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

Marcos, Ana T.; Kosalková, Katarina; Cardoza, Rosa E.; Fierro, Francisco; Gutiérrez, Santiago; Martı́n, Juan F.

doi:10.1007/s004380000363

Cited by 11 publications

(11 citation statements)

References 24 publications

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“…These results indicated that those transformants that suffered the canonical recombination process lost the mecB gene in the gene replacement process. These results confirmed that there is a single copy of the mecB gene in A. chrysogenum, as was proposed before on the basis of Southern hybridization evidence (19).…”

Section: Vol 183 2001supporting

confidence: 91%

“…However, this mutant might have contained a second mutation affecting cephalosporin production and, unfortunately, the CIBA-Geigy mutant is not available for further studies (J. Heim, personal communication). In this article, we have inactivated the mecB gene, which encodes cystathionine-␥-lyase activity in A. chrysogenum (19). The in- activation of the mecB gene does not affect cephalosporin biosynthesis when the sulfur source is sulfate, i.e., cysteine for cephalosporin biosynthesis may be synthesized from either sulfate or methionine.…”

Section: Discussionmentioning

confidence: 99%

“…The mecB gene of A. chrysogenum has recently been cloned and characterized in our laboratory (19). This gene encodes a cystathionine-␥-lyase activity which splits cystathionine into cysteine and ␣-ketobutyrate.…”

Section: ␤-Lactam Biosynthesis Inmentioning

confidence: 99%

“…Sawada and coworkers established that methionine increases isopenicillin N synthase, deacetoxycephalosporin C synthase (30), and ␣-aminoadipyl-cysteinyl-valine synthetase activities (38) in Acremonium cultures. Later, Velasco et al (35) showed that methionine induces the transcription of three of the four known cephalosporin biosynthetic genes.Therefore, methionine has presumably a double effect on cephalosporin biosynthesis: (i) it could be the main supplier of cysteine via the reverse transsulfuration pathway and (ii) it has an induction effect on cephalosporin biosynthetic genes.The mecB gene of A. chrysogenum has recently been cloned and characterized in our laboratory (19). This gene encodes a cystathionine-␥-lyase activity which splits cystathionine into cysteine and ␣-ketobutyrate.…”

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confidence: 99%

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Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-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

et al. 2001

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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 doublemarker 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-␥-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 (SH 2 ) 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-␥-lyase. ␤-Lactam biosynthesis inAcremonium chrysogenum (formerly known as Cephalosporium acremonium) begins with the nonribosomal condensation of the three precursor amino acids L-␣-aminoadipic acid, L-cysteine, and L-valine (1, 21, 23). There are two ways to synthesize cysteine in filamentous fungi (35,26). One pathway, the so-called autotrophic pathway, converts inorganic sulfur to cysteine via serine O-acetyltransferase and O-acetylserine sulfhydrilase (Fig. 1). In the second route, cysteine can be obtained via the reverse transsulfuration pathway, in which the sulfur atom of methionine is transferred to cysteine through S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, and cystathionine as intermediates.The predominant route of the cysteine supply for ␤-lactam biosynthesis depends on the producer microorganism (6). Cysteine for penicillin biosynthesis in Penicillium chrysogenum is obtained mainly from sulfate reduction (31). In this microorganism, the transsulfuration pathway has been considered to have a minor role in penicillin biosynthesis. On the other hand, the sulfur atom for ␤-lactam biosynthesis by A. chrysogenum is believed to derive preferentially from methionine via the reverse transsulfuration pathway (3, 24). However, there is no genetic evidence for, or against, this hypothesis.DL-Methionine is known to stimulate cephalosporin biosynthesis in A. chrysogenum (7, 16). For many years it was unclear whether the stimulatory effect of DL-methionine was due to a precursor effect (one providing cysteine) or to an inducing effect (8,20,23). Sawada and coworkers established that methionine increases isopenicillin N synthase, deacetoxycephalosporin C synthase (30), and ␣-...

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Section: Vol 183 2001supporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: ␤-Lactam Biosynthesis Inmentioning

confidence: 99%

mentioning

confidence: 99%

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Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-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

et al. 2001

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“…Some bacterial species, including B. subtilis, can also synthesize Cys by the reverse trans-sulfuration pathway using Met as a precursor (71,86). The two genes involved in these reactions are yrhA (encoding cystathionine ␤-synthase) and yrhB (encoding cystathionine ␥-lyase), which are commonly found in the same transcriptional unit.…”

Section: Pathways For Synthesis Of the Sulfur-containing Amino Acids mentioning

confidence: 99%

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

Gutiérrez-Preciado

Henkin

Grundy

et al. 2009

Microbiol Mol Biol Rev

140

230

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SUMMARY The T-box mechanism is a common regulatory strategy used for modulating the expression of genes of amino acid metabolism-related operons in gram-positive bacteria, especially members of the Firmicutes. T-box regulation is usually based on a transcription attenuation mechanism in which an interaction between a specific uncharged tRNA and the 5′ region of the transcript stabilizes an antiterminator structure in preference to a terminator structure, thereby preventing transcription termination. Although single T-box regulatory elements are common, double or triple T-box arrangements are also observed, expanding the regulatory range of these elements. In the present study, we predict the functional implications of T-box regulation in genes encoding aminoacyl-tRNA synthetases, proteins of amino acid biosynthetic pathways, transporters, and regulatory proteins. We also consider the global impact of the use of this regulatory mechanism on cell physiology. Novel biochemical relationships between regulated genes and their corresponding metabolic pathways were revealed. Some of the genes identified, such as the quorum-sensing gene luxS, in members of the Lactobacillaceae were not previously predicted to be regulated by the T-box mechanism. Our analyses also predict an imbalance in tRNA sensing during the regulation of operons containing multiple aminoacyl-tRNA synthetase genes or biosynthetic genes involved in pathways common to more than one amino acid. Based on the distribution of T-box regulatory elements, we propose that this regulatory mechanism originated in a common ancestor of members of the Firmicutes, Chloroflexi, Deinococcus-Thermus group, and Actinobacteria and was transferred into the Deltaproteobacteria by horizontal gene transfer.

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Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation

Özcengiz

Demain

2013

Biotechnology Advances

115

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

Cited by 11 publications

References 24 publications

Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-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 Inactivation of the mecB Gene, Encoding Cystathionine-γ-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

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation

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