Industrial Biology and Biotechnology

180 Agrobacterium tumefaciens-mediated expression of cry genes in Trichoderma hazianum. Hoang Quoc-Khanh, Cao Cuong and Le Tan Duc. Institute of Tropical Biology, Microbiology, Ho Chi Minh City, VN, Vietnam

Agrobacterium tumefaciens transfers a part of its Ti plasmid, the T-DNA to plant cells during tumorigenesis. Not restricted to plant species, A. tumefaciens is able to transfer T-DNA to yeasts and filamentous fungi. A highly efficient and reproducible procedure for the transformation of the antagonistic Trichoderma harzianum by A.tumefaciens-mediated homologous recombination is reported. Transformation were carried out with plasmids carrying cry genes, which encode Bacillus thuringiensis insecticidal crystal proteins.

181Heterologous protein production in Aspergillus niger. Jesper Mogensen, Bettina Andreasen, and Hans van den Brink. Chr. Hansen A/S, Genetics, Hørsholm, Denmark

Chr. Hansen uses Aspergillus niger to produce bovine chymosin. To get more insight in the processes involved in chymosin secretion we decided to use GFP as a tool. Two constructs were made, both using the glucoamylase (glaA) promoter. In the first construct the gfp gene was directly fused to the glaA promoter, resulting in cytoplasmic localisation of GFP. In the second construct a glaA-gfp fusion gene, resembling the expression cassette used for chymosin production, was made. Both constructs were transformed to a chymosin producing A. niger strain and compared to a laboratory strain (AB4.1) carrying similar constructs. Studying these strains we made two interesting observations, which will be discussed on our poster; 1. In a single hyphal tip both active and non active compartments can be found 2. GFP is liberated from the glucoamylase carrier intracellularly even though no processing site is present in the construct.

182 Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori. B. Christien Lokman, Vivi Joosten, Robin J. Gouka, Ernst-Jan Rutjes, Jacqueline Hovenkamp, and Cees A.M.J.J. van den Hondel. TNO Voeding, P.O. Box 360, 3700 AJ Zeist, the Netherlands.

Peroxidases are produced by a wide variety of organisms including plants, animals, and microorganisms. The specificity and biological functions of peroxidase vary with the source of the enzyme. Arthromyces ramosus peroxidase (ARP) is a 41 kD monomeric glycoprotein containing one mole of protoheme IX per mole of enzyme as a prosthetic group. ARP has a broad specificity for phenolic and anilinic hydrogen donors which makes the enzyme interesting for industrial applications. Until now, the heterologous expression of peroxidases has been studied only with limited success. In these studies heme availability has been suggested to be a possible bottleneck for the overproduction. In our study we analysed the expression of the gene encoding ARP in the filamentous fungus Aspergillus awamori. Under control of the endoxylanase promoter secretion of active ARP was achieved up to 0.6 g/L in shake flask cultures. Westernblot analysis showed an rARP product of the correct size. Furthermore, our results suggest that there is no heme limitation during overproduction of ARP in A. awamori.

183 Cloning and expression of the cyanide-insensitive alternative oxidase gene ( aox1) from a citric acid-producing strain Aspergillus niger WU-2223L. Masashi Yoda, Kohtaro Kirimura, Kiyotake Kamigaki, Kuniki Kino, and Shoji Usami. Department of Applied Chemistry, School of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo, 169-8555, Japan.

In Aspergillus niger WU-2223L, a cyanide (CN)- and antimycin A- insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists and is catalyzed by the alternative oxidase (AOX), besides the CN- sensitive cytochrome pathway. Both the CN-insensitive and SHAM-sensitive respiration and the activity of AOX, as duroquinol oxidase activity, were shown to be localized in mitochondria. Such respiration and specific activity in purified mitochondria increased by addition of methanol, an inducer of citric acid production, or antimycin A, both accompanying the increase of citric acid productivity. On the other hand, when WU-2223L was cultivated with SHAM, the AOX activity decreased accompanying the decrease of citric acid production drastically although mycelial growth was not affected. The complementary DNA (cDNA) and chromosomal DNA encoding the AOX were cloned. One full-length cDNA was obtained and sequenced to reveal that the clone contained an ORF encoding a polypeptide of 351 amino acid. The deduced amino acid sequences revealed that there are two long hydrophobic regions regarded as membrane-spanning regions of the protein and two iron-binding motifs as a reactive center. When the whole ORF was introduced and expressed in Escherichia coli, the transformant harboring pKAOX1 containing the ORF gene showed cyanide-insensitive and SHAM- sensitive respiration, and the expression was induced to two folds by addition of IPTG. The chromosomal DNA encoding AOX gene (aox1) was also cloned from a chromosomal DNA library of A. niger and a 2856 bp-long DNA fragment was isolated. The aox1 contains two introns, and two TATA boxes in untranslated regions. Southern hybridization analysis with the cDNA and aox1 as probes revealed that there is only one copy of aox1 in the chromosome of A. niger WU-2223L. The motifs for carbon catabolite repressor (CREA) and two nitrogen metabolite repressor (AREA) binding sites were also found in upstream region of the ORF, suggesting that the carbon catabolism and nitrogen metabolism regulation might be involved in the transcription of aox1. The Northern blot analysis was done on the total RNA extracted from the mycelia cultivated under citric acid producing-conditions with 2% methanol (v/v). The transcription activity was highest for the mycelia cultivated for 2 days, the lowest for 4 days, and maintained at almost constant level through 6 to 8 days, and the correlation between the mRNA levels and AOX activities was observed.

184 MstA: an Aspergillus niger high affinity transporter of glucose and mannose. Patricia A. vanKuyk¹, Jasper Diderich³, Andrew P. MacCabe², Oscar Hererro², George J. G. Ruiter¹, and Jaap Visser¹. ¹ Molecular Genetics of Industrial Microorganisms. Wageningen University. Dreijenlaan Nl-6703 HA Wageningen. The Netherlands. ² Consejo Superior de Investigaciones Cientificas. Instituto de Agroquimica y Tecnologica de Alimentos. P.O. Box 73. Burjassot. Valencia 46100. Spain. ³ Swammerdam Institute for Life Sciences. BioCentrum. University of Amsterdam. Plantage Muidergracht 12. NL-1018 TV Amsterdam. The Netherlands.

