Search Results (12)

Hypocrellin A (HA) represents a pharmaceutically important perylenequinone photosensitizer produced by Shiraia bambusicola. However, submerged fermentation remains constrained by filamentous morphological characteristics and inherent mass transfer limitations. Although microparticle-enhanced cultivation (MPEC) has demonstrated efficacy in filamentous fungal systems, the molecular mechanisms by which physical cues, such as microparticle-induced shear stress, reprogram fungal metabolism remain largely unexplored. This work systematically optimizes SiO₂-based MPEC parameters for S. bambusicola GDMCC 60438, including particle dimensions, temporal addition protocols, and solid loading. Mechanistic investigations integrated pellet morphology analysis, membrane lipid composition, intracellular redox status, energy/precursor markers, and RNA-seq transcriptomic profiling with qRT-PCR validation. Under optimized conditions (10% w/v SiO₂, 30 mesh, added at 6 h), HA yield reached 41.76 ± 5.02 mg/L, representing a 3.65-fold increase over controls. MPEC shifted morphology toward smaller, more porous pellets with denser internal structure, accompanied by increased membrane fluidity (unsaturated/saturated fatty acid ratio from 1.54 to 2.63), elevated ROS levels with antioxidant enzyme activation, and enhanced acetyl-CoA and ATP accumulation. Transcriptomic analysis identified 206 differentially expressed genes enriched in oxidative phosphorylation, carbon metabolism, and stress responses, with upregulation of PKS-related biosynthetic genes and major facilitator superfamily transporters. This work establishes an integrated mechanistic framework linking particle-induced morphological changes to metabolic reprogramming through oxidative stress and subsequent transcriptional activation of the HA biosynthetic pathway, providing rational design principles for MPEC strategies in filamentous fungi. Full article

Hypocrellin A (HA), a photodynamic perylenequinone pigment from Shiraia fruiting bodies, functions as an efficient photosensitizer for clinical photodynamic therapy. Glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), governs carbon flux into NADPH production. This study elucidates G6PDH’s regulatory role in HA biosynthesis in Shiraia sp. S9. Bamboo polysaccharide (BPS) elicitation (100 mg/L) significantly enhanced HA production to 428.1 mg/L, 1.6-fold higher than controls after 5 days. We cloned the G6PDH gene and demonstrated that BPS upregulated its expression and activity, concomitant with increased reactive oxygen species (ROS; H₂O₂ and O₂^•−) and nitric oxide (NO) generation. ROS production was mediated by NADPH oxidase induction, while NO generation was attributed to elevated nitric oxide synthase and nitrate reductase activities. Critically, the G6PDH inhibitor glucosamine (1.0 mM) suppressed both H₂O₂ and NO production. These ROS/NO signals upregulated key HA biosynthetic (PKS, Omef) and transport (MFS) genes. Our findings establish G6PDH as a central regulator of BPS-induced HA biosynthesis via ROS/NO signaling, revealing novel metabolic crosstalk between the PPP and fungal perylenequinone biosynthesis. This work presents BPS elicitation as a biotechnological strategy for scalable HA production in Shiraia mycelium cultures. Full article

Perylenequinonoid compounds, represented by photosensitive therapeutic agents such as hypocrellins and elsinochromes, demonstrate extensive potential across biomedical, agricultural, and food industrial applications. Nevertheless, their restricted biosynthesis remains a critical bottleneck for commercial exploitation. This study implemented UV mutagenesis to enhance perylenequinone production in fungal strains of Pseudoshiraia conidialis, achieving significant yield improvements at the 120 J/m² and 150 J/m² irradiation intensities. Through systematic optimization of the HPLC analytical platform, we established the precise quantification of five distinct perylenequinonoid derivatives: hypocrellin A, hypocrellin B, shiraiachrome A, elsinochrome A, and elsinochrome B. The mutant strain Z2-1 demonstrated a remarkable biosynthetic capacity with the total perylenequinonoid yields reaching 2101.6 mg/L, representing a 705.70% enhancement over the parental strain zzz816 (260.84 mg/L). Particularly noteworthy was the hyperproduction of hypocrellin A at 1100.7 mg/L, corresponding to a 1208.02% increase from the baseline yield (84.15 mg/L). Furthermore, this work reports the first successful generation of an elsinochrome A-overproducing strain, achieving a 312.68 mg/L output (429.25% increase from 59.08 mg/L). Intriguingly, different mutant strains exhibited distinct production profiles for specific compounds, revealing biosynthetic preference variations among derivatives. These findings emphasize the necessity for comprehensive metabolite profiling during fermentation process optimization to maximize the target compound yields. Full article

