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TheScientificWorldJournal 2014The halotolerant fungus Aspergillus glaucus CCHA was isolated from the surface of wild vegetation around a saltern with the salinity range being 0-31%. Here, a...
The halotolerant fungus Aspergillus glaucus CCHA was isolated from the surface of wild vegetation around a saltern with the salinity range being 0-31%. Here, a full-length cDNA library of A. glaucus under salt stress was constructed to identify genes related to salt tolerance, and one hundred clones were randomly selected for sequencing and bioinformatics analysis. Among these, 82 putative sequences were functionally annotated as being involved in signal transduction, osmolyte synthesis and transport, or regulation of transcription. Subsequently, the cDNA library was transformed into E. coli cells to screen for putative salt stress-related clones. Five putative positive clones were obtained from E. coli cells grown on LB agar containing 1 M NaCl, on which they showed rapid growth compared to the empty vector control line. Analysis of transgenic Arabidopsis thaliana lines overexpressing CCHA-2142 demonstrated that the gene conferred increased salt tolerance to plants as well by protecting the cellular membranes, suppressing the inhibition of chlorophyll biosynthesis. These results highlight the utility of this A. glaucus cDNA library as a tool for isolating and characterizing genes related to salt tolerance. Furthermore, the identified genes can be used for the study of the underlying biology of halotolerance.
Topics: Arabidopsis; Aspergillus; Carbohydrates; Caseins; Escherichia coli; Gene Expression Regulation, Fungal; Gene Library; Lipids; Plant Proteins, Dietary; Plants, Genetically Modified; Salinity; Salt Tolerance; Signal Transduction; Sodium Chloride; Stress, Physiological
PubMed: 25383373
DOI: 10.1155/2014/620959 -
Food Additives & Contaminants. Part A,... 2015The growth of toxigenic fungi can adversely affect grain quality and even produce mycotoxins of food safety concern, which should be sensitively monitored and controlled...
The growth of toxigenic fungi can adversely affect grain quality and even produce mycotoxins of food safety concern, which should be sensitively monitored and controlled during grain storage. To establish the relationship between the growth of toxigenic fungi and their carbon dioxide (CO2) production, the pattern of CO2 concentration changes was studied during the fungal growth in grain. The results showed the CO2 concentrations increased exponentially (r ≥ 0.96) during the growth of toxigenic fungi Aspergillus flavus, Penicillium sp. and Aspergillus ochraceus, which was different from the linear increase of CO2 concentration produced by the non-toxigenic xerophilic fungi Aspergillus glaucus and Aspergillus restrictus. The acceleration of CO2 concentration was found much earlier than the growth of toxigenic fungi, which would be useful for the prevention of grain spoilage. In addition, the CO2 concentration changes were also determined in storage containers loaded with grain of different moisture content and significant correlation (p < 0.05) was found between changes of CO2 concentration and fungal growth as well as mycotoxin production. The nonlinear increase of CO2 concentration in stored grains could be considered as an indication of the rapid growth of toxigenic fungi and greater risk of microbial spoilage of grains. The results can provide a valid foundation for the prevention of toxigenic fungi and mycotoxin production in stored grains through monitoring the CO2 concentration changes.
Topics: Aspergillus; Carbon Dioxide; Edible Grain; Food Contamination; Food Microbiology; Food Storage; Mycotoxins; Oryza; Penicillium; Triticum; Zea mays
PubMed: 25254604
DOI: 10.1080/19440049.2014.968221 -
Journal of Agricultural and Food... Aug 2014Six analogues of natural trans-4-butyl-cis-3-oxabicyclo[4.3.0]nonan-2-one (3) and three derivatives, 11, 12, and 13, of Vince lactam (10) were synthesized and tested as...
