| Jasmonic acid is a cyclopentanone compound derived from fatty acids and has broad application prospects in agriculture,flavors and fragrances,and pharmaceutical industries.At present,the synthesis pathway of jasmonic acid in plants is relatively clear,but the research in fungi is still limited to the isotope tracer method and the detection of related precursors/metabolites.In the early works of our laboratory,Lasiodiplodia iranensis producing jasmonic acid by fermentation was screened.In order to improve jasmonic acid production,we took L.iranensis as the research object in this paper and initially determined that 10R-lipoxygenase is a key enzyme in the synthesis of jasmonic acid by GC-MS analysis of fermentation products,genome sequencing and analysis,excavation of key enzymes related to synthetic pathways and catalytic verification in vitro.By adding the precursor?-linolenic acid during fermentation,the production of jasmonic acid can be more than doubled.Details are as follows:?1?Detection of jasmonates in fermentation broth.Methylated fermentation broth was detected by the GC-MS method.The maximum proportion of jasmonates was 80.26%,of which jasmonic acid was the main component with maximum proportion of 78.05%,followed by dihydrojasmonic acid with maximum proportion of 2.21%,the maximum proportion of cis-jasmone was very small,only 0.17%.?2?Analysis of L.iranensis genome sequencing results.Using the Illumina HiSeq sequencing platform to de novo sequence for L.iranensis,43.01M genomic sequence was obtained with GC content of 54.82%,78 scafolds,N50 of 1.08M.Using the software Prokka combined with the annotation file of the swiss-prot library,11224 genes were predicted.Through comparison with the GO,COG,and KEGG databases,7360 genes were annotated,with an annotation rate of 65.57%.Four candidate genes for jasmonic acid synthesis were mined in the annotation data:one lipoxygenase?10R-LOX?,three?-oxidases?3-ketoacyl-CoA-thiolase?KAT1,KAT2?,Acetyl-CoA oxidase?ACX??.Phylogenetic and domains analysis indicated that the annotation information of KAT1,KAT2,and ACX were accurate,10R-LOX is a fusion protein with peroxidase and p450 domains.?3?Verification of candidate genes by transcriptional expression analysis.Based on the gene sequences of the four target genes,primers were designed.All four target genes were transcribed by RT-PCR analysis.EF1?was selected as the internal reference gene for qRT-PCR experiment to quantify gene expression.The results showed that the expression levels of the four target genes during the fermentation of L.iranensis were positively related to jasmonic acid production.Expression of 10R-LOX had the strongest correlation to jasmonic acid production,with a correlation coefficient of 0.96.?4?Functional verification for 10R-LOX.10R-LOX was cloned and expressed in E.coli BL21.The supernatant?soluble expression?was used to catalyze?-linolenic acid.The product was analyzed by LC-MS,and showed the presence of hydroperoxide and???-ketone?generated by AOS catalyzed hydroperoxide?.In addition,there are conserved motifs of 10R-DOX and AOS in the enzyme domain.It was speculated that this enzyme catalyzes the first two steps in the synthesis pathway of jasmonic acid.?5?L.iranensis fermentation test for producing jasmonic acid.30 g·L-1 sucrose were supplemented at 96 h during static fermentation of L.iranensis,which incresed the yield of jasmonic acid to 1.99 g·L-1;3 g·L-1?-linolenic acid were added at 72 h during static fermentation of L.iranensis,which incresed the yield of jasmonic acid to 2.9 g·L-1.Which were1.28 and 1.63 times higher than the control,respectively.L.iranensis can be repeated for two batches of fermentation.Jasmonic acid production in the second batch was 101.5%of the first batch,and the fermentation cycle was shortened. |
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