Arthur F. J. Ram

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Agrobacterium-mediated transformation as a tool for functional genomics in fungi.

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.

Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red.

The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4–6 h (excluding the incubation time required for fungal growth).

Agrobacterium -mediated transformation of the filamentous fungus Aspergillus awamori

Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori. This protocol has been used to determine the function of Agrobacterium virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate A. awamori gene knockout transformants. This protocol in not only applicable to A. awamori, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced Agrobacterium, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200–250 transformants per 1 × 106 conidiospores. Using this protocol, transformants can be obtained within 10–12 d.

Glucoamylase : green fluorescent protein fusions to monitor protein secretion in Aspergillus niger

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses

Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here, an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However, ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails. Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation. Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches.

Aspergillus as a multi-purpose cell factory: current status and perspectives

Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts.

Fungal gene expression on demand: An inducible, tunable and metabolism-independent expression system for Aspergillus niger

ABSTRACT Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined.

Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription

Analysis of genes involved in yeast cell wallβ-glucan assembly has led to the isolation ofEXG1,PBS2 andPTC1.EXG1 andPBS2 were isolated as genes that, when expressed from multicopy plasmids, led to a dominant killer toxin-resistant phenotype. ThePTC1 gene was cloned by functional complementation of the calcofluor white-hypersensitive mutantcwh47-1.PTC1/CWH47 is the structural gene for a type 2C serine/threonine phosphatase,EXG1 codes for an exo-β-glucanase, andPBS2 encodes a MAP kinase kinase in the Pbs2p-Hog1p signal transduction pathway. Overexpression ofEXG1 on a 2μ plasmid led to reduction in a cell wallβ1,6-glucan and caused killer resistance in wild type cells; while theexg1Δ mutant displayed modest increases in killer sensitivity andβ1,6-glucan levels. Disruption ofPTC1/CWH47 and overexpression ofPBS2 gave rise to similarβ-glucan related phenotypes, with higher levels ofEXG1 transcription, increased exo-β-glucanase activity, reducedβ1,6-glucan levels, and resistance to killer toxin. Genetic analysis revealed that loss of function of thePBS2 gene was epistatic toPTC1/CWH47 disruption, indicating a functional role for the Ptc1p/Cwh47p phosphatase in the Pbs2p-Hog1p signal transduction pathway. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in cell wall glucan assembly, and that this is effected in part by modulating Exg1p activity.