The Effects Of Melatonin And Laccase On The Growth And Hypocrellin Biosynthesis Of Shiraia Bambusicola

Shiraia bambusicola is a traditional Chinese medicinal fungus to treat rheumatoid arthritis,gastritis,vitiligo,psoriasis and other skin diseases.S.bambusicola is of a variety of biologically active metabolites such as polysaccharides,laccase and hypocrellins.Hypocrellins,photosensitive perylenequinones,are the main active constituents of S.bambusicola,which have anti-tumor,antiviral and antibacterial activity.Laccase,a polyphenol oxidase is a member of the gene cluster for hypocrellin biosynthesis,and is used widely in several fields including pulp bleaching,sewage treatment,dye decolorization,food addition and drug synthesis.During the infestation of S.bambusicola on the host bamboos,the fungus produces hypocrellins and releases,reactive oxygen species(ROS)under daylight to facilitate the fungal infestation,which also result in a higher oxidative statue in fungus.Recently,melatonin has been reported as a new physiological signal in animals and plants to regulate stress resistance and antioxidant capacity.However,there is less report on the effects of laccase and melatonin on secondary metabolites in fungi,especially for hypocrellin biosynthesis in S.bambusicola.Hereby we reported the production of endogenous melatonin during different growth and development stages of S.bambusicola for the first time.The relationship among melatonin,fungal redox state and hypocrellin biosynthesis was revealed by adding exogenous melatonin.Additionally,we detected the laccase production in S.bambusicola and cloned laccase gene lcc1.The role of laccase in hypocrellins biosynthesis was explored by the addition of laccase and the inhibitors.The major results were presented as follows:(1)We detected the presence of endogenous melatonin in S.bambusicola S8 for the first time.The content of melatonin was 5.6-27.1 mg/g dry weight(DW).The HA production decreased after the addition of exogenous melatonin.Melatonin at 100 μM reduced the HA production by 89%,and inhibited the spore formation and germination.Moreover,melatonin is an effective ROS scavenger.After 24 h of 100 μM melatonin treatment,the content of H2O2 and O2-decreased by 43.4%and 51.9%,respectively.Further analysis showed that melatonin could promote the activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)by up-regulating the expression of these antioxidant enzyme genes,thereby reducing the level of reactive oxygen species and leading to a decrease in HA accumulation.On the one hand,melatonin could also down-regulate the expression of multiple genes for HA biosynthesis gene cluster such as polyketide synthase,FAD/FMN-dependent oxidoreductase and so on,thereby reducing the synthesis of intracellular HA.(2)We also cloned the full-length cDNA of laccase gene lcc1.The 1785 bp open reading frame(ORF)encoded a protein Lcc1(594 amino acids).Phylogenetic tree analysis revealed that Lcc1 was the most closely to that of Stagonospora sp.with 99%similarity,while it was distantly related to that of Shiraia sp.SUPER-H168 with 65%similarity,indicating Lcc1 was a new laccase in S.bambusicola.(3)The transcriptional analysis demonstrated that the relative expression of laccase gene reached the highest level in the maturity stage of the fruiting body.And after adding 200 U/L laccase extract to the medium,the intracellular HA production was 1.52-fold higher while the laccase inhibitors L-cys(1 mM)and NaN3(0.1 mM)decreased HA production by 46.4%and 94.5%,respectively.These results preliminarily revealed that laccase may play an important role in the fungal development and HA biosynthesis.(4)La3+,as a rare earth element,could induce laccase production.After 1.0 g/L La3+ treatment,the extracellular laccase activity could reach 2.72×104 U/L,about 10-fold over the control after 10-day old cultures.La3+ ireatment had no significant effects on the growth and spore germination.In the process of La3+-induced laccase production,the increase of cell membrane permeability and laccase activity are closely related to ROS and NO signals.Meanwhile,La3+ could up-regulate the expression of lcc1,thereby stimulating the biosynthesis of laccase at the transcriptional level.This is the first report on the regulation of melatonin and laccase on fungal secondary metabolism.We detected the time profiles of the endogenous melatonin content and laccase production in S.bambusicola S8,and explored the effect of melatonin and laccase on the growth and HA biosynthesis.Laccase gene lcc1 was cloned and analyzed,and the effective condition of La3+-induction laccase production was optimized.The mechanisms of melatonin regulation of HA biosynthesis and lanthanum regulation of laccase production were preliminarily explored.This research not only develops a novel method for improving the production of industrial laccase,but also provides a basis for understanding HA biosynthesis pathways and regulatory factors.