Industrial Biology and Biotechnology

401. Efficient production by Aspergillus awamori of a Llama antibody fragment fused to a peroxidase. B. Christien Lokman¹, Vivi Joosten¹, Marc Roelofs¹, Niels van den Dries¹, Robin J. Gouka², C. Theo Verrips², and Cees A.M.J.J. van den Hondel¹. ¹TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, the Netherlands. ²Unilever Research Vlaardingen, P.O. Box 114, 3130 AC Vlaardingen, the Netherlands

The development of fusion proteins consisting of antibody fragments and enzymes is of great medical and industrial importance. Previously, our group has demonstrated that single chain Fv antibody fragments (scFv) could be efficiently produced in Aspergillus awamori (Frenken et al., 1998). Recently, we have studied the production of Llama heavy-chain antibody fragments (VHH ) by A. awamori. The advantage of VHH over ScFv antibody fragments is that VHHs are devoid of light chains (Figure 1). Furthermore, VHH lack the hydrophobic regions that are normally facing the variable domain of the light chain and are therefore suggested to be better secreted than scFv fragments. A suitable enzyme for industrial applications is Arthromyces ramosus peroxidase (ARP). This 41 kD monomeric glycoprotein has a broad specificity for phenolic and anilinic hydrogen donors. In previous studies we have demonstrated that there is no heme limitation during overproduction of ARP in A. awamori. Under control of the endoxylanase promoter secretion of active ARP was achieved up to 0.8 g/L in shake flask cultures. Fusions between enzymes and VHHs permit interesting applications due to the fact that VHHs direct the enzymes to the place where they should act. As an example we studied the integrity of N-terminal and C-terminal fusions of ARP with a VHH fragment against the azo-dye RR6 after production in A. awamori.

402. Identification of Manganese-inducible genes in Aspergillus niger using suppression subtractive hybridization. Ziyu Dai, Jon Magnuson and Linda Lasure. Processing Science, Pacific Northwest National Laboratory, Richland WA.

Morphology of filamentous fungi in fermentation processes is critical to optimal product output. The proper morphology of citric acid production strains of Aspergillus niger is regulated by manganese (Mn²⁺) and other factors. Detailed examination of the effects of Mn²⁺ on A. niger morphology formation shows that filamentous hyphae of A. niger developed rapidly at Mn²⁺ concentrations of 14 ppb or higher, while citric acid production dramatically decreased. Molecular mechanisms involved in Mn²⁺ induction of morphology formation in A. niger have not been well defined. Here, suppression subtractive hybridization identified fifteen genes differentially induced when A. niger grew at 1000 ppb Mn²⁺ and seven genes differentially expressed at 10 ppb Mn²⁺. Among the fifteen filamentous form associated genes, nine are unknown and six have some degree of identity to genes in other organisms. Similarly, six non-filamentous form associated genes are unknown, but one clone, Arsa-1, exhibits homology to a yeast gene. Elevated mRNA levels in ten filamentous form associated genes were observed 40 and 120 min after the addition of 1000 ppb Mn²⁺ to 12 hr cultures, while the mRNA of all seven non-filamentous form associated genes were rapidly degraded. Transcription of the other five filamentous form associated genes were dramatically increased during the first 40 min following Mn²⁺ induction and thereafter rapidly degraded. These findings create the opportunity for detailed studies on genes and enzymes involved in morphology determination. These results also reveal that SSH is a powerful and sensitive technique for the detection of differential gene expression in A. niger morphology formation.

403. Global Expression Profiling of the Lignin Degrading Fungus Ceriporiopsis subvermispora for the Discovery of Novel Enzymes. Debbie S. Yaver, Barbara Weber and Jeff Murrell. Novozymes Biotech, Davis, CA.