Six analogues of natural trans-4-butyl-cis-3-oxabicyclo[4.3.0]nonan-2-one (3) and three derivatives, 11, 12, and 13, of Vince lactam (10) were synthesized and tested as fungistatic agents against Botrytis cinerea AM235, Penicillium citrinum AM354, and six strains of Aspergillus. Moreover, bioresolution carried out by means of whole cell microorganisms and commercially available enzymes afforded opposite enantiomerically enriched (-) and (+) isomers of Vince lactam (10), respectively. The effect of compound structures and stereogenic centers on biological activity has been discussed. The highest fungistatic activity was observed for four lactones: 3, 4, 7, and 8 (IC50 = 104.6-115.2 μg/mL) toward B. cinerea AM235. cis-5,6-Epoxy-2-aza[2.2.1]heptan-3-one (13) indicated significant fungistatic activity (IC50 = 107.1 μg/mL) against Aspergillus glaucus AM211. trans-4-Butyl-cis-3-oxabicyclo[4.3.0]nonan-2-one (3) and trans-4-butyl-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (7) exhibited high fungistatic activity (IC50 = 143.2 and 110.2 μg/mL, respectively) against P. citrinum AM354 as well.
Topics: Aspergillus; Botrytis; Fungicides, Industrial; Lactams; Lactones; Molecular Structure; Penicillium
PubMed: 25110806
DOI: 10.1021/jf502148h -
Applied Biochemistry and Biotechnology Oct 2014The production of cellulase from Aspergillus glaucus HGZ-2 was improved by using genome shuffling. The starting populations, obtained by UV irradiation, were subjected...
The production of cellulase from Aspergillus glaucus HGZ-2 was improved by using genome shuffling. The starting populations, obtained by UV irradiation, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 7 mg/ml snailase and 5 mg/ml cellulase at 34 °C for 3.0 h using 0.7 M NaCl as an osmotic stabilizer. The protoplasts were inactivated under UV for 30 min or heated at 50 °C for 50 min, and a fusant probability of about 100 % was observed. The positive colonies were created by fusing the inactivated protoplasts. The optimal conditions for protoplast fusion were PEG6000 concentration of 35 %, CaCl2 concentration of 0.02 M, and incubation time of 12 min. After two rounds of genome shuffling, one strain (Y) was obtained. Its filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) activity reached 71 and 70 U/ml, respectively, which were increased by 1.95-fold and 1.72-fold in comparison with that of its ancestor strain. The results indicated that genome shuffling was an efficient means for the improved production of cellulases by A. glaucus HGZ-2.
Topics: Aspergillus; Cellulase; DNA Shuffling; Fungal Proteins; Genome, Fungal; Polyethylene Glycols; Ultraviolet Rays
PubMed: 25099375
DOI: 10.1007/s12010-014-1102-0 -
Microbial Cell Factories May 2014For filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in...
BACKGROUND
For filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process.
RESULTS
Hyphal morphology shaping by modifying polarized growth genes of A. glaucus was applied to reduce its shear-sensitivity and enhance aspergiolide A production. Degenerate PCR and genome walking were used to obtain polarized growth genes AgkipA and AgteaR, followed by construction of gene-deficient mutants by homologous integration of double crossover. Deletion of both genes caused meandering hyphae, for which, ΔAgkipA led to small but intense curves comparing with ΔAgteaR by morphology analysis. The germination of a second germ tube from conidiospore of the mutants became random while colony growth and development almost maintained the same. Morphology of ΔAgkipA and ΔAgteaR mutants turned to be compact pellet and loose clump in liquid culture, respectively. The curved hyphae of both mutants showed no remarkably resistant to glass bead grinding comparing with the wild type strain. However, they generated greatly different broth rheology which further caused growth and metabolism variations in bioreactor fermentations. By forming pellets, the ΔAgkipA mutant created a tank environment with low-viscosity, low shear stress and high dissolved oxygen tension, leading to high production of aspergiolide A (121.7 ± 2.3 mg/L), which was 82.2% higher than the wild type.
CONCLUSIONS
A new strategy for shaping fungal morphology by modifying polarized growth genes was applied in submerged fermentation in bioreactor. This work provides useful information of shaping fungal morphology for submerged fermentation by genetically modification, which could be valuable for morphology improvement of industrial filamentous fungi.
Topics: Anthraquinones; Antineoplastic Agents; Aspergillus; Batch Cell Culture Techniques; Fungal Proteins; Mutation; Open Reading Frames; Polyketides
PubMed: 24886193
DOI: 10.1186/1475-2859-13-73 -
Applied and Environmental Microbiology Jul 2014Ribosomal proteins are highly conserved components of basal cellular organelles, primarily involved in the translation of mRNA leading to protein synthesis. However,...