A gene encoding a putative sugar transporter was cloned from an A. niger gDNA library using the A. nidulans putative sugar transporter, mstA, as a probe. The gene identified has been designated mstA_{(A. niger)}. The derived amino acid sequence of MstA has the highest similarity to the amino acid sequence of Amantia muscaria MST-1, as well as to a number of yeast sugar sensors including Saccaromyces cerevisiae Snf3. mstA is expressed under conditions of carbon starvation indicating that this gene is subject to carbon catabolite repression. Expression levels of mstA in a wildtype strain are influenced by extracellular pH. A full length cDNA clone of A. niger mstA has been isolated and sequenced. This cDNA has been inserted into a shuttle vector in which the expression of mstA is under the control of a copper inducible promoter that is functional in S. cerevisiae. After transformation into a S. cerevisiae strain in which a number of sugar transporters have been disrupted (hxt1delta-hxt7delta gal2delta) a copper inducible partial restoration of growth was observed when glucose, fructose, or mannose is the sole carbon source. These results imply that MstA_{(A. niger)} is able to transport glucose, fructose, and mannose. Uptake experiments using ¹⁴C labelled sugars as the substrate for the yeast strain expressing mstA_{(A. niger)} confirmed that MstA transports glucose, fructose, and mannose. The Km value obtained for fructose was 4 mM, whilst the Km values for glucose and mannose are at least 10 fold lower. This result, in conjunction with the expression data, suggests that A. niger MstA is a hexose transporter with high affinity for glucose and mannose.

185 A Fruitbody tissue method for efficient Agrobacterium-mediated genetic transformation of Agaricus bisporus. Xi Chen, Michelle Stone, Carl Schlagnhaufer and C. Peter Romaine. Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 16802 USA

We have devised a highly efficient and convenient Agrobacterium-mediated genetic transformation method for the mushroom, Agaricus bisporus. Transformation was carried out using a binary plasmid vector (pBGgHg) comprised of a pCAMBIA1300 backbone with the hygromycin resistance (hph) gene controlled by the A. bisporus glyceraldehyde 3-phosphate dehydrogenase promoter. Gill tissue pieces from fruitbodies were cocultivated for 3 days in the presence of acetosyringone with A. tumefaciens carrying pBGgHg, and then transferred to a hygromycin-containing medium. Antibiotic-resistant cultures appeared after 7 to 28 days from ca. 35% of the tissue pieces. The choice of promoter, bacterial strain and fruitbody tissue was critical for maximal transformation efficiency. Transformants had from one to four copies of the hph gene integrated at random sites in the genome. The antibiotic resistance trait was stably maintained, being expressed by the first-generation fruitbodies and basidiospores. Our method creates new prospects for using transgenic technology in the genetic improvement of this commercial mushroom, and represents an important tool for the genetic analysis of biological processes in this species.

186 Expression of Mn-peroxidase gene from Coriolus versicolor in transgenic tabacco for remediation. Iimura, Y.¹, Ikeda, S.², Sonoki, T.², Hayakawa, T.³, Kinbara, K.³, Kajita, S.², Katayama, Y.² and Tatsumi, K.¹. ¹ Natl Inst Res & Env, AIST, MITI, Hydrospheric Environ Prot, Tsukuba, Ibaraki 3058569, Japan; ²Tokyo Univ Agri &Tech, BASE, Koganei Tokyo 184-8588 Japan; ³Railway Tech Res Inst, , Koganei Tokyo 1858540 Japan

Plants offer many advantages over bacteria as agents for remediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Biodegradative abilities of plants are less impressive than those of adapted bacteria and fungi. But these disadvantages are balanced by the large amounts of plant biomass that can easily be sustained in the field. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both system. We genarated transgenic tobacco expressing Mn-peroxidase gene from Coriolus versicolor .

187 Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus. Beom-Gi Kim, Young-Bok Yoo and Suk-Tae Kwon¹. Division of Applied Microbiology, National Institute of Agricultural Science and Technology, Suwon, 441-707 Korea. ¹Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea.

Uracil auxotrophs of Pleurotus ostreatus were isolated using resistance to 5' fluoro-orotic acid (5'-FOA). Uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Normal fruiting bodies were induced in hybrid to cross uracil auxotroph with wild-type monokaryon, and the basidiospores collected from this fruiting body showed a biased segregation rate to prototrophy. Uracil auxotrophic monokaryons were crossed each other and dikaryotic auxotrophic strains were generated. We will use these strains as host for transformation.

188 Hop, an active MuDR-like element in the filamentous fungus Fusarium oxysporum. Fabienne Chalvet¹, Fiona Kaper¹, Thierry Langin² and Marie-Josée Daboussi¹. ¹Université Paris Sud, IGM, Orsay, France; ²Université Paris Sud, IBP, Orsay France

Four different class II transposons have been identified in the genome of the fungal plant pathogen Fusarium oxysporum. All are active since they have been trapped through their transposition into the nitrate reductase structural gene (niaD) used as a target. Three belong to the Tc1-mariner superfamily, and the last one, presented here, is related to the MuDR family. Two insertions located in the last exon of the niaD gene, at the same nucleotidic site and in opposite orientation, have been characterized. These elements, called Hop, are 3299 bp long with perfect inverted terminal repeats of 99 pb. Partial sequencing of genomic copies reveals a 9 bp target site duplication and no apparent sequence specificity at the insertion sites. Sequencing of a cDNA indicates that Hop does not contain any intron and encodes a putative transposase of 836 AA. Comparison of Hop transposase to those from MuDR-like elements (maize, rice, Arabidopsis) reveals conserved domains. The structural features (length, ITRs size, 9 bp duplication) as well as the presence of conserved domains in the transposase strongly suggest that Hop is a MuDR-like element. This is the first active MuDR-like element found outside maize. Moreover, Hop excises from the niaD gene with a rather high frequency in spite of its location in an exon. This suggests that Hop is very active and thus represents a promising tool for developing an efficient insertional mutagenesis system.

189 Expression and production of Llama variable heavy-chain antibody fragments (V_HHs) by Aspergillus awamori. Vivi Joosten, Cees A.M.J.J van den Hondel and B.Christien Lokman. TNO Voeding, P.O. Box 360, 3700 AJ Zeist, the Netherlands.

