Lysine Regulation of Penicillin Biosynthesis in Low-producing and Industrial Strains of Penicillium chrysogenum

Luengo, José M.; Revilla, Gloría; Villanueva, Julio R.; Martı́n, Juan F.

doi:10.1099/00221287-115-1-207

Cited by 59 publications

(32 citation statements)

References 6 publications

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“…After this time, 0.16 M D-glucose was added, and the suspension was incubated for 4 h to allow germ tubes to protrude. Under these conditions DNA synthesis began synchronously just before germ tube formation (20 (12)(13)(14).…”

Section: Methodsmentioning

confidence: 99%

Glucokinase-deficient mutant of Penicillium chrysogenum is derepressed in glucose catabolite regulation of both beta-galactosidase and penicillin biosynthesis

Barredo

Álvarez

Cantoral

et al. 1988

Antimicrob Agents Chemother

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One glucokinase-deficient mutant (glkl) of Penicillium chrysogenum AS-P-78 was isolated after germ tube-emitting spores were mutated with nitrosoguanidine and selected for growth on lactose-containing medium in the presence of inhibitory concentrations of D-2-deoxyglucose (3 mM). Penicillin biosynthesis was greatly reduced (55%) in D-glucose-grown cultures of the parental strain, but this sugar had no repressive effect on the rate of penicillin biosynthesis in the mutant glkl. This mutant was deficient in ATP-dependent glucokinase and showed a greatly reduced uptake of D-glucose. The parental strain P. chrysogenum AS-P-78 showed in vitro ATP-dependent phosphorylating activities of D-glucose, D-2-deoxyglucose, and D-galactose. The glkl mutant was deficient in the in vitro phosphorylation of D-glucose and D-2-deoxyglucose but retained a normal D-galactose-phosphorylating activity. D-Glucose repressed both ,-galactosidase and isopenicillin-Nsynthase but not acyl coenzyme A:6-aminopenicillanic acid acyltransferase in the parental strain. The glucokinase-deficient mutant was simultaneously derepressed in carbon catabolite regulation of I-galactosidase and isopenicillin-N-synthase, suggesting that a common regulatory mechanism is involved in carbon catabolite regulation of both sugar utilization and penicillin biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Glucokinase-deficient mutant of Penicillium chrysogenum is derepressed in glucose catabolite regulation of both beta-galactosidase and penicillin biosynthesis

Barredo

Álvarez

Cantoral

et al. 1988

Antimicrob Agents Chemother

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“…P. chrysogenum AS-P-78, a high-penicillin-producing strain (donated by Antibi6ticos, S.A., Le6n, Spain), was used in this study. 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: 99%

“…Uptake of L-[U-14C]valine and [a-6-14C]aminoadipic acid were as reported before (13,14). In 25°C to induce the transport system before addition of the labeled amino acid.…”

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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“…P. chrysogenum Wis-54-1255, a strain producing low levels of penicillin, was obtained from A. L. Demain (Massachusetts Institute of Technology, Cambridge). Mutants npel to npe8, which are blocked in penicillin biosynthesis, were obtained by mutation of spores of culture was added to 50 ml of complex production (CP) medium (17) in 500-ml baffled flasks. The cultures were incubated (250 rpm) at 25°C for 96 to 120 h. Penicillin G was determined in culture broths after centrifugation at 15,000 x g for 10 min.…”

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

confidence: 99%

“…The cultures were incubated (250 rpm) at 25°C for 96 to 120 h. Penicillin G was determined in culture broths after centrifugation at 15,000 x g for 10 min. Microbiological assays and colorimetric determinations of penicillin G were carried out as described before (17). 6-APA acyltransferase assay.…”

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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Lysine Regulation of Penicillin Biosynthesis in Low-producing and Industrial Strains of Penicillium chrysogenum

Cited by 59 publications

References 6 publications

Glucokinase-deficient mutant of Penicillium chrysogenum is derepressed in glucose catabolite regulation of both beta-galactosidase and penicillin biosynthesis

Glucokinase-deficient mutant of Penicillium chrysogenum is derepressed in glucose catabolite regulation of both beta-galactosidase and penicillin biosynthesis

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

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