The unique ability of white rot fungi to degrade all components of wood, including lignin, has attracted considerable biotech interest for several decades. Ceriporiopsis subvermispora is among the most selective lignin-degraders. Pretreatment of wood chips with C. subvermispora prior to mechanical pulping has been shown to reduce energy consumption by 30-40%. Using DNA microarray technology, global gene expression profiling of C. subvermispora was used to discover novel enzymes whose expression is induced during growth on mechanical pulp. Plasmid DNA was isolated from 50,000 independent clones of a random genomic library containing 2 to 3 kb genomic fragments and spotted on poly-lysine coated glass slides at a density of 10,000 per slide. Hybridization probes were prepared from RNA which was isolated from thirty day old cultures grown on either minimal medium or hardwood mechanical pulp labeling them with either Cy-3 or Cy-5 dyes. Hybridization to the first 20,000 clones identified 20 clones whose expression is induced due to growth on pulp. Sequence analysis has shown that the 20 clones actually represent 11 unique clones. One of these clones has significant homology to manganese peroxidases from white rot fungi including those previously identified from C. subvermispora; however, it is novel. We will report the heterologous expression and characterization of the peroxidase as well as the characterization of the other pulp-induced clones.

404. Targeted Illegitimate Recombination in A. nidulans. R. David Laidlaw and Jay D. Keasling. Department of Chemical Engineering, University of California at Berkeley, Berkeley CA, USA.

Metabolic engineering of fungal species often suffers from low transformation efficiencies, a low number of selectable markers, difficulty controlling insertion sites for transformed fragments and the possibility that DNA will insert into multiple ectopic locations. Assembling heterologous pathways into stable isogenic production strains presents a plethora of technical challenges. Elegant systems based on traditional homologous recombination have been developed to simplify insertion or disruption of gene-cassettes in filamentous fungal hosts such as N. crassa and A. nidulans. Few possibilities currently exist for single-step insertion of large multi-gene pathways to a single location within the genome. Here, we introduce a technique we have named 'Targeted Illegitimate Recombination' as a strategy for inserting DNA into the genome independent of a need for homology. We have develloped a modified REMI approach to insert DNA fragments at an engineered genomic site within A. nidulans. To reduce the uncontrolled nature by which REMI insertions typically occur, we have spliced a silent recognition sequence for the commercially-available homing endonucleaseI-SceI into a selectable marker gene. We present results demonstrating the usefulness of this technique for inserting DNA fragments into A. nidulans as the first step towards a suite of possibilities for metabolic engineering tools. Funding for this project provided by Merck and Co.

405. Modification of the N-glycosylation pathway of lower eukaryotes to a mammalian type. Vervecken, W., Callewaert, N., Geysens, S. and Contreras, R. Fundamental and Applied Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium.

Lower eukaryotes only synthesise N-glycans of the high-mannose type, whereas human glycoproteins have a very divers, complex type of N-glycans. Redirection of the fungal pathway, even to a simple hybrid or complex mammalian type requires several genetic interventions such as gene knock-outs and heterologous expression of mammalian glycosyl transferases. Furthermore, additional in vitro enzymatic manipulations may be required. In general, it seems that humanising N-glycans from filamentous fungi is an easier task than modifying yeast protein linked carbohydrates because the long alfa-1,6-arm apparently is absent. The problem is reduced to importing an efficient alfa-1,2-mannosidase and addition of complex sugar glycosyl transferases. We started a strategy to humanise the N-glycosylation pathway in the filamentous fungus Aspergillus niger NW195. In a first step a HDEL tagged a-1,2-mannosidase from Trichoderma reesei was introduced. The over expression of this enzyme lead to the conversion of the majority of the N-glycans to Man5GlcNAc2 (ca. 80%). In a second step the over expression of human Nacetylglucosaminyltransferase I lead to the detection of GlcNAcMan5GlcNAc2 structures. The percentage conversion of Man5GlcNAc2 to GlcNAcMan5GlcNAc2 was inversely related to the amount of protein that was synthesised, ranging from ca. 40% to merely all. Ultimately, the terminal sialic acid should be added using in vitro procedures.

406. Genomics of Fusarium venenatum: An Alternative Fungal Host for Making Enzymes. Randy M. Berka, Beth A.Nelson, Elizabeth J. Zaretsky, Wendy T. Yoder, and Michael W. Rey. Novozymes Biotech, Davis, CA.

Fusarium venenatum A3/5 (formerly F. graminearum Schwabe A3/5) has been used since 1985 as the commercial source of Quorn^TM mycoprotein, a processed form of fungal mycelia applied in several human food products to simulate chunks of chicken or beef. Regulatory approval of the organism for human consumption made it an attractive candidate to consider as a host for the production of industrial and food grade enzymes. Systems for genetic manipulation and transformation of F. venenatum cells have been developed together with several strong promoters and selectable markers for the introduction and expression of heterologous genes. Recent marketing of a heterologous xylanase and a fungal trypsin have provided a "proof of concept" for F. venenatum as a useful alternative to more traditional fungal hosts such as Aspergillus niger or A. oryzae. However, compared to the latter organisms and well-studied model fungi such as Neurospora crassa and A. nidulans, information regarding the genomics of F. venenatum is inadequate. This poster provides one of the first overviews of F. venenatum genomic information based on a compilation of expressed sequence tags and chromosomal gene sequences to initiate momentum for more comprehensive genome sequencing efforts.

