Fungal Transformation Research Articles

Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter

We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79% of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.

Microbial transformation of anti-cancer steroid exemestane and cytotoxicity of its metabolites against cancer cell lines

BackgroundMicrobial transformation of steroids has been extensively used for the synthesis of steroidal drugs, that often yield novel analogues, not easy to obtain by chemical synthesis. We report here fungal transformation of a synthetic steroidal drug, exemestane, used for the treatment of breast cancer and function through inhibition of aromatase enzyme.ResultsMicrobial transformation of anti-cancer steroid, exemestane (1), was investigated by using two filamentous fungi. Incubation of 1 with fungi Macrophomina phaseolina, and Fusarium lini afforded three new, 11α-hydroxy-6-methylene-androsta-1, 4-diene-3,17-dione (2), 16β, 17β-dihydroxy-6-methylene-androsta-1, 4-diene-3-one (3), and 17β-hydroxy-6-methylene-androsta-1, 4-diene-3, 16-dione (4), and one known metabolites, 17β-hydroxy-6-methylene-androsta-1, 4-diene-3-one (5). Their structures were deduced spectroscopically. Compared to 1 (steroidal aromatase inactivator), the transformed metabolites were also evaluated for cytotoxic activity by using a cell viability assay against cancer cell lines (HeLa and PC3). Metabolite 2 was found to be moderately active against both the cell lines.ConclusionsBiotransformation of exemestane (1) provides an efficient method for the synthesis of new analogues of 1. The metabolites were obtained as a result of reduction of double bond and hydroxylation. The transformed product 2 exhibited a moderate activity against cancer cell lines (HeLa and PC3). These transformed products can be studied for their potential as drug candidates.

Biotransformation of vinblastine to vincristine by the endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus

A fungal transformation method for the production of the less abundant but more valuable anticancer drug vincristine using the more abundant anticancer drug vinblastine by an endophytic fungus Fusarium oxysporum isolated from the plant Catharanthus roseus is described. Vinblastine dissolved in sterile water when incubated with the fungal mycelial mass was transformed into vincristine at room temperature. The biotransformation process was monitored by periodic sampling of aliquots that were extracted with ethyl acetate. The crude transformed compounds were analysed using TLC where the Rf value of the spot obtained on the 8th day was found to be similar to that of standard vincristine (Rf 0.74). Purification by HPLC using a C 18 symmetry column showed a peak with RT (34.9 min) similar to that of standard vincristine. Further characterization with the help of ESI-MS showed a peak at m/z 825 attributed to the (M + H)+ ion. This indicated that the fungus Fusarium oxysporum had converted vinblastine to vincristine.

Fungal transformation of cedryl acetate and α-glucosidase inhibition assay, quantum mechanical calculations and molecular docking studies of its metabolites

The fungal transformation of cedryl acetate (1) was investigated for the first time by using Cunninghamella elegans. The metabolites obtained include, 10β-hydroxycedryl acetate (3), 2α, 10β-dihydroxycedryl acetate (4), 2α-hydroxy-10-oxocedryl acetate (5), 3α,10β-dihydroxycedryl acetate (6), 3α,10α-dihydroxycedryl acetate (7), 10β,14α-dihydroxy cedryl acetate (8), 3β,10β-cedr-8(15)-ene-3,10-diol (9), and 3α,8β,10β -dihydroxycedrol (10). Compounds 1, 2, and 4 showed α-glucosidase inhibitory activity, whereby 1 was more potent than the standard inhibitor, acarbose, against yeast α-glucosidase. Detailed docking studies were performed on all experimentally active compounds to study the molecular interaction and binding mode in the active site of the modeled yeast α-glucosidase and human intestinal maltase glucoamylase. All active ligands were found to have greater binding affinity with the yeast α-glucosidase as compared to that of human homolog, the intestinal maltase, by an average value of approximately −1.4 kcal/mol, however, no significant difference was observed in the case of pancreatic amylase.

