P. J. Punt

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.

Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3 end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed.

Expression of the Escherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi

SummaryA chimaeric β-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E. coli uidA gene. Cotransformation of this vector into A. nidulans, A. niger and the tomato pathogen Fulvia fulva (syn. Cladosporium fulvum (Cooke)) resulted in the expression of β-glucuronidase. GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts. Expression of the gene in F. fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity. These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi.

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5CTCCGHGG3. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

Molecular genetic strain improvement for the overproduction of fungal proteins by Filamentous fungi

During the last 5 years, strain improvement by genetic engineering has been established as a good alternative for traditional methods of strain improvement such as mutagenesis and genetic recombination. Considerable success has been achieved in the overproduction of a variety of fungal proteins. However, from the available data a limitation at the level of transcription is evident. A more detailed analysis of this transcription limitation was carried out for the overproduction of glucoamylase in A. niger. This analysis revealed that both the site of integration of introduced gene copies and the available amount of trans-acting regulatory proteins were at the root of the observed limitation. Based on these results, approaches for the construction of a new generation of protein-overproducing strains are suggested.

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.

Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway of Aspergillus niger

ABSTRACT Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene,pdiA, encoding PDIA was previously isolated fromAspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A.pdiA also complemented PDI function in aSaccharomyces cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

Transformation of Fulvia fulva, a fungal pathogen of tomato, to hygromycin B resistance

SummaryA transformation system for the tomato pathogen Fulvia fulva has been developed. Hygromycin B resistant colonies were obtained after treatment of protoplasts with a plasmid containing an E. coli hygromycin B phosphotransferase gene fused to an Aspergillus nidulans promoter. The DNA was stably integrated into the genome. The number and sites of integrations varied among transformants. The demonstration of transformation opens the way for the molecular genetic analysis of the interaction of Fulvia with tomato.

A vector for Aspergillus transformation conferring phleomycin resistance.

Recently, transformation of Aspergillus species with vector pAN7-1, conferring resistance to hygromycin B was reported (Punt et al. 1987 Gene 56:117-124). Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol35/iss1/13 TABLE 1. Numbers of stable transformants (± one standard deviation) obtained when 2 x 10^7 spheroplasts were transformed as described in the text with 1 ug of cosmid DNA prepared wither by CsCl gradient or by alkaline lysis followed by purification through LMP agarose. Numbers reported are pooled from many transformations. The variability among LMP agarose preparations is partially due to the use of different sources of DNA (different cosmid pools). Inaccuracies in the estimation of DNA concentration also contributes to variability, since estimates based on fluorescence in agarose gels cannot be extremely precise Sources of cosmid DNA LMP agarose CsCl gradient bd A 1450 ± 619 1800 ± 307 Spheroplasts bd al-2 arg-13 a 1670 ± 776 1940 ± 394 Purification of miniprep DNA through LMP agarose has several advantages: (1) it is rapid, with no need of an ultracentrifuge; (2) DNA so prepared efficiently transforms Neurospora; (3) DNA so prepared can be used for a number of other purposes without further purification: it can be used for restriction and modification reactions, bacterial transformations (Struhl, K. 1985 Biotechniques 3:452), and radioactive labelling (Feinberg, A.P. and B. Vogelstein. 1984 Anal. Biochem. 137:266-267). Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03756 Mattern. I.E., P.J. Punt and C.A.M.J.J. Van den Hondel A vector of Aspergillus transformation conferring phleomycin resistance. expressed A. nidulans gpd gene, Recently, transformation of Aspergillus species with vector pAN7-1, conferring resistance to hygromycin B was reported (Punt et al. 1987 Gene 56:117-124). Here we describe a transformation vector (pAN8-1, Fig. 1) containing the Streptococcus hindustanus phleomycin resistance gene (obtained from G. Tiraby, Toulouse, France) flanked by the promoter region of the highly and the terminator region of the A. nidulans trpC gene. Transformation of A. nidulans and A. niger was achieved with this vector at frequencies of 1 to 20 transformants per ug pAN8-1 DNA. These frequencies are similar to those found for transformation with pAN7-1. Transformants could be selected at low concentrations of phleomycin (5-10 ug/ml for A. niger, 10-20 ug/ml for A. nidulans). A. oryzae, which cannot be transformed with pAN7-1 because of its innate insensitivity to hygromycin B, is inhibited in its growth at 50-100 ug/ml phleomycin. Phleomycin resistant transformants were obtained by cotransformation of an A. oryzae pyrG mutant with pAB4-1 (containing the A. niger pyrG gene) and pAN8-1 (Mattern et al. 1987, MGG 210:460-461). Experiments are in progress to achieve direct selection of phleomycin resistant transformants of A. oryzae. Figure 1. Vector pAN8.1. A 0.4 kb NcoI-StuI fragment from pUT701 (G. Tiraby, unpublished) containing the coding region of the S. hindustandus phleomycin resistance gene was ligated into pAN52-3, which was cut with HindIII, treated with T4 polymerase and subsequently cut with NcoI. Vector pAN52-3 is a derivative of pAN52-1 (Punt et al. 1987 Gene 56:117-124) in which the unique BamHi site was converted into a Hind III site by site directed mutagenesis. NcoI Medical Biological Laboratory TNO, P.O. Box 45, 2280 AA Rijswijk, The Netherlands

The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins.

We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori. The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids. The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5- and 3- noncoding regions. Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5 non-transcribed regions of both genes. The expression of the A. niger bipA gene is increased by heat shock and tunicamycin treatment.