ABSTRACT
This study evaluated the effects of supplementing the diet of lactating cows with Acremonium terrestris culture (ATC) on milk production, serum antioxidant capacity, inflammatory indices, and serum lipid metabolomics. Over 90 days, 24 multiparous Chinese Holstein cows in mid-lactation (108 ± 10.4 days in milk, 637 ± 25 kg body weight, 30.23 ± 3.7 kg/d milk yield) were divided into either a control diet (CON) or a diet supplemented with 30 g of ATC daily. All the data were analyzed using Student's t test with SPSS 20.0 software. The results showed that compared with CON feeding, ATC feeding significantly increased milk yield, antioxidant capacity, and immune function. Lipidome screening identified 143 lipid metabolites that differed between the two groups. Further analysis using "random forest" machine learning revealed three glycerophospholipid serum metabolites that could serve as lipid markers with a predictive accuracy of 91.67%. This study suggests that ATC can be a useful dietary supplement for improving lactational performance in dairy cows and provides valuable insights into developing nutritional strategies to maintain metabolic homeostasis in ruminants.
Subject(s)
Acremonium , Dietary Supplements , Lactation , Lipidomics , Tandem Mass Spectrometry , Animals , Cattle , Female , Lipidomics/methods , Acremonium/metabolism , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid , Lipids/blood , Milk/chemistry , Milk/metabolism , Animal Feed/analysis , Antioxidants/metabolismABSTRACT
Argemone mexicana belonging to family Papaveraceae is a traditional medicinal plant widely utilized by tribal people in India for treating various ailments like skin infections, wounds and inflammation. This plant is very rich in alkaloidal content, which has a great potential in the treatment of anti-inflammatory disorders. Therapeutically promising bioactive molecules are often produced by endophytic fungi associated with medicinal plants. In this investigation, endophytic fungi were isolated from various parts of A. mexicana and screened for alkaloidal content. Among these, one of the fungal isolate, Acremonium alternatum AMEF-5 producing maximum alkaloids showed significant anti-inflammatory activity. Fractionation of this crude fungal extract through column chromatography yielded eight fractions, which were further screened for anti-inflammatory activities. Fraction 3 exhibited significant anti-inflammatory activity by the inhibition of lipoxygenase enzyme (IC50 15.2 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50 11.38 ± 0.35 µg/ml), protein denaturation (IC50 14.93 ± 0.4 µg/ml), trypsin inhibition (IC50 12.06 ± 0.64 µg/ml) and HRBC stabilization (IC50 11.9 ± 0.22 µg/ml). The bioactive alkaloid in fraction 3 was identified as aconitine which was confirmed by UV, FTIR, HPLC, HRMS, 1H NMR, and 13C NMR analysis. This study demonstrates that endophytic fungi serve a potential source for sustainable production of therapeutically important alkaloids.
Subject(s)
Aconitine , Acremonium , Anti-Inflammatory Agents , Endophytes , Acremonium/metabolism , Acremonium/chemistry , Anti-Inflammatory Agents/pharmacology , Aconitine/pharmacology , Aconitine/chemistry , Endophytes/metabolism , Endophytes/chemistry , Endophytes/isolation & purification , Animals , Nitric Oxide/metabolism , Mice , Alkaloids/pharmacology , Lipoxygenase/metabolism , RAW 264.7 Cells , IndiaABSTRACT
While endophytic fungi offer promising avenues for bolstering plant resilience against abiotic stressors, the molecular mechanisms behind this biofortification remain largely unknown. This study employed a multifaceted approach, combining plant physiology, proteomic, metabolomic, and targeted hormonal analyses to illuminate the early response of Brassica napus to Acremonium alternatum during the nascent stages of their interaction. Notably, under optimal growth conditions, the initial reaction to fungus was relatively subtle, with no visible alterations in plant phenotype and only minor impacts on the proteome and metabolome. Interestingly, the identified proteins associated with the Acremonium response included TUDOR 1, Annexin D4, and a plastidic K+ efflux antiporter, hinting at potential processes that could counter abiotic stressors, particularly salt stress. Subsequent experiments validated this hypothesis, showcasing significantly enhanced growth in Acremonium-inoculated plants under salt stress. Molecular analyses revealed a profound impact on the plant's proteome, with over 50% of salt stress response proteins remaining unaffected in inoculated plants. Acremonium modulated ribosomal proteins, increased abundance of photosynthetic proteins, enhanced ROS metabolism, accumulation of V-ATPase, altered abundances of various metabolic enzymes, and possibly promoted abscisic acid signaling. Subsequent analyses validated the accumulation of this hormone and its enhanced signaling. Collectively, these findings indicate that Acremonium promotes salt tolerance by orchestrating abscisic acid signaling, priming the plant's antioxidant system, as evidenced by the accumulation of ROS-scavenging metabolites and alterations in ROS metabolism, leading to lowered ROS levels and enhanced photosynthesis. Additionally, it modulates ion sequestration through V-ATPase accumulation, potentially contributing to the observed decrease in chloride content.