Hypocrellins (HYPs), naturally occurring 3,10-xylene-4,9-anthracene derivatives sourced from Shiraia bambusicola and Hypocrella bambusae, exhibit significant photobiological activities. Despite their capability for generating a high yield of reactive oxygen species, including singlet oxygen and superoxide anion radical, their application in photodynamic therapy (PDT) is constrained. This limitation is due to their low dark phototoxicity, weak absorption within the therapeutic window of PDT (600–900 nm), and inherent hydrophobicity, which hinder their immediate use in amphipathic PDT applications. This review comprehensively discusses the research advancements in the bioactivities and biosynthesis of HYPs, alongside the reported chemical and physical modifications that enhance their water solubility and extend their therapeutic window. Additionally, it explores potential strategies for developing pharmaceuticals, photocatalytic agents, and photosensitive pesticides based on HYPs. Full article

Perylenequinones (PQs) are important natural compounds that have been extensively utilized in recent years as agents for antimicrobial, anticancer, and antiviral photodynamic therapies. In this study, we investigated the molecular mechanisms regulating PQ biosynthesis by comparing Shiraia sp. Slf14 with its low PQ titer mutant, Slf14(w). The results indicated that the strain Slf14 exhibited a higher PQ yield, a more vigorous energy metabolism, and a more pronounced oxidation state compared to Slf14(w). Transcriptome analysis consistently revealed that the differences in gene expression between Slf14 and Slf14(w) are primarily associated with genes involved in redox processes and energy metabolism. Additionally, reactive oxygen species (ROS) were shown to play a crucial role in promoting PQ synthesis, as evidenced by the application of ROS-related inhibitors and promoters. Further results demonstrated that mitochondria are significant sources of ROS, which effectively regulate PQ biosynthesis in Shiraia sp. Slf14. In summary, this research revealed a noteworthy finding: the higher energy metabolism of the strain Slf14 is associated with increased intracellular ROS accumulation, which in turn triggers the activation and expression of gene clusters responsible for PQ synthesis. Full article

Hypocrellin A (HA) is a valuable pigment with promising applications in biotechnology and pharmaceuticals. The submerged cultivation of Shiraia bambusicola offers a strategic opportunity to enhance HA production. This study investigates the regulatory mechanisms for HA biosynthesis through urea supplementation and presents a strategy to increase HA yield. In the absence of urea, S. bambusicola (GDMCC 60438) does not synthesize HA. However, the addition of 40 g/L urea 12 h into the fermentation process results in a final HA production of 46.7 ± 8.2 mg/L. Morphological analysis reveals an optimized environment for HA synthesis, characterized by a densely intertwined and reticular hyphal structure with minute pores. RNA sequencing shows significant upregulation of genes involved in DNA repair, recombination, and metabolism. Conversely, genes related to cellular homeostasis, cell-wall chitin, and amino polysaccharide metabolism are downregulated. Urea supplementation facilitates the upregulation of amino acid metabolism and the cysteine desulfurase gene, enhancing acetyl-CoA accumulation within the mycelium and providing the necessary precursor materials for HA synthesis. Our work underscores the pivotal role of urea in regulating HA biosynthesis and proposes a practical approach to enhance HA production. The findings contribute novel insights to the fields of biotechnology for pharmaceuticals. Full article

Melatonin (MLT), an evolutionarily conserved pleiotropic molecule, is implicated in numerous physiological processes in plants and animals. However, the effects of MLT on microbes have seldom been reported. In this study, we examined the influence of exogenous MLT on the growth and hypocrellin biosynthesis of bambusicolous fungus Shiraia sp. S9. Hypocrellin A (HA) is a photoactivated and photoinduced perylenequinone (PQ) toxin in Shiraia. Exogenous MLT at 100.00 μM not only decreased fungal conidiation and spore germination but inhibited HA contents significantly in fungal cultures under a light/dark (24 h:24 h) shift. MLT treatment was associated with higher activity of antioxidant enzymes (superoxide dismutase, catalase and peroxidase) and a marked decline in reactive oxygen species (ROS) production in the mycelia. Moreover, MLT induced endogenous nitric oxide (NO) production during the culture. The NO donor sodium nitroprusside (SNP) potentiated MLT-induced inhibition of O₂⁻ production, but NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) enhanced O₂⁻ production, whereas MLT-induced NO level was increased by the ROS scavenger vitamin C (Vc). The changes in NO and H₂O₂ were proved to be involved in the MLT-induced downregulation of the expressions of HA biosynthetic genes, leading to the suppression of HA production. This study provides new insight into the regulatory roles of MLT on fungal secondary metabolism activities and a basis for understanding self-resistance in phototoxin-producing fungi. Full article

