Study On The Biosynthesis Of Hypocrellin A And 20-hydroxyecdysone On The Basis Of RNA-Seq Dependent Transcriptional Analysis

Medicinal plants and fungi are main sources of drugsThe secondary metabolites from them are valuable bank for drug development.To improve the production of medicinal components,it is necessary to clarify the biosynthesis of seconsray metablites and their regulation.Functional genetic approaches,especially transcriptome analysis,are effective tools wildly used toinvestigate pathway of secondary metabolism.In this study,we investigated the biosynthesis of hypocrellin A?HA?in medicinal fungus Shiraia bambusicola and 20-hydroxyecdysone?20E?in medicinal plant Cyanotis arachnoidea on the basis of RNA-Seq dependent transcriptional analysis.?HA?is the main bioactive compound isolated from medicinal fungus S.bambusicola and has been widely used for the treatment of different skin diseases Furthermore,as a new type of non-porphyrin photosensitizer,HA has gained considerable attention because of its marked anticancer and antiviral activities with its higher photodynamic therapy?PDT?effects but super low dark toxicity.It was reported that adding surfactants is a simpler and effective strategy to enhance production of secondary metabolites in submerged cultures of fungi.In this work,we tried to stimulate the biosynthesis HA by adding surfactants in submerged cultures of S.bambusicola.Furthermore,the transcriptomic and physiological responses of S.bambusicola to the addition of Triton X-100 in submerged cultures were also investigated.1 Screening the surfactantsTo stimulate HA production,eight surfactants?Pluronic F68,Pluronic F-127,Tween-40,Tween-80,SDS,Brij 52,Span 80 and Triton X-100?were added to the culture.Only Triton X-100 could induce HA production.Triton X-100 as the stimulatory additive for HA production was further tested at various concentrations?0.5-3.0%,w/v?and different addition time?0-72 h?.When Triton X-100 at 2.5%?w/v?was applied at 36 h of the initial cultures,it not only induced the biosynthesis of HA in mycelia?1.88 mg/g DW?,but also stimulated the release of HA into the medium at 70.9 mg/L with the highest total production?96.9 mg/L?on day 8.The result indicated that the enhancement of HA production was mainly due to both the stimulated cellular biosynthesis,and the enhanced membrane permeability.2 Transcriptomic response of S.bambusicola to the addition of Triton X-100In order to better understand the molecular mechanism of Triton X-100 action and HA biosynthesis,cDNA samples extracted from Triton X-100 treated and untreated mycelia were sequenced.Finally,a total of 27,728 unigenes were assembled,with an average length of 1,025 bp.Functional annotation revealed that 12,903 unigenes were similar to known genes.After the treatment of Triton X-100,a total of 2,461 DEGs was identified,among which 1,304 unigenes were up-regulated while 1,157 unigenes were down-regulated.GO classification of DEGs revealed that‘transmembrane transport'?GO:0055085?,‘oxidoreductaseactivity'?GO:0016491?,‘monooxygenase activity'?GO:0004497?,‘integral component of membrane'?GO:0016021?,‘plasma membrane'?GO:00058860?and‘endoplasmic reticulum membrane'?GO:0005789?were significantly affected during Triton X-100 assition.Based on the results of GO classification,the genes involved in transmembrane transport,redox changes and related to cell membrane were further investigated.3 The mechanism of stimulation of HA biosynthesis by Triton X-100With the addition of Triton X-100,the composition of saturated fatty acids of fungal cell memberane was significantly decreased,whereas the composition of unsaturated fatty acids was dramatically increased.The ratio of unsaturated/saturated fatty acids in the mycelia increased,indicating the increased permeability of mycelia cells.Subsequently,we investigated the transcription changes of genes involved in transmembrane transport.As a result,a total of 24 unigenes encoding major facilitator superfamily?MFS?transporters and13 unigenes encoding ATP-binding cassette?ABC?transporters were up-regulated after Triton X-100 treatment.These results indicated the stimulating effects of Triton X-100 on HA release maybe based on the enhanced permeability of the mycelial cell membrane and activation of ABC and MFS transporters.Finally,we detected reactive oxygen species?ROS?generation in S.bambusicola.After Triton X-100 treatment,both H₂O₂ and O₂^-content were enhanced at the end of the culture period?6-10 d?.We suggested that Triton X-100 may cause ROS induction and some other defense responses leading to HA biosynthesis.4 Candidate genes involved in HA biosynthesisA total of 23 unigenes involved in HA biosynthesis was identified in S.bambusicola transcriptome database,including two unigenes annotated as polyketide synthase,18unigenes annotated as monooxygenase and three unigenes annotated as O-methyltransferase.C.arachnoidea C.B.Clarke,a perennial grass from the family Commelinaceae,has been used in traditional Chinese medicine as a therapeutic for curing limb numbness,rheumatoid arthritis or as a tonic for promoting the circulation of blood,relaxing the muscles and joints.C.arachnoidea contains a rich source of bioactive phytoecdysteroids including 20E as a major one.20E is not only widely used in sericulture,but also has many pharmacological activities.It can stimulate the synthesis of protein and nucleic acid of vertebrates,promote the metabolism of carbohydrates and lipids,and regulate the immunological function and the central nervous system.However,due to its complex structure,the chemical synthesis cannot be used in its commercial production.Since the supply of 20E from the plants is in great shortage,its application is always restricted.Thus,it is meaningful to study the biosynthesis of 20E in plants.The contents of 20E in C.arachnoidea were detected by HPLC.The 20E contents in roots were much higher than in leaves by 5.90-fold.Spatial distribution of 20E indicates a root-preferential expression pattern of genes involved in 20E biosynthesis.We generated a unique transcript library for C.arachnoidea,containing 79,835 unigenes with an average length of 894 bp.A total of39,425?49.38%?unigenes were annotated and 1,070 unigenes were involved in secondary metabolism pathways according to the KEGG pathway analysis.Moreover,we identified a total of 54 unigenes related to 20E biosynthesis,including 15 unigenes involved in mevalonate?MVA?pathway,19 unigenes involved in steroid biosynthesis and 20 unigenes encoding CYP450s.Besides,transcription factors?TFs?were searched in C.arachnoidea transcriptome and a total of 1,312 TFs were found.Furthermore,148 TFs were DEGs,which might regulate the biosynthesis of 20E.Simple sequence repeats?SSRs?in C.arachnoidea were analyzed based on the transcriptome data.Finally,19,158 SSRs were identified,which might be helpful in plant breeding and construction of genetic map.