根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

A method for the large scale isolation of high transformation efficiency fungal genomic DNA

Abstract A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau 3AI partially digested DNA showed high transformation efficiency (10⁶ / 100ng DNA) when ligated to ZAP-express λ vector.     

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.     

EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.     

Sensitivity of Various Bacteria, Including Actinomycetes, and Fungi to Cadmium and the Influence of pH on Sensitivity

A variety of microorganisms, including gram-negative and gram-positive eubacteria, actinomycetes, yeasts, and filamentous fungi, were tested for their sensitivity to cadmium (Cd). In general, the actinomycetes were more tolerant to Cd than were the eubacteria; gram-negative eubacteria were more tolerant to Cd than were gram-positive eubacteria. The period of exponential growth of the eubacteria and actinomycetes was extended in the presence of Cd. Wide extremes in sensitivity to Cd were noted among the fungi; there was no correlation between the class of fungus and tolerance to Cd. Fungal sporulation was more sensitive to Cd than was mycelial growth, as spore formation was inhibited at Cd concentrations that were noninhibitory to mycelial proliferation. The toxicity of Cd to the eubacteria, actinomycetes, and fungi appeared to be pH dependent, as toxicity was generally potentiated at pH 8 or 9.     

Autonomous Plasmid Replication inAspergillus nidulans:AMA1 and MATE Elements

With few exceptions, in eukaryotic organisms the presence of a chromosomal replicator on a circular vector molecule is not sufficient to confer on it the ability to persist and replicate extrachromosomally. However, it is possible to isolate from genomes of some filamentous fungi DNA fragments which can provide extrachromosomal maintenance of plasmids. InAspergillus nidulans,two functional classes of such sequences can be distinguished: effective plasmid replicators (e.g., AMA1) and transformation enhancers (e.g., ANS1 or MATEs), which apparently are able to initiate aberrant replication, leading to vector rearrangement and multimerization and eventually resulting in chromosomal integration. We discuss the similarity of these events to DNA amplification in other eukaryotes. A model is suggested which accounts for the formation of effective replicating plasmids as a result of sequence amplification. The model is based on the observation that in some organisms, includingA. nidulansandSchizosaccharomyces pombe,duplication of an inefficient replicator enhances its efficiency dramatically. Some structural traits of transformation enhancers inA. nidulansimply a role for topoisomerases in amplification and replication of circular DNA molecules. We discuss practical applications of replicative vectors for gene cloning and expression studies.     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

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.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Transformation systems of non-Saccharomyces yeasts

This review describes the transformation systems including vectors, replicons, genetic markers, transformation methods, vector stability, and copy numbers of 13 genera and 31 species of non-Saccharomyces yeasts. Schizosaccharomyces pombe was the first non-Saccharomyces yeast studied for transformation and genetics. The replicons of non-Saccharomyces yeast vectors are from native plasmids, chromosomal DNA, and mitochondrial DNA of Saccharomyces cerevisiae, non-Saccharomyces yeasts, protozoan, plant, and animal. Vectors such as YAC, YCp, YEp, YIp, and YRp were developed for non-Saccharomyces yeasts. Forty-two types of genes from bacteria, yeasts, fungi, and plant were used as genetic markers that could be classified into biosynthetic, dominant, and colored groups to construct non-Saccharomyces yeasts vectors. The LEU2 gene and G418 resistance gene are the two most popular markers used in the yeast transformation. All known transformation methods such as spheroplast-mediating method, alkaline ion treatment method, electroporation, trans-kingdom conjugation, and biolistics have been developed successfully for non-Saccharomyces yeasts, among which the first three are most widely used. The highest copy number detected from non-Saccharomyces yeasts is 60 copies in Kluyveromyces lactis. No general rule is known to illustrate the transformation efficiency, vector stability, and copy number, although factors such as vector composition, host strain, transformation method, and selective pressure might influence them.     

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.     

Applying dimorphic yeasts as model organisms to study mycelial growth: Part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative and qualitative studies of the microflora of barley malt production

Populations of aerobic heterotrophic bacteria, mycelial fungi and yeasts occurring in malting barley were estimated by a plate technique and scanning electron microscopy. There was an increase in the total number of micro-organisms during germination, although populations declined after kilning. Bacteria dominated on all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious spatial distribution of micro-organisms on the samples although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of the kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicum, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filamentous fungi were identified as Aiternaria alternata, Aspergillus glaucus (group), Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, C. vini, Debaryomyces hansenii, Hansenula polymorpha, Kloeckera apiculata, Rhodotorula mucilaginosa, Sporobolomyces roseus and Trichosporon beigelii.     

Development of new tools for studying gene function in fungi based on the Gateway system

Genomic information of many fungi has been released but large scale functional genomic studies are still limited by a lack of high-throughput methods. The low rates of homologous recombination and low rates of transformation are limiting steps in filamentous fungi, but the molecular tools are also lagging behind. In this paper we describe two new high-throughput functional genomic tools for filamentous fungi that are based on the Gateway technology. One system is the Gateway RNAi vector for fungi that allows gene silencing in a high-throughput manner. The other system is a high-throughput deletion construct system. These systems were tested using the PAC1 gene of Colletotrichum gloeosporioides. Using these types of approaches, large scale functional genomics experiments can be performed in filamentous fungi.     

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.     

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process

Summary We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.     

Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.