Reduction of preg-robbing by biomodified carbonaceous matter – A proposed mechanism

The carbonaceous matter (CM) in gold ores behaves similarly to activated carbon in its ability to adsorb aurocyanide ions. This phenomenon is termed preg-robbing as the adsorbed gold cannot be recovered from the CM. The mechanism of gold adsorption depends, among other things, on the surface functional groups, pore structure and maturity of CM, and hence the development of graphitic structure. In this study, the fungus Phanerochaete chrysosporium was used to biomodify the surface and alter the gold adsorption ability of anthracite coal, which was used as surrogate for CM. Characterization, employing FTIR, XANES, Raman and TEM analyses, showed more oxygen-containing groups and enhancement in the amorphous nature of carbon, following fungal transformation. As well, pore structure measurement indicated drastic reduction in surface area and micro pore volume, and enlargement in average pore diameter. The results suggest a number of theories by which P. chrysosporium biotransforms CM: (a) surface oxidation, which disrupts the continuous graphitic structure, thus decreasing the active sites necessary for adsorption; and (b) reduction in surface area via (i) plugging of pores possibly by fungal biomass and/or slimy substances formed through fungal interaction with the growth medium, and hence decreasing accessibility of gold to the adsorption sites, and (ii) cleavage of bonds leading to pore enlargement, thus rendering the pores too large for adsorption of the aurocyanide ion which are known to adsorb into micropores.

Geomycology: Fungi as Agents of Biogeochemical Change

Geomycology is the study of the roles of fungi in geological processes. The biogeochemical importance of fungi is significant in several areas, including nutrient and element cycling, rock, mineral and metal transformations, bioweathering and mycogenic biomineral formation. Such processes can occur in aquatic and terrestrial habitats, but it is the terrestrial environment where fungi probably have the greatest geochemical influence. Of special significance are the mutualistic relationships with photo trop hie organisms: lichens (algae, cyanobacteria) and mycorrhizas (plants). Central to many geomycological processes are transformations of metals and minerals, and fungi possess many properties that effect changes in metal speciation, toxicity and mobility, as well as mineral formation or mineral dissolution or deterioration. Some fungal transformations have potential applications in environmental biotechnology, e.g. metal and radionuclide leaching, recovery, detoxification and bioremediation, and in the production or deposition of biominerals or metallic elements with catalytic or other properties. Metal and mineral transformations may also result in adverse effects when these processes result in spoilage and destruction of natural and synthetic materials, rock and mineral-based building materials (e.g. concrete), biocorrosion of metals, alloys and related substances, and adverse effects on radionuclide speciation, mobility and containment.

Performance Testing of Thermal Cutting Systems for Sweet Pepper Harvesting Robot in Greenhouse Horticulture

This paper proposes design of end-effector and prototype of thermal cutting system for harvesting sweet peppers. The design consists of two parallel gripper bars mounted on a frame connected by specially designed notch plate and operated by servo motor. Based on voltage and current, two different types of thermal cutting system prototypes; electric arc and temperature arc respectively were developed and tested for performance. In electric arc, a special electric device was developed to obtain high voltage to perform cutting operation. At higher voltage, electrodes generate thermal arc which helps to cut stem of sweet pepper. In temperature arc, nichrome wire was mounted between two electrodes and current was provided directly to electrodes which results in generation of high temperature arc between two electrodes that help to perform cutting operation. In both prototypes, diameters of basic elements were varied and the effect of this variation on cutting operation was investigated. The temperature arc thermal system was found significantly suitable for cutting operation than electric arc thermal system. In temperature arc thermal cutting system, 0.5 mm nichrome wire shows significant results by accomplishing harvesting operation in 1.5 seconds. Also, thermal cutting system found suitable to increase shelf life of fruits by avoiding virus and fungal transformation during cutting process and sealing the fruit stem. The harvested sweet peppers by thermal cutting system can be preserved at normal room temperature for more than 15 days without any contamination.

