Characterization of the biosynthesis in vivo of ?-aminoadipyl-cysteinyl-valine inPenicillium chrysogenum

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

doi:10.1007/bf00582418

Cited by 54 publications

(32 citation statements)

References 21 publications

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“…The growth characteristics, penicillin production kinetics, and lysine regulation of penicillin biosynthesis in this strain have been previously reported (13,14). Conidia obtained from sporulation medium PM1 (11) were kept at -20°C in 20% glycerol.…”

Section: Methodsmentioning

confidence: 91%

“…Peptides from mycelium of P. chrysogenum were extracted, oxidized to their sulfonic derivatives with performic acid, purified by chromatography on Dowex 5OW x 4 (H+ form), and separated by electrophoresis at pH 1.8 by a modification of the Arnstein and Morris method (2) as previously reported (11). Incorporation of L-[U-14C]valine into ACV was carried out as described by L6pez-Nieto et al (11).…”

Section: Methodsmentioning

confidence: 99%

“…Pure ACV (dimer form) and isopenicillin N were kindly provided by P. van (Fig. 2, peaks 1 and 2) and the accumulation of penicillaminic acid (peak 4), previously identified as a degradation product of isopenicillin N and penicillin G (11).…”

Section: Analysis Of Intracellular Concentration Of Amino Acidsmentioning

confidence: 99%

“…The recent development of reliable assay methods to quantify the tripeptide ACV (11) and to measure the activities of isopenicillin N synthase (21) and penicillin acyltransferase (E. Alvarez and J. F. Martin, unpublished data) prompted us to study the effect of glucose on the synthesis * Corresponding author. and activity of the different enzymes involved in penicillin biosynthesis, as well as on the availability of precursor amino acids for tripeptide biosynthesis.…”

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confidence: 99%

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Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Revilla¹,

Ramos²,

López-Nieto³

et al. 1986

J Bacteriol

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The content of a-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into a-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of a-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing at-aminoadipic acid accumulation. The repression of a-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of oa-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts a-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.Glucose exerts a negative control on the biosynthesis of penicillin in Penicillium chrysogenum (17,22) similar in many aspects to carbon catabolite regulation of the biosynthesis of cephalosporins in Acremonium chrysogenum (3,24) and cephamycins in Streptomyces clavuligerus (1,23) and Streptomyces lactamdurans (6). Glucose (28 to 140 mM) produces a concentration-dependent repression of the incorporation of ['4C]valine into penicillin but does not have an inhibitory effect on such incorporation by enzymes formed before glucose addition (22). The total activity of the penicillin-synthesizing enzymes in P. chrysogenum is repressed by glucose (22). Derepression of penicillin biosynthesis occurs after depletion of glucose. However, the overall biosynthetic activity of the pathway in vivo is determined by the availability of precursors of the L-a-aminoadipyl-Lcysteinyl-D-valine (ACV) tripeptide and by the activities of the (at least) three enzymes involved in penicillin biosynthesis.The first well-established intermediate in the penicillin biosynthetic pathway, ACV (2, 11), appears to be formed by a sequential condensation of the three precursor amino acids (8) (Fig. 1). ACV is then cyclized to form isopenicillin N by the action of isopenicillin N synthase, an enzyme that has been purified to near homogeneity from extracts of P. chrysogenum (19,21). In the last step of penicillin biosynthesis, the a-aminoadipyl side chain of isopenicillin N is exchanged for phenylacetic acid, which is previously activated in the form of phenylacetyl coenzyme A (CoA) (20; B. Spencer and C. Maung, Biochem. J. 118:29p-30p, 1970).The recent development of reliable assay methods to quantify the tripeptide ACV (11) and to measure the activities of isopenicillin N synthase (21) and penicillin acyltransferase (E.

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Section: Methodsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Intracellular Concentration Of Amino Acidsmentioning

confidence: 99%

mentioning

confidence: 99%

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Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Revilla¹,

Ramos²,

López-Nieto³

et al. 1986

J Bacteriol

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“…chrysogenum TD189, a disrupted strain lacking isopenicillin epimerase activity, 15 and P. chrysogenum strains ABC and npe6 pyrG, both lacking the penDE gene, 10,16,17 were used. Micrococcus luteus ATCC 9341, an IPN sensitive bacterial strain, [18][19][20] was used for bioassays in each step of purification. b-lactamase from Bacillus cereus UL1 was used to check the presence of penicillin compound in these assays.…”

Section: Methods Microorganismsmentioning

confidence: 99%

A preparative method for the purification of isopenicillin N from genetically blocked Acremonium chrysogenum strain TD189: studies on the degradation kinetics and storage conditions

et al. 2011

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A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and À75 1C with a half-life of 770 days.

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Biosynthetic concepts for the production of β‐lactam antibiotics in Penicillium chrysogenum

Weber

Bovenberg

Driessen

2011

Biotechnology Journal

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Industrial production of β-lactam antibiotics by the filamentous fungus Penicillium chrysogenum is based on successive classical strain improvement cycles. This review summarizes our current knowledge on the results of this classical strain improvement process, and discusses avenues to improve β-lactam biosynthesis and to exploit P. chrysogenum as an industrial host for the production of other antibiotics and peptide products. Genomic and transcriptional analysis of strain lineages has led to the identification of several important alterations in high-yielding strains, including the amplification of the penicillin biosynthetic gene cluster, elevated transcription of genes involved in biosynthesis of penicillin and amino acid precursors, and genes encoding microbody proliferation factors. In recent years, successful metabolic engineering and synthetic biology approaches have resulted in the redirection of the penicillin pathway towards the production of cephalosporins. This sets a new direction in industrial antibiotics productions towards more sustainable methods for the fermentative production of unnatural antibiotics and related compounds.

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Characterization of the biosynthesis in vivo of ?-aminoadipyl-cysteinyl-valine inPenicillium chrysogenum

Cited by 54 publications

References 21 publications

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

A preparative method for the purification of isopenicillin N from genetically blocked Acremonium chrysogenum strain TD189: studies on the degradation kinetics and storage conditions

Biosynthetic concepts for the production of β‐lactam antibiotics in Penicillium chrysogenum

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