Characterisation of a monooxygenase in Shiraia bambusicola.[Pubmed: 30028664]

A monooxygenase-encoding gene (Mono) is located in the hypocrellin gene cluster of Shiraia sp. SUPER-H168 and was targeted by a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. The ΔMono mutant abolished hypocrellin production, whereas the ΔMono complement mutant restored hypocrellin production. Relative expression levels of the Mono and its adjacent genes were abolished in the ΔMono mutant compared with the wild-type strain. These results indicate the essential role of Mono in hypocrellin biosynthesis. The Mono gene of Shiraia bambusicola was further expressed in Pichia pastoris and salicylate monooxygenase activity was detected, which suggested that this monooxygenase has the ability to catalyse decarboxylative hydroxylation. The relative growth ratio of the ΔMono mutant was significantly improved compared with the wild-type strain. In contrast to the wild-type strain, the ΔMono mutant also represented excellent oxidative stress tolerance after exposure to high concentrations of H2O2 (16 mM) based on the increasing activities of superoxide dismutase, catalase, and glutathione peroxidase. These results suggest that ΔMono mutants could be used as microbial cell factories to produce metabolites that will cause oxidative stress. This study also enhances our understanding of hypocrellin biosynthesis and opens an avenue for decoding the hypocrellin pathway.

Enhanced hypocrellin production via coexpression of alpha-amylase and hemoglobin genes in Shiraia bambusicola.[Pubmed: 29721676]

Shiraia bambusicola is an important and valuable macrofungus and hypocrellins are its main secondary metabolites which have been widely applied in many medical fields. However, during SSF process of this filamentous fungus, use ratio of corn substrate and dissolved oxygen supply are two main limiting factors, which influence production cost, yield and product quality. To solve these problems, overexpressions of amy365-1 and vgb in S. bambusicola were investigated and three overexpression transformants were constructed. Results demonstrated that expressions and coexpression of AMY365-1 and VHb not only increased the productions of biomass, amylase, hypocrellin, but also up-regulated relative expression levels of four central carbon metabolism genes (pdc, ald, acs, acc) and seven hypocrellin biosynthesis genes (fad, mono, zftf, omef, msf, pks, mco). Furthermore, expression of VHb decreased SSF period. When amy365-1 and vgb were coexpressed, relative expression levels of zftf and pks reached their highest levels at 72 h under liquid fermentation, hypocrellin production reached the highest level 75.85 mg/gds which was 2.99-fold compared with wild type strain within 11 days under SSF, and residual starch of solid substrates was decreased from 35.47 to 14.57%.

Improved hypocrellin A production in Shiraia bambusicola by light-dark shift.[Pubmed: 29656218]

Hypocrellin A (HA) is a major bioactive perylenequinone from the fruiting body of Shiraia bambusicola used for the treatment of skin diseases and developed as a photodynamic therapy (PDT) agent against cancers and viruses. The mycelial culture of S. bambusicola under dark is a biotechnological alternative for HA production but with low yield. In this study, light and dark conditions were investigated to develop effective elicitation on HA production in the cultures. Our results showed the constant light at 200 lx stimulated HA production without any growth retardation of mycelia. A light/dark shift (24: 24 h) not only increased HA content in mycelia by 65%, but stimulated HA release into the medium with the highest total HA production 181.67 mg/L on day 8, about 73% increase over the dark control. Moreover, light/dark shifting induced the formation of smaller and more compact fungal pellets, suggesting a new effective strategy for large-scale production of HA in mycelium cultures. The light/dark shift up-regulated the expression levels of two reactive oxygen species (ROS) related genes including superoxide-generating NADPH oxidase (Nox) and cytochrome c peroxidase (CCP), and induced the generation of ROS. With the treatment of vitamin C, we found that ROS was involved in the up-regulated expression of key biosynthetical genes for hypocrellins and improved HA production. These results provide a basis for understanding the influence of light/dark shift on fungal metabolism and the application of a novel strategy for enhancing HA production in submerged Shiraia cultures.

