Poster Abstracts, Biotechnology

Endoproteolytic Processing of Fusion Proteins by Aspergillus niger

David Archer1, Donald MacKenzie1, Andrew Spencer1,4, David Jeenes1, MarieFrangoise Le Gal-Coeffet1,2, James Kraunsoe3, Gordon Lowe3, Donald Haynie4 and Christopher Dobson4. 1Institute of Food Research,Norwich, UK, 2Department of Molecular Biology and Biotechnology, University of Sheffield, 3Dyson Perrins Laboratory and 4New Chemistry Laboratory, University of Oxford, UK.

Optimised secretion of heterologous proteins from filamentous fungi increasingly resorts to the use of translational fusion constructs whereby the gene encoding the target protein is fused downstream of the gene encoding a naturally well-secreted carrier protein such as glucoamylase. The increased yields that result are thought to be due to a combination of factors relating to the carrier protein, e.g. improved mRNA stability and eased passage of the protein through the secretary pathway. A successful strategy for separation of the target protein from its carrier protein has been the incorporation of a DNA sequence encoding a dibasic amino acid recognition site for a kexin-like endoprotease at the fusionjunction. Cleavage occurs intracellularly and the two proteins are secreted separately. We have examined the specificity of this cleavage in Aspergillus niger in order to be able to predict the chance of processing at the correct site when a new target gene is expressed in a fusion construct. We used a truncated glucoamylase (sequence to amino acid 498) as the carrier protein, with a dibasic cleavage site Lys Arg at the junction with three different target proteins: hen egg white lysozyme (HEWL), the glucoamylase starch-binding domain (SBD) and bovine pancreatic trypsin inhibitor (BPTI). Cleavage sites were determined by a combination of Nterminal sequencing and electrospray mass spectrometry. The SBD was cleaved from the fusion entirely at the correct site. BPTI cleavage occurred at three sites in addition to the target processing site. The proportion of correct cleavage was increased by expressing mutant BPTI genes designed to make the BPTI resemble anti-elastase, suggesting that BPTI binds to the (major) kexin protease and interferes with its activity. Wild-type HEWL was cleaved entirely at the correct site. However, shortening the region of HEWL immediately Nterminal of the first et-helix by two amino acids caused the site of cleavage to be moved two (56%) and three (44%) residues away from the HEWL Nterminus. This suggests that correct processing requires a minimal distance away from a region of ordered structure such as an ot-helix, in addition to a preferred dibasic amino acid sequence. Cleavage of other mutant HEWLs is being studied to test this model.

Approaches Towards Cloning of Genes from Claviceps purpurea Expressed during Alkaloid Metabolism

Claudia Arntz and Paul Tudzynski, Institut fur Botanik, Westfalische Wilhelms-Universitat, Schlo garten 3, 48149 Munster, Germany

During the infection process C. purpurea forms sclerotia on cereals, e.g. rye. Sclerotia are dormant structures which contain pharmacologically active ergot alkaloids. These compounds are used in the treatment of a variety of clinical conditions as for example migraine and Parkinsons disease. The genetics of the biosynthesis is not known in detail. In order to isolate genes involved in ergot alkaloid biosynthesis a differential screening of a cDNA library was performed with cDNA from alkaloid producing and non-producing mycelia (cDNA+/cDNA-) as probes, resp..Several genes could be isolated by this method: e.g. the Dimethylallyltryptophan Synthase which catalyses the first pathway specific step of ergot alkaloid biosynthesis (1); a gene showing homology to a circadian clock regulated, light and stress induced gene (ccg-1) of Neurospora crassa (2); a gene (isolated several times) showing significant homology to hydrophobins or hydrophobic proteins, resp., like cryparin, an abundant cell-surface protein from Cryphonectria parasitica (3) and QID 3, a cell-wall surface protein of Trichoderma harzianum (4). These results show that this method yields not only genes of the alkaloid pathway, but obviously also genes which might be involved in the differentiation process accompanying induction of alkaloid synthesis, i.e. formation of "sclerotial" hyphae.

(1)Tsai H-F, Wang H, Gebler JC, Poulter CD, Schardl CL (1995) The Claviceps purpurea gene encoding Dimethylallyltryptophan Synthase, the committed step for ergot alkaloid biosynthesis. Biochem Biophys Res Corn 216:119-125

(2)Loros JJ, Denome SA, Dunlap JC (1 989) Molecular cloning of genes under control of the circadian clock in Neurospora. Science 243:385-388

(3)Zhang L, Villalon D, Sun Y, Kazmiercak P, van Alfen N K (1 994) Virus-associated downregulation of the gene encoding cryparin, an abundant cell-surface protein from thb chestnut blight fungus, Cryphonectria parasitica. Gene 139:59-64

(4)Lora JM, de la Cruz J, Benitez T, LLobell A, Pintor-Toro JA (1 994) A putative cataboliterepressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum. Mol Gen Genet 242:461-466

Probes for Fungal Polyketide Synthase Genes

Lewis E.H. Bingle and Colin M. Lazarus. School of Biological Sciences, University of Bristol, Bristol BS8 1UG, U.K.

Polyketides are natural products derived from the successive condensation of small carboxyhc acids; assembly of the initial carbon skeleton is catalysed by an enzyme known as a polyketide synthase (PKS). Many of the polyketide metabolites so far identified are products of the fungi, particularly of the Deuteromycetes. Several of these compounds are of pharmacological interest or mycotoxins and some fungal melanins have a polyketide origin. All fungal PKS enzymes characterised have been large multi-functional proteins (MV I PKS).

We observed that the available fungal PKS gene sequences could be arranged into two subgroups on the basis of amino acid sequence conservation in functional domains. Two pairs of PCR primers (LCl/2c and LC3/5c) were designed to bind to regions where amino acid sequence was conserved within these subgroups but not between them. These primers amplified corresponding fragments containing the condensing domains from a range of fungal PKS genes in Deuteromycete genomes. The products from one primer pair hybridise strongly to each other, but not to products from the other primer pair. Each type of PCR product shows a different pattern of homologous hybridisation to the fungal genome from which it was derived. This pattern of hybridisation may indicate two subclasses of PKS genes, both widely distributed among the filamentous fungi. The PCR products obtained using the LC primers described above should provide useful homologous probes for cloning novel fungal PKS genes.

Development of Heterologous Protein Secretion Systems in Filamentous Fungi

Martine Parriche1, Jean-Christophe Bousson1, Michel Baron2 and Girard Tiraby2. 1CAYLA Ltd, Av, de Larrieu, 31093 Toulouse, France. 2Laboraloire de Microbiologie et Genetique Appliques, Universite Paul Sabatier, 118 Rte de Narbonne, 31062 Toulouse, France.

