Expression of fungal genes involved in penicllin biosynthesis (original) (raw)
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FEMS Microbiology Letters, 1995
Transcription of the Aspergillus nidulans ipn4 gene is under carbon regulation. Loss-of-function mutations in creB or creC do not cause full derepression of ipnA transcript levels in sucrose-grown mycelia and do not elevate repressed penicillin levels, indicating that neither of these genes plays a major regulatory role in penicillin biosynthesis. However, these mutations reduce external pH acidification, accelerate sucrose degradation and result in extracellular accumulation of resulting D-glucose and D-fructose. These effects would explain the partial elevation of carbon-repressed ipnA transcript levels observed in strains carrying creB_ or creC-mutations.
BMC Microbiology, 2009
Background: Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.
Journal of Biological Chemistry, 1993
Nine mutants of Penicillium chrysogenum (npel to npe8 and npel0) impaired in penicillin biosynthesis were screened after nitrosoguanidine mutation. Mutants npel, npe4, npe5, npe6, npe7, npe8, and npelQ failed to synthesize significant levels of penicillin, whereas strains npe2 and npe3 synthesized about 20% of the penicillin level produced by the parental strain. Mutants npe5 and npelO did not show a-aminoadipylcysteinyl-valine (ACV) synthetase activity in vitro and did not form ACV in vivo. Immunoblotting analysis of the different mutants using antibodies raised against Aspergillus nidulam ACV synthetase showed that mutants npe5 and npelO lacked this multienzyme protein, which in the parental strain had a molecular mass of about 420 kDa, and mutants npe2 and npe3 formed reduced level of this protein. All mutants showed normal levels of isopenicillin N synthase, as shown by Western blot analysis and enzyme assays (except npelQ that lacked this enzyme and npe2 and npe3 that formed reduced levels); npel, npe4, npe6, npe7, npe8, and npelO lacked isopenicillin N acyltransferase. Southern hybridizations of total DNA of the parental strain and mutants npe6, npe6, npe8, and npelO with probes internal to the pcbAB, pcbC, and penDE genes showed that mutants npe5, npe6, and npe8 had the same arrangement of the penicillin gene cluster carrying probably point mutations, but mutant npelQ lacked the three penicillin biosynthetic genes, suggesting that it had suffered a deletion of the entire penicillin cluster. Southern hybridization with a pyrG probe as control and fingerprinting analysis of total DNA of npelQ as compared to several P. chrysogenum strains and other Penicillium and Aspergillus species, confirmed that npel0 is a deletion mutant of P. chrysogenum that had lost the penicillin biosynthetic genes. The penicillin biosynthetic pathway has been largely elucidated in the last decade (see reviews in Refs. 1-3). Penicillin is formed from three precursor amino acids L-a-aminoadipic acid, L-cysteine, and L-valine, which are activated and condensed to form the tripeptide &(L-a-aminoadipy1)-L-cysteinyl-D-valine (ACV)' in which the valine has been racemized
Journal of Bacteriology, 2000
Genes for the biosynthesis of secondary metabolites are usually arranged in clusters (15, 59) together with genes for resistance to the toxic action of secondary metabolites on the producer organisms (20) and sometimes with genes for biosynthesis of antibiotic precursors (54). The penicillin biosynthesis cluster consists of three genes pcbAB, pcbC, and penDE (58) and are arranged the same in Penicillium chrysogenum, Aspergillus nidulans (1), and Penicillium nalgiovense (51) (Fig. 1). The pcb genes encode enzymes involved in penicillin and cephalosporin biosynthesis, whereas pen genes are specific for the penicillin pathway. The pcbAB and pcbC genes are expressed from a 1.16-kb bidirectional promoter region in opposite directions (6, 7, 21, 63). The expression of these genes is subject to sophisticated controls by both nutritional and developmental factors (1, 8, 9, 55, 60). Multiple DNA-binding proteins appear to bind to different regions of the pcbAB-pcbC bidirectional promoter. Proteins that interact with the pcbAB-pcbC intergenic control region have been found by DNA mobility shift and DNA footprinting assays (17, 29;
Characterization of an Autoinducer of Penicillin Biosynthesis in Penicillium chrysogenum
Applied and Environmental Microbiology, 2011
Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of -lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by ␥-butyrolactones, jasmonic acid, or the penicillin precursor ␦-(L-␣-aminoadipyl)-L-cysteinyl-Dvaline. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.
Applied and Environmental Microbiology, 2002
Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.
Transcript analysis of penicillin genes from Penicillium chrysogenum
Current Genetics, 1992
The presence of a transcriptional control simultaneously affecting the expression of the three penicillin biosynthetic genes, pcbAB, pcbC, and penDE (pen genes), was demonstrated in Penicillium chrysogenum. Using probes specific to each gene, it was observed that the highest level of expression of the pen genes occurred during exponential growth, in both the original ancestral strain (NRRL1951) and a high-penicillin producing strain P2. Expression also occurred in the presence of high concentrations of glucose, indicating that carbon catabolite repression was not directly involved in the regulation. Transcription of the pen genes appeared to cease as the growth rate decreased. Growth was limited in a fermenter by the rate of oxygen transfer. The phosphoglycerate kinase gene (pgk), used as a control, was strongly induced by the reduced oxygen levels, suggesting a stress-related response. By maintaining optimum growth conditions in fermenters, no induction of the pgk gene was observed whereas expression of the pen genes could be maintained. It was also possible to re-establish expression of the pen genes, after normal cessation, by the addition of cycloheximide to the culture medium.