High level production of antibody fragments is of industrial importance. Previous studies on the production of single chain (scFv) fragments in Saccharomyces cerevisiae demonstrated that these fragments accumulated in the Endoplasmic Reticulum (ER) and vacuole. It was postulated that the secretion of scFv might be hampered by improper folding of these fragments, or by the formation of large aggregates via interactions of the hydrophobic regions on the VH and VL domains (Frenken et al, 1994). Recent investments in Aspergillus awamori showed that scFv could be produced up to 200mg/l (Frenken et al, 1998). However, this production level is still too low for a cost effective large scale process. Serum of Camelidae (camels and llamas) contains IgG immunoglobulins that are devoid of light chains and therefore are referred as 'heavy-chain' antibodies. Since the variable domains of these heavy-chain antibodies (V_HHs) lack the hydrophobic regions that are normally facing the variable domain of the light chain, it is suggested that these fragments are better secreted than the scFv fragments. In S. cerevisiae antigen-specific V_HHs were secreted at levels over 100 mg/l in shake flask cultures. It is expected that the production level of V_HH by A. awamori will be even higher. In this study the production of four llama V_HHs (R2, R5, R7 and R9) raised against the azo-dye RR6, was analysed in A. awamori. Therefore, PCR fragments encoding these V_HHs were cloned in an Aspergillus expression vector containing the expression signals of the highly inducible endoxylanase gene. Northern analysis under inducing conditions revealed mRNAs of the expected size. Furthermore, Western analysis demonstrated that the four V_HHs are secreted in the culture medium. Intra- and extracellular protein levels were determined.

190 Molecular genetic diversity of fungal communities in petroleum-contaminated soil using terminal restriction fragment patterns (TRFP). N. S. Lord, P. Shank, L. Martinez, C. Reimers, K. Palmer, C. Kaplan, C. L. Kitts, S. L. Elrod. California Polytechnic State University, Biology, San Luis Obispo, CA, USA

Terminal restriction fragment pattern (TRFP) analysis was performed on DNA extracted from sand samples taken from a petroleum-contaminated pilot scale land treatment unit (LTU) on a dune system on the Central California Coast. The LTU consisted of three different treatment cells. Cell one was a control, cell two was amended with corn steep liquor, and cell three was amended with glucose. In addition, each cell was amended with nitrogen and phosphorous as well as aerated and hydrated on a regular basis. In order to assess temporal changes in fungal molecular genetic diversity during petroleum degradation, community sand DNA from each LTU cell was PCR amplified using primers to conserved regions of fungal 18S or ITS ribosomal regions; one primer was fluorescently-labeled. Amplicons were then digested separately with MspI, HaeIII, and DpnII and terminal restriction fragments (TRFs) were detected by capillary gel electrophoresis. Cells 1, 2 and 3 each returned unique TRFPs over the course of pilot-scale treatment. In addition, ITS TRFPs demonstrated a greater genetic richness (i.e. a larger number of peaks) than 18S TRFPs. These data suggest this TRFP method can be used to detect changes in the genetic diversity of fungal communities. Further analysis to identify the source of ITS TRFP richness (e.g. species or ribosomal gene copy number) is in progress.

191 Characterisation of a gene involved in protein secretion in Aspergillus niger. Peter J. Punt, Eric Record^a, Vivi Joosten and Cees A.M.J.J. van den Hondel. Department of Applied Microbiology and Gene Technology, TNO Nutrition and Food Research Institute Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands, ^aLaboratoire de Biotechnologie des Champignons Filamenteux, Faculte des Sciences de Luminy INRA, CP 925, 163 Avenue de Luminy - 13288 Marseille Cedex 09, France

A major part of our research on filamentous fungi is dedicated to the characterization of the fungal secretion pathway. The ultimate aim of this research is to resolve bottlenecks for the efficient production of proteins in these organisms. Besides a more systematic approach, based on knowledge available from S.cerevisiae, in which specific secretion related genes were cloned and functionally characterized, we have also followed a more random approach based on selection of fungal mutant strains with perturbed secretion. Using a secretion reporter approach based on the E.coli glucuronidase (GUS) A. niger mutant strains were obtained with altered secretion characteristics. One of these mutants, GUS15.1#16, was unable to secrete the reporter protein, but also did not secrete significant amounts of glucoamylase. The strain also showed severely altered hyphal morphology, resulting in a hyper- branching phenotype. Using its restricted growth phenotype, the gene corresponding to the mutation was cloned by phenotypic complementation. For this purpose a cosmid library based on an autonomously replicating fungal transformation vector was generated and introduced in the mutant strains. Characterization of the complementing gene revealed that this gene showed significant homology with a sub-unit of a global transcriptional complex in S. cerevisiae.

192 Production of proteins under the control of the trypsin-like promoter from F. oxysporum is not growth associated. Natalie Farnworth, M.G.Wiebe, G.D.Robson and A.P.J.Trinci. School of Biological Sciences, University of Manchester, Manchester, M13 9PT.

A trypsin-like protease, with commercial value, has been identified in Fusarium oxysporum. The promoter from this protease has been used to drive the production of recombinant glucoamylase (GAM) in Fusarium venenatum. Trypsin-like protease was only produced in stationary phase in batch culture and the specific production rate did not increase with increasing specific growth rate. Similarly, native trypsin-like protease activity in F. venenatum was produced during stationary phase and was negatively correlated with growth rate. Further studies have shown that when GAM was produced in a F. venenatum transformant under control of the F. oxysporum trypsin-like protease promoter, it was only detected in culture supernatants after biomass increase (and therefore hyphal extension) had ceased. Western blot analysis suggested de novo synthesis of GAM occurred during stationary phase. This and other recombinant constructs thus provide a useful system for studying the production and secretion of proteins which are independent of hyphal tip extension.

193 Characterization of beta-endoglucanase genes of industrial fungus Aspergillus kawachii. Kiyoshi Ito, Yumi Hinoki, and Yukari Hara National Research Institute of Brewing, Genetic Engineering, Higashihirosima, Hiroshima, Japan

Filamentous fungus Aspergillus kawachii is a nearly related strain to A. awamori and it is widely used for shochu (a Japanese traditional spirit) fermentation. A. kawachii produces many kinds of cellulolytic enzymes along with sacchalolytic enzymes, so the enzymes from this strain can digest the cell wall of plants efficiently. We have cloned many genes of cellulolytic enzymes from A. kawachii. From them, we will present the characterization of three endoglucanase genes. EglA was an endoglucanase containing cellulose binding domain (CBD) at C-terminus with a very long linker region. The catalytic domain of EglA showed homology with family-5 endoglucanases. EglB had also CBD at C-terminus. The catalytic domain of EglB showed no homology with any cellulases but showed low homology with Cellulose-growth-specific protein (Cel1) from Agaricus bisporus. EglC showed high homology with the catalytic domain of EglA but had no CBD and linker. The transformed Saccharomyces cerevisiae with eglA, eglB, and eglC cDNA showed endoglucanase (CMCase) activity. The enzymatic properties were examined using the enzymes produced by yeast. EglA bound tightly to microcrystalline cellulose.