This work aims to develop efficient heterologous protein secretion systems in filamentous fungus Trichoderma reesei . This can only be achieved by use of high performance strains and expression vectors. Suitable host strains have been obtained after several rounds of chemical mutagenesis and selection for high level secretion of various heterologous proteins. Vector construction strategy is based on the use of fusion proteins which is the most efficient system to date for secretion of heterologous proteins. The model systems in this study use A. niger glucoamylase (GlaA), T. reesei cellobiohydrolase I (CBHI) or the prokaryotic phleomycin resistance protein (Sh-ble) fused in 5' to human lysozyme (HLys). These carrier proteins have already been shown to be efficiently secreted by fungi. GlaA::HLys, CBHI::HLys and Sh - ble::HLys fusion vectors have been constnicted with or without introduction of modifications to the carrier protein. HLys secretion efficiency has been assessed in T. reesei strains endowed with different properties with respect to secretion. Findings could be applied to the production of other human proteins, commercially valuable, in T. reesei as well as in other fungi.

Expression of the Extracellular Aspartic Proteinase in Penicilljum roqueforti

Stehanie Bruyant, Nathalie Poussereau and Michel Fevre. Laboratoire de Biologie Cellulaire Fongique CGMC-CNRS UMR 106. Universite Claude Bernard Bat 405. 43 Bd du II novembre 1918 69622 Villeurbanne Cedex. France

Filamentous fungi can grow under very varying environmental conditions and utilize a wide variety of compounds as nutrients. The acquisition and metabolism of the large number of different carbon and nitrogen sources are under sophisticated regulatory controls governed by several regulatory circuits, involving both wide-domain and pathway-specific regulatory genes. Penicillium roqueforti, an economically-important fungus secretes a number of enzymes including endo and exopeptidases. The aspartic proteinase is the major extracellular proteolytic activity in Penicillium roqueforti. We have cloned the gene aspA encoding the aspartic proteinase using a PCR probe. Nucleotide sequence data revealed that aspA is composed of three exons of 325, 526 and 340 bp. Two introns which interrupt the coding sequence are 57 and 67 bp in length. The deduced amino acid sequence of aspA presents a high degree of homology to other fungal aspartic proteinase and indicates that this aspartic proteinase is synthesized as a zymogen containing an Nterminal prepro-region of 71 amino acids followed by a mature protein of 326 amino acids. Southern blot experiments reveal that the aspA gene is present as a single copy in the Penicillium roqueforti genome.

Regulation of the aspartic proteinase production was investigated using biochemical and Northern analyses. Our results suggest that aspA expression is submitted to transcfiptionnal regulations. ASPA is not controlled by glucose and not produced in presence of ammoniac in the culture medium. The pH of the culture medium plays also a major role in the regulation of -this gene since it was completely turned off under alkaline conditions.

Analysis of the promoter sequence revealed the presence of putative AREA and PACC binding sites. Electrophoretic mobility shift assay experiments indicate that different transcription factors are involved according to the culture conditions. The hierarchy existing among the different regulatory systems will be discussed.

Isolation and Characterisation of the Aspergillus nidulans Sulphate Reduction Pathway Gene Adenosyl Phosphosulphate Kinase

David L. Clarke, Roger W. Newbert and Geoffrey Turner. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, SI 0 2UH England

Many microorganisms, including the filamentous fungi Aspergillus nidulans and Penicillium chrysogenum, are able to assimilate sulphur by the uptake and reduction of inorganic sulphate, the suiphide thus produced being incorpotated into methionine and cystine. The sulphate reduction pathway required sulphate permease (sB), ATP sulphurylase (sC) APS kinase (sD), PAPS reductase (sA) and sulfite reductase. The sC and sA genes of A. nidulans have already been characterised as detailed by M.I. Borges-Walmsley et al, Mol Gen Genet (1995) 247:423-429.

It is proposed to isolate the remaining genes of the pathway in order to gain a more complete understanding of its control mechanisms. Such dats would not only be of academic interest but may also have inplications in the commercial procuction of -lactam antibiotics in which industrial production strains place a heavy burden on their cysteine pools.

By a process of heterologous colony hybridisation we have isolated the APS kinase gene from an A.nidulans cosmid library. A subclone of the cosmid isolated was shown, by transformation, to complement an A.nidulans sD mutant strain. The identity of the gene has been further verified by DNA sequence analysis displaying 59% identity with the MET14 gene of Saccharomyces cerevisiae.

Work is proceeding to complete the sequence analysis of the A. nidulans sD gene and to iso!ate the remaining pathway genes from both A. nidulans and P.chrsyogenum.

Approach to Structural Determination of Cyp P450 and Cyp P450 Reductase in Filamentous Fungus Cochliobolus lunatus

Katja Drobnic, Marko Vitas, Damjana Rozman, Robert van Gorcom* and Radovan Komel. Institute of Biochemistry, Medical Faculty, Vrazov trg 2, 61000 Ljubljana, Slovenia; * TNO Nutrition and Food Research, Department of Molecular Genetics and Gene Technology, 2280 HVRijswijk, The Netherlands

Steroid hydroxylations by filamentous fungi such as Cochliobolus lunatus, including 11 -hydroxylation, are economically important for the production of corticosteroids. According to CO and substrate binding spectra, the enzymes responsible for these reactions have been proposed to a family of cytochrome P450 monooxygenases as found in higer eucaryotes. The general unit of every microsomal cytochrome P450containing monooxygenase system is composed of two functional components- cyp P450 and cyp P450 reductase as electron-donor. Despite their importance little is known of their genes structure or genome organisation in fungi. We describe here the use of two DNA fragments which served as DNA probes for screening the genomic library in E coli, prepared in ZAPII vector. The first fragment was obtained by using a pair of oligonucleotide primers based on the conserved domain of the NADPH-cytochrome reductase (cprA) from Aspergillus niger. After PCR from C. lunatus genomic DNA, we amplified a 429 bp fragment which showed 74% amino acid homology with the corresponding cprA gene. The same approach was used in experiment looking for strain-specific cytochrome(s) P450. PCR was done with a pair of primers designed according to two structurally conserved domains as found in different cyp P450 from lower eucaryotes. We isolated a 273 bp fragment which showed 72% homology with the benzoate-para-hydroxylase gene from Aspergillus niger belonging to the cyp 53 family. DNA sequencing of the positive clones is in progress and the latest results will be presented.

Expression of a Synthetic Gene Encoding the Sweet-tasting Protein Thaumatin in Filamentous Fungi

Icinacio Faus1, Cristina Patino1, Jose Luis del Rio1, Catalina del Moral2 and Victor Rubio2. 1Laboratory of Biotechnology, URQUIMA,S.A. (URIACH GROUP),Arnau de Vilanova, 22-42, 08105 Sant Fost de Campsentelles(Barcelona), Spain.

2Centro Nacional de Biotecnologia (CSIC-UAM), Campus Canto Blanco, 28049 Madrid, Spain.