Ribosomal proteins are highly conserved components of basal cellular organelles, primarily involved in the translation of mRNA leading to protein synthesis. However, certain ribosomal proteins moonlight in the development and differentiation of organisms. In this study, the ribosomal protein L44 (RPL44), associated with salt resistance, was screened from the halophilic fungus Aspergillus glaucus (AgRPL44), and its activity was investigated in Saccharomyces cerevisiae and Nicotiana tabacum. Sequence alignment revealed that AgRPL44 is one of the proteins of the large ribosomal subunit 60S. Expression of AgRPL44 was upregulated via treatment with salt, sorbitol, or heavy metals to demonstrate its response to osmotic stress. A homologous sequence from the model fungus Magnaporthe oryzae, MoRPL44, was cloned and compared with AgRPL44 in a yeast expression system. The results indicated that yeast cells with overexpressed AgRPL44 were more resistant to salt, drought, and heavy metals than were yeast cells expressing MoRPL44 at a similar level of stress. When AgRPL44 was introduced into M. oryzae, the transformants displayed obviously enhanced tolerance to salt and drought, indicating the potential value of AgRPL44 for genetic applications. To verify the value of its application in plants, tobacco was transformed with AgRPL44, and the results were similar. Taken together, we conclude that AgRPL44 supports abiotic stress resistance and may have value for genetic application.
Topics: Aspergillus; DNA, Fungal; Fungal Proteins; Gene Expression Regulation, Fungal; Gene Library; Osmotic Pressure; Plants, Genetically Modified; Promoter Regions, Genetic; Ribosomal Proteins; Saccharomyces cerevisiae; Sequence Alignment; Sequence Analysis, DNA; Sodium Chloride; Stress, Physiological; Nicotiana; Transcriptome
PubMed: 24814782
DOI: 10.1128/AEM.00292-14 -
Journal of Asian Natural Products... Sep 2013Two new benzyl derivatives, aspergentisyl A (1) and aspergentisyl B (2), as well as one new naphthoquinone derivative, aspergiodiquinone (3), together with seven known...
Two new benzyl derivatives, aspergentisyl A (1) and aspergentisyl B (2), as well as one new naphthoquinone derivative, aspergiodiquinone (3), together with seven known prenylated benzaldehyde derivatives (4-10) were isolated from the marine-derived fungus Aspergillus glaucus HB1-19. The structures of these compounds were characterized based on 1D and 2D NMR spectra analyses and comparison with those reported in the literature. In addition, each isolate was tested for its 1,1-diphenyl-2-picrylhydrazyl radical-scavenging property and all these compounds except compound 3 exhibited strong radical-scavenging activity.
Topics: Aspergillus; Benzyl Compounds; Biphenyl Compounds; Free Radical Scavengers; Marine Biology; Molecular Structure; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Picrates; Polyketides
PubMed: 23947932
DOI: 10.1080/10286020.2013.826205 -
Folia Microbiologica Nov 2013Effects of different light conditions on development, growth, and secondary metabolism of three marine-derived filamentous fungi were investigated. Darkness irritated...
Effects of different light conditions on development, growth, and secondary metabolism of three marine-derived filamentous fungi were investigated. Darkness irritated sexual development of Aspergillus glaucus HB1-19, while white, red, and blue lights improved its asexual behavior. The red and blue lights improved asexual stroma formation of Xylaria sp. (no. 2508), but the darkness and white light inhibited it. Differently, development of Halorosellinia sp. (no. 1403) turned out to be insensitive to any tested light irradiation. Upon the experimental data, no regularity was observed linking development with secondary metabolism. However, fungal growth showed inversely correlation with productions of major bioactive compounds (aspergiolide A, 1403C, and xyloketal B) from various strains. The results indicated that aspergiolide A biosynthesis favored blue light illumination, while 1403C and xyloketal B preferred red light irradiation. With the favorite light sensing conditions, productions of aspergiolide A, 1403C, and xyloketal B were enhanced by 32.9, 21.9, and 30.8 % compared with those in the dark, respectively. The phylogenetic analysis comparing the light-responding proteins of A. glaucus HB 1-19 with those in other systems indicated that A. glaucus HB 1-19 was closely related to Aspergillus spp. especially A. nidulans in spite of its role of marine-derived fungus. It indicated that marine fungi might conserve its light response system when adapting the marine environment. This work also offers useful information for process optimization involving light regulation on growth and metabolism for drug candidate production from light-sensitive marine fungi.