Conditions and Regulation of Mixed Culture to Promote Shiraia bambusicola and Phoma sp. BZJ6 for Laccase Production.[Pubmed: 29259312]

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Genome editing in Shiraia bambusicola using CRISPR-Cas9 system.[Pubmed: 28690135]

Shiraia bambusicola can produce a type of hypocrellin, which is applied in antibacterial, antitumoral, and antiviral areas. Studies on the hypocrellin pathway have not been confirmed due to the deficiency of suitable genetic methods. We constructed a clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system in Shiraia sp. SUPER-H168 and targeted a polyketide synthase (SbaPKS). No hypocrellin production was detected in the ΔSbaPKS mutant. Relative expression levels of SbaPKS and its adjacent genes were extremely down-regulated in the ΔSbaPKS mutant compared to those in the wild strain. Subsequent pathogenicity assays showed that deletion of SbaPKS attenuated virulence on bamboo leaves. In contrast, restored hypocrellin in a SbaPKS overexpression strain generated necrotic lesions on bamboo leaves. These results suggest that SbaPKS is involved in hypocrellin biosynthesis and hypocrellin has an essential role in the virulence of S. bambusicola on bamboo leaves. The CRISPR/Cas9 system in Shiraia sp. will open an avenue for decoding the hypocrellin pathway and genome editing of other filamentous fungi. Strategies that disrupt hypocrellin biosynthesis may reduce the detriment of S. bambusicola.

Transcriptomic responses involved in enhanced production of hypocrellin A by addition of Triton X-100 in submerged cultures of Shiraia bambusicola.[Pubmed: 28685359]

The addition of surfactant is a useful strategy to enhance the product yield in submerged fermentation process. In this study, we sought to explore the mechanism for the elicitation of Triton X-100 on production of hypocrellin A (HA) in cultures of Shiraia bambusicola through transcriptomic analysis. Triton X-100 at 2.5% (w/v) not only induced HA biosynthesis in mycelia, but also stimulated the release of HA into the medium. We found 23 of 2463 transcripts, possible candidate genes for HA biosynthesis under Triton X-100 induction. Gene ontology (GO) analysis showed Triton X-100 treatment changed expression of genes involved in transmembrane transport and oxidation-reduction process, indicating that enhanced HA production was mainly due to both elicited biosynthesis in mycelium and the increased membrane permeability for HA release. These data provided new insights into elicitation of surfactants in submerged cultures of fungi.

Enhanced production of hypocrellin A by ultrasound stimulation in submerged cultures of Shiraia bambusicola.[Pubmed: 28633821]

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Characterization of a major facilitator superfamily transporter in Shiraia bambusicola.[Pubmed: 28549739]

Reactive oxygen species (ROS) generated by photo-activated hypocrellin from Shiraia bambusicola are detrimental to cellular macromolecules. However, S. bambusicola can still maintain excellent morphology during continuous hypocrellin production, indicating an extraordinary autoresistance system that protects against the harmful ROS. In this study, a major facilitator superfamily transporter (MFS) was isolated from S. bambusicola and deleted using the clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system. The ΔMFS mutant abolished hypocrellin production and was slightly sensitive to 40-μM hypocrellin, while the ΔMFS compliment strain restored hypocrellin production and resistance. Hypocrellin treatment also enhanced the relative expression of MFS in wild-type S. bambusicola. Subsequent pathogenicity assays showed that MFS deletion reduced damage to bamboo leaves. By contrast, restoration of hypocrellin production in the MFS compliment strain generated similar necrotic lesions on bamboo leaves to those observed with the wild-type strain. These results revealed that the identified MFS is involved in efflux of hypocrellin from cells, which reduces the hypocrellin toxicity. Furthermore, hypocrellin contributed to the virulence of S. bambusicola on bamboo leaves. These findings could help to reduce plant loss by disrupting hypocrellin biosynthesis in S. bambusicola, or overexpressing the associated resistance gene in transgenic plants.

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions.[Pubmed: 28050855]

Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek-Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

Adaptive Responses to Oxidative Stress in the Filamentous Fungal Shiraia bambusicola.[Pubmed: 27563871]

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.