The thaumatins are proteins with a very sweet taste and the ability to increase the palatability (upgrading or improving other flavours) of food; in industry they are currently extracted from the arils of the fruit of the plant Thaumatoccocus daniellii Benth. Thaumatins I and II, which predominate in the arils and have very similar sequences of amino acids, are much sweeter than saccharose (100,000 times sweeter according to one estimate). Besides being natural products, thaumatins I and II are non-toxic, making them a good substitute for common sweeteners in the animal and human food industries.

In this study we prepared a synthetic gene encoding thaumatin II, and used it to transform filamentous fungi. The recombinant strains obtained secrete thaumatin II into the culture medium. The recombinant protein is sweet.

Analysis of Heterologous Protein Production in Defined Recombinant Aspergillus awamori Strains

Robin J. Gouka, Peter J. Punt, Johanna (H.) G. M. Hessing and Cees A.M.J.J. van den Hondel. TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 5815, 2280 HV Rijswijk, the Netherlands

Filamentous fungi, especially members of the genus Aspergillus, are able to secrete large amounts of homologous proteins into the medium which makes them attractive as a host for protein production. In contrast, heterologous proteins are very poorly produced and secreted.

The objective of our research is to obtain more insight in the parameters that influence heterologous protein production in Aspergillus awamori. To investigate this, a systematic analysis was carried out in which the expression levels of a number of different fungal and non-fungal genes were analyzed. This method is based on the single copy integration of different expression cassettes at the pyrG locus of A. awamori. Differences in expression mainly occured at the steady state mRNA level, varying from high mRNA levels for genes of fungal origin to low levels for genes of non-fungal origin. With one gene, encoding plant Cyamopsis tetragonoloba -galactosidase, no full length mRNA could be detected. With RT-PR and nuclear run-on transcription assays it could be demonstrated that incorrect processing of full length mRNA was probably occurring, resulting in the lack of about 900 nt in the mRNA. By changing the DNA sequence of the gene improved levels of full length mRNA could be obtained. In most cases the protein levels corresponded to the amount expected on basis of the mRNA levels. Only in the case of human interleukin-6, relatively high mRNA levels were obtained, whereas, only very low amounts of protein could be detected.To further investigate the problems observed for plant agalactosidase and human IL6, gene fusions with the A. niger glucoamylase gene (glaA) were constructed. Data on improved mRNA and protein levels will be presented.

Studies on The Production of Single-chain Antibody Fragments by Aspergillus Species

Hanny (J.)G..M. Hessing1, Leon G.J. Frenken2 Marian van Muijlwijk-Hartveld1, Wouter Musters2 and Cees A.M.J.J.

van den Hondel1. 1Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research,

PO Box 5815, 2280 HVRijswijk. 2 Unilever Research Laboratorium, Vlaardingen, The Netherlands.

Filamentous fungi, such as Aspergillus niger and Aspergillus awamori, are able to produce extracellularly significant amounts of homologous proteins. Furthermore, they have the capacity to secrete heterologous proteins although to a much lesser extent. We investigated their capacity for production of single-chain antibody (scFv-) fragments. These fragments comprise the variable fragment of an antibody heavy chain connected via a linker peptide to a variable fragment of a light chain. We studied a.o. the production of scFv-anti Hen egg white lysozyme [scFv-LYS; (1)] as a model. For expression of the scFv encoding sequence expression cassettes were made in which the gene to be expressed was flanked by efficient expression signals such as the A.niger glucoamylase promoter (2) or signals derived from the A.awamori 1,4-B-endoxylanase A gene (3). Transformants comprising multiple copies of the expression cassette produced up to 10 mgA scFvLYS in the presence of a signal sequence. However, when scFv-LYS was expressed as a fusion protein with glucoamylase - a protein which is well secreted by A. niger- production levels increased at least five-fold. To obtain mature scFv-LYS, a KEX2 cleavage site was inserted between the glucoamylase and the scF-v-1,YS sequences. More data on the production of scFv-LYS and on the production of other scFv-antibody fragments will be presented.

1. Ward S. et al; Nature 341 (1989) 544-546; 2. Verdoes J.C et al; J. of Biotechnology 36 (1994) 165-175

3. J.G.M. Hessing et al,- Curr. Genet. 26 (1994) 228-232

Isolation and Characterisation of PDI-family Genes from Asperglllus niger

David Jeenes, Celina Ngiam and David Archer. Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich, UK.

Filamentous fungi, such as Aspergillus niger, secrete high levels of native protein making them attractive hosts for the production of recombinant proteins. Secreted yields of heterologous protein are often significantly lower however. Data suggest that a major problem precluding high yields occurs during transit of heterologous proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, a step intimately associated with folding. The in vivo role of ER-specific chaperones and folding catalysts, such as protein disulfide isomerase (PDI), is poorly understood although their manipulation has increased yields of heterologous secreted proteins in Saccharomyces cerevisiae. In Vitro experiments show that foldases can often act synergistically to increases both the rate and the yield of folded end product. A family of ER-specific proteins which share active site homology with PDI has been identified from a variety of other systems. Several of these proteins are stress inducible, specifically by agents which cause malfolding of proteins in the ER. Here we report the isolation of two PDI-family genes from A.niger. Using a PCR based approach, redundant primer mixes designed against known ER retention signals and the PDI active site were used to isolate genomic and partial cDNA clones of a gene encoding a 359 aa protein. This ORF comprises a 19aa ER-targetting secretion signal and a 340 aa mature protein which includes a KDEL-type ER retention signal. It also contains two putative thiol oxidoreductase active sites with a CGHC motif, the most distal of which is created by excision of a 66bp intron. Both CT- and TATA-boxes can be distinguished in the putative promoter region and cDNA data from 2 independent clones confirm intron splice signals and define the site for polya tailing. Southern and dot blot analysis suggest the gene is present in single copy. Transcript sizing by northern analysis supports the postulated start codon. Analysis of the ORF reveals significant differences from PDI itself, the active sites are too closely spaced and there is no peptide binding site. Database searches have identified a stress-inducible homologue, GI protein, from alfalfa. Preliminary data suggest that overexpression of heterologous proteins do not induce this gene in A.niger however. Data on the effects of agents known to cause protein misfolding and a preliminary functional analysis will be reported. A second gene has been isolated using the S. cerevisiae PDI gene as probe. Further characterisation of this gene will also be presented.

Vacuolar Targeting in Aspergillus niger

Anders Kassow, Peter J. Punt and Cees A.M.J.J. van den Hondel. Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research, PO Box 5815, 2280 HV Rijswijk, The Netherlands.