194 Characterization of alpha-amylase genes of industrial fungus Aspergillus kawachii. Toshikazu Sugimoto, Kenji Murashima, and Kiyoshi Ito. Kyowa Hakko Kogyo, Ibaraki, Japan. Natl Res Inst of Brewing, Hiroshima,

Japan Filamentous fungus Aspergillus kawachii is a nearly related strain to A. awamori and it is widely used for shochu (a Japanese traditional spirit) fermentation. Alpha-amylase is one of the most important enzymes in industrial process, so we examined the expression of alpha-amylase genes. A. kawachii produces two types of alpha-amylase, acid stable amylase (AA) and neutral amylase (NA). NA is almost the same as Taka-amylase (TA) of A. oryzae. NA was produced with glycerol or glucose as a carbon source, besides the common inducers such as starch or maltose. In the promoter region of NA gene, about 60 bp was depleted comparing the promoter of TA gene. Consequently, we considered that the constitutive expression of NA gene was due to the depletion of this region. AA was not produced in liquid culture but produced in solid-state culture. In liquid culture using maltose as a carbon source, strong signal of AA mRNA was detected by Northern analysis and strong GUS activity was detected by reporter gene analysis, in spite of the no AA activity. In this condition, no AA protein was detected inside and outside of the cell. Consequently, it was considered that the solid-state specific AA production was regulated at (post)- translational level.

195 Control of hyphal morphology in Aspergillus niger by regulated expression of the cotA gene. Shirley Burrow¹, Sarah Pollerman¹, Nigel Dunn-Coleman² and Geoffrey Turner¹. ¹Department of Molecular Biology and Biotechnology, University of Sheffield, UK, ²Genencor International, Palo Alto, CA

Strains of Aspergillus niger and the related species A. awamori are used in the commercial production of secreted native and heterologous enzymes. Limitations on enzyme yield in fermenter growth are influenced by mycelial morphology, which affects mycelial density, energy input and oxygenation. While mutations affecting morphology can arise during strain improvement programmes, the ability to manipulate morphology during fermenter growth by controlled expression of a specific gene would be advantageous, since an appropriate vector could be introduced into any strain of A. niger by transformation. In order to demonstrate the feasibility of this approach, we have cloned part of the cotA gene of A. niger, a homologue of the Neurospora crassa cot-1 gene, and placed it under the control of the regulatable glaA promoter. The cot-1 mutation of Neurospora crassa is a temperature sensitive mutation in a serine-threonine protein kinase, and growth at the non-permissive temperature leads to loss of polar growth of hyphae and to hyperbranching as a result of loss of function. Using a vector designed for promoter replacement, transformants of A. niger were selected on maltose as carbon source, and were screened for growth morphology on xylose. Approximately 60% of transformants showed a compact morphology on xylose, which represses glaA, and were purified for further analysis. These transformants also showed a compact growth form in xylose liquid medium in shake flasks, but were normal in maltose medium. These strains will be analysed for growth characteristics in fermenters.

196 Structural features of maltose utilization gene clusters in Aspergillus oryzae. Masahiro Takizawa¹, Takeshi Akao², Osamu Akita², and Katsuya Gomi¹. ¹Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. ²National Research Institute of Brewing, Higashi-Hiroshima, Japan.

Aspergillus oryzae produces a copious amount of amylolytic enzymes such as a-amylase, glucoamylase, and a-glucosidase, which are most important in sake brewing. Recently, a transcriptional activator gene, amyR, involved in the amylolytic gene expression has been cloned (1). The amyR gene disruptants showed significantly poor growth on starch medium, but showed normal growth on maltose medium, indicating the existence of the maltose utilization enzymes, production of which might not be regulated by the amyR. An EST clone homologous to yeast maltase gene (MAL62) was found in the EST database constructed by Aspergillus EST sequencing consortium of Japan. Then two different gene clusters probably involved in maltose utilization have been isolated from an A. oryzae genomic library. One consists of MAL62 homologue itself, designated malT, and a gene homologous to yeast maltose permease gene (MAL61), designated malP. In addition, a putative transcriptional regulator gene which has a typical fungal zinc finger motif at N-terminus is located at downstream of the malT, although this regulator gene, DmalR, is unlikely functional because of truncation in the C terminal region. Another gene cluster is highly homologous to the sugar utilization gene cluster in A. parasiticus recently reported (2). Nucleotide sequence analysis of about 20-kb fragment encompassing the cluster revealed that the gene cluster has genes encoding a-glucosidase (glcA), a sugar transporter (hxtA), and a transcriptional activator (sugR) and is located at one end of the aflatoxin biosynthetic gene cluster, as in A. parasiticus. (1)Gomi et al, Biosci. Biotechnol. Biochem., 64, 816-827 (2000). (2)Yu et al, Biochim. Biophys. Acta,

197 Isoenzyme multiplicity and characterization of recombinant manganese peroxidases (rMnPs) from Ceriporiopsis subvermispora and Phanerochaete chrysosporium. Luis F. Larrondo¹, Sergio Lobos², Phillip Stewart³, Dan Cullen³ and Rafael Vicuña¹. ¹ Departamento de Genetica Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Catolica de Chile, Santiago, Chile, and Millennium Institute for Fundamental and Applied Biology, Santiago, Chile. ² Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile. ³ USDA Forest Products Laboratory, Madison, Wisconsin 53705.

We expressed cDNAs coding for manganese peroxidases from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under the control of the a-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (Cs-rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. IEF analysis of rH4 showed several isoforms with pIs between 4.83 and 4.06, one of which coincides with the pI (4.6) described for H4 isolated from P. chrysosporium. IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, closely resembling the pattern observed with MnPs isolated from C. subvermispora grown on solid state cultures. The thermostability of both recombinant enzymes was also analyzed. We compared the rMnPs ability to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium. Acknowledgment: This work was financed by grants 8990004 and 2000076 from FONDECYT-Chile and by U.S. Dept. of Energy grant DE-FG02-87ER13712. L.F.L is a Predoctoral Fellow of Fundación Andes.

