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Journal of Bacteriology, March 2001, p. 1765-1772, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1765-1772.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Gang Liu,1 Javier Casqueiro,1,2 Oscar Bañuelos,1 Rosa E. Cardoza,2 Santiago Gutiérrez,1,2 and Juan F. Martín1,2,*

Area of Microbiology, Faculty of Biology, University of León, 24071 León,1 and Institute of Biotechnology (INBIOTEC), Science Park of León, 24006 León,2 Spain

Received 26 October 2000/Accepted 14 December 2000

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous 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 analysis and no cystathionine-gamma -lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH2) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma -lyase.

* Corresponding author. Mailing address: Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain. Phone: 34 987 291505. Fax: 34 987 291506. E-mail: degjmm{at}unileon.es.

Journal of Bacteriology, March 2001, p. 1765-1772, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1765-1772.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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