The filamentous fungus Aspergillus niger, is able to secrete large amounts of proteins into the culture medium. When expressing heterologous genes, however, the production level is often much lower than for homologous proteins, even when the same expression signals are used. Incorrect folding of the proteins and intracellular degradation of these are likely to be one of the explanations for this. The fungal vacuole, which contains the protease machinery for such degradation, may well be involved in this proces. In the yeast Saccharomyces cerevisiae the targeting and function of the molecular machinery of the vacuole has been studied in detail, but hardly any information is available on this subject in filamentous fungi. We have now started research on vacuolar targeting in Aspergillus niger.

We are currently studying putative vacuolar targeting signals from A.niger genes encoding vacuolar proteases homologous to S.cerevisiae CarboxyPeptidase Y and Proteinase A. The functionality of these targeting signals are analysed by fusion to reporter proteins. In first instance we have chosen two homologous reporter genes encoding secreted proteins, -galactosidase A and Glucoamylase A. Biochemical data on the location of these reporter proteins in A.niger strains expressing the fusions will be presented.

Ultrastructural Studies on the Localization of the Secretory Pathway of Glucoamylase in Aspergillus niger

I.Keizer-Gunnink, P.J.Punt, M. Veenhuis and CA.M.J.J. v.d.Hondel. Laboratory for Electron Microscopy, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

We study the secretary pathway in hyphae of A. niger at the subcellular level using glucoamylase (GLA) as a model protein. In order to facilitate directed sectioning of the hyphae the cells were grown between two apposed dialysis membranes, layered on solid agar plates. After two days of growth under GLAinducing conditions the colonies were flat embedded and sections could be prepared from defined parts of the colony. Specific morphological differences were observed between cells from leading hyphae and sub-apical branching cells. Leading hyphae were highly elongated (5-10 um thick and over 200 um long). The apical zone (1-3 um) was crowded with various types of vesicles including 'Spitzenkorper"; the sub-apical part contained numerous mitochondria, microbodies, vesicles, ER and 'tubular vesicular complexes" (TVC), which are probably derived from the ER. In branching cells the apical zone was defined by the presence of vesicles. However, accumulation of vesicles and other subcellular organelles, as in apical cells, was not observed.

GLA protein was localized in these cells immunocytochemically, using monoclonal anti-GLA and secondary goat-anti-mouse antibodies coupled with 15 nm gold particles. In top cells of the leading hyphae, including the apical vesicles, labeling was never observed. Instead specific labeling was confined to the sub-apical cells and branches, including the top cells of the branches. The labeling was localized on endo-membrane structures, resembling ER and TVC, and also on the nuclear envelope. Plasma membranes, nuclei and miitochondria were not labeled. Control cells, grown at GLA-repressing conditions and cells of the glaa-deletion mutant lacked labeling, indicating that the labeling on the induced cells was specific.

The localization of the gold particles indicated that GLA is transported via an endo-membranous system, most probably derived/related to the ER. The secretion pathway of GLA will be further studied in depth using genetically constructed mutants which are blocked at different stages of GLA secretion.

Characterisation of 5' and 3' UTR Sequence of an Alpha-amylase Hyper-expressing Gene from Aspergillus oryzae

S.Khanapur1, S.Padmanabhan1, S.Suryanarayan2, G, Das1, 1Department of Molecular Biology, Syngene International Pvt. Ltd. 2Research and Development Division, Biocon India Limited, Bangalore, India.

The filamentous fungus, Aspergillus oryzae has received increased attention as a favorable host for the production of heterologous proteins because of its ability to secrete large amounts of proteins. We have an alpha-amylase hyper -expressing Aspergillus oryzae strain which has been obtained by routine strain improvement methods. In order to characterise the alpha-amylase gene of this strain, the 5'UTR (618bp) and 3'UTR (709bp) regions were PCR ampfified, cloned and sequenced, The 5'UTR sequence does not show any significant changes from the reported sequence. However, the 3'UTR sequence shows more than forty alterations, some of which could play a significant role in the stability of the mRNA. The significance of these changes is yet to be established. Currently, efforts are under way to develop an expression vector in Aspergillus oryzae utilizing these elements.

Mutagenesis and Transformation of Aspergillus terreus Based on the Nitrate Reductase Pathway

Eiza Ferraz, Nelson Lima and Manuel Mota. Centro de Engenharia Biologica, Universidade do Minho, Largo do Paco, 4709 Braga Codex, Portugal

Aspergillus terreus is an efficient producer of different extracellular enzymes which are used in food industries. Thus, we are interested in developping a genetic transformation system based on the nitrate structural gene niaD. In order to develop this system the nitrate assimilation pathway in A. terreus was studied and spontaneous mutants defective in the genes required for nitrate assimilation were obtained on the basis of chlorate resistance. Of particular interest were niaD mutants which failed to grow on nitrate but grew as wild type on other sole nitrogen sources.

Afterwards the transformation system was developed for mutants defective in nitrate reductase of A. terreus using pSTA10 vector containing niaD gene from A. niger. The transformation frequency obtained was c.a. 0.5 per ug DNA. The vector appeared to be mitotically stable and Southern hybridization analysis of transformants showed that transformation events occurred by integration into to the recipient genome. To assess the amount of product made by the transformants, nitrate reductase assays were carried out under inducing conditions.

Experiments to improved transformation frequency and to characterize the integration are now in progress.

Regulation of Recombinant Protein Production in Saccharomyces cerevisiae by the Adhl Promoter

Robert Weik1, Gerald Striedner1, Lars Tollner1, Andrej Francky2, Peter Raspor 2 , Karl Bayer1 and Diethard Mattanovich1.

1Institut fur Angewandte Mikrobiologie, Universitat fur Bodenkultur, Nu dorfer Lande 1 1, A-1 1 90 Vienna, Austria

2Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia

We have cloned alliin lyase from garlic (Allium sativum) and expressed it in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. This enzyme catalyzes the degradation of sulphurous amino acids, mainly alliin, resulting in the characteristic odour of garlic and other Allium species. Several physiological effects are linked to the product allicin, among them antimicrobial action, lowering of cholesterol levels and increase of fibrinolytic activity. Significant levels of expression could be detected in all three systems.

In S. cerevisiae, the gene for alliin lyase was set under control of the alcohol dehydrogenase 1 (ADH 1) promoter. Transcription from this promoter has been described to be enhanced by glucose and decreased by ethanol. Therefore the regulation of product formation will be closely interrelated with the cultivation strategy, namely carbon source supply.

Results will be presented concerning the optimization of the feeding strategy to harmonize supply with carbon source, aerobic ethanol formation and regulation of product formation.

Expression of Human Insulin Gene in Aspergillus

Silvija Mestric1 , Peter J. Punt2, Radovan Valinger1 and Cees A.M.J.J. van den Hondel2. IPLIVA Research Institute, 10000 Zagreb, Prilaz baruna Filipovica 25, Croatia. 2 Department of Molecular Genetics and Gene-technology, TNO Nutrition and Food Research Institute, PO Box 5815, 2280 HV Rijswijk, The Netherlands.