Agrobacterium tumefaciens-mediated genetic transformation of the Taxol-producing endophytic fungus Ozonium sp EFY21

An efficient Agrobacterium tumefaciens-mediated genetic transformation method was successfully established for a newly isolated Taxol-producing fungus, Ozonium sp EFY21. A specific hygromycin B resistance expression vector, pCAMBIA1304'AN7-1, was constructed for fungal transformation. Key factors affecting transformation efficiency were thoroughly investigated and optimized. PCR amplification and Southern hybridization were used to verify the transformation events. This study should pave the way for future genetic modification studies of Ozonium sp EFY21.

Synthesis of some potent immunomodulatory and anti-inflammatory metabolites by fungal transformation of anabolic steroid oxymetholone

BackgroundBiotransformation of organic compounds by using microbial whole cells provides an efficient approach to obtain novel analogues which are often difficult to synthesize chemically. In this manuscript, we report for the first time the microbial transformation of a synthetic anabolic steroidal drug, oxymetholone, by fungal cell cultures.ResultsIncubation of oxymetholone (1) with Macrophomina phaseolina, Aspergillus niger, Rhizopus stolonifer, and Fusarium lini produced 17β-hydroxy-2-(hydroxy-methyl)-17α-methyl-5α-androstan-1-en-3-one (2), 2α,17α-di(hydroxyl-methyl)-5α-androstan-3β,17β-diol (3), 17α-methyl-5α-androstan-2α,3β,17β-triol (4), 17β-hydroxy-2-(hydroxymethyl)-17α-methyl-androst-1,4-dien-3-one (5), 17β-hydroxy-2α-(hydroxy-methyl)-17α-methyl-5α-androstan-3-one (6), and 2α-(hydroxymethyl)-17α-methyl-5α-androstan-3β-17β-diol (7). Their structures were deduced by spectral analyses, as well as single-crystal X-ray diffraction studies. Compounds 2–5 were identified as the new metabolites of 1. The immunomodulatory, and anti-inflammatory activities and cytotoxicity of compounds 1–7 were evaluated by observing their effects on T-cell proliferation, reactive oxygen species (ROS) production, and normal cell growth in MTT assays, respectively. These compounds showed immunosuppressant effect in the T-cell proliferation assay with IC50 values between 31.2 to 2.7 μg/mL, while the IC50 values for ROS inhibition, representing anti-inflammatory effect, were in the range of 25.6 to 2.0 μg/mL. All the compounds were found to be non-toxic in a cell-based cytotoxicity assay.ConclusionMicrobial transformation of oxymetholone (1) provides an efficient method for structural transformation of 1. The transformed products were obtained as a result of de novo stereoselective reduction of the enone system, isomerization of double bond, insertion of double bond and hydroxylation. The transformed products, which showed significant immunosuppressant and anti-inflammatory activities, can be further studied for their potential as novel drugs.

Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

BackgroundSpecies of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential.ResultsWe established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi.ConclusionThe neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

Copper induction of enhanced green fluorescent protein expression inPleurotus ostreatusdriven by laccasepoxa1bpromoter

In silico analyses of several laccase promoter sequences have shown the presence of many different responsive elements differentially distributed along the promoter sequences. Analysis of Pleurotus ostreatus laccase promoter poxa1b extending around 1400-bp upstream of the start codon showed the presence of several putative response elements, such as 10 metal-responsive elements. Development of a system for in vivo analysis of P. ostreatus laccase promoter poxa1b by enhanced green fluorescent protein expression was carried out, based on a polyethylene glycol-mediated procedure for fungal transformation. Quantitative measurement of fluorescence expressed in P. ostreatus transformants grown in the presence and in the absence of copper sulfate was performed, demonstrating an increase in expression level induced by the metal.

Enhanced production of Aspergillus niger laccase‐like multicopper oxidases through mRNA optimization of the glucoamylase expression system

In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields.