Current Genetics, 2003
Non-ribosomal peptide synthetases, polyketides and fatty acid synthetases have a modular organisation of multi-enzymatic activities. In all of them, the acyl or peptidyl carrier proteins have 4'-phosphopantetheine (P-pant) as an essential prosthetic group. This is added by 4'-phosphopantetheinyl transferases (PPTases) that derive the P-pant group from coenzyme A. While many PPTases of varying specificity have now been isolated from a number of bacteria, a filamentous fungal PPTase has yet to be characterised. Through database searching of the Aspergillus fumigatus genome sequence against Sfp from Bacillus subtilis, we identified a unique sequence which appears to encode a PPTase, as deduced from conserved residues considered important in PPTases. The PPTase candidate was used to search the NCBI data base and an unexpected homologue in A. nidulans was identified as npgA. Mutations in this gene (cfwA/npgA) were identified previously as leading to defects in growth and pigmentation. To test whether the temperature-sensitive cfwA2 mutation impairs penicillin biosynthesis, which is dependent on the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase, bioassays with B. calidolactis were set up at permissive and non-permissive temperatures. The cfwA2 mutant did not produce penicillin at the non-permissive temperature. Since no other PPTase homologue has been detected in the A. fumigatus genome to date, the data suggest that a single enzyme may be able to transfer the cofactor to a broad range of enzymes with acyl or peptidyl carrier protein domains.
Carbon catabolite repression of penicillin biosynthesis by Penicillium chrysogenum
The Journal of Antibiotics, 1984
The addition of glucose to batch cultures of Penicillium chrysogenun: ASP -78 reduced the biosynthesis of penicillin. This regulatory effect was also observed in penicillin biosynthesis by nitrogen-limited resting cells when cultures were previously grown in high concentrations of glucose. The effect of glucose was concentration-dependent in the range of 28-j140 mm. Incorporation of L-[U-"C]valine into penicillin in nitrogen-limited resting cultures was reduced by 70% when cells were grown on 140 mm glucose, as compared with that grown on lactose. It was not affected when the sugar was added to the resting cell system, in which penicillin biosynthesis took place without growth. Fructose, galactose and sucrose exerted the regulatory effect to the same extent as glucose (64 to 70%). Lactose did not exert suppression of penicillin biosynthesis. Penicillin-synthesizing activity in control cultures with lactose reached a peak at 24 hours of incubation and decreased slowly thereafter, as studied with resting cell cultures in which further protein synthesis was blocked with cycloheximide. Glucose repressed the formation of penicillin-synthesizing enzymes, but had no effect on the activity of these enzymes. These results suggest that glucose represses but does not inhibit penicillin biosynthesis. Glucose is the best carbon and energy source for the growth of most antibiotic-producing microorganisms. However, rapid utilization of glucose decreases the biosynthesis of many antibiotics'). In a medium containing glucose plus a second carbon source glucose is generally used first, thereby suppressing antibiotic biosynthesis. When glucose is depleted (or its concentration falls below a repressing threshold) the second carbon source is used and antibiotic formation occurs. This phenomenon, which is similar to the initially named "glucose effect'12), is now understood in terms of carbon catabolite regulation','). Carbon catabolite regulation of antibiotic biosynthesis is a general mechanism controlling the biosynthesis of antibiotics belonging to different biosynthetic groups. These include actinomycin production by Streptomyces antibioticus'), puromycin by Streptomyces alboniger'), cephalosporin by Cephalosporium acremonium7'9'8) and Streptomyces clavuligerus10® and penicillin by Penicillium chrysogenum"I. Early studies on media development for penicillin production indicated that di-, oligo-and polysaccharides were better carbon sources than glucose for penicillin production''). Industrial production is therefore carried out using lactose which is slowly utilized. Carbon catabolite regulation of penicillin biosynthesis is bypassed when glucose is slowly fed to the culture"). The same occurs in cephalosporin production'). These results suggest that carbon catabolite regulation is exerted by an effector formed during the transport or catabolism of glucose. The precise molecular mechanism of carbon catabolite regulation of penicillin biosynthesis is unknown, although there appears to exist a close relationship with the energy metabolism of the cells".
Folia Microbiologica, 2010
The transcription start points of the penicillin biosynthesis genes from Penicillium chrysogenum were mapped using the primer extension method. For each of the three genes consensus sequences of the core promoter elements were identified, supporting the notion that the basal transcription of these genes is mediated separately. Interestingly, transcription start of the pcbC gene is located within the potential Inr element with no TATA box-like sequence being found at expected position. This is in contrast to pcbAB and penDE genes with proposed TATA boxes or even to Aspergillus nidulans ipnA (pcbC) gene indicating possible differences in basal transcription regulation. Using the quantitative RT-PCR analysis the expression of all three biosynthesis genes was monitored in both the high and low production strain of P. chrysogenum during a 3-d cultivation under production conditions. The differences were found between the strains in time regulation and transcript levels of the biosynthesis genes. Furthermore, we showed that the effect of higher gene dosage on productivity in the production strain is amplified by more efficient transcription of the biosynthesis genes with the RNA levels ≈37- and 12-times higher, respectively, than in a low production strain.