Isopenicillin N epimerase activity in a high cephalosporin-producing strain of Cephalosporium acremonium

Lübbe, Claus; Wolfe, Saul; Demain, Arnold L.

doi:10.1007/bf00257034

Cited by 10 publications

(7 citation statements)

References 4 publications

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“…Bioinformatic analysis of CefD1 and CefD2 amino acid sequences showed that both enzymes contain a peroxisomal targeting signal. CefD1 has Moreover, in early biochemical studies [68], it was reported that pH 7.0 is the optimum pH for conversion of IPN into PenN in A. chrysogenum cell-free extracts, which is coincident with the estimated pH of the peroxisomal lumen [139]. The presence of CefD1-and CefD2-related proteins (although not strict orthologs) in other fungi lacking the cephalosporin cluster suggests that A. chrysogenum has recruited and adapted these genes from other fungal functions or perhaps from nonfungal organisms by a horizontal gene transfer mechanism [43,74].…”

Section: Peroxisomal Enzymes Involved In Cephalosporin Biosynthesismentioning

confidence: 99%

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Martı́n

Ullán

Garcı́a-Estrada

2012

Journal of Industrial Microbiology and Biotechnology

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Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H(2)O(2) (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillin N acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillin N) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.

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Section: Peroxisomal Enzymes Involved In Cephalosporin Biosynthesismentioning

confidence: 99%

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Martı́n

Ullán

Garcı́a-Estrada

2012

Journal of Industrial Microbiology and Biotechnology

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“…The IPN epimerase assay was performed as described by Lu¨bbe et al (1986) and modified by Ulla´n et al (2002). IPN epimerase activity was determined by measuring the IPN-dependent coupled conversion of IPN into CPC in vitro.…”

Section: Ipn Epimerase Activity Assaymentioning

confidence: 99%

“…The IPN is later converted into penicillin N (PenN) by an epimerase system that has remained uncharacterized for many years (Lu¨bbe et al 1986;Ulla´n et al 2002). After the epimerization step, PenN is converted into deacetoxycephalosporin C (DAOC) and deacetylcephalosporin C (DAC) by the action of a bi-functional enzyme with thiazolidine ring-expanding activity (expandase) and 3¢-hydroxylase activity.…”

Section: Introductionmentioning

confidence: 99%

Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

et al. 2004

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The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72-96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression.

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“…IPN Epimerase Assay-The IPN epimerase assay was performed as described by Lü bbe et al (34). IPN epimerase activity was determined by measuring the in vitro conversion of isopenicillin into penicillin N. The results were quantified by a coupled reaction by determining the conversion of IPN into cephalosporin C (CPC).…”

Section: Methodsmentioning

confidence: 99%

A Novel Epimerization System in Fungal Secondary Metabolism Involved in the Conversion of Isopenicillin N into Penicillin N inAcremonium chrysogenum

Ullán

Casqueiro

Bañuelos

et al. 2002

Journal of Biological Chemistry

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The epimerization step that converts isopenicillin N into penicillin N during cephalosporin biosynthesis has remained uncharacterized despite its industrial relevance. A transcriptional analysis of a 9-kb region located downstream of the pcbC gene revealed the presence of two transcripts that correspond to the genes named cefD1 and cefD2 encoding proteins with high similarity to long chain acyl-CoA synthetases and acylCoA racemases from Mus musculus, Homo sapiens, and Rattus norvegicus. Both genes are expressed in opposite orientations from a bidirectional promoter region. Targeted inactivation of cefD1 and cefD2 was achieved by the two-marker gene replacement procedure. Disrupted strains lacked isopenicillin N epimerase activity, were blocked in cephalosporin C production, and accumulated isopenicillin N. Complementation in trans of the disrupted nonproducer mutant with both genes restored epimerase activity and cephalosporin biosynthesis. However, when cefD1 or cefD2 were introduced separately into the double-disrupted mutant, no epimerase activity was detected, indicating that the concerted action of both proteins encoded by cefD1 and cefD2 is required for epimerization of isopenicillin N into penicillin N. This epimerization system occurs in eukaryotic cells and is entirely different from the known epimerization systems involved in the biosynthesis of bacterial ␤-lactam antibiotics.The molecular genetics of cephalosporin biosynthesis is an excellent model for secondary metabolism, since considerable information on the enzymology (reviewed in Refs. 1 and 2), molecular genetics, and gene expression mechanisms (3-5) has accumulated in the last few years. The biosynthesis of cephalosporins by Acremonium chrysogenum and cephamycins by Amycolatopsis (Nocardia) lactamdurans and Streptomyces clavuligerus (reviewed in Refs. 6 and 7) begins with the formation1 by the ACV synthetase (8), followed by cyclicization of ACV to isopenicillin N (IPN). IPN is later converted into penicillin N by an epimerase activity that has remained uncharacterized so far in A. chrysogenum. After the epimerization step, penicillin N is transformed by a deacetoxycephalosporin C synthase (expandase) into deacetoxycephalosporin C, and finally, deacetoxycephalosporin C is converted into deacetylcephalosporin C (DAC) and cephalosporin C by DAC synthase (hydroxylase) and DAC acetyltransferase, respectively (Fig. 1).The genes pcbAB (9) and pcbC (10) encoding ACV synthetase and IPN synthase are linked together in chromosome VII in the so-called "early cephalosporin gene cluster" (11). The genes cefEF, encoding the bifunctional expandase-hydroxylase (12), and cefG, encoding the DAC acetyltransferase (13,14), are linked together in the so-called "late cephalosporin cluster" in chromosome I (11).The isopenicillin N epimerization step still remains unclear. Demain and co-workers (15, 16) reported that isopenicillin N was converted into penicillin N by extracts of A. chrysogenum, although the epimerizing enzyme was extremely labile (17, 18), preventing ...

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Isopenicillin N epimerase activity in a high cephalosporin-producing strain of Cephalosporium acremonium

Cited by 10 publications

References 4 publications

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

A Novel Epimerization System in Fungal Secondary Metabolism Involved in the Conversion of Isopenicillin N into Penicillin N inAcremonium chrysogenum

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