Overexpression Of Llm1 Affects The Secondary Metabolites And Development Of Eurotium Cristatum

As the dominant fungus during the fermentation of Fu-brick tea,the extracellular enzymes and secondary metabolites secreted by Eurotium cristatum affects the quality and taste of tea.There are a large number of synthetic silencing gene clusters of secondary metabolites with unknown functions in the genome of E.cristatum.However,the expression of silencing gene clusters is low or even not expressed under conventional laboratory culture conditions.In order to make full use of the potential of secondary metabolites produced by E.cristatum,the strategy of overexpression of global secondary metabolite regulators was used to activate silent gene clusters.Based on the genomic and transcriptome data of E.cristatum,a methyltransferase gene llm1 gene similar to LaeA was found and identified in the genome of E.cristatum by local blast.The overexpression mutant of E.cristatum llm1 gene was successfully constructed by seamless cloning,Agrobacterium transformation and real-time quantitative PCR(RT-q PCR).The colony morphology and asexual development of overexpression mutant and wild-type strain were compared,observed and counted.The maps of fermentation products of overexpression mutant and wild-type(WT)were compared by high performance liquid chromatography(HPLC),the differential metabolites were analyzed,and then the gene expression profiles of overexpression mutant and WT were analyzed by transcriptome sequencing.The main results are as follows:1.A putative methyltransferase gene llm1 was successfully cloned from the genome of E.cristatum.The ORF of the gene is 1116 bp and encodes 371 nucleotides.It is predicted that the molecular weight of the protein encoded by llm1 is 42345.88 k D,the molecular formula was C₁₈₉₃H₂₈₀₁N₅₀₅O₅₈₁S₁₃,the theoretical isoelectric point is 4.77,it is a hydrophilic protein and does not contain signal peptide.Seamless cloning and Agrobacterium tumefaciens transformation were used to successfully transform the vector into E.cristatum,and the overexpression mutant was verified by resistance screening,vector fragment amplification and RT-q PCR.2.Overexpression of llm1 strain resulted in a significant decrease in the number of conidia of E.cristatum,and this regulation mechanism was not affected by light.At the same time,overexpression of llm1 resulted in the reduction of oxidative stress tolerance of E.cristatum,which showed that the overexpression mutant grew slowly,indicating that llm1 may negatively regulate the oxidative tolerance of E.cristatum.Interestingly,the color of the fermentation broth of the overexpression mutant was significantly deeper than that of WT,it was speculated that llm1 was involved in the accumulation of melanin,yellow pigment and other pigment substances of E.cristatum,and showed negative regulation.The metabolite fingerprints based on HPLC showed that there were significant differences between llm1 gene overexpression mutant and wild-type strain,which showed the changes of the content of most main metabolites in the fermentation products.Among them,three new chromatographic peaks were detected in the overexpression mutant,indicating that overexpression of llm1 may activate the silencing gene cluster of secondary metabolites of E.cristatum,resulting in the production of new compounds.3.Transcriptome sequencing was used to further analyze the effect of overexpression of llm1 gene on the secondary metabolic synthesis gene cluster of E.cristatum.The results showed that a total of 600 differentially expressed genes were screened,of which 256 genes were up-regulated and 354 genes were down regulated.The GO database was used to enrich and analyze the significantly differentially expressed genes(DEGs).The results show that the main enrichment pathway of DEGs is related to the transmembrane transport of substances and the synthesis of secondary metabolites,including phenolic substances,isoprenoid biosynthesis and terpene metabolism,indicating that the pathway significantly affected by overexpression of llm1 was related to secondary metabolites.4.The transcriptome data were further annotated based on anti SMASH database.A total of 582 genes encoding secondary metabolites were annotated,of which 64 genes were significantly differentially expressed,accounting for 11.01%of all secondary metabolite genes.A total of 17 core regulatory genes in each secondary metabolite synthesis gene cluster were significantly differentially expressed,of which 11 core genes were significantly up-regulated and 6 genes were significantly down regulated.Differential genes are annotated as non-ribosomal polypeptide synthase,polyketide synthase and mono oxidase,which are related to the synthesis and modification of secondary metabolites.Interestingly,the expression levels of 4 core regulatory genes in secondary metabolite synthesis gene cluster 32 were significantly up-regulated,and 50%of the genes in this gene cluster were significantly up-regulated.The final results showed that overexpression of llm1gene activated the expression of secondary metabolic gene cluster of E.cristatum.