| Grifola frondosa is a common used medicinal fungus,containing a variety of natural active products and nutrients,with the functions of anti-oxidation,anti-tumor,antihypertension and anti-inflammatory pressure.Ergothioneine is an amino acid derivative with special oxidizing activity.It is mainly used to reduce the symptoms of cell aging,counteract oxidative stress,and remove ultraviolet rays.G.frondosa is rich in ergothioneine,but the synthesis mechanism in the cell has not been reported yet.In recent years,research on the biosynthetic pathways of ergothioneine has developed rapidly.There are two different biosynthetic pathways.One ergothioneine synthetic pathway that exists in a variety of prokaryotes,represented by Mycobacterium smegmatis,is constructed by five synthetic enzymes Egt ABCDE together;another kind of ergothioneine synthesis exists in most eukaryotes,represented by Neurospora crassa,and constitutes with only two synthases Egt1 and Egt2.Most of edible fungus and N.crassa are filamentous fungus,thus their ergothioneine synthetic pathway can be similar with N.crassa.However,the studies on ergothioneine of edible fungus mostly focus on extraction and purification,and there was only one report about Flammulina velutipes for the function research of biosynthetic genes.Therefore,this article used the Saccharomyces cerevisiae exogenous expression system to express the ergothioneine synthesis genes obtained from G.frondosa in vivo,and analyzed the production of ergothioneine by chromatography and mass spectrometry.The main results were as follows:(1)Acquisition and informatics analysis of Gf Egt1 and Gf Egt2.In this study,the Blast platform was used to compare the genome and its protein annotation data of G.frondosa with N.crassa ergothioneine synthase Nc Egt1 and Nc Egt2 as comparison sources,and three unidentified proteins were found.Subsequently,using G.frondosa c DNA as a template,two gene sequences were amplified and named Gf Egt1 and Gf Egt2,and the corresponding proteins were named Gf Egt1 and Gf Egt2.Later,the phylogeny of G.frondosa was found to be similar to that of Trametes versicolor,Ganoderma sinense,and Wolfiporia cocos by constructing evolutionary tree.Then,analyzed the structure of Gf Egt1 and Gf Egt2 proteins,and preliminary explored the binding methods of the target proteins with substrate and coenzyme.In this study,two ergothioneine synthesis genes of G.frondosa were obtained for the first time,and evolutionary tree analysis was performed with unidentified ergothioneine synthesis proteins of edible fungus.(2)Verification of ergothioneine synthesis function of Gf Egt1 and Gf Egt2.By expressing Gf Egt1 and Gf Egt2 in S.cerevisiae,and comparing with recombinant strains containing empty vectors and wild-type strains to comparative analyze the HPLC detection of their extraction,indirectly speculated that Gf Egt1 and Gf Egt2 had corresponding enzymatic activities.ESI-MS identification of the extraction confirmed that the products contained ergothioneine.In this study,the two ergothioneine synthases of G.frondosa were expressed and their activities were identified by HPLC and ESI-MS for the first time.(3)Optimization of ergothioneine synthesis of yeast engineering strains.By adjusting the medium and expression elements,it was concluded that the best yeast engineering strain chose the TEF1 p promoter for protein expression,activated by the medium with glucose as the sole carbon source,and fermented in the medium with glycerol as the sole carbon source.After 7 days fermentation,the system can stably produce 11-13 mg / L ergothioneine.If3 m M histidine were contained in medium,the ergothioneine concentration can reach up to17 mg / L.Besides,it was found that the addition of amino acids did not have an excessive effect on the synthesis of ergothioneine in yeast engineering strains.This study found for the first time that unconventional carbon sources can effectively increase the production of ergothioneine by yeast engineering strain,with a relative glucose increase of 3 to 4 times. |
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