The latest data on hcterologous gene expression in fungi indicate that fungi express and sccrete significant amounts of heterologous protein under appropriate culture conditions, e.g. (1). This encouraged us to start our research on human insulin production in Aspergillus. The first aim of our work was to synthesize the human proinsulin gene and to clone it in fungal expression vector. Various mutations were introduced in the sequence of human proinsulin gene to study the (hopefully) positive effects of these mutations on protein secretion.

The human proinsulin gene was synthesized by PCR based on overlapping, complementary oligonucleotides used as templates and primers at the same time. The synthesis was performed in fragments of approximately 100-200 bp, which were joined together in pUC19 resulting in the following proinsulin genes: 1) wt proinsulin, using codons favored for efficient expression in A. niger; 2) proinsulin with a C-chain carrving a N-glycosylation consensus; 3) proinsulin from which most of the C-chain is deleted and 4) proinsulin from which the complete C-chain is deleted. Different genes were cloned into fungal expression vectors based on either the glaA promoter and signal sequence or the glaA promoter and the entire glucoamylase coding region (as secretion carrier). The various insulin expression vectors were introduced in A. niger and insulin activity was determined in culture supernatants of obtained transformants.

(1)M. P. Brockhuijsen et al (1993) J. Blotechnol. 31: 135-145.

Molecular Approaches Against Mushroom Browning

Anna Moller, Miranda van de Rhee, Odette Mendes, Marc Werten, Dorine Donkers, Harry Wichers and Hans Mooibroek

Agrotechnological Research Institute ATO-DLO, P.O.Box 17, 6700 AA Wageningen, The Netherlands

Browning is an important aspect of common mushroom (Agaricus bisporus) quality loss, which is caused by mechanical injury, Pseudomonas tolaasii infection and by senescense. Brown discolouration has been associated with the transition of latent to active tyrosinase, possibly mediated by serine proteinase. Tyrosinase catalyses the oxidation of phenolic substrates mainly L-tyrosinase, p-amino-phenol (pAP) and -glutamylhydroxy benzene (GHB) to melanins.

Mushroom tyrosinase has been isolated before and was characterized as a monomeric protein of 43 kDa under denaturing conditions and 47 kDa under native conditions.

Work was carried out to clarify which one of the following parameters: tyrosinase activity, phenolic substrate content or protease activity has the highest correlation with the colour and discolouration of mushrooms. Preliminary statistical analyses show that tyrosinase activity significantly contributes to the browning reaction.

Two partial tyrosinase cDNA sequences (600 hp) from A. bisporus encompassing the CuA and CuB binding domains of the enzyme have been isolated by PCR. The fragments (Abtyr1 and Abtyr2) have been included in transformation constructs to attempt gene disruption or antisense inhibition. Transformation is based on hygromycin B resistance (E.coli hpt gene) driven by the A. bisporus gpd2-promoter (van de Rhee et al. in press).

Over a hundred putative co-transfonnants have been produced and are being analysed at the level of the homokaryotic transgenic mycelium, heterokaryotic mycelium (after mating) and fruitbodies.

Results will be presented on the analysis of the transformants and on the possible effect on browning phenomena.

Kinetics of Protein Secretion in Trichoderma reesei

Tiina M. Pakula, Katri Salonen, Jaana Uusitalo, Markku Saloheimo, Robert Aarts, and Merja Penttila. VTT, Biotechnology and Food Research, P.O. Box 1500, FIN-02044 V7T, Finland

In order to obtain knowledge of the protein secretion process in filamentous fungi, and to study the rate-limiting steps in protein production, we have developed methodology for measuring the kinetics of protein production and secretion by metabolic labelling in Trichoderma. The synthesis of the 35S- labelled proteins and their transport into the growth medium was monitored by 2D gel electrophoresis and/or immunoprecipitation. The major cellulose of T. reesei, cellobiohydrolase I (CBHI), was used as a model protein in the studies. To study the protein production kinetics in a controlled physiological state, a chemostat cultivation system was set up for the fungus. In the chemostat cultivations, the intracellular production rate of labelled CBHI and the accumulation rate of the protein into the culture medium was measured at different dilution rates, and the corresponding specific steady-state mRNA levels were determined. With this methodology we have been able to follow the time course of protein synthesis, glycosylation and transport into the medium, and obtain information on the synthesis vs. secretion capacity of the fungus in relation to growth rate.

Investigation of the Genetic System of the Xylose Fermenting Yeast Pichia stipitis

Tsega Melake, Volkmar Passoth and Ulrich Klinner. Lehr und Forschungsgebiet Angewandte Mikrobiologie, RWTH Aachen, Germany

There is only poor knowledge on genetics of the xylose fermenting yeast P stipitis. We investigated the life cycle and some aspects of extrachromosomal inheritance of this yeast. Mutant frequencies indicated that wild type strains of P stipitis are haploid. Stable diploids could be obtained by protoplast fusion or sexual hybridization. Sexual mating was induced by a shift from complete medium to nutritionally poorer conditions. Conjugation was followed immediately by sporulation. Mitotically stable diploids could be isolated by transferring the nascent zygotes to complete medium before meiosis had started. Segregation patterns of auxotrophic markers indicated an ordered meiosis although asci contained in most cases only dyads. Further, we describe isolation and characterization of presumptive mitochondrial mutants in this petite negative yeast.

Characterization of a Protease Deficient Mutant of Penicillium roqueforti Generated by Heterologous Transformation

Nathalie Poussereau and Michel Fevre. Laboratoire de Biologie Cellulaire Fongique CNRS UMR 106. Universite Claude Bernard. Bat 405, 43 Boulevard du II Novembre 1918. 69622 Villeurbanne. France

Filamentous fungi are identified as potentially excellent hosts for the expression of recombinant proteins of interest To date, an increasing range of proteins from a variety of fungal and non fungal origins have been produced in fungi. However, the product yields are low in comparison with the yields of the native proteins. Among different factors affecting heterologous protein production i.e. vector construction, transformation, gene expression, glycosylalion and secretion, degradation of recombinant proteins by native proteases can be overcomed. We have characterized a Penicillium protease deficient strain following insertionnal mutagenesis . A strain of Penicillium roqueforti was transformed with a plasmid which confers resistance to phleomycin. Stable transformants exhibiting high resistance to the antibiotic were selected. One transformant was isolated as a proteolytic deficient mutant unable to degrade casein. The extracellular proteins profile of the strain reveals the absence of a 43 kDa polypeptide corresponding probably to the aspartyl protease of Penicillium roqueforti. The mutant is characterized by a tandem integration of the transformant vector in one site of the genome. The asp A gene which encodes aspartyl protease of Penicillium roqueforti is not expressed in the transformant and Southern analyses show that the asp A gene is not disrupted by the transformation vector.

