Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum , which encodes a functional cystat
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O R I GI N A L P A P E R
A. T. Marcos á K. Kosalkova á R. E. Cardoza F. Fierro á S. GutieÂrrez á J. F. Martõ n
Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-c-lyase Received: 20 March 2000 / Accepted: 30 August 2000 / Published online: 18 October 2000 Ó Springer-Verlag 2000
Abstract In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in kEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-c-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-c-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays con®rmed that the cloned mecB gene encodes a cystathionine-c-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-®eld 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 levels were not signi®cantly aected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-c-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis. Communicated by C. A. M. J. J. van den Hondel A. T. Marcos (&) á K. Kosalkova á R. E. Cardoza á F. Fierro S. GutieÂrrez á J. F. Martõ n Instituto de Biotecnologõ a INBIOTEC, Parque Cientõ ®co de LeoÂn, Avda. del Real, 1, 24006 Leon, and Universidad de LeoÂn, Facultad de Biologõ a, 24071 LeoÂn, Spain E-mail: [email protected] Tel.: +34-987-291505 Fax: +34-987-291506
Key words Cystathionine á Cysteine á Methionine á Cystathionine-c-lyase á Transsulfuration pathway
Introduction In Saccharomyces cerevisiae and ®lamentous fungi cysteine is synthesized by two pathways (Ono et al. 1994; Paszewski et al. 1994) (Fig. 1). One (the so-called autotrophic pathway) converts inorganic sulfur to cysteine by the action of serine O-acetyltransferase and O-acetylserine sulfhydrylase (Fig. 1). The second cysteine biosynthetic pathway in fungi is the reverse transsulfuration pathway, which converts methionine into cysteine using cystathionine b-synthase and cystathionine-c-lyase (Cherest and Surdin-Kerjan 1992). Recent evidence based on gene disruption studies indicates that in S. cerevisiae the rev
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