407. Expression of laccase gene from Coriolus versicolor in transgenic tabacco for remediation. Yosuke Iimura¹, Seiichirou Ikeda², Tomonori sonoki², Shinya Kajita², Kenji Tatsumi¹, and Yoshihiro Katayama². ¹National Institute of Advanced Industrial Science & Technology, Ibaraki, Japan. ²Tokyo University of Agriculuture & Technology, Tokyo, 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 laccase gene from Coriolus versicolor .

408. The influence of AmyR on extracellular enzyme production by Aspergillus niger is NOT limited to enzymes involved in starch degradation. P.A. vanKuyk¹, J. Visser¹, A.M. Levin², H.A.B. Wösten², Ronald P. de Vries^1,2. ¹MGIM, Wageningen University, Wageningen, The Netherlands; ²Microbiology, Utrecht University, Utrecht, The Netherlands

The role of AmyR in the regulation of starch degradation has been studied in detail in A. oryzae and A. nidulans. AmyR regulates genes encoding glucoamylase, alpha-amylase and alpha-glucosidase resulting in production and secretion of the enzymes. A. niger wild type, and amyR disruptant and multicopy strains were compared for gene expression, protein secretion, extracellular enzyme activity, morphology and growth. Production of starch degrading enzymes was strongly reduced in the amyR disruptant and increased in the amyR multicopy strains. Surprisingly, production of some extracellular enzymes, not involved in starch degradation(e.g. beta-galactosidase) is influenced in a similar manner. This was confirmed by protein secretion profiles. In addition, the expression of sugar transporter encoding genes was influenced by AmyR. No strain differences were observed with respect to growth and morphology during growth on D-fructose, D-xylose, or glycerol. However, on starch, maltose, cellulose and lactose growth was strongly reduced for the amyR knockout, while the amyR multicopies showed enhanced growth, but reduced sporulation. This indicates that AmyR not only affects production of starch degrading enzymes, but also of other extracellular enzymes involved in hydrolysis of other oligo- and polysaccharides.

409. Aspergillus sojae, a new system for efficient protein production. Margreet Heerikhuisen, Anneke Drint-Kuyvenhoven, Alwin Albers, Nick van Biezen, Cees van den Hondel, Peter Punt. TNO Nutrition and Food Research, Department of Applied Microbiology and Gene Technology, Utrechtseweg 48, Zeist 3704 HE, The Netherlands.

Aspergillus species are known as high-level expression hosts for the production of enzymes or metabolites. In the last two decades for the industrially used species forAspergillus niger, A. foetidus, A. tubigensis, and A. oryzae expression systems have been developed. We describe the development of an expression system based on the koji mold Aspergillus sojae for the production of homologous and heterologous proteins. Transformation based on auxotrophic (pyrG, niaD) and/or dominant markers (amdS), was used to introduce the genes of interest. To improve the yield of produced protein, Aspergillus sojae mutants with lower protease activity, either by gene disruption or by UV mutagenesis, were isolated. To improve the fermentation yields of Aspergillus sojae also morphological and so-called fermentor adapted mutants with lower viscosity, were isolated. As one of the examples for heterologous protein production the production of human interleukin 6 was used. References: Heerikhuisen, M. et al. (2001) World Patent Application WO 01/09352

410. Optimization of the Agrobacterium-mediated Fruiting Body Tissue Method for the Transformation of Agaricus bisporus. Michelle Stone,¹ Carl Schlagnhaufer,¹ Lori A. MacDonald,² Mark P. Wach,² and C. Peter Romaine.^{1 1}Department of Plant Pathology, Penn State University, University Park, PA and ²Sylvan Inc., Kittanning, PA.