Heterologous protein degradation was tested and demonstrated that proteins from, different origins were not degraded by the culture filtrate of the protease deficient mutant contrarly to the wild type. Result anticipate that this strain will be adequate for heterologous expression of most proteins.

Towards an Analysis System of the Secretion Pathway of Aspergillus niger

Peter. J. Punt, Gerrit Veldhuisen, Anneke Kuijvenhoven and Cees A.M.J.J. van den Hondel. Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research, PO Box 5815, 2280 HV Rijswijk, The Netherlands.

Filamentous fungi, such as Aspergillus niger, are able to secrete large amounts of proteins into the culture medium (1). However, hardly any information is available on the molecular mechanisms of the process of protein secretion. Therefore, two lines of research have been initiated towards a systematic analysis of the mechanism of protein targeting and secretion by A.niger (2).

I To identify the different compartments of the secretion pathway and to study the routing of proteins to these compartments, several genes encoding compartment-specific proteins have been isolated. The cloning and characterisation of the A. niger bipA gene will be presented together with initial results aimed at the analysis of the BipA protein and its targeting using specific antibodies.

II Based on research carried out in Saccharomyces cerevisae, defined (conditional) mutations in the secretion pathway will provide important information about the Organisation of the pathway. Therefore, the cloning of several secretion genes has been started. We have focused on the isolation of various genes encoding GTP-binding proteins involved in the vesicle transport between the different compartments of' the secretion pathway, using heterologous hybridisation with cloned S. pombe genes as probes. The successful cloning of the SAR1-homologous A. niger sarA gene and initial resulted on A. niger sarA mutants will be presented.

1. Verdoes J C et al (1995) Appl Microbiol Biotechnol 43:195-205

2. Punt PJ et al (1994) Anthony van Leeuwenhoek 65:211-216

Heterologous Gene Expression and Secretion of a Eukaryotic Protein by Acremonium chrysogenum

Renate Radzio and Ulrich Kuck. Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat Bochum, 44780 Bochum, Germany

Filamentous fungi are frequently used as host organisms for the expression of heterologous genes or for the overproduction of homologous proteins. One major advantage is their ability to secrete large quantities of protein, which facilitates the purification procedure of the protein of interest (for a review, see [1]).

Acremonium chrysogenum is an industrially important producer of the -lactam antibiotic cephalosporin C, for which fermentation technology has already been established. Therefore it is a favourable host for the production of heterologous proteins.

We have developed a system allowing the production and secretion of heterologous proteins by A. chrysogenum. The expression is controlled by the promoter of the homologous pcbC gene that encodes isopenicillin N synthetase, a key enzyme of the cephalosporin biosynthesis. This promoter, which has previously been shown to mediate a relatively high level of expression [2], was fused to a gene encoding a small eukaryotic coagulation inhibitor. In order to achieve the secretion of this heterologous protein, we used two different signal sequences from Fusarium and from A. chrysogenum, respectively. Transformation of these expression vectors into A. chrysogenum resulted in the efficient secretion of the heterologous protein.

[1] van den Hondel CAMJJ, Punt PJ, van Gorcom RFM (1991) in: Bennett JW, Lasure LL (eds), More gene manipulations in fungi, Academic Press, San Diego CA, pp 396 - 428

[2] Menne S, Waiz, M, Kilck U (1994) Appi Microbiol Biotechnol 42: 57-66

Characterization of the SAR1 Gene from the Filamentous Fungus Trichoderma reesei

Saloheimo, M1, Punt, P.J.2, van den Hondel, C.A.M.J.J.2 and Penttila, M.1. 1VTT Biotechnology and Food Research, P.O.Box 1500, 02044 VTT, Finland. 2TNO Nutrition and Food Research Institute, P.O.Box 5815, 2280 Rijswijk, The Netherlands

Filamentous fungi have a long history in production of extracellular enzymes and are well known for their high capacity in protein secretion. The largest reported amount of secreted protein from the cellulolytic fungus Trichoderina reesei is about 40 g/l. Despite the above, the protein secretion machineries of filamentous fungi are poorly known. As one of the first studies in this field, we report here the characterization of the sarl gene from Trichoderma reesei.

The Saccharomyces cerevisiae SAR1 gene product is a small GTP-binding protein involved in the ER to Golgi step of protein secretion. The Sar 1p is thought to be regulating the budding of vesicles from the endoplasmic reticulum. The corresponding gene, sarA, has been isolated from the fungus Aspergillus niger (1). Using this gene as a probe, we isolated the cDNA and chromosomal copies of sarl gene of Trichoderma reesei. The Trichoderma sequence shows 72 % conservation with yeast SAR1 and 86 % conservation with Aspergillus sarA at the amino acid level. The regions responsible for GTP binding are well conserved. The exon-intron structure of Trichoderma sarl shows some divergence when compared to that of Aspergillus sarA. Complementation experiments with yeast sarl mutants will be


(I)Punt, P.J., Veldhulsen, G. and van den Hondel, C.A.M.J.J., 1994. Antonie van Leeuwenhoek 65, 211-216

Cloning of the Isocitrate Lyase Gene of Ashbya gossypii by Saccharomyces cerevisiae Complementation

K.-Peter Stahmann. Institute of Biotechnology, Research Center Julich GmbH, 52428 Julich, Germany

Ashbya gossypii is a filamentous hemiascomycete used for industrial riboflavin production. Growth experiments on glucose or soybean oil as carbon source revealed a correlation of isocitrate lyase (ICL) specific activity and riboflavin formation. Mutants resistant to a specific ICL inhibitor showed an increased ICL specific activity and an enhanced riboflavin production1. To investigate the role of ICL in riboflavin production on molecular level the respective gene was cloned. For this purpose a plasmid library of genomic A. gossypii DNA was constructed with YEp 352, a yeast/E. coli shuttle vector, and used to transform S. cerevisiae icl1 d ura3 (fs)2 which is not able to grow on ethanol. Colonies obtained by selection for ura3 complementation were replica-plated on minimal medium with ethanol as sole carbon source. Two out of 3000 clones showed the expected phenotype i.e. grew on ethanol. Curing from the plasmid, retransformation, and detection of ICL activity indicated that the ICL-gene is located on a 2.9 kb Sphl fragment. Sequencing revealed an open reading frame of 1680 bp showing 65 % identity to the ICL1-gene of S. cerevisiae and 75 % identity to the predicted amino acid sequence. The conserved hexapeptide KKCGHM, possibly confined to the catalytic domain, as well as the C-terminal tripeptide AKL, described as targeting signal for peroxisomes, were found.

