Functional Analysis Of The Key Genes In Huperzine A Biosynthesis Of Endophytic Fungi In Huperzia Serrata

Huperzine A?Hup A?,discovered by Chinese scientists,is one of lycopodium alkaloid to treat mild-to-moderate Alzheimer's disease.However,it is difficult to chemically synthesize Hup A,to artificially cultivate and tissue culture Huperziaceae plants.The limited resource of Hup A,which is mainly isolated from decreasing Huperzia serrata and some other species in Traditional Chinese medicine resources?such as Huperziaceae?in low quantity,restricts its pharmaceutical production.In this study,we isolated two HupA-producing fungi Colletotrichum gloeosporioides Cg01 and Penicillium polonicum hy4 from H.serrata and did whole-genome sequencing and transcriptome sequencing.Based on comparative genomics and transriptomics,we obtained a series of candidate genes,including lysine decarboxylase?LDC?,copper amine oxidase?CAO?,polyketides synthases?PKS?,histone methyltransferase?HMT?,histone acetyltransferase?HAT?,histone deacetylase?HDAC?,et al.We analyzed the relationship between candidate genes and hup A biosynthesis in endophytic fungi of H.serrata by gene-knockout.This study will provide insights into the mechanism of Hup A biosynthesis and regulation,be of great importance to biosynthesize HupA,consequently resolving the problem of limited natural resources and realizing sustainable development.The main results were as following:?1?Thirty-six strains of endophytic fungi were isolated and identified from the stem?25 strains?and leaves?11 strains?of H.serrata.It covered 3 class,6 order,11 family and 15 genera,five incertae sedis.No hupA was identified in hyphae in all isolated endophytic fungi,and 21.04 ?g/L hupA was detected in broth of C.gloeosporioides Cg01.Inducers were screened;only extracts of H.serrata could induce the hupA biosynthesis,while ethanol,methyl jasmonate and salicylic acid could not induce the hupA biosynthesis.The content of hupA in C.gloeosporioides Cg01 fermentation broth was up to 556.21 ?g/L,increasing almost 26 folds,when the extracts of H.serrata added.The P.polonicum hy4 could biosynthesize hupA when the extracts of H.serrata added,and the content was up to 535.54 ?g/L.The hupA content in the fermentation broth of the isolated endophytic fungi in this study was higher than that in the other reported endophytic fungi.?2?The genomes of these two endophytic fungi were sequenced by Illumina Hiseq 2000 with the strategy of paired-end and mate-pair.The reads were assembled by Allpaths-LG and the genes were predicted by Genemark-ES v4.3 and Augustus v2.7.The genome of P.polonicum hy4 was 32.35 Mb,with average GC content of 46.9%,scaffold N50 of ?4.0 Mb,N90 of ?1.1 Mb.It encoded 11,696 genes,including 1 LDC gene,6 CAO genes,23 PKS genes,9 HMT genes,17 HAT genes,13 HDAC genes.The genome of C.gloeosporioides Cg01 was larger?53.19 Mb?,with average GC content of 53.2%,scaffold N50 of ?1.7 Mb,N90 of ?0.5 Mb.It encoded 17,713 genes,including 3 LDC gene,12 CAO genes,44 PKS genes,8 HMT genes,17 HAT genes,13 HDAC genes.We did homologous analysis between the two endophytic fungi and found 7940 orthologous genes in each fungus.In addition,the mitochondrial genomes of hy4 and Cg01 were completed and the evolutionary status of hy4 and Cg01 were confirmed by phylogenetic analysis with the whole mitochondrial genome.?3?The differentially expressed genes were analyzed by Cufflinks,and the GO and KEGG enrichments were analyzed by GOseq and KOBAS.According to the KEGG pathway enrichment and GO functional cluster enrichment by differentially expressed genes with similar tendency between two endophytic fungi,the differentially expressed genes were focused on the carbon and nitrogen metabolism,nucleolus,ribosome and the process of rRNA.We screened the candidate genes associated with hupA biosynthesis by differentially expressed genes and orthologous genes with similar express tendency.It included biosynthesis genes,such as LDC genes Cg01_LDC₀9672-RA,Cg01_LDC₀2374-RA,Cg01_LDC₀2554-RA,CAO genes Cg01_CAO₁1135-RA,Cg01_CAO₁4540-RA,Cg01_CAO₁7078-RA,Cg01_CAO₀6279-RA,PKS genes Cg01_PKS₀0212-RA,Cg01_PKS₁4302-RA,Cg01_PKS₀7270-RA,Cg01_PKS₀9514-RA;epigenetic regulation genes,such as HMT gene Cg01_HMT₁2377-RA,HAT genes Cg01_HAT₀4293-RA,Cg01_HAT₁6852-RA,HDAC gene Cg01_HDAC₁0899-RA,Cg01_HDAC₁2662-RA,Cg01_HDAC₁2969-RA.?4?We knock-out the candidate biosynthesis genes individually,and the hupA content of each mutant fermentation broth was not changed.However,both knock-out of LDC genes Cg01_LDC₀9672-RA and Cg01_LDC₀2374-RA,the hupA content was increased by 83%.At the same time,the expression of Cg01_LDC₀2554-RA was decreased.According to these,we proposed the hupA biosynthesis was not by the pathway of lysine decarboxylate to cadaverine,which might be the branch of precursors of hupA biosynthesis,reducing the flow distribution of hupA precursor in hupA biosynthesis.?5?We knock-out the candidate regulation genes successfully except Cg01_HAT₀4293-RA,Cg01_HDAC₁2662-RA.The hupA content of mutant strain ?Cg01_HAT₁6852-RA was not changed,while that of mutant strains ?Cg01_HMT₁2377-RA,?Cg01_HDAC₁0899-RA,?Cg01_HDAC₁2969-RA were reduced by 13.46%,22.26%,18.37%,respectively.It reveals that the HMT genes and HDAC genes associated with histone modification were involved in the regulation of hupA biosynthesis in endophytic fungi.In conclusion,we isolated and identified two hupA-producing endophytic fungi P.polonicum hy4 and C.gloeosporioides Cg01 from H.serrata,and found that the addition of extracts of H.serrata could efficiently induce the biosynthesis of hupA.We identified the genome features of P.polonicum hy4 and C.gloeosporioides Cg01 by genome sequencing.The transcriptome analysis revealed that the induced biosynthesis of hupA might be associated with carbon and nitrogen metabolism,nucleolus,ribosome and the process of rRNA.The function of candidate genes including biosynthesis genes and histone modification genes were identified by homologous deletion.We proposed that the biosynthesis pathway in endophytic fungi might be different from that in the plant H.serrata.The histone modification genes Cg01_HMT₁2377-RA,Cg01_HDAC₁0899-RA,Cg01_HDAC₁2969-RA were related with the regulation of the hupA biosynthesis.