Topics: Anthraquinones; Aquatic Organisms; Biological Products; Cluster Analysis; Darkness; Fungi; Gene Expression Regulation; Light; Phylogeny; Pyrans; Secondary Metabolism
PubMed: 23546832
DOI: 10.1007/s12223-013-0242-x -
Journal of Applied Genetics Aug 2012Filamentous fungi from the marine environment have shown great potential as cell factories for the production of pharmacologically active metabolites, but extremely low...
Filamentous fungi from the marine environment have shown great potential as cell factories for the production of pharmacologically active metabolites, but extremely low frequency of homologous recombination brings difficulty to further molecular biology studies. To bypass this problem and develop a highly efficient gene targeting system in marine-derived filamentous fungus Aspergillus glaucus, LigD, a homolog of Neurospora crassa Mus-53 which is considered to play a significant role in nonhomologous end joining (NHEJ), was coloned and deleted, and frequency of targeted gene replacement (TGR) increased dramatically from <2% to 85% in comparison with that in the wild type, when containing 1000 bp of homologous flanking sequence. Such results strongly indicate that AgLigD is indeed involved in the repair of NHEJ in A. glaucus and functions in this pathway. Furthermore, the AgLigD-defective mutant has no discernible differences with wild type regarding sensitivity to mutagens and UV, growth characteristics and transformation frequency. The AgligD-deficient transformant, as the first NHEJ-defective mutant in the field of marine-derived filamentous fungus, will help in expediting studies of molecular biology of marine-derived microorganisms.
Topics: Aquatic Organisms; Aspergillus; DNA End-Joining Repair; Gene Targeting; Genes, Fungal; Genetic Loci; Hydroxyurea; Methyl Methanesulfonate; Mutation; Phenotype; Sequence Homology, Nucleic Acid; Transformation, Genetic; Ultraviolet Rays
PubMed: 22562375
DOI: 10.1007/s13353-012-0095-z -
Marine Biotechnology (New York, N.Y.) Dec 2012An integrated control strategy of pH, shear stress, and dissolved oxygen tension (DOT) for fermentation scale-up of the marine-derived fungus Aspergillus glaucus HB...
An integrated control strategy of pH, shear stress, and dissolved oxygen tension (DOT) for fermentation scale-up of the marine-derived fungus Aspergillus glaucus HB 1–19 for the production of the anti-cancer compound aspergiolide A was studied. Keeping initial pH of 6.5 and shifting pH from 6.0 to 7.0 intermittently during the production phase greatly facilitated biosynthesis of aspergiolide A in shake flask cultures. Thus, a pH-shift strategy was proposed that shifting pH to 7.0 once it went lower than 6.0 by pulsed feeding NaOH solution during the production phase in bioreactor fermentation of A. glaucus HB 1–19. As a result, aspergiolide A production in a 30-L bioreactor was increased to 37.6 mg/L, which was 48.6% higher than that in 5-L bioreactor without pH shift. Fermentation scale-up was then performed in a 500-L bioreactor on the basis of an integrated criterion of near-same impeller tip velocity of early phase, DOT levels, and pH shift. The production of aspergiolide A was successfully obtained as 32.0 mg/L, which was well maintained during the process scale-up. This work offers useful information for process development of large-scale production of marine microbial metabolites.
Topics: Anthraquinones; Antineoplastic Agents; Aquatic Organisms; Aspergillus; Batch Cell Culture Techniques; Bioreactors; Culture Media; Feedback, Physiological; Fermentation; Oceans and Seas; Pilot Projects; Species Specificity; Systems Integration
PubMed: 22286337
DOI: 10.1007/s10126-012-9435-6