1) Schmidt G, Stahmann KP, Kaesler B and Sahm H (1996)Correlation of isocitrate tyase activity and riboflavin formation

inthe riboflavin overproducer Ashbya gossypii, Microbiology, in press

2)Fernandez E, Moreno F, Rodicio R (1992) The ICL1 gene of Saccharomyces cerevisiae, Eur. J. Biochem. 204, 983-990

Cloning of Specific Genes of the Gibberellin Pathway from Gibberella fujikuroi

Veronika Homann, Katrin Mende, Solveig Woitek, Pia Linnemannstons and Bettina Tudzynski. Westfilische Wilheims-Universitat, Institut fur Botanik, Schlossgarten 3, 48149 Munster, Germany

The gibberellins are a group of natural plant hormones with various effects on growth and differentiation of plants. The rice pathogen Gibberella fujikuroi accumulates large amounts of Giberellic acid ( GA3 ) and some other gibberellins which induce the superelongation disease on infected rice seedlings. The biosynthetic pathway for gibberellins has been established from the identification of intermediates and by using mutants affected in the gibberellin biosynthesis. However, genetics of gibberellin formation and the relationship to the biosynthetic pathway in higher plants are not well understood until now. Therefore, we have started to isolate and characterize some of the versatile genes of the central isoprenoid pathway which are involved in the biosynthesis of sterols, carotenoids and gibberellins.

So far, the genes coding for HMG-CoA-reductase, farnesylpyrophosphate synthetase and geranylgeranylpyrophosphate synthetase were isolated by screening genomic and expression libraries of this fungus with heterologous probes or with PCR-fragments synthesized on the basis of sequence homologies to other fungal prenyltransferases. The sequence comparison with analogous genes of other fungi and plants showed that the pathway must be highly conserved.All the three genes isolated from Gibberella fujikuroi were found as single copy genes which are not linked to each other.

Beside those genes from the central terpenoid pathway, we are trying to isolate the kaurene synthetase gene and the C20-oxidase gene from the gibberellin-specific part of the pathway using the recently isolated corresponding plant genes ( Sun and Kamiya,1994; Lange et al.,1994) as heterologous probes and designed specific PCR-primers on the basis of sequence comparison between these plant genes in order to amplify parts of the corresponding Gibberelia genes.Additionally, REMI mutagenesis was used to isolate tagged gibberellin-defective mutants.

1.Sun,T. and Kamiya,Y. (I 994) Plant Cell 6,1509-1518.

2.Lange,T.,Heden,P. and Graebe,J.E. (1994) Proc.Natl. Acad Sci. USA 91:8552-8556.

Heterologous Expression ofthe Fusarium solani pisi cutinase gene in Aspergillus awamori

I.A. van Gemeren1 , A. Beijersbergen2, W. Musters2, R. Gouka3, C.A.M.J.J. van den Hondel3 , C.T. Verrips1,2. 1Dept. of Molecular and Cellular Biology, University of Utrecht. NL. 2Unilever Research Laboratory, Vlaardingen, NL, 3TNO Nutrition and Food Research, Rijswijk, NL

Filamentous fungi are used as hosts for the commercial production of heterologous proteins because they have an enormous capacity for secretion. The production of many hetcrologous proteins are, however, rather low compared with the high levels which are obtained with homologous proteins such as glucoamylase. This study hits been started to sort out the bottlenecks in the production of heterolagous proteins, and subsequently to identify the factors, involved in these ratelimiting pathways. Therefore, the effect of different pro- and presequences and the presence of multiple copies of the gene on the production of heterologous proteins is studied. The cutinase protein from Fusarium solani pisi is used as a model for the production of heterologous proteins.

A synthetic copy of the cutinase cDNA was constructed and expressed under the control of the endoxylanase II expression signals from A. awamori. Four different constructs were used to test the effect of different pre- and pro-sequences. A single copy of these expression cassettes was integrated at the pyrG locus of A. awamori. Shake flask induction experiments revealed that the pre-sequences used were equally efficient in the production of extracellular cutinase. The absence of a pro-sequence, howcver, resulted in a two-fold increase in extracellular cutinase. To study the effect of multi copy gene expression, one of the constructs was integrated in multiple copies into the genome of A.awamori. There was no linear correlation between copy number and extracellular cutinase production, but the amount of active enzyme produced correlated with the level of cutinase specific mRNA. These data and the fact that a relatively small amount of cutinase was found inside The cell suggest that there is no limitation in the secretion of this protein by A.awamori.

Improvement of Cytochrome P450 Activities in Aspergillus niger

Hans (J.) M. van den Brink1, Hans (J.) G.M. van Nistelrooy2 , Maarten A. de Waard2, Cees A.M.J.J. van den Hondel1 and Robert FM. van Gorcom1. 1TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene technology, PO Box 5815, NL-2280 HV Rijswijk, The Netherlands. 2Department of Phytopathology, Wageningen Agricultural University, PO Box 8025, NL-6700 EE Wageningen, The Netherlands.

Recently the gene encoding cytochrome P450 reductase (cprA) was cloned from the filamentous fungus A. niger. CPR functions as an electron donor for many different cytochrome P450 enzymes and is thus essential for P450 activity.

We used the cprA gene to generate A. niger strains with increased cytochrome P450 activities. For this purpose, strains were constructed which contain multiple copies of the cprA gene, multiple copies of a cytochrome P450 encoding gene or multiple copies of both genes. T\vo fungal cytochrome P450 encoding genes were used in this study: the A. niger bpha (cyp53) gene, encoding benzoate para-hydroxylase, and the P. italicum cyp5l gene, encoding eburicol 14 -demethylase.

For both sets of transformants it was found that the highest cytochrome P450 activity was detected in strains which contained multiple copies of both genes. Increasing the copy number of only one of the components of the enzyme system (the P450 encoding gene or the cprA gene) resulted in a significant, but much smaller increase in cytochrome P450 activities as compared to wildtype.

These results show that for an optimal increase of cytochrome P450 activity in filamentous fungi simultaneous overexpression of CPR and of the cytochrome P450 is needed.

Transport Steps Involved in Penicillin Biosynthesis by Penicillium chrysogenum

Mart van de Kamp. Dirk J. Hillenga, Hanneke J.M. Versantvoort, Arnold J.M. Driessen and Wil N. Konings, Department of Molecular Microbiology, University of Groningen and Groningen Biomolecular Sciences and Biotechnology Institute, Biological Centre, Kerklaan 30, NL-9751 NN Haren, The Netherlands.

The filamentous fungus Penicillium chrysogenum is well known for its ability to produce penicillin. The biosynthesis of penicillin involves three steps which are catalyzed by three different enzymes, -aminoadipyl-cysteinylvaline synthetase (ACVS), isopenicillin N synthetase (IPNS), and isopenicillin N acyltransferase (AT) (1). Evidence exists that the activity of the different enzymes is confined to specific intracellular compartments (2-5). This sublocalisation, together with the import of precursor molecules and the secretion of the produced penicillin, imposes the need for several active and/or passive transport steps.

