  4.4.1.36 | more |
construction of an egt2+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. Generation of a egt2+ deletion mutant, DELTAegt2, by replacing the target loci in the wild-type 972 strain with the kanamycin resistance marker (kanMX). Mutant DELTASPBC660.12c, designated DELTAegt2, shows a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibit no phenotypic defects during vegetative growth or quiescence. Construction of multiple deletion mutants of SPBC660.12c (egt2+) with the other candidate EgtE homologues, but even in successfully constructed double and triple mutants, a significant amount of EGT still remains, because hercynylcysteine sulfoxide can spontaneously convert into ergothioneine in the presence of pyridoxal 5'phosphate. Mutant DELTAegt1 strain shows no growth defects during cultivation in either rich (YE) or minimal (EMM2) culture media, deletion of gene egt1+ causes no significant perturbation to the intracellular metabolome of quiescent cells |
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