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%. Full article

The main active ingredients of the fruiting bodies of Shiraia bambusicola and Rubroshiraia bambusae are Hypocrellins, belonging perylenequinones with potential photodynamic activity against cancer and microbial diseases. However, the strains of S. bambusicola and R. bambusae do not produce hypocrellins in culture, so resource exploitation of natural products was seriously restricted. In this study, a series of novel Shiraia-like fungal endophyte strains, with varying sporulation ability and synthesizing diverse secondary metabolites, was isolated from different bamboos. Based on phylogenetic analyses and morphological characteristics of the endophytes, Pseudoshiraia conidialis gen. et sp. nov. is proposed. The secondary metabolites of different fruiting bodies and strains have been comprehensively analyzed for the first time, finding that the endophytic strains are shown not only to produce hypocrellins, but also other perylenequinonoid compounds. It was noteworthy that the highest yield of total perylenequinone production and hypocrellin A appeared in P. conidialis CNUCC 1353PR (1410.13 mg/L), which was significantly higher than any other wild type P. conidialis strains in published reports. In view of these results, the identification of Shiraia-like endophytes not only confirm the phylogenetic status of similar strains, but will further assist in developing the production of valuable natural products. Full article

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. Full article

Shiraia bambusicola can retain excellent physiological activity when challenged with maximal photo-activated hypocrellin, which causes cellular oxidative stress. The protective mechanism of this fungus against oxidative stress has not yet been reported. We evaluated the biomass and hypocrellin biosynthesis of Shiraia sp. SUPER-H168 when treated with high concentrations of H₂O₂. Hypocrellin production was improved by nearly 27% and 25% after 72 h incubation with 10 mM and 20 mM H₂O₂, respectively, while the inhibition ratios of exogenous 20 mM H₂O₂ on wild S. bambusicola and a hypocrellin-deficient strain were 20% and 33%, respectively. Under exogenous oxidative stress, the specific activities of catalase, glutathione reductase, and superoxide dismutase were significantly increased. These changes may allow Shiraia to maintain normal life activities under oxidative stress. Moreover, sufficient glutathione peroxidase was produced in the SUPER-H168 and hypocrellin-deficient strains, to further ensure that S. bambusicola has excellent protective abilities against oxidative stress. This study creates the possibility that the addition of high H₂O₂ concentrations can stimulate fungal secondary metabolism, and will lead to a comprehensive and coherent understanding of mechanisms against oxidative stresses from high hydrogen peroxide concentrations in the filamentous fungal Shiraia sp. SUPER-H168. Full article

Shiraia bambusicola is a species of the monotypic genus Shiraia in the phylum Ascomycota. In China, it is known for its pharmacological properties that are used to treat rheumatic arthritis, sciatica, pertussis, tracheitis and so forth. Its major medicinal active metabolite is hypocrellin A, which exhibits excellent antiviral and antitumor properties. However, the genes involved in the hypocrellin A anabolic pathways were still unknown due to the lack of genomic information for this species. To investigate putative genes that are involved in the biosynthesis of hypocrellin A and determine the pathway, we performed transcriptome sequencing for Shiraia bambusicola S4201-W and the mutant S4201-D1 for the first time. S4201-W has excellent hypocrellin A production, while the mutant S4201-D1 does not. Then, we obtained 38,056,034 and 39,086,896 clean reads from S4201-W and S4201-D1, respectively. In all, 17,923 unigenes were de novo assembled, and the N50 length was 1970 bp. Based on the negative binomial distribution test, 716 unigenes were found to be upregulated, and 188 genes were downregulated in S4201-D1, compared with S4201-W. We have found seven unigenes involved in the biosynthesis of hypocrellin A and proposed a putative hypocrellin A biosynthetic pathway. These data will provide a valuable resource and theoretical basis for future molecular studies of hypocrellin A, help identify the genes involved in the biosynthesis of hypocrellin A and help facilitate functional studies for enhancing hypocrellin A production. Full article