Transformation in fungi

[This corrects the article on p. 159 in vol. 53.].     

根癌农杆菌介导真菌遗传转化的研究进展

根癌农杆菌介导的真菌遗传转化是近年来发展的一种新方法 ,与其它方法相比 ,该方法具有操作简便、转化效率高和易得到稳定转化子等特点。目前 ,在根癌农杆菌介导下已实现了多个属种真菌的遗传转化 ,显示出良好的应用前景。综述了根癌农杆菌介导真菌遗传转化的转化机理和T DNA在真菌细胞中的存在方式等方面的研究结果 ,并展望这一方法的应用前景。     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.     

Transformation of vanadinite [Pb5(VO4)3Cl] by fungi

Saprotrophic fungi were investigated for their bioweathering effects on the vanadium‐ and lead‐containing insoluble apatite group mineral, vanadinite [Pb₅(VO₄)₃Cl]. Despite the insolubility of vanadinite, fungi exerted both biochemical and biophysical effects on the mineral including etching, penetration and formation of new biominerals. Lead oxalate was precipitated by Aspergillus niger during bioleaching of natural and synthetic vanadinite. Some calcium oxalate monohydrate (whewellite) was formed with natural vanadinite because of the presence of associated ankerite [Ca(Fe²⁺,Mg)(CO₃)₂]. Aspergillus niger also precipitated lead oxalate during growth in the presence of lead carbonate, vanadium(V) oxide and ammonium metavanadate, while abiotic tests confirmed the efficacy of oxalic acid in solubilizing vanadinite and precipitating lead as oxalate. Geochemical modelling confirmed the complexity of vanadium speciation, and the significant effect of oxalate. Oxalate–vanadium complexes markedly reduced the vanadinite stability field, with cationic lead(II) and lead oxalate also occurring. In all treatments and geochemical simulations, no other lead vanadate, or vanadium minerals were detected. This research highlights the importance of oxalate in vanadinite bioweathering and suggests a general fungal transformation of lead‐containing apatite group minerals (e.g. vanadinite, pyromorphite, mimetite) by this mechanism. The findings are also relevant to remedial treatments for lead/vanadium contamination, and novel approaches for vanadium recovery.     

Denitrification by fungi

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.     

Biotransformation of cycloalkenones by fungi Baeyer-Villiger oxidation of bicycloheptenone by dematiaceous fungi

Summary The biotransformation by ring expansion of a bicyclo [3.2.0]-alkenone has been demonstrated in 29 species of dematiaceous fungi. One of these biocatalysts,Curvularia lunata NRRL 2380 has been shown to produce synthetically-important chiral lactones in high yields.     

Biotransformations by fungi

Summary The regio-and stereoselective characteristics of biotransformations involving oxidative ring expansion of bicyclo[3.2.0]hept-2-en-6-one have been characterised in various dematiaceous fungi of the generaCurvularia andDrechslera.     

Dissimilation of methionine by fungi

Soil fungi that attacked methionine required a utilizable source of energy such as glucose for growth. This is an example of co-dissimilation. Experiments with one of the fungi, representative of the group, are reported. In the absence of glucose, pregrown mycelium, even when depleted of energy reserves, oxidatively deaminated methionine with accumulation of α-keto-γ-methyl mercapto butyric acid and α-hydroxy-γ-methyl mercapto butyric acid. When glucose was provided, all of the sulfur of methionine was released as methanethiol, part of which was oxidized to dimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected. Evidence was obtained that deaminase and demethiolase were constitutive. Deamination preceded demethiolation and α-keto butyric acid accumulated as a product of the two reactions. Other carbon residues were α-hydroxy butyric acid and α-amino butyric acid. Inability of the fungus to metabolize α-keto butyrate was responsible for its inability to utilize methionine as a source of carbon and energy. Several other fungi isolated from soil grew on α-amino butyrate but could not grow on methionine owing to inability to demethiolate it.     

Biotransformation of N-acetylphenothiazine by fungi

Cultures of the fungi Aspergillus niger, Cunninghamella verticillata, and Penicillium simplicissimum, grown in a sucrose/peptone medium, transformed N-acetylphenothiazine to N-acetylphenothiazine sulfoxide (from 13% to 28% of the total) and phenothiazine sulfoxide (from 5% to 27%). Phenothiazin-3-one (4%) and phenothiazine N-glucoside (4%) were also produced by C. verticillata. The probable intermediate, phenothiazine, was detected only in cultures of P. simplicissimum (6%). Received: 15 January 1999 / Received revision: 7 May 1999 / Accepted: 21 May 1999     

Glycosylation of xanthohumol by fungi

A screening test on 29 microorganisms for transformation of xanthohumol led to the selection of twelve fungal strains. One of them, Beauveria bassiana AM278, converted xanthohumol into a glucosylated derivative. This product was identified as xanthohumol 4'-O-beta-D-4'-methoxyglucopyranoside.