High level of cephalosporin biosynthesis in Acremonium chrysogenum C10 is dependen on the transsulfuration pathway: targeted inactivation of the mecB gene
Liu G.1, Casqueiro J.1,2, Ullán R.V.1, Bañuelos O.1, Cardoza R.E.2, Gutiérrez S.1,2, Naranjo L.1, and Martín J.F.1,2.
1Area of Microbiology, Faculty of Biology, University of León, 24071 León and 2Institute of Biotechnology (INBIOTEC), Avda del Real Nº1, 24006 León, Spain.
Acremonium
chrysogenum is industrially used as cephalosporin C producer. Cysteine
is a precursor molecule in antibiotic biosynthesis and there are two ways
to synthesize cysteine in filamentous fungi microorganisms. One, the so-called
autotrophic pathway, converts inorganic sulphur to cysteine via the serine
O-acetyltransferase and O-acetylserine sulfhydrilase. In the second one, cysteine
can be obtained via the reverse transsulfuration pathway in which the sulphur
atom of the methionine is transferred to cysteine through s-adenosylmethionine,
s-adenosylhomocysteine, homocysteine and cystathionine as intermediates. We
have studied the contribution of the transsulfuration pathway to cephalosporin
C biosynthesis by targeted inactivation of the mecB gene encoding cysthathionine-g-lyase.
We increased 10 times the gene disruption eficiency in Acremonium chrysogemum
by adapting the double marker technique to filamentous fungi. Disruption of
the mecB gene with the double marker technique was obtained in 5% of
the transformants screened. Mutants T6 and T45 that showed by Southern blot
analysis to have the mecB inactive, lacked cystathionine-g-liase
activity. These mutants also exhibited a lower cephalosporin production as
compared to the control strain A. chrysogenum C10, in MDFA plus 3 g/L
of methionine. However, there was no difference in cephalosporin biosynthesis
between A. chrysogenum C10 and the mutants T6 and T45 in MDFA without
methionine, showing that for high cephalosoporin biosynthesis the supply of
cysteine through the transsulfuration pathway is required.
