Genome Analysis Of Bamboo Yellow And Study On The Synthesis And Metabolism Of Perylenequinone Compounds

Shiraia bambusicola is a rare resource of medicinal fungi,distributed only in southern provinces of China and Japan.Hypocrellin A is the main active ingredient of Shiraia bambusicola which belongs to perylenequinones.It has antimicrobial,antiviral and antineoplastic activities.As an excellent photosensitizer,hypocrellin A can be used in clinical photodynamic therapy.Shiraia bambusicola are facing the situation that wild resources are less and less,and the active metabolites of strain fermentation,are low and cannot be produced on a large scale.Therefore,it is extremely urgent to study the biosynthesis pathway of hypocrellin A and to conduct directed evolution on wild strains of Shiraia bambusicola by means of molecular biology to obtain strains with high yield.At present,there are few reports on molecular biology of hypocrellin A,and the biosynthetic genes,pathways and regulation are still unclear.The aim of this study is to reveal the genomic characteristics of Shiraia bambusicola by genome sequencing,bioinformatics and molecular biology methods,to understand the ability of Shiraia bambusicola to synthesize secondary metabolites,to identify the hypocrellin A biosynthetic gene cluster,and to study its biosynthetic pathway and regulation pathway.In this study,Illumina Hi Seq and Pac Bio platforms were used to sequence the genome of Shiraia bambusicola.The final Shiraia bambusicola S4201 genome assembly included32 Mb in 57 contigs?scaffolds?,with a contig?scaffold?N50 of 1,565,644 bp,a total of11,332 genes were predicted.To conduct functional annotation of the S4201 gene model,The Shiraia bambusicola S4201 genome encodes 414 putative CAZymes,including 166glycoside Hydrolases,52 glycosyl Transferases,9 polysaccharide Lyases,13 carbohydrate Esterases,72 auxiliary Activities and 102 carbohydrate-binding modules.Shiraia bambusicola S4201,together with Arthrobotrys oligospora ATCC 24927,tended to show a similar carbohydrate degradation pattern.Eight hundred ninety genes in Shiraia bambusicola S4201 were putatively involved in pathogenicity and virulence when analyzed with the PHI database,and this accounts for 7.85%of the total predicted genes.One thousand twenty-six putative P450s?9.05%?were predicted in Shiraia bambusicola S4201.In total,74 Major Facilitator Superfamily transporters and 46 ATP-Binding Cassette transporters were predicted;Synteny analysis showed that Shiraia bambusicola S4201 and Parastagonospora nodorum genome structures have many large areas of synteny and they also share more homology between proteins.Comparative analysis of core and pan genes among the four species showed that Shiraia bambusicola S4201 has the fewest specific genes.The taxonomic status analysis revealed that Shiraia bambusicola S4201 belongs amongst the species of Pleosporales,Dothideomycetes;Using the scaffolds as the query sequences for the anti SMASH 4.1.0 platform,73 putative secondary biosynthetic gene clusters were predicted.In total,15 polyketide synthases?PKS,14 T1PKS and one T3PKS?,six non-ribosomal peptide synthases?NRPS?,one linaridin,two terpenes,one indole,one cf?fatty?acid,28 cf?putative and two other gene clusters were identified in the Shiraia bambusicola S4201 genome.It shows that Shiraia bambusicola has great potential for the development of secondary metabolites.Some hypocrellin A biosynthetic genes in the cluster we predicted by anti SMASH differed from the ones in Shiraia sp.slf14?Gen Bank:KM434884.1,unpublished results?.Therefore,we verified these genes at the transcriptional level and preliminarily identified the genes that may be involved in the biosynthesis of hypocrellin A.Based on the biosynthesis pathway of cercosporin and elsinochrome C,the biosynthesis pathway of hypocrellin A was speculated.In order to study the function of Shiraia bambusicola gene,the genetic transformation system of Shiraia bambusicola was constructed.The effects of some antibiotics and herbicides commonly used in other fungi?hygromycin B,geneticin,zeocin,kanamycin,glufosinate,glyphosate,benomyl,carboxin,5-fluoroorotic acid?on the growth of Shiraia bambusicola were examined,and the selection marker benomyl suitable for Shiraia bambusicola was found.Finally,the screening concentration of benomyl on PDA medium was determined to be 10?g/m L.Orthogonal experiment was used to study the effects of different factors?mycelial age,enzymolysis time,enzymolysis temperature,enzymolysis speed?on protoplast yield.The results showed that the highest protoplast yield was obtained when culture time was 14 hours and enzymolysis time was 3.5 hours in a shaker at 34°C and 200 rpm.In order to study the role of zftf in hypocrellin A biosynthesis gene cluster,the overexpression plasmid p OE-zftf was constructed and randomly inserted into the genome of Shiraia bambusicola with transformation.The mutant strains were screened by PCR amplification.The results showed that zftf annotated as a transcription factor of Zn?II?Cys₆could regulate the core gene cluster of hypocrellin A,enhance the expression of related genes and increase the production of hypocrellin A.According to previous studies and RNA-Seq data,complex cell signal cascades are involved in the biosynthesis of hypocrellin A.In this study,hydrogen peroxide?H₂O₂?and nitric oxide?NO?were suspected to be involved in the biosynthesis and regulation of hypocrellin A as signaling molecules.Liquid co-culture experiments showed that 0.01 m M H₂O₂ and 0.01 m M SNP?NO donor?can increase perylenequinones production and are independent of exogenous oxidative stress.Activation or inhibition of H₂O₂ and NO signals in vitro indicates that there is a cascade relationship between H₂O₂ and NO,and NO may act as a downstream signaling molecule of H₂O₂ to regulate the synthesis of perylenequinones.To further explore the protential mechanisms by which H₂O₂ and NO inducing increased production of perylenequinones,we evaluated the transcriptional response of the hypocrellin A biosynthesis gene.The results showed that H₂O₂ and NO increased the transcription level of the biosynthetic gene of hypocrellin A and promoted the accumulation of elsinochrome A and hypocrellin A.In summary,the whole genome of Shiraia bambusicola S4201 was sequenced and compared with other fungal species.The pathogenicity,taxonomic status and secondary metabolites of Shiraia bambusicola were analyzed,and the biosynthesis pathway of hypocrellin A was speculated.The genetic transformation system of Shiraia bambusicola S4201 was constructed to overexpress transcription factor in the hypocrellin A biosynthetic gene cluster,regulate the expression of other genes in the gene cluster,and increase the yield of hypocrellin A.The synthesis of perylenequinones induced by H₂O₂ and NO was studied.The interaction between H₂O₂ and NO signal was investigated.The reason for the increase of hypocrellin A production was analyzed at the transcriptional level.It provided a theoretical reference for the biosynthesis of elsinochrome A and hypocrellin A.