198 Heterologous expression and secretion of an acetyl xylan esterase (AXE II) from Penicillium purpurogenum in Saccharomyces cerevisiae and Aspergillus nidulans. Luis F. Larrondo, Francisco Herrera, Soledad Quiroz, Marcela Colombres, Alessandra Peirano and Jaime Eyzaguirre. Laboratorio de Bioquímica, Universidad Católica de Chile, Santiago, Chile.

P. purpurogenum secretes several esterases to the culture medium, among them AXE II. This enzyme was purified, sequenced and its three-dimensional structure determined by X-ray diffraction. Site-directed mutagenesis studies require a heterologous expression system. Here we describe the results obtained in the expression of the wild type enzyme in two different hosts. The coding region of the mature cDNA of AXE II was cloned in the yeast expression vector pBS24.1. The resultant plasmid (pFH5) was used to transform Saccharomyces cerevisiae AD3. Positive clones were selected and protein secretion was achieved by growing the transformants in YEPD with 2% glucose. Expression in Aspergillus nidulans was accomplished by fusing the cDNA of AXE II, including its signal peptide, with the A. oryzaeTaka amylase promoter and the A. awamori terminator (pSQ1). Spheroplasts were co-transformed with pSQ1 and ppyrG, which confers prototrophy to uridine. Positive clones were grown in YEM with 2% maltose to induce the expression of the recombinant protein. rAXE II was followed by activity, western blots and SDS-PAGE in culture supernatants. Saccharomyces transformants secreted active enzyme, but several bands appeared in SDS-PAGE, while two bands of similar intensity were observed in western blots, one corresponding to the mature AXE II and another slightly larger, suggesting incomplete processing of the signal peptide. Aspergillus transformants secreted active enzyme and only one band identical in size to native AXE was observed in SDS PAGE and immunoblots. One of the selected clones produced up to18 mg/L of protein and 3.5 U/mg of AXE. In conclusion, Aspergillus nidulans is a good host for the expression of AXE II; rAXE II is secreted in an active form and in significant levels, permitting rapid detection and purification. This will facilitate the expression and characterization of site-directed mutants of AXE II. Acknowledgment: Work supported by DIPUC and by grants 8990004 and 2000076 from FONDECYT, Chile. L.F.L is a Predoctoral Fellow of Fundación Andes.

199 Cloning the gene encoding acetyl xylan esterase from Aspergillus ficuum and its expression in Pichia pastoris. Hea-Jong Chung, Seung-Moon Park*, Moon-Sik Yang and Dae-Hyuk Kim. Chonbuk National University, Biological Sciences, Chonju, Chonbuk, Korea. Chonbuk National University, *Basic Science Research, Chonju, Chonbuk, Korea

Acetyl esterases are important in the complete degradation of acetylated polysaccharides, such as pectins and xylans. We isolated the gene encoding acetyl xylan esterase (AfAXE) from a genomic library of Aspergillus ficuum. We cloned the corresponding cDNA by RT-PCR. The Afaxe gene contained two introns, one TATAA box, and two CAAT-like boxes. The transcription initiation site was 61 bp upstream of the start codon. The deduced amino acid sequence consisted of a putative 28-amino acid leader peptide and a mature protein with an estimated molecular mass of 29.5 kDa. The nearest homolog of the cloned gene was acetyl xylan esterase of A. niger. The cloned gene was placed in a Pichia expression vector and expressed in Pichia pastoris. The culture filtrate of the transformant liberated acetyl moieties from p-nitrophenyl acetate and its activity reached 75.8 IU/ml, which was over 100-fold greater than the activity of the native enzyme expressed in A. ficuum. The native and recombinant acetyl xylan esterases were purified from the culture filtrates of A. ficuum and P. pastoris, respectively. Both enzymes had approximately the same optimal temperature (37 C) and pH (7.0). The recombinant protein had greater tolerance for alkaline conditions (> pH 7.0), but was less thermostable above 55 C. The cloned enzyme catalyzed the release of acetic acid from acetylated hardwood xylan, confirming that the cloned gene encoded an acetyl xylan esterase of A. ficuum. This work was supported by 2000's Korea Research Foundation Grant.

200 The foldase, CYPB, from Aspergillus niger contains a novel endoplasmic reticulum retention signal and its overexpression improves yields of secreted glucoamylase. Patrick M.F. Derkx, Harm J. Mulder and Susan M. Madrid. Danisco Cultor Innovation, Langebrogade 1, DK 1001, Copenhagen, Denmark

Screening of Aspergillus niger libraries with degenerate oligo nucleotides encoding the endoplasmic reticulum (ER) retention signal HDEL, led to the isolation of the cypB gene. The cypB gene encodes a peptidyl prolyl cis-trans isomerase (PPIase) belonging to the cyclophilins and contains an ER targeting signal and a novel ER retention signal. The ER retention signal, HEEL was shown to be capable of retaining the green fluorescent protein (GFP) within the ER. The expression of cypB was upregulated by the unfolded protein response and heat shock. Escherichia coli expressed and purified CYPB-(His)₆ is capable of isomerising a substrate peptide in vitro. The intracellular level of CYPB in A. niger was increased by introducing a plasmid allowing constitutive expression of cypB. This enhanced the yield of native secreted glucoamylase 3 to 10 fold. Our results suggest that CYPB is a rate limiting step in the secretory pathway of A. niger. In the ongoing process of the improvement of A. niger as a production organism for homologous and heterologous proteins, the identification of bottlenecks in protein secretion is of considerable biotechnological interest.

201 Development of ABC transporter mutants of Aspergillus nidulans as innovative tools in drug discovery. Alan C. Andrade, Ciska Braam and Maarten A. De Waard. Wageningen University, Lab. of Phytopathology, Wageningen, The Netherlands

Antimicrobial resistance is a global problem that undoubtedly concerns modern society. The World Health Organization together with public health authorities worldwide, are already developing action plans with strategies and guidelines to combat this growing problem. Drug resistance is not a new phenomenon but has evolved since man began to use antibiotics. However, the development of new families of antimicrobials in previous decades made us believe that we could always remain ahead of the pathogens. Concern emerged in the late 1990s that resistance was accumulating without the discovery of new antimicrobials. A traditional way to discover these agents is broad-based whole cell screening with libraries of chemicals or natural extracts. A general problem facing this type of screening is that the ambient concentration of drugs is prone to be low. This problem is amplified by the function of multidrug pumps which occur in all living organisms. These pumps reduce the accumulation of drugs inside the test organism and will inevitably diminish the chances of the discovery of new drugs. Mutants of micro-organisms lacking multidrug-efflux pumps have significant potential for enhancement of the sensitivity to antimicrobial agents. Our major goal is to develop such mutants of Aspergillus nidulans. Using a combination of classical and molecular genetics, multiple knock-out mutants of ABC transporters (atr) genes from this fungus are being generated in different combinations. Results indicate that these mutants indeed display increased sensitivity to a broad range of toxicants. Hence, these mutants are usefull tools in screening programs of new lead compounds with antifungal activity.

