a signal molecule. In the future, the yield of perylenequinones can be increased by adding exogenous nitric oxide in fermentation.

Antifibrotic activity of Hypocrellin A united LED red light irradiation on Keloid Fibroblasts through counteracting TGF-β/Smad/autophagy/apoptosis signaling pathway

Keloid disease is characterized by abnormal proliferation of fibroblast and continuous deposition of extracellular matrix (ECM) components. More and more attention in dermopathic is paid to photodynamic therapy (PDT) with visible light. The natural photosensitizer Hypocrellin A (HA) is identified to develop a splendid light induced anticancer, antimicrobial and antiviral activity. In this experiment, we investigated the impacts of HA united light-emitting diode (LED) red light irradiation on human keloid fibroblast cells (KFs). Our results showed that HA united red light irradiation treatment (HA-R-PDT) decreased KFs viability, reduced KFs collagen production and ECM accumulation, inhibited cell proliferation, suppressed cell invasion and induced cell apoptosis. Moreover, our observations demonstrated that TGF-β/Smad signaling pathway and autophagy were restrained by HA-R-PDT. TGF-β1 could accommodate autophagy in KFs through both Smad and ERK pathway, while inhibiting autophagy could mediate TGF-β1 level by the negative feedback. Therefore, HA-R-PDT could suppress cell hyper-proliferation, collagen synthesis and ECM accumulation in KFs via regulating TGF-β1-ERK-autophagy-apoptosis signaling pathway. HA-R-PDT deserves systematic investigation as a potential therapeutic strategy for keloid treatment and autophagy might be a promising candidate in the therapy of KFs.

Nitric Oxide and Hydrogen Peroxide Signaling in Extractive Shiraia Fermentation by Triton X-100 for Hypocrellin A Production

Shiraia mycelial culture is a promising biotechnological alternative for the production of hypocrellin A (HA), a new photosensitizer for anticancer photodynamic therapy (PDT). The extractive fermentation of intracellular HA in the nonionic surfactant Triton X-100 (TX100) aqueous solution was studied in the present work. The addition of 25 g/L TX100 at 36 h of the fermentation not only enhanced HA exudation to the broth by 15.6-fold, but stimulated HA content in mycelia by 5.1-fold, leading to the higher production 206.2 mg/L, a 5.4-fold of the control on day 9. After the induced cell membrane permeabilization by TX100 addition, a rapid generation of nitric oxide (NO) and hydrogen peroxide (H2O2) was observed. The increase of NO level was suppressed by the scavenger vitamin C (VC) of reactive oxygen species (ROS), whereas the induced H2O2 production could not be prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), suggesting that NO production may occur downstream of ROS in the extractive fermentation. Both NO and H2O2 were proved to be involved in the expressions of HA biosynthetic genes (Mono, PKS and Omef) and HA production. NO was found to be able to up-regulate the expression of transporter genes (MFS and ABC) for HA exudation. Our results indicated the integrated role of NO and ROS in the extractive fermentation and provided a practical biotechnological process for HA production.