We previously described a highly effective and convenient transformation system for the button mushroom, Agaricus bisporus, involving Agrobacterium-mediated delivery of DNA to fruiting body tissue (Chen et al. 2000. Appl. Environ. Microbiol. 66:4510). Herein, we have explored several parameters of the method, which when optimized have provided upwards of 100% transformation efficiency (TE) as measured by fraction of the fruiting body tissue pieces expressing a hygromycin resistance (hph) selectable marker gene. Experiments were conducted using an A. tumefaciens strain (AGL-1) carrying a binary plasmid vector (pBHg) containing the hph gene controlled by the A. bisporus promoter for the glyceraldehyde 3-phosphate dehydrogenase gene. In two independent research laboratories, reproducibly high TEs in the range of 30-100% were obtained with (1) induction of the bacterium for 2 to 24 hr at 20-25 C, and (2) co-cultivation of the bacterium and fruiting body tissue for 3 to 4 days at 18-26 C. Further, there was a trend towards higher rates of transformation using gill tissue (73% TE) as opposed to the spongy tissue derived from the caps and stems (44% TE) of fruiting bodies. Comparably high efficiencies were observed using either gill or spongy tissue from a sporeless mushroom strain (~90% TE) and either undeveloped or developed gill tissue of several commercial hybrid strains (~70% TE). The described method represents a facile tool for introducing genes into A. bisporus and may well be applicable to other fungi bearing fleshy fruiting bodies.

411. A Metabolic Engineering Tool for the Filamentous Fungus Aspergillus nidulans. Guang Yi Wang and Jay D. Keasling. Department of Chemical Engineering, University of California at Berkeley, Berkeley CA, USA.

Filamentous fungi are a prolific source of secondary metabolites. This feature along with their physiological and metabolic diversity and available fermentation protocols makes fungi a promising metabolic engineering host for the production of valuable pharmaceutical agents from fungi and other eukaryotic organisms. Nevertheless, metabolic engineering of filamentous fungi often requires introduction of multi-gene pathways under the control of fungal regulatory elements into auxotrophic strains. On the other hand, increases in numbers of genes and regulatory elements in the transformation vector always enhance the high efficiency of ectopic integrations and greatly decrease the targeted homologous recombination and transformation efficiency in DNA-mediated transformation. To overcome these obstacles, we have adapted a cosmid mediated transformation approach (NAR, 28:E97) to introduce multiple isoprenoid-gene pathways into the fungal genome. The comsid carrying the multiple isoprenoid genes was engineered with the A. nidulans cosmid carrying ArgB gene and the engineered isoprenoid gene expression cassettes using the Red system developed in E. coli (PNAS, 97:6640-6645). We present results of using this approach to introduce isoprenoid-gene pathways into A. nidulans for the production of isoprenoids.

412. Cloning and Expression of Glycosyl Hydrolase Cel7 family members. Paulien Neefe¹, Vicky Huynh², Peter Gualfetti² and Frits Goedegebuur¹. ¹ Genencor International BV, Archimedesweg 30, 2333CN Leiden, the Netherlands. ² Genencor International Inc., 925 Page Mill Road, Palo Alto, Ca 94304, USA

The enzymatic degradation of cellulosic biomass to glucose is an important step for utilizing renewable resources for the creation of bioproducts. This process is performed in nature by a complex mixture of cellulolytic enzymes. Within the cellulase mixture produced by Trichoderma reesei, CBHI (Hypocrea jecorina Cel7A) is ~50% of the secreted protein and is an essential enzyme for cellulose degradation. To improve this molecule, a program has been started to identify mutants with changes in activity and in thermal stability. In addition, many previously identified CBHI homologs have been cloned, sequenced and expressed in a heterologous host. Those Cel7 proteins that were successfully expressed were purified and characterized in terms of their stability and activity.

413. Cloning of the Chrysosporium lucknowense CBH I (Cel7A) gene and characterisation of the encoded enzyme. Alexander V. GUSAKOV*, Tatyana N. SALANOVICH*, Fedor E. BUKHTOJAROV*, Alexander V. MARKOV*, Boris B. USTINOV*, Cora van ZEIJL**Peter PUNT**, Richard BURLINGAME† and Arkady P. SINITSYN*. *Department of Chemistry, M. V. Lomonosov Moscow State University, Moscow 119899, Russia, **TNO Nutrition and Food Research, P. O. Box 360, 3700 AJ Zeist, The Netherlands, †Dyadic International, Inc., