Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-gamma-lyase

In Acremonium chrysogenum, biosynthesis of cysteine for the formation of ce phalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 ge nomic library in lambda EMBL3-ble. The cloned DNA fragment encodes a protei n of 423 amino acids with a deduced molecular mass of 45 kDa that shows gre at similarity to cystathionine-gamma -lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional beca use it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma -lyase. The cloned gen e did not complement A. nidulans mecA or metG mutants. Enzyme activity assa ys confirmed that the cloned mecB gene encodes a cystathionine-gamma -lyase activity. The mecB gene is present in a single copy in the wild-type A. ch rysogenum (Brotzu's strain) and also in the A. chrysogenum strain CIO, a hi gh cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb ), as shown by hybridization to A. chrysogenum chromosomes resolved by puls ed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript leve ls were not significantly affected by addition of DL-methionine to the cult ure, indicating that expression of this gene is not regulated by methionine . The availability of this gene provides a very useful tool for understandi ng the proposed role of cystathionine-gamma -lyase in splitting cystathioni ne to supply cysteine for cephalosporin biosynthesis.