Effects Of Lanthanum Chloride On Hypocrellin A Biosynthesis In Shiraia Bambusicola

Zhu huang is the fruit-body of a pathogenic fungus Shiraia bambusicola of bamboos,which has been used in Chinese traditional medicine to treat rheumatic pain,stomachache,vitiligo and psoriasis.Hypocrellins are perylenequinonoid pigments from the fruit-bodies.The highest component among hypocrelllins is hypocrellin A(HA),which attracted intense interest as clinically useful photosensitizers in photodynamic therapy(PDT)for different skin diseases including white lesions of the vulva,lichen amyloidosis and for unique antiviral,anticancer properties.Due to the limitation of the wild sources of the fruit-bodies and complexity of chemical synthesis of HA,the mycelium culture of Shiraia bambusicola is becoming a biotechnological alternative for HA production.However,HA is always produced by mycelium cultures with lower contents,which limited biotechnological production of HA.Rare earth elements(REEs)have been widely applied as efficient elicitors to improve production of biologically important secondary metabolites in plants.However,there are less reports on the effects of REEs on fungal metabolites.In this paper,the effects of LaC13 on the fungal growth and HA biosynthesis,were investigated.The physiological changes in S.bambusicola in response to lanthanum were found,emphasizing the lanthanum elicitation on HA production via ROS mediation.Our results provided a foundation and reference for theoretical study and industrial application for the subsequent large-scale fermentation of Shiraia bambusicola cultures.We screened the effects of metal ions(Ca2+,Fe3+,Co3+,Cu2+and La3+)on HA biosynthesis and found that Ca2+,Fe3+and La3+enhanced HA production significantly.2 g/L of Ca2+ obviously enhanced the HA content in mycelia,2.36-fold higher than that in control group.Fe3+at 0.2 g/L stimulated S.bambusicola cultures for 8 days to prodcuce 24.17 mg/g HA in mycelia,3.3-fold of the control group.Furthermore,La3+at 1.0 g/L not only increased HA content by 2.50-fold in mycelia,but stimulated HA exudation to the medium with the highest production(225.05 mg/L)on day 6.Glucose-6-phosphorate dehydrogenase(G6PDH)is the first and critical rate-limiting enzyme in the pentose phosphate pathway(PPP).It is involved in the synthesis of NADPH and ribose-5-phosphate and plays an important role in response to biotic and abiotic stresses.We succeeded in cloning the full length cDNA of G6PDH of S.bambusicola(SbG6PDH),encoding 509 amino acids.A phylogenic tree revealed that SbG6PDH belongs to the homolog of G6PDH in fungi and the amino acid sequences of SbG6PDH gene are the most closely related in species of Parastagonospora nodorum with 82.9%similarity.In La3+-induced cultures,HA was improved simultaneously with the increased expression of G6PDH.The G6PDH inhibitor(glucosamine,GlcN)could obviously suppressed the activity and expression of G6PDH and HA accumulation.Real time-qPCR showed that GlcN reduced gene expression of polyketide synthase(PKS),O-methyltransferase(Omef),FAD/FMN-dependent oxidoreductase(FAD)in HA biosynthetic pathway.Further analysis showed that La3+ could induce the intracellular reactive oxygen species(ROS)accumulation with 2.33-fold in mycelium.HA production induced by La3+treatment could be suppressed by ROS scavengers.Further analysis revealed that the elicitation of La3+ on HA accumulation was mediated by ROS generation to enhance expression of HA biosynthetical genes.The full length cDNA encoding ATP-binding cassette transporter gene(SbABC)was cloned firstly.The 3,828 bp open reading frame(ORF)encoded a protein of 1,275 amino acids.In addition,A phylogenic tree revealed that SbABC belongs to the protein family of ABC transporter in fungi and the amino acid sequences of SbABC gene are the most closely related in species of Stagonospora sp.(OAK96363.1)with 86.7%similarity.The expression of SbABC for HA exudation was also regulated partially via ROS generated by NADPH oxidase.These results indicated that the enhanced HA production by La3+ might be mediated by ROS signaling pathway.This is the first report on the eliciting role of REEs on fungal secondary metabolite production.We explored the effects of La3+ on the growth and HA biosynthesis of S.bambusicola.This study revealed that the mechanism of regulation of HA biosysnthesis induced by La3+ via variation of oxidative stress,the expression of genes associated with biosynthesis of HA.Last but not least,present research also provided theoretical and applicable basis for the following HA production of S.bambusicola in large-scale.