Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamen tous fungus, Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysi s and no cystathionine-gamma -lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C 10, in MDFA medium supplemented with methionine, However, there was no diff erence in cephalosporin production between parental strain A. chrysogenum C 10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine, These results indicate that the supply of cysteine thro ugh the transsulfuration pathway is required for high-level cephalosporin b iosynthesis but not for low-level production of this antibiotic in methioni ne-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesi s in A. chrysogenum derives from the autotrophic (SH2) and the reverse tran ssulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB m utants, indicating that the induction effect is not mediated by cystathioni ne-gamma -lyase.