Theo Goosen

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

SummaryA homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5′-phosphate-decarboxylase gene. A. niger Pyr− mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/μg DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA+ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.

Genomic analysis of the secretion stress response in the enzyme-producing cell factory Aspergillus niger.

BackgroundFilamentous fungi such as Aspergillus niger have a high capacity secretory system and are therefore widely exploited for the industrial production of native and heterologous proteins. However, in most cases the yields of non-fungal proteins are significantly lower than those obtained for fungal proteins. One well-studied bottleneck appears to be the result of mis-folding of heterologous proteins in the ER during early stages of secretion, with related stress responses in the host, including the unfolded protein response (UPR). This study aims at uncovering transcriptional and translational responses occurring in A. niger exposed to secretion stress.ResultsA genome-wide transcriptional analysis of protein secretion-related stress responses was determined using Affymetrix DNA GeneChips and independent verification for selected genes. Endoplasmic reticulum (ER)-associated stress was induced either by chemical treatment of the wild-type cells with dithiothreitol (DTT) or tunicamycin, or by expressing a human protein, tissue plasminogen activator (t-PA). All of these treatments triggered the UPR, as shown by the expression levels of several well-known UPR target genes. The predicted proteins encoded by most of the up-regulated genes function as part of the secretory system including chaperones, foldases, glycosylation enzymes, vesicle transport proteins, and ER-associated degradation proteins. Several genes were down-regulated under stress conditions and these included several genes that encode secreted enzymes. Moreover, translational regulation under ER stress was investigated by polysomal fractionation. This analysis confirmed the post-transcriptional control of hacA expression and highlighted that differential translation also occurs during ER stress, in particular for some genes encoding secreted proteins or proteins involved in ribosomal biogenesis and assembly.ConclusionThis is first genome-wide analysis of both transcriptional and translational events following protein secretion stress. Insight has been gained into the molecular basis of protein secretion and secretion-related stress in an effective protein-secreting fungus, and provides an opportunity to identify target genes for manipulation in strain improvement strategies.

Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene

SummaryConidial protoplasts of an A. nidulans amdS deletion strain (MH1277) have been transformed to the AmdS+ phenotype with a plasmid carrying the wild type gene (p3SR2). Optimalisation of transformation and plating conditions now has resulted in frequencies of 300–400 transformants per μg of DNA.Analysis of DNA from AmdS+ transformants of MH1277 showed that transformation had occurred by integration of vector DNA sequences into the genome. In virtually all these transformants multiple copies of the vector were present in a tandemly repeated fashion, not preferentially at the resident, partially deleted amdS gene. It is suggested that the observed integration phenomena are dependent on the genetic background of the A. nidulans strain, used for transformation. A model to explain the tandem type of integration is proposed.

Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans

We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans.

Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway

BackgroundThe filamentous fungus, Aspergillus niger, responds to nutrient availability by modulating secretion of various substrate degrading hydrolases. This ability has made it an important organism in industrial production of secreted glycoproteins. The recent publication of the A. niger genome sequence and availability of microarrays allow high resolution studies of transcriptional regulation of basal cellular processes, like those of glycoprotein synthesis and secretion. It is known that the activities of certain secretory pathway enzymes involved N-glycosylation are elevated in response to carbon source induced secretion of the glycoprotein glucoamylase. We have investigated whether carbon source dependent enhancement of protein secretion can lead to upregulation of secretory pathway elements extending beyond those involved in N-glycosylation.ResultsThis study compares the physiology and transcriptome of A. niger growing at the same specific growth rate (0.16 h-1) on xylose or maltose in carbon-limited chemostat cultures. Transcription profiles were obtained using Affymetrix GeneChip analysis of six replicate cultures for each of the two growth-limiting carbon sources. The production rate of extracellular proteins per gram dry mycelium was about three times higher on maltose compared to xylose. The defined culture conditions resulted in high reproducibility, discriminating even low-fold differences in transcription, which is characteristic of genes encoding basal cellular functions. This included elements in the secretory pathway and central metabolic pathways. Increased protein secretion on maltose was accompanied by induced transcription of > 90 genes related to protein secretion. The upregulated genes encode key elements in protein translocation to the endoplasmic reticulum (ER), folding, N-glycosylation, quality control, and vesicle packaging and transport between ER and Golgi. The induction effect of maltose resembles the unfolded protein response (UPR), which results from ER-stress and has previously been defined by treatment with chemicals interfering with folding of glycoproteins or by expression of heterologous proteins.ConclusionWe show that upregulation of secretory pathway genes also occurs in conditions inducing secretion of endogenous glycoproteins – representing a more normal physiological state. Transcriptional regulation of protein synthesis and secretory pathway genes may thus reflect a general mechanism for modulation of secretion capacity in response to the conditional need for extracellular enzymes.

Cotransformation of Aspergillus nidulans: A tool for replacing fungal genes.

SummaryWhen a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS+ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS+ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS+, LacZ+ transformants with a Trp− mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC+ transformants which, with a low frequency, had a LacZ− phenotype. These latter transformants had also lost the AmdS+ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping.

SummaryTheAspergillus nidulans gene coding for acetamidase (amdS) was introduced intoA. niger by transformation. Twelve Amd+ transformants were analysed genetically. TheamdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed)A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for theamdS insert have the Amd+ phenotype. We exploited the opportunity for two-way selection inA. niger: transformants can be isolated based on the Amd+ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologousamdS gene inA. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI betweenpabA1 andcnxA1, providing evidence for a single transformational insert. The results also show that theamdS transformants ofA. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use ofamdS transformants for mitotic gene mapping in other fungi.

Transformation of Claviceps purpurea using a bleomycin resistance gene

SummaryTo develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleoR) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.

Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

Abstract Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-based liquid medium. Hybridization of A. oryzae RNAs to a macroarray bearing cDNAs isolated from a library representing at least 16% of the total number of A. niger genes identified 14 differentially expressed cDNA clones, showing that heterologous macroarray analysis with an A. niger cDNA library can be used to identify regulated gene transcripts in the related species A. oryzae. Moreover, Northern analysis with a selection of eight probes for A. niger genes encoding proteins involved in morphological development and cell wall biosynthesis identified five more differentially expressed genes. A suppression subtractive hybridization procedure revealed another 12 differentially expressed genes. The results presented show that, of the 29 identified genes which are expressed at higher levels during growth on wheat kernels, six encode proteins that are functionally related to polarized growth, four encode products known to be involved in morphogenesis, three code for proteins related to cell wall composition, and nine of the cDNA clones encode novel proteins. These findings pinpoint genes associated with the changes in cellular morphogenesis seen in A. oryzae grown on wheat kernels as opposed to wheat-based liquid medium.

Is Integration Essential for Mu Development

The genome of bacteriophage Mu can integrate randomly in the chromosome of its host Escherichia coli, thereby causing mutations (Taylor, 1963). Random integration seems a more widespread phenomenon, since it was found that other DNA elements also show a more or less random integration: insertion sequences (IS) and transposons (Tn).