Yue Wang,Xiang Zhang,Cansong Lu,Xinping Li,Jianqin Zhou,Jianwen Wang
Fungal laccases are industrially important inducible enzymes extensively used in the delignification of lignocellulosics, detoxification of environmental wastes and decolorization of textile dyes. The discovery of new inducers is crucial for laccase productivity by filamentous fungi. In this study, a novel laccase-producing strain S8 from a bambusicolous fungus Shiraia bambusicola was identified by using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as laccase secretion indicator and native polyacrylamide gel electrophoresis . The corresponding full-length cDNA of laccase was cloned and characterized. The effect of lanthanum (LaCl 3 ) on extracellular laccase activity was tested at the concentration from 0.2 to 2.0 g/L and at different addition time (day 1–4) in the mycelium culture. In the presence of 1.0 g/L La 3+ at the beginning of the mycelium cultures, the highest laccase activity (2.7 × 10 4 U/L) is reached after 10 days of cultivation, about tenfold higher compared with non-induced cultures. La 3+ is shown to be a very strong inducer for fungal laccase activity with no inhibitory effect on fungal growth at the optimized concentration. In vivo , La 3+ added to the mycelium culture not only promotes a continuous and high expression of laccase gene ( lcc1 ), but also induces a rapid generation of signaling molecules including reactive oxygen species (ROS) and nitric oxygen (NO). Both the NO donor sodium nitroprusside (SNP) and exogenous hydrogen peroxide (H 2 O 2 ) potentiate La 3+ -induced laccase activity and increase membrane permeability of hyphal cells. Moreover, the scavengers such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) for NO and vitamin C for ROS suppress the induction. These results suggest that these signals can mediate La 3+ -induced laccase biosynthesis and its secretion. Our study provides a basis for understanding the induction mechanism of La 3+ on laccase and a practical strategy for enhanced fungal laccase production.
Background Hypocrellin A (HA) is a perylene quinone pigment with high medicinal value that is produced by Shiraia bambusicola Henn. (S. bambusicola) and Hypocrella bambusae (Berk. & Broome) Sacc. (Ascomycetes) with great potential in clinical photodynamic therapy. Submerged cultivation of S. bambusicola is a popular technique for HA production. However, there is not much research on how temperature changes lead to differential yields of HA production.Results The temperature regulation of submerged fermentation is an efficient approach to promote HA productivity. After a 32 °C fermentation, the HA content in the mycelia S. bambusicola (GDMCC 60438) was increased by more than three- and fivefold when compared to that at 28 °C and 26 °C, respectively. RNA sequencing (RNA-seq) analysis showed that the regulation of the expression of transcription factors and genes essential for HA biosynthesis could be induced by high temperature. Among the 496 differentially expressed genes (DEGs) explicitly expressed at 32 °C, the hub genes MH01c06g0046321 and MH01c11g0073001 in the coexpression network may affect HA biosynthesis and cytoarchitecture, respectively. Moreover, five genes, i.e., MH01c01g0006641, MH01c03g0017691, MH01c04g0029531, MH01c04g0030701 and MH01c22g0111101, potentially related to HA synthesis also exhibited significantly higher expression levels. Morphological observation showed that the autolysis inside the mycelial pellets tightly composted intertwined mycelia without apparent holes.Conclusion sThe obtained results provide an effective strategy in the submerged fermentation of S. bambusicola for improved HA production and reveal an alternative regulatory network responsive to the biosynthesis metabolism of HA in response to environmental signals.
Rare earth elements (REEs) have been widely applied as efficient elicitors to improve production of biologically important secondary metabolites in plants. However, their effects on microbial secondary metabolites were seldom reported. In this study, lanthanum (LaCl 3 ) was used as an elicitor in mycelium cultures of Shiraia bambusicola to stimulate the production of hypocrellin A (HA), a promising photosensitizer for anticancer photodynamic therapy. La 3+ at 1.0 g/L not only increases HA content by 2.50-fold in mycelia, but stimulates HA exudation to the medium with the highest production (225.05 mg/L) on day 6. Further analysis shows that La 3+ can induce the intracellular ROS (reactive oxygen species) accumulation with 2.33-fold higher contents of H 2 O 2 in mycelium. HA production induced by La 3+ treatment can be suppressed by ROS scavengers. Further analysis reveals that the elicitation of La 3+ on HA accumulation is mediated by ROS generation to enhance expression of HA biosynthetical genes. The expression of ATP-binding cassette transporter gene ( SbABC ) for HA exudation is also regulated partially via ROS generated by NADPH oxidase . These results indicate that the enhanced HA production by La 3+ might be mediated by ROS signaling pathway. Our results provide a basis for understanding REE elicitation on fungal metabolites and a practical biotechnological process for enhanced production of HA.
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