Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N

Ramos, Filomena R.; López-Nieto, Manuel J.; Martı́n, Juan F.

doi:10.1128/aac.27.3.380

Cited by 129 publications

(12 citation statements)

References 22 publications

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“…3 ). Upon growth of these cells in medium containing AAA, intracellular accumulation of a β-lactam antibiotic (presumably IPN) could be demonstrated in a bioassay using the β-lactam sensitive indicator strain Micrococcus luteus [7] ( Fig. 5A ).…”

Section: Resultsmentioning

confidence: 99%

An Engineered Yeast Efficiently Secreting Penicillin

et al. 2009

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This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's.

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Section: Resultsmentioning

confidence: 99%

An Engineered Yeast Efficiently Secreting Penicillin

et al. 2009

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“…We have determined the enzyme activity of IPNS produced in methanol grown cells of strain H. polymorpha IPNS 4.2 at different growth temperatures via a bioassay that utilizes the β-lactam sensitive bacterium Micrococcus luteus (ATCC 9341) as indicator strain [ 14 ]. Cells of the H. polymorpha host strain and P. chrysogenum served as controls.…”

Section: Resultsmentioning

confidence: 99%

Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

et al. 2008

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Background: β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha.

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“…IPNS activity was measured by monitoring the formation of isopenicillin N (IPN) from Bis‐ACV as previously described [33] and IPN was quantified by bioassay against Micrococcus luteus . DAC‐AT activity was measured monitoring by HPLC in vitro conversion of DAC and acetyl‐CoA into cephalosporin C as described [5].…”

Section: Methodsmentioning

confidence: 99%

Strain improvement for cephalosporin production byAcremonium chrysogenumusing geneticin as a suitable transformation marker

RodrÃguez-SÃ¡iz¹,

Lembo²,

Bertetti³

et al. 2004

FEMS Microbiology Letters

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An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production. Best results were obtained with cefEF and cefG genes, selecting transformants with increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants, designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains. Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptII gene from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette Pgdh-nptII-trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker.

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Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N

Cited by 129 publications

References 22 publications

An Engineered Yeast Efficiently Secreting Penicillin

An Engineered Yeast Efficiently Secreting Penicillin

Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

Strain improvement for cephalosporin production byAcremonium chrysogenumusing geneticin as a suitable transformation marker

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