Enhanced itaconic acid production in Aspergillus niger using genetic modification and medium optimization

BackgroundAspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. Expressing the CAD gene from Aspergillus terreus opened the metabolic pathway towards itaconic acid in A. niger. In order to increase the production level, we continued by modifying its genome and optimizing cultivation media.ResultsBased on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde −3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger. Besides, new media were designed based on a reference medium for A. terreus. To analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production. Using 20 different media, copper was discovered to have a positive influence on itaconic acid production. Effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation.ConclusionsThe performance of gpdA and hbd1 transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. Medium optimization showed that, copper was positively correlated with improved itaconic acid production. Interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production.

Enhanced Green Fluorescent Protein Expression in Pleurotus ostreatus for In Vivo Analysis of Fungal Laccase Promoters

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

Fungal Transformation and Schistosomicidal Effects of Pimaradienoic Acid

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100 μM. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100 mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

The potential of Cyathus africanus for transformation of terpene substrates

The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced (4R)-9α-hydroxycadin-10(15)-en-3-one, while the latter gave 2β-hydroxyaromadendr-1(10)-en-9-one, 2α-hydroxyaromadendr-1(10)-en-9-one and 10α-hydroxy-1β,2β-epoxyaromadendran-9-one. The bioconversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto unreported 8α,13-dihydroxysantonin, while cedrol yielded 3β,8β-dihydroxycedrane and 3α,8β-dihydroxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to the lupane family, was supplied to the fungus 18α-ursane and 18α-oleanane derivatives, namely 19β-hydroxy-3-oxo-18α-oleanan-28-oic acid and 19α-hydroxy-3-oxo-18α-ursan-28-oic acids, were generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal rearrangement occurred. All substrate administration experiments were done in the presence of the terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase - pulse feed method.

Antispasmodic activity of pimaradienoic acid derivatives obtained by microbial transformation

Pimaradienoic acid (PA, 1) is a pimarane-type diterpene which is associated with a wide spectrum of biological activities, including its great ability to inhibit rat aorta contraction [1]. In the present work, the fungal transformation of PA (0.1g/L) was performed using culture of Aspergillus niger (1.5×107 spores/mL) during 9 days of incubation. After this period, the culture was filtered and the aqueous layer was extracted with ethyl acetate to furnish the extract codified as AtPA. Chemical and NMR studies of AtPA allowed us to isolate and to identify four PA derivatives (2–5). The chemical structures of 2, 4 and 5 are described for the first time in the scientific literature.

Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid

Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells.

Transformation and attachment of Beauveria bassiana conidia on the cuticle of Tribolium confusum and Sitophilus oryzae in conjunction with diatomaceous earth

Agrobacterium tumefaciens-mediated transformation (AMT) was used in order to create fluorescent conidia of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales). From the fungal transformants, one Enhanced Green Fluorescent Protein B. bassiana strain indicated stability (maintained the GFP characteristics) in fluorescent signal, while its pathogenicity did not differ significantly from the wild B. bassiana type. After exposure on wheat treated with the transformed conidia, fluorescence was evident on all body parts but especially on metasternon of adults of Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and on elytra in of Sitophilus oryzae (L.) (Coleoptera: Curculionidae). No significant differences were noted in fluorescence levels in the case of T. confusum between the transformed and the wild strain. However, the addition of diatomaceous earth (DE) increased attachment, especially at increased intervals after exposure. For S. oryzae, fluorescence of the transformed strain was decreased with the increased post-exposure time, but the simultaneous presence of DE increased fluorescence. The results of this study demonstrate that fluorescent conidia of B. bassiana can be utilized as a tool for the evaluation of parameters that affect the attachment of entomopathogenic fungi in insects’ cuticle.

A split luciferase complementation assay for studying in vivo protein–protein interactions in filamentous ascomycetes

Protein-protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein-protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein-protein interactions in filamentous ascomycetes.