202 Chrysosporium lucknowense, a new fungal host for protein production. Cora van Zeijl¹,Peter Punt¹, Mark Emalfarb², Rich Burlinghame³, Arkady Sinitsyn⁴, Martine Parriche⁵, Jean-Christophe Bousson⁵, and Cees van den Hondel¹. ¹TNO Nutrition and Food Research Institute, Applied Microbiology and Gene Technology, Zeist, The Netherlands; ²Dyadic International. Inc, Jupiter, Florida, USA; ³Bio-Technical Resources, Manitowoc, Wisconsin, USA; ⁴FermTech Ltd., Moscow, Russia; ⁵Cayla,Toulouse, France

Chrysosporium lucknowense, an ascomycetous fungus, not closely related to Aspergillus or Trichoderma, is developed as a new fungal host for protein production. This thermophilic fungus is able to secrete large amounts of (hemi)cellulases. Mutant strains of C. lucknowense have been isolated with 5-50 fold improved protein yields (per gram biomass) compared to available Aspergillus and Trichoderma strains. Large- scale fermentations were developed up to 150,000 litre cultures. Growth conditions of Chrysosporium strains are very versatile from acid to alkaline pH and at temperatures of 25-43 C. Several transformation systems have been developed for this fungus, based on nutritional or dominant selection markers. The use of this fungal system for the production of fungal and non- fungal proteins will be discussed.

203 Seeing the light with quantum dots. S.T. Merino¹, T. Prendergast², S. Nie² and M.E. Zolan¹. Department of Biology¹ and Chemistry², Indiana University, Bloomington, Indiana

Our research focus is studying genes involved in DNA repair and meiosis in the fungus Coprinus cinereus. One commonly used assay is Fluorescence in situ Hybridization (FISH), which is used to examine pairing between homologs. Our lab is also using immunolocalization to examine protein localization in wildtype strains and meiotic mutants. Currently, we are attempting to modify these fluorescent techniques by using Quantum Dot technology. Quantum Dots (QDs) are cadmium selenide nanocrystals, capped with a layer of zinc sulphide. Luminescent QDs can be coupled to biological molecules. In comparison to organic dyes such as rhodamine, QDs are 20 times as bright, 100 times as stable, and one-third as wide in spectral linewidth. Using a streptavidin-conjugated QD, which will bind to biotinylated DNA probes, QDs can be used for FISH analysis. Using QD technology results in less background and brighter signal, which is especially useful in examining sister-chromatid cohesion. For immunolocalization, a QD is covalently coupled to an antibody, resulting in a fluorescent tag with stronger, specific signal. The potential use for Quantum Dot technology in fungal research appears unlimited.

204 Analysis of the penicillin biosynthetic gene cluster: directed strain improvement of Penicillium chrysogenum. Marco van den Berg, Richard Kerkman, Ilja Westerlaken, Chris Leeflang, Eric Koenhen and Roel Bovenberg. DSM Life Sciences, Division Anti-Infectives (624-0270), PO Box 1, 2600 MA Delft, The Netherlands.

Within the various business groups of the DSM Life Sciences Cluster research efforts are focussed on the development and improvement of microbial strains and production processes. Filamentous fungi like Aspergillus niger and Penicillium chrysogenum are two of our preferred organisms, the so-called pluGbugs, for enzyme and B-lactam antibiotics production, respectively. To optimize the production processes it is essential to have detailed knowledge on these organisms. For years we have for filled this with classical biochemical and genetical analyses of strains and fermentations. Recent additions to the molecular and biochemical toolkit rapidly increased the possibilities: genome sequencing, DNA arrays, proteomics, NMR, Green Fluorescent Protein, etc. In this presentation I want to present the most recent results obtained at our laboratories by applying these techniques to one of our production organisms: Penicillium chrysogenum. In previous studies we have shown that is possible to increase penicillin production of the laboratory strain Wisconsin54-1255, by transforming it with different combinations of the penicillin biosynthetic genes (1). A detailed analysis revealed a complex regulatory system (2). Recently, it was demonstrated that not only the penicillin biosynthetic enzymes determine the production rate, but regulatory circuits in primary metabolism (e.g. NADPH- balans) have a distinct influence (3). To get more insight in the processes involved we determined the complete sequence (60 kb) of the penicillin biosynthetic gene cluster. These gene cluster was shown to be amplified in industrial production strains (4). A detailed (functional) analysis of all ORFs will be discussed.

1). Van den Berg, M.A. et al. (1999) Antonie van Leeuwenhoek 75:155-61 2). Theilgaard, H. A. et al. (2000) in press 3). Van Gulik, W.M. et al. (2000) Biotechnol Bioeng 68:602-18 4). Fierro, F. et al. (1995) Proc Natl Acad Sci USA 92:6200-6204

205 Characterisation of the translation initiation factors of Trichoderma reesei. Anne Huuskonen¹, Edward Alatalo, Markku Saloheimo¹, Merja Penttil ¹, and Joop van der Laan². ¹VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland. ²Genencor International B.V., P.O. 218, Leiden, The Netherlands

Protein translation is regulated in eukaryotes in response to various stress conditions. Inhibition of protein secretion leads to reduced translation initiation rates in mammalian cells. Our aim has been to characterise translation initiation factors of the filamentous fungus Trichoderma reesei both at gene and protein levels and to address possible feedback control between secretion and translation initiation in filamentous fungi. The genes encoding the translation initiation factors eIF2alpha, eIF2beta and eIF4A have been isolated and sequenced from T. reesei. Based on the results from the Southern hybridisation all three genes are present in the Trichoderma genome in one copy. The eIF2beta gene contains three introns which can be differentially spliced generating a population of mRNAs with different ORFs. The expression levels of the eIF genes have been examined in mycelia treated with different chemical agents known to have influence on protein translation and/or secretion. These agents include dithiotreitol (DTT) and Brefeldin A (BFA). Of the chemicals studied the reducing agent DTT appears to have the most drastic effect on all the three genes. DTT's effect is most pronounced in the case of the eIF2beta where the drug has been shown to alter the ratio of the different mRNA forms. The effects of the different drugs on the translation initiation factor protein levels has also been under investigation. To obtain more information on the effects of the eIF2alpha, one of the key regulatory factors in translation initiation, two different strains expressing a mutant form of this gene have been constructed. One strain is expressing the non-phosphorylative form of this protein and the other strain mimics the constitutively phosphorylated form of the protein. The mutations introduced to the gene appear to have only minor effects on the protein production and growth, possibly on the germination stage.