Recently, we reported the isolation and characterisation of plasma membranes obtained from low- and high-producing strains of P. chrysogenum (6). Subsequent studies showed that the uptake of the penicillin G side-chain precursor phenylacetic acid across the plasma membrane occurs by passive diffusion (7). Further studies are being directed at transport steps across the vacuolar membrane. The vacuole has been implicated to play various roles, e.g. as a storage compartment for (basic) amino acids and/or as the compartment where the first reaction of the penicillin biosynthesis, i.e. the formation of the tripeptide a-aminoadipyl-cysteinyl-valine by the enzyme ACVS, takes place (5). First results from experiments that are eventually aimed at elucidating the role of the vacuole in penicillin biosynthesis will be presented.

1) Martin JF and Gutierrez S (1995) Ant van Leeuwenhoek 67, 181-200. 2) Kurzatkowski W et al (1991) Appl Microbiol Biotechnol 35, 517-520. 3) Muller WH et al (1991) EMBO J 10, 489-495. 4) Muller WH et al (1992) Biochim Biophys Acta 11 16, 210-213. 5) Lendenfeld T et al (1993) J Biol Chem 268, 665-67 1. 6) Hillenga DJ et al (1994) Eur J Biochem 224, 581-587. 7) Hillenga DJ et al (1995) Appl Environm Microbiol 61, 2589-2595.

Development of a Virus-resistant Strain of Agaricus bisporus

Ted R. van der Lende and Joseph G. H. Wessels. Dept of Plant Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands.

Fruit bodies of Agaricus bisporus affected by La France disease contain a set of nine unique dsRNA molecules (Harmsen et al., 1989). In addition to the nucleic acid sequences of the L3, M1 and M2 dsRNAs determined by Harmsen et al. (1991), the sequences of the L1 and L5 dsRNAs have now been detemined (Van der Lende et al., 1996; in press). These dsRNAs are contained in 34 nm virus particles that also contain at least three immunologically unrelated proteins of 120, 115, and 90 kDa (Van der Lende et al., 1994). The virion-associated protein of 115 kDa proved to be the RNA-dependent RNA polymerase encoded by the L1 dsRNA, whereas the virion-associated protein of 90 kDa is encoded by the L3 dsRNA. In vitro translation experiments of the individual dsRNA segments indicate that L2 dsRNA encodes the protein of 120 kDa. Antibodies raised against a protein encoded by M2 dsRNA, reacted with a protein present in diseased fruit bodies but not with any of the virion-associated proteins (Van der Lende et al., 1996; in press). A number of essential virus genes have now been characterised. Current research is directed towards the development a virus resistant strain of Agaricus bisporus using pathogen-derived-resistance (PDR). For this purpose the development of an efficient transformation protocol for commercial strains is in progress. In addition, different expression vectors are being developed in order to express viral sense RNAs and antisense RNAs and/or viral proteins in Agaricus which may inhibit viral replication as has been shown for virus-resistant transgenic plants.

Harrnsen, M.C., Van Griensven, L.J.L.D. & Wessels, J.G.H. (1989). Journal of General Virology 70, 1613-1616.

Harrnsen, M.C., Tolner, B., Kram, A., Go, S.J., De Haan, A. & Wessels, J.G.H. (1991). Current Genetics 20, 137-144.

Van der Lende, T.R., Harinsen, M.C. & Wessels, J.G.H. (1994). Journal of General Virology 75, 2533-2536.

Van der Lende, T. R., Duitman, E. H., Gunnewijk, M. G. H., Li, Y. & Wessels, J.G.H. (1996). Virology (in press).

Secretion of Mammalian Glycosyltransferases by Aspergillus

Katherine Kodama, Enrique Baliu, Anita van Kimmenade, Scott Power, Jody Schultz* Mark Williams* Michael Ward

Genencor International, 180 Kimball Way, South San Francisco, CA 94114, USA and * Cytel Corporation, 3525 John Hopkins Court, San Diego, CA 92121, USA.

We have investigated the production and secretion of mammalian glycosyltransferases by Aspergillus niger. These enzymes are involved in the glycosylation of proteins and are normally located within the secretary apparatus of the cell. They typically possess a transmembrane anchor sequence which serves to localize them on the membrane. Recently, there has been interest in the use of these enzymes for the enzymatic synthesis of therapeutic complex carbohydrates. However, production of these enzymes at commercially viable levels has been difficult. We tested secretion of rat alpha 2,3 sialyltransferase (ST) in A. niger by production of a fusion protein between native glucoamylase and a truncated form of ST (without the transmembrane domain). A KEX2 cleavage site was optionally incorporated between glucoamylase and ST. Either full-length or truncated glucoamylase (lacking the starch binding domain) was employed. Initial yields were low and inconsistent and ST purification was not possible due to interaction with media components. However, media optimization gave increased and consistent yields and allowed ST purification. Yields of 1700 U/L (85 mg/L) were obtained with A. niger compared to 7 and 0.2 U/L obtained from Baculovirus and mammalian cell expression systems respectively. A similar strategy also allowed a much higher level of expression of human alpha 1,3 fucosyltransferase (FIF) in A. niger compared to the Baculovirus and mammalian cell systems. The yield and quality of both ST and FT produced in A. niger are sufficient to allow their commercial use in synthesis of complex carbohydrates.

Trehalose-6-phosphate Synthetase Activity of Aspergillus niger Is Encoded by Two Differentially Exxpressed Genes

Markus F. Wolschek and Christian P. Kubicek. Abteilung fur Mikrobielle Biochemie, Institut fur Biochemische Technologie und Mikrobiologie, TU Wien, A-1060 Wien, Austria

Aspergillus niger is of major industrial importance because of its production of several hydrolytic enzymes as well as organic acids. All these processes are strongly influenced by the carbon source supplied. In order to investigate a possible glucose signalling mechanism in A. niger we have cloned the homologue of the yeast GGS1/TPS1 gene, which encodes a trehalose-6-phospate synthase. The GGS1 gene product has been reported to be involved in the transduction of several glucose dependent regulatory phenomena in Saccharomyces cerevisiae and Kluyveromyces lactis.

The A. niger ggsA gene contains four introns and its deduced aa-sequence shows 65.2 % and 64.5 % identity to the corresponding gene products of S cerevisiae and K. lactis, respectively. Northern analysis demonstrated that its expression is growth correlated. Neither gene disruption nor gene amplification had a significant effect on the glycolytic flow in A. niger. Disruption of ggsA gene reduced most of the trehalose-6-phosphate synthase (TPS)activity of growing mycelia, whereas mycelia exposed to heat shock still exhibited high TPS-activity, suggesting the existence of at least one further isoenzyme. Using PCR, we amplified a genomic fragment, whose nt-sequence showed areas of high similarity to ggsA, yet is clearly distinct and therefore encodes ggsb. Its nt-sequence and expression pattern are currently investigated.

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