Gliotoxin, a redox-active metabolite, is produced
by the opportunistic fungal pathogen Aspergillus
fumigatus, and its biosynthesis is directed by the
gli gene cluster. Knowledge of the biosynthetic
pathway to gliotoxin, which contains a disulfide
bridge of unknown origin, is limited, although L-Phe
and L-Ser are known biosynthetic precursors. Deletion
of gliG from the gli cluster, herein functionally
confirmed as a glutathione S-transferase, results in
abrogation of gliotoxin biosynthesis and accumulation
of 6-benzyl-6-hydroxy-1-methoxy-3-methylenepiperazine-
2,5-dione. This putative shunt metabolite
from the gliotoxin biosynthetic pathway contains an
intriguing hydroxyl group at C-6, consistent with
a gliotoxin biosynthetic pathway involving thiolation
via addition of the glutathione thiol group to a reactive
acyl imine intermediate. Complementation of gliG
restored gliotoxin production and, unlike gliT, gliG
was found not to be involved in fungal self-protection
against gliotoxin