Direct Enzymatic Synthesis of Penicillin G Using Cyclases of Penicillium chrysogenum and Acremonium chrysogenum

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

doi:10.1038/nbt0186-44

Cited by 35 publications

(20 citation statements)

References 21 publications

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“…Baffled flasks (500 ml) containing 50 ml of complex production medium were inoculated with 2.5 ml of the seed culture and incubated at 25°C, and penicillin was determined by bioassay (22). Control and ,-lactamase-treated samples of the filtered broth were assayed by high-pressure liquid chromatography to determine the formation of penicillin G or isopenicillin N, using a Varian 5000 high-pressure liquid chromatograph equipped with a micropack mCH-10 column as described elsewhere (12,23).…”

Section: Methodsmentioning

confidence: 99%

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: 99%

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 reaction product was identified as penicillin G by (i) microbial susceptibility tests, (ii) degradation with penicillinase, and (iii) thin-layer chromatography and high-pressure liquid chromatography after the reaction product was extracted with solvent. Microbial susceptibility tests against M. luteus ATCC 9341 E. coli ESS 22-31, Bacillus subtilis ATCC 6633, and Klebsiella pneumoniae ATCC 29665 were carried out as described before (16,26,27). The antibiotic that was formed showed activity against B. subtilis, M. luteus, and E. coli, but not against the penicillin-resistant strain of K. pneumoniae.…”

Section: The 3-lactam-thiazolidine Nucleus Of Penicillins Is Formed Bmentioning

confidence: 99%

“…The penicillinase destroyed the antibiotic that was formed. Further characterization of the reaction product by thin-layer chromatography and high-pressure liquid chromatography was carried out as described before (16). In the thin-layer chromatographic procedure, the reaction product comigrated with pure penicillin G. In the high-pressure liquid chromatographic procedure the antibiotic activity eluted in a peak with a retention time of 12.3 min, which is identical to that of authentic penicillin G.…”

Section: The 3-lactam-thiazolidine Nucleus Of Penicillins Is Formed Bmentioning

confidence: 99%

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum

Álvarez

Cantoral

Barredo

et al. 1987

Antimicrob Agents Chemother

108

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The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-pQlyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 ± 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25°C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloroipercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.The 3-lactam-thiazolidine nucleus of penicillins is formed by cyclization of the tripeptide 8-(L-a-aminoadipyl)-Lcysteinyl-D-valine to form isopenicillin N (IPN), an intermediate in the biosynthetic pathway that has an L-aaminoadipyl side chain (Fig.

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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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Direct Enzymatic Synthesis of Penicillin G Using Cyclases of Penicillium chrysogenum and Acremonium chrysogenum

Cited by 35 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

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of 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

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