Subject(s)
Acremonium , Homeostasis , Oxidation-Reduction , Plant Growth Regulators , Salt Tolerance , Signal Transduction , Acremonium/metabolism , Acremonium/physiology , Plant Growth Regulators/metabolism , Salt Tolerance/physiology , Brassica napus/microbiology , Brassica napus/metabolism , Brassica napus/physiology , Brassica napus/drug effects , Salt Stress/physiology , Plant Proteins/metabolism , Plant Proteins/genetics , Abscisic Acid/metabolism , PhotosynthesisABSTRACT
Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA . Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high-yielding strains AG-6, AG-18, and AG-41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac-∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression.
Subject(s)
Acremonium , Cephalosporins , Cephalosporins/metabolism , Transcriptome , Acremonium/genetics , Acremonium/metabolism , Metabolic EngineeringABSTRACT
Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.
Subject(s)
Acremonium , Alkyl and Aryl Transferases , Sesquiterpenes , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sesquiterpenes/chemistry , Acremonium/genetics , Acremonium/metabolism , Genome, Fungal , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolismABSTRACT
In the present work, the potential of Cephalosporium strain in degrading the pre-treated (ultraviolet irradiation followed by nitric acid treatment) low-density polyethylene and high-density polyethylene films was investigated. Our observations revealed a significant weight reduction of 24.53 ± 0.73% and 18.22 ± 0.31% in pre-treated low-density polyethylene and high-density polyethylene films respectively, after 56 days of incubation with the Cephalosporium strain. Changes in the physicochemical properties of the mineral salt medium (MSM) were studied to assess the extent of biodegradation. The pH of the MSM decreased gradually during the incubation period, whereas its total dissolved solids and conductivity values increased steadily. Fourier transform infrared spectroscopy (FTIR) indicated the formation of hydroxyl and C = C groups in biodegraded low-density polyethylene films, while in the case of biodegraded high-density polyethylene films it indicated the [Formula: see text]CH2 stretching. Furthermore, the thermogravimetric analysis (TGA) revealed an enhancement in the thermal stabilities of both the LDPE and HDPE films post the biodegradation. Modifications in the polymer surface morphologies after UV irradiation, chemical treatment, and biodegradation steps were visualized via scanning electron microscopy (SEM) analysis. All our observations confirm the ability of the Cephalosporium strain in biodegrading the pre-treated LDPE and HDPE films.
Subject(s)
Acremonium , Polyethylene , Polyethylene/chemistry , Acremonium/metabolism , Environmental Monitoring , Biodegradation, Environmental , Spectroscopy, Fourier Transform InfraredABSTRACT
The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N1-acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.