This study has been made within the Eurofung project funded by the EU.

206 Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase. Frits Goedegebuur¹*, Timothy Fowler** Jay Phillips#, Pim van der Kley¹, Piet van Solingen¹, Lydia Dankmeyer¹, and Scott D. Power². ¹Genencor International B.V., Archimedesweg 30, 2333CN Leiden, The Netherlands. ²Genencor International Inc., 925 Page Mill Road, Palo Alto, CA 94304-1013, U. S. A. # Current address: JPhillips@microcide.com ** Current address: tim@bainbridge.net

Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as either endoglucanases (EGs) or cellobiohydrolases (CBHs). This poster describes molecular screening for EGs from the GH family 12. Using three homologous boxes deduced from 5 previously known members of the family we analysed cellulase producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions (PCRs) using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologs. Fragments from these PCRs were used as templates for a second semi nested PCR. Gene specific primers were developed to determine the up and downstream sequences. Rapid Amplification of Genomic Ends (RAGE), a PCR based genome- walking technique was used to elucidate full-length gene sequences. Several fungi showed the presence of multiple versions of the gene while sequence analysis showed a diversity in 15 novel members of the family ranging from 27 to 94% similarity.

207 Role of virulence proteins in Agrobacterium tumefaciens-mediated transformation of Aspergillus awamori. C.B. Michielse, A.F.J. Ram, H. van Attikum, P. Bundock, P.J.J. Hooykaas, C.A.M.J.J. van den Hondel. Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

Agrobacterium tumefaciens, a plant pathogen, is widely used for the transformation of plants, yeast and filamentous fungi (de Groot et al. 1998). A. tumefaciens transfers a part of its tumor inducing plasmid (T-DNA) to the host, where it integrates into the genome. The tumor inducing plasmid also contains a virulence region encoding proteins, which are involved in generation, transfer and integration of the T-DNA. The integration of T-DNA is a random process and therefore Agrobacterium can be used as a tagging system. The critical parameters involved in Agrobacterium-mediated transformation of Aspergillus awamori were identified and used to optimize the transformation protocol, resulting in a highly reproducible and efficient transformation system. To assess the role of A. tumefaciens virulence proteins on T-DNA transfer, several A. tumefaciens mutants were tested in their ability to transform A. awamori. These experiments showed that host range factors, like VirH and VirF, are not important for A. awamori transformation. Mutation in the regulatory proteins (VirA, VirG), transport pore proteins (VirB) and proteins involved in generation of the T- strand (VirD1, VirC2) reduces or abolishes the formation of transformants.

1. de Groot et al. (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi, Nature Biotechnology 16: 839-842.

208 ER stress response: The A. niger transcription factor HacAp mediates the upregulation of the molecular chaperone bipA and the peptidyl prolyl cis-trans isomerase cypB. Harm J. Mulder, Patrick M. F. Derkx and Susan M. Madrid. Danisco Cultor Innovation, Langebrogade 1, DK 1001, Copenhagen, Denmark.

The accumulation of unfolded proteins within the lumen of the ER results in the activation of an intracellular signaling pathway leading from the ER to the nucleus. This signaling pathway is known as the Unfolded Protein Response (UPR) and results in an increased transcription of genes encoding ER resident proteins. One of the key elements in the UPR is HacAp, a bZIP family transcription factor that binds to the promoters of the target genes when the UPR is induced. The promoters of bipA and cypB contain several putative UPR elements (UPRE) based on sequence similarity to the yeast and mammalian UPRE as well as a heat shock element. We have used transcriptional fusion between varying lengths of 5' upstream region and the E.coli GUS gene to delineate the unfolded response elements of the genes encoding the ER resident proteins: cypB and bipA. In addition, gel mobility band shift assays were performed using purified HacA protein and the different putative UPREs. The regulation of the different UPR target genes (bipA, cypB) was studied using different conditions of HacA overexpression. The transcriptional regulation of HacA was altered by fusing the hacA gene to a strong inducible glucoamylase promoter (GLA) and the constitutive GPDA promoter. The overexpressing HacA transformants were analysed by northern and western-immunoblot analyses.

209 Aminopeptidase Y and carboxypeptidase Y of Aspergillus niger are targeted to the vacuole. Basten E.J.W., Muller Y., Visser J., Schaap P.J.. Molecular Genetics of Industrial Microorganisms Wageningen University, Wageningen, The Netherlands

The fungal vacuole is thought to have several important physiological functions. Degradation is one obvious function but it is also a site for storage of metabolites. The vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Most of the vacuolar enzymes are delivered to the vacuole via the early compartments of the secretory pathway and the endosome. In yeast several vacuolar proteases have been described. Most of these proteases are synthesized as inactive precursors that are transported to the vacuole. Since both, proteins that are secreted and proteins destined for the vacuole use the same parts of the secretory pathway, there must be signals within the proteins themselves that allows them to be sorted and targeted in a precise and efficient manner. After delivery to the vacuole the vacuolar proteases are matured, by removal of the propeptide, by the protease PrB. The propeptide also functions as an autoinhibitor. In the past vacuolar proteases of Aspergillus niger have been cloned and characterized, however little is known about maturation and vacuolar targeting of these proteases. To study in more detail, the maturation and vacuolar targeting of aminopeptidase Y in Aspergillus niger, a fusion construct was made with GFP. Results on the subcellular location and maturation of the (fusion) protein will be presented.

