Rapid screening of glutamate-producing macrofungi

Abstract:

[Background] Macrofungi are important food and drug resources, which can produce a variety of nutrients including essential amino acids. Glutamate, one of the important functional molecules of edible macrofungi, presents umami taste and is an important index to measure the nutritional value of edible fungi. [Objective] We evaluated the resources of macrofungal strains containing glutamate based on the conserved homologous genes of glutamate synthase in Agaricales, aiming to guide the exploitation of wild macrofungal resources and the manufacture of glutamate products. [Methods] We compared the glutamate synthase gene sequences from 25 strains and analyzed the conserved exon region. Two pairs of degenerate primers, GS Ag_les F1/R1 and GS Ag_les F2/R2, were designed for the conserved exon region of glutamate synthase and used for PCR screening of 65 strains belonging to 13 families of Agaricales. In addition, phenylisothiocyanate derivatization was combined with LC-MS and HPLC to quantify the glutamate produced by the 65 strains, and then the screening efficiency of this method was evaluated. [Results] The screening efficiency of primers designed in this study was 86.2%, and the screening efficiency of the primer pair GS Ag_les F1/R1 was significantly better than that of GS Ag_les F2/R2. The fermentation conditions of Agaricales for the production of glutamate were optimized as follows:LMM medium, 28℃, 200 r/min and 2-4 days. The results of PCR and HPLC showed that the primer screening method in this study had a true positive rate of 81.5%, and the application scope involved multiple families including Tricholomataceae,Physalacriaceae, Agaricaceae, Cortinariaceae, Bolbitiaceae, Lyophyllaceae, Pleurotaceae, Psathyrellaceae and Nidulariaceae of Agaricales. [Conclusion] The primers obtained in this study exhibit a good application prospect in the screening of glutamate-producing strains of Agaricales.