Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the β-lactam ring in Aspergillus nidulans

Ramón, Daniel; Carramolino, Laura; Patiño, Cristina; Sánchez, Flora; Peñalva, Miguel

doi:10.1016/0378-1119(87)90120-x

Cited by 123 publications

(50 citation statements)

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“…Most of these enzymes are 2OG dependent, including hyoscyamine 6b-hydroxylase (Matsuda et al, 1991) and desacetoxyvindoline-4-hydroxylase (Vazquez-Flota et al, 1997), which are involved in the biosynthesis of tropane and monoterpenoid indole alkaloids, respectively, and anthocyanidin synthase (Davies, 1993), flavonol synthase (Holton et al, 1993), and flavanone 3b-hydroxylase (Britsch et al, 1993), functioning in flavonoid metabolism. A few notable exceptions that do not require 2OG include 1-aminocyclopropane-1-carboxylate oxidase (Holdsworth et al, 1987) and fungal isopenicillin N-synthase (Ramó n et al, 1987). Cj NCS1 was reported to require Fe(II) ion but was not dependent on 2OG or O 2 and was proposed to lack a 2OG binding domain (Minami et al, 2007).…”

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confidence: 99%

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

2010

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Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate-dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type-specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde.

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Section: Introductionmentioning

confidence: 99%

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

2010

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“…1 kb) (25,26,39,47,48). The nucleotide sequence of the 872-bp intergenic region of A. nidulans between acvA and ipnA contains the transcription start point of acvA and presumably of ipnA (26,33).…”

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Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose

1992

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Expression of the Aspergilus nidulans penicillin biosynthesis genes acvA and ipnA, encoding 8-(L-aaminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, respectively, was analyzed. The intergenic region carrying the divergently oriented promoters was fused in frame in both orientations to Escherichia coli lacZ and E. coli uidA4 reporter genes. Each construct permits simultaneous expression studies of both genes. Transformants of A. nidulans carrying a single copy of either plasmid integrated at the chromosomal argB locus were selected for further investigations. Expression of both genes was directed by the 872-bp intergenic region. ipnA-and acvA-derived gene fusions were expressed from this region at different levels. ipnA had significantly higher expression than did acvA. Glucose specifically reduced the production of penicillin and significantly repressed the expression of ipnA but not of acvA gene fusions. The specific activities of isopenicillin N synthetase, the gene product of ipnA, and acyl coenzyme A:6-aminopenicillanic acid acyltransferase were also reduced in glucose-grown cultures.The P-lactam antibiotic penicillin is synthesized by filamentous fungi of the genera Penicillium and Aspergillus (17, 32). It has been produced industrially by Penicillium chrysogenum for 50 years (20). Despite the worldwide usage of this antibiotic over a long period of time, little is known about the genetic regulation of its biosynthesis. This is in part because P. chrysogenum lacks a sexual cycle, and hence the genetics of this organism is poorly developed. In contrast, although producing only modest amounts of penicillin, Aspergillus nidulans, because of its sexual cycle, is well characterized genetically (12). A wide variety of genetic techniques are available, making identification of regulatory mechanisms feasible (2, 13, 51). The genetic system of A. nidulans permits manipulation of the genome with a high precision, providing an attractive model system in which to investigate genetic control of the biosynthesis of the secondary metabolite penicillin.In the first step of the penicillin biosynthetic pathway, the (5, 6, 11, 26, 31,

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“…Cloning, sequencing, and expression in E. coli of pcbC genes from Aspergillus nidulans (17,25) and S. lipmandi (25) have recently been reported. DNA sequence information from the previously cloned fungal genes allowed the isolation of another fungal pcbC gene by cross-hybridization.…”

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Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

et al. 1989

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Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.

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Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the β-lactam ring in Aspergillus nidulans

Cited by 123 publications

References 20 publications

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

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