210 Recombinant t-PA production in Aspergillus niger and Trichoderma reesei. Karin Lanthaler, Atul Karandikar, Marilyn G. Wiebe, Geoff D. Robson and Anthony P. J. Trinci. University of Manchester, Microbiology, Manchester, Manchester, United Kingdom

Recombinant t-PA has been produced in two filamentous fungi, Aspergillus niger and Trichoderma reesei. t-PA production was studied in batch and chemostat cultures of A. niger and in chemostat cultures of T. reesei. The concentration of t-PA produced by A. niger was enhanced by addition of soya peptone to a defined medium. In batch cultures, 1.9 mg total t-PA [g biomass]^-1 was produced in this medium at pH 6.0, of which only 4.3 mg [g biomass]^-1 was active. Active and total t-PA were rapidly lost from culture supernatant during stationary phase. However, 2.7 mg total t-PA [g biomass]^-1 was produced in glucose-limited chemostat cultures (D = 0.07 h^-1, pH = 6.0, 25 C) for at least 140 h of continuous medium flow. t-PA could also be produced in chemostat cultures of the Trichoderma reesei transformant. Addition of soya peptone to the medium did not substantially increase the amount of t-PA produced by this strain. In lactose-limited chemostat cultures 0.14 mg total t-PA [g biomass]^-1 was produced for up to 140 h of continuous medium flow.

211 Characterisation of KexB, the kexin-like maturase of Aspergillus niger. Ruud Jalving, Peter van de Vondervoort, Jaap Visser and Peter Schaap. Section Molecular Genetics of Industrial Microorganisms, Wageningen University and Research Centre, Wageningen, The Netherlands.

In filamentous fungi an effective method to enhance the yields of secreted foreign proteins is the use of a translational fusion between the target protein and an endogenous secreted carrier protein. Removal of the carrier protein is usually achieved in vivo through cleavage of an engineered KEX2 endoprotease recognition site at the fusion junction. We have cloned the kexin- encoding gene of Aspergillus niger (kexB). Disruption of kexB resulted in a transformant with a hyper-branching morphology. Using fluorogenic substrates kexin-like activity was measured in membrane-protein fractions of the wild type strain and a KexB overexpressing strain. In contrast, no kexin specific activity was detected in similar protein fractions of the kexB-disrupted strain. Expression in this loss of function strain of a glucoamylase human interleukin-6 fusion protein with an engineered KEX2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein.

212 het-gene homologs in filamentous fungi. Theo van der Lee, Cees Waalwijk. Plant Research International, P.O. box 16, 6700 AA Wageningen, The Netherlands.

In filamentous fungi fusion of hyphae of the same individual generates a network of hyphae or mycelium. Fusion of somatic hyphae between genetically distinct individuals of the same species is usually restricted by vegetative incompatibility, a process of cell death and growth inhibition that occurs after fusion (reviewed in 1). This process is governed by het-genes and combinations of incompatible het-genes can be either allelic or non- allelic. The number of different functional alleles of het-gene loci is limited, but the number of these loci and their allele frequencies imply that under natural conditions, somatic hyphal fusion between different genotypes is rare (1). A number of het-genes have been cloned and they encode a variety of proteins. How these proteins trigger cell death and how het- genes evolved is unknown. The het-c gene of Neurospora crassa encodes a glycine rich protein that is probably located in the cell wall (2). Searches in both genomic (3) and EST databases revealed the presence of a homolog of het-c in Neurospora crassa. This homolog is distinct from the het-c alleles previously described. It lacks some highly variable regions but still has highly significant homology at the protein level. Searches in databases from other fungi and PCR with degenerate primers show homologs in a wide range of fungi. Interestingly, the homologs of other fungi show more homology to the newly found het-c homolog in Neurospora crassa, indicating that this may be the ancient gene from which the het-genes originated. Paralogs and probable orthologs of other het-genes were also found in filamentous fungi but not in other organisms like yeast, plants and animals, suggesting that these homologs are general only to filamentous fungi. The het-gene homologs may help to identify how these genes evolved and thus, might offer new insight in the process of vegetative incompatibility.
(1) Glass et al. (2000) Ann. Rev. Genet. 34:165-186 (2) Saupe et al. (1996) Genetics 143: 1589-1600
(3) http://www.mips.biochem.mpg.de/proj/neurospora

213 Creation of a federated database for Magnaporthe grisea integrating genetic, physical, and BAC end sequence data. Stan Martin, Sheila Dunn, Barbara Blackmon, T.D. Houfek, Rod Wing and Ralph A. Dean. Fungal Genomics Laboratory, North Carolina State University, Raleigh NC USA

We are in the process of sequencing the genome of Magnaporthe grisea , the causal agent of rice blast disease. This information will be useful for understanding the mechanisms of fungal infection and for developing strategies to counteract disease caused by this fungus. We have created a searchable database integrating end sequence data from BAC clones, genetic marker data, and contig assembly data that represent the majority of the M. grisea genome. A previously generated library of BAC clones representing a 25-fold coverage of the entire genome was end sequenced and fingerprinted by a HindIII digestion. The Image/FPC software package was then used to generate an assembly of ~188 contigs covering >95% of the genome. The database contains the results of this assembly and previously generated RFLP data. We used the RFLP data to physically anchor most of the FPC contigs onto one of the seven chromosomes in the M. grisea genome. We used AceDB for our core database engine and used the MySQL relational database, which we populated with numerical representations of sequence information, to create appropriately scaled images. The database will facilitate efforts to fill in the gaps in the parts of the genome that remain to be sequenced. The database also allows researchers attempting to map known genes, or other sequences of interest, rapid and easy access to the fundamental organization of the M. grisea genome. Repetitive sequences such as MAGGY and Pot2, are prevalent within the genome, and may potentially undermine the fidelity of the contig assembly. In order to minimize the impact of this source of potential error, we have undertaken an extensive analysis of the distribution of these repeats.

214 Diversification of heterologous DNA in Neurospora. J.P. Rasmussen¹, P.J. Yeadon¹, F.J. Bowring¹, E. Cambereri², W.D. Stuart² and D.E.A. Catcheside¹. ¹ School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia 5001. ² Neugenesis Corporation, 871 Industrial Road, San Carlos, California 94070.

Recombination hotspots active in meiosis provide a means of diversifying pairs of DNA sequences differing at multiple sites. We have constructed plasmids that permit targeted transfection of heterologous genes such that they are located between his-3 and the cog hotspot in Neurospora crassa. This positioning enables enrichment for sequences which experience exchanges in the heterologous DNA during meiosis by selecting progeny that are recombinant at his-3. We have used this system to shuffle human immunoglobulin kappa chain sequences and also sequences from other fungi. Our system provides a novel method for accelerated evolution of genes in which new gene variants are expressed and the products secreted from cells without further manipulation.

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