Subject(s)
Acremonium , Spermidine , Spermidine/metabolism , Acremonium/genetics , Acremonium/metabolism , Cephalosporins/metabolismABSTRACT
Cephalosporins are currently the most widely used antibiotics in clinical practice. The main strain used for the industrial production cephalosporin C (CPC) is Acremonium chrysogenum. CPC has the advantages of possessing a broad antibacterial spectrum and strong antibacterial activity. However, the yield and titer of cephalosporins obtained from A. chrysogenum are much lower than penicillin, which is also a ß-lactam antibiotic produced by Penicillium chrysogenum. Molecular biology research into A. chrysogenum has focused on gene editing technologies, multi-omics research which has provided information on the differences between high- and low-yield strains, and metabolic engineering involving different functional genetic modifications and hierarchical network regulation to understand strain characteristics. Furthermore, optimization of the fermentation process is also reviewed as it provides the optimal environment to realize the full potential of strains. Combining rational design to control the metabolic network, high-throughput screening to improve the efficiency of obtaining high-performance strains, and real-time detection and controlling in the fermentation process will become the focus of future research in A. chrysogenum. This minireview provides a holistic and in-depth analysis of high-yield mechanisms and improves our understanding of the industrial value of A. chrysogenum. KEY POINTS: ⢠Review of the advances in A. chrysogenum characteristics improvement and process optimization ⢠Elucidate the molecular bases of the mechanisms that control cephalosporin C biosynthesis and gene expression in A. chrysogenum ⢠The future development trend of A. chrysogenum to meet industrial needs.
Subject(s)
Acremonium , Acremonium/genetics , Acremonium/metabolism , Anti-Bacterial Agents/metabolism , Cephalosporins , Fermentation , PenicillinsABSTRACT
Petroleum-based polymers are not susceptible to microorganisms because of its high molecular weight. Acid treatments convert the polymers into a more oxidized form having low molecular weight. The present in-vitro degradation study focuses on the potential of Cephalosporium species to degrade acid-treated polystyrene (PS) and low-density polyethylene (LDPE) films. A weight loss of around 12% and 13% was achieved for PS and LDPE films respectively in eight weeks of treatment with Cephalosporium species. Fourier transform infrared spectroscopy analysis showed the formation of hydroxyl and carbonyl groups in nitric acid treated PS and LDPE films, respectively. Scanning electron microscopy indicated modifications in the surface morphology of PS and LDPE films after chemical and microbial treatment. An increase in crystallinity of pre-treated polymer samples was observed after fungal treatment. The observations of present study confirmed the enzymatic deterioration and assimilation of pre-treated PS and LDPE samples by the microbial species.
Subject(s)
Acremonium , Polyethylene , Acremonium/metabolism , Biodegradation, Environmental , Nitric Acid , Polyethylene/metabolism , Polystyrenes/metabolism , Spectroscopy, Fourier Transform InfraredABSTRACT
Cephalosporin C (CPC) production is often accompanied by a typical morphological differentiation of Acremonium chrysogenum, involving the fragmentation of its hyphae into arthrospores. The type I integral plasma membrane protein Axl2 is a central component of the bud site selection system (BSSS), which was identified as the regulatory factor involved in the hyphal septation process and arthrospore formation. Using CRISPR/Cas9 technology and homologous recombination (HR), we inserted an egfp donor DNA sequence into the Acaxl2 locus, causing the generation of the deletion strain Ac-ΔAcaxl2:eGFP from Acremonium chrysogenum FC3-5-23, the industrial producer of CPC. The mycelial morphology of the deletion strain Ac-ΔAcaxl2:eGFP was mainly composed of arthrospores with a characteristic diameter of 2-8 µm, which increased from 75% at 48 h to 90% at 72 h post culture and were maintained until the end of the fermentation process. However, the deletion strain showed accelerated production of CPC, and the final titer was 5573 µg/ml, which was nearly three times higher than that of the control strain FC3-5-23. The up-regulation of genes related to the biosynthesis gene cluster in Ac-ΔAcaxl2:eGFP, especially the "late" genes, was one reason why its CPC production was higher than that of the original strain. Furthermore, compared with FC3-5-23, the more significant increase of genes involved in the BSSS (Acbud3 and Acbud4) in Ac-ΔAcaxl2:eGFP in the late stage of fermentation, may be responsible for this increase in arthrospore formation. Similarily, the transcription of the regulatory factors AcFKH1 and CPCR1 were also markedly increased at this time and may be the factors responsible for the regulation of CPC synthesis. These results indicated that Acaxl2 plays an important role in both arthrospore formation and CPC production, strongly implicating these regulatory factors as having pivotal links between mycelial morphology and secondary metabolite production in high-yielding A. chrysogenum. To the opposite, the axl2 gene knockout of wild strain CGMCC 3.3795 did not significantly influence the CPC production, which reflected the complexity of the secondary metabolic process and the differences in the function of axl2 gene in high- and low-yielding strains.
