The Research On The Function Of Ergothioneine Biosynthetic Genes And G Protein-coupled Receptor Genes In Flammulina Velutipes

Flammulina velutipes is delicious and rich in nutrients and bioactive compounds.Therefore,long-term consumption of F.velutipes can prevent diseases such as cancer,high blood pressure,ischemic heart disease,high blood lipids,diabetes and gallstones.Ergothioneine is a natural antioxidant that has been used in health care,food,cosmetics and pharmaceuticals due to its anti-inflammatory,anti-oxidant,and cell-protection against UV damage and cardiovascular protection.At present,the biosynthesis pathway of ergothioneine in F.velutipes remains unknown.In recent years,the industrialization of F.velutipes industry has developed rapidly.In recent years,the total annual output of F.velutipes grew by about 20%.However,F.velutipes is a kind of low-temperature sturdy fungi.Hyphae needs to be induced at low temperature before they can form primordia and develop into fruit bodies,which resulting in high energy consumption and high cost in the current industrial production.G protein-coupled receptors are a class of protein receptors with seven transmembrane structures on cell membranes.Studies have shown that G protein-coupled receptors in fungi were involved in the growth of mycelium,differentiation of primordia and the formation of fruit body.Therefore,in this paper,we explored the functions of ergothioneine biosynthesis genes and G protein-coupled receptor genes in F.velutipes by enzymatic reactions in vitro and gene knockouts in vivo.The main findings are as follows:(1)Expression and functional identification of ergothioneine synthetic genes 1)Expression and functional identification of Fvegt1 gene.In this study,we used Blast p homologous alignment to obtain the ergothioneine biosynthesis gene Fvegt1.The ergothioneine biosynthetic gene Fvegt1 was cloned by overlap-derivatization PCR and the recombinant enzyme FvEgt1 was obtained in E.coli BL21(DE3).HPLC and ESI-MS analysis showed that the recombinant enzyme FvEgt1 could catalyze the formations of S-adenosyl-L homocysteine and hercynlcysteine sulfoxide with histidine,cysteine and S-adenosylmethionine as substrates.This study first defined the function of the Fvegt1 gene in F.velutipes.The recombinant FvEgt1 expressed in this gene not only acted as a methyltransferase but also a 5’-histidylcysteine sulfoxide synthase.2)Expressions and functional identifications of Fvegt2 and Fvegt3 genes.In this study,we used Blast p homologous alignment to obtain the ergothioneine biosynthesis genes Fvegt2 and Fvegt3.The ergothioneine biosynthetic genes Fvegt2 and Fvegt3 were cloned by total gene synthesis.After expression in E.coli BL21(DE3),recombinant enzymes FvEgt2 and FvEgt3 were obtained.HPLC and ESI-MS analysis showed that the recombinase FvEgt2 and FvEgt2 can catalyze the formation of ergothioneine from hercynlcysteine sulfoxide.This study for the first time established a biosynthetic pathway for ergothioneine in basidiomycotina F.velutipes other than bacteria and ascomycetes.3)Study on the mechanism of action of FvEgt2 and FvEgt3.In this study,the two cysteine desulfurase were found for the first time in F.velutipes,namely FvEgt2 cysteine-cysteine desulfurase and FvEgt3 pyridoxal phosphate-cysteine desulfurase.Both enzymes were involved in the formation of ergothioneine.Binding to cysteine could activate SH on cysteine to form sulfur anion,whereas FvEgt3 catalyzes the formation of ketimine adduct with pyridoxal phosphate and hercynlcysteine sulfoxide.The former sulfur anion attacked the ketimine adduct to form ergothioneine.(2)The putative G protein-coupled receptor genes were obtained.Two hypothetical G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in F.velutipes were obtained by amino acid homologous comparison(Blast P)and transcriptome data of mycelium phase of mycelial mushroom and cold-induced primordial transcriptome analysis.Among them,the expression level of Fvgpcr1 in the primordial phase was upregulated compared to the mycelial phase,whereas the expression level of Fvgpcr2 was down-regulated.(3)CRISPR/Cas9 knockout vector was constructed.Four CRISPR/Cas9 knockout vectors containing hph,sgRNA,and Fvcas9 were constructed by restriction endonuclease digestion.They were: pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA1 pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA2,pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1,pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA2.(4)Optimized Agrobacterium-mediated system for genetic transformation of F.velutipes.A large number of transformants could be obtained by using fermentation broth of F.velutipes mycelium as a transformation receptor.This is the first time to use fermentation broth as transformation receptor.(5)Identification of F.velutipes strain containing FvCas9 protein.Four CRISPR/Cas9 knockout vectors were transformed into F.velutipes by agrobacterium-mediated genetic transformation.Thirteen transformants targeting Fvgpcr1 and 33 transformants targeting Fvgpcr2 were obtained after hygromycin selection and PCR identification.Targeted mutation site sequencing results showed that the target site was not mutated.RT-qPCR and Western blot results showed that Fvcas9 gene has been successfully integrated into the F.velutipes genome and can be normally transcribed and translated into FvCas9 protein.In this study,F.velutipes strains stably expressing FvCas9 protein were obtained.