Subject(s)
Acremonium , Acremonium/genetics , Acremonium/metabolism , Cephalosporins/metabolism , Hyphae/metabolismABSTRACT
Six new 16-residue peptaibols, acremopeptaibols A-F (1-6), along with five known compounds, were isolated from the cultures of the sponge-associated fungus Acremonium sp. IMB18-086 grown in the presence of the autoclaved bacterium Pseudomonas aeruginosa on solid rice medium. The peptaibol sequences were established based on comprehensive analysis of 1D and 2D NMR spectroscopic data in conjunction with HRESIMS/MS experiments. The configurations of the amino acid residues were determined by advanced Marfey's analysis. Compounds 1-6 feature the lack of the highly conserved Thr6 and Hyp10 residues in comparison with other members of the SF3 subfamily peptaibols. A plausible biosynthetic pathway of compounds 1-6 was proposed on the basis of genomic analysis. Compounds 1, 5, 7, and 10 exhibited significant antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans. Compounds 7-10 showed potent cytotoxicities against the A549 and/or HepG2 cancer cell lines.
Subject(s)
Acremonium/metabolism , Peptaibols/isolation & purification , Porifera/microbiology , Pseudomonas aeruginosa/metabolism , A549 Cells , Animals , Biosynthetic Pathways , Hep G2 Cells , Humans , Peptaibols/chemistry , Peptaibols/pharmacologyABSTRACT
Small cell lung cancer (SCLC) was defined as a recalcitrant cancer, and novel therapies are urgently needed. Marine natural products (MNPs) may bring continuing hope for treatment of SCLC. In this study, 3-bromoascochlorin (BAS), an MNP isolated from the coral-derived fungus Acremonium sclerotigenum GXIMD 02501, was primarily screened out with antiproliferative activity towards SCLC cell lines. Then western blot analysis (WB) and flow cytometry were conducted, and we found BAS could induce the apoptosis of H446 and H69AR cells. Besides, BAS could suppress the invasion and migration of H446. In an SCLC xenograft mice model, BAS inhibited the growth of tumor without affecting the body weight of mice. Finally, the underlying mechanisms were preliminarily explored. According to the results of RNA-seq, reverse transcription-quantitative polymerase chain reaction, and WB, our results revealed that BAS exerted antitumor activity via inhibiting mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) pathway. Collectively, these results indicated that BAS can be used as a promising compound for the treatment of human SCLC.
Subject(s)
Acremonium/metabolism , Biological Products/pharmacology , Small Cell Lung Carcinoma/drug therapy , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Lung Neoplasms/pathology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Male , Mice , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Small Cell Lung Carcinoma/metabolism , Small Cell Lung Carcinoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.
Subject(s)
Acremonium/metabolism , Epithelial Cells/drug effects , Epithelium, Corneal/cytology , Keratitis/microbiology , Lectins/toxicity , Apoptosis , Cell Line , Cell Proliferation , Humans , Keratitis/pathology , Lectins/immunology , Myeloid Differentiation Factor 88ABSTRACT
Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.
Subject(s)
Biological Products/metabolism , Bridged-Ring Compounds/metabolism , Fungi/chemistry , Heterocyclic Compounds, 4 or More Rings/metabolism , Pyrrolidinones/metabolism , Acremonium/chemistry , Acremonium/metabolism , Biological Products/chemistry , Bridged-Ring Compounds/chemistry , Catalysis , Cycloaddition Reaction , Fungi/metabolism , Heterocyclic Compounds, 4 or More Rings/chemistry , Hypocreales/chemistry , Hypocreales/metabolism , Molecular Conformation , Oxidation-Reduction , Oxidoreductases/metabolism , Pyrrolidinones/chemistry , StereoisomerismABSTRACT
This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.
Subject(s)
Acremonium/metabolism , Chickens/metabolism , Gastrointestinal Tract/metabolism , Muramidase/metabolism , Peptidoglycan/metabolism , Animals , Hydrolysis , Male , Peptidoglycan/chemistry , Peptidoglycan/isolation & purificationABSTRACT
The present study approaches the capability of Cephalosporium strain NCIM 1251 to degrade pre-treated polystyrene films. Polystyrene was initially treated with UV for the introduction of oxygen molecules in pure polystyrene samples. UV treatment inserts aliphatic ketones functional group in polystyrene whereas it created C-C stretching after chemical treatment in UV-treated polystyrene as analyzed by Fourier-transform infrared spectroscopy (FTIR). The gravimetric study confirmed a decline in the weight of the pre-treated polystyrene by 20.62 ± 1.47% after 8 weeks of the incubation period. pH, total dissolved solids (TDS), and conductivity of mineral salt media were correlated with the extent of biodegradation. Treatment with UV and acid increased the thermal stability of pure polystyrene, whereas thermal stability decreased in pre-treated polystyrene after incubation with Cephalosporium strain NCIM 1251 as studied by Thermogravimetric analysis (TGA). Scanning Electron Microscopy (SEM) analysis observed revisions in the morphology and surface patterns in pre-treated polystyrene after inoculation with Cephalosporium strain NCIM 1251. The observed findings suggest that the Cephalosporium strain NCIM 1251 could be efficient for the decomposition of pre-treated polystyrene.
Subject(s)
Acremonium/metabolism , Biodegradation, Environmental , Polystyrenes/metabolism , Ultraviolet Rays , Ketones/chemistry , Microscopy, Electron, Scanning , Polystyrenes/chemistry , Spectroscopy, Fourier Transform InfraredABSTRACT
The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.
Subject(s)
Acremonium/metabolism , Cell Membrane/metabolism , Cephalosporins/biosynthesis , Cephalosporins/metabolism , Proton-Translocating ATPases/metabolism , Adenosine Triphosphatases/metabolism , Culture Media/metabolism , Fermentation/physiology , Gene Expression Regulation, Fungal/physiology , beta-Lactams/metabolismABSTRACT
In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.
Subject(s)
Acremonium/metabolism , Cephalosporins/biosynthesis , Penicillins/biosynthesis , Penicillium chrysogenum/metabolism , Reactive Oxygen Species/metabolism , Acremonium/drug effects , Biosynthetic Pathways , Fermentation , Gene Expression Regulation, Fungal , Hydrogen Peroxide/pharmacology , Penicillium chrysogenum/drug effects , Reactive Oxygen Species/pharmacology , Secondary MetabolismABSTRACT
Routine microbial identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been achieved based on the spectra of ribosomal proteins with molecular masses between 2000 and 20000Da. It is a rapid, cost-effective, and simple method to characterize different species of microorganisms. But for some subspecies of molds, there are high similarities between their spectra in 2000-20000Da, it makes them indistinguishable in this mass range. Based on the specialized metabolite production, there are obvious differences between the high resolution spectra of the same samples in 600-2000Da. It allows the rapid discrimination of these microbial subspecies. The ability of the method to discriminate microbial subspecies was demonstrated by characterizing three different Aspergillus niger strains. Furthermore, this approach has been applied to discriminate two different Acremonium alternatum strains which were collected from mildew plants. It demonstrated the applicability of the method to the actual samples. The high resolution MS in the range of 600-2000Da was presented as a complementary approach for the routine method in 2000-20000Da. The molds could be identified into species-level group by the spectra between 2000 and 20000Da and the strains within each group could be further discriminated based on differences in metabolites. The spectra between 2000 and 20000Da and the spectra between 600 and 2000Da were obtained from the same samples, which extracted with the same method. There is no need of additional pre-processing to obtain the high resolution spectra. It potentially provides a powerful tool for the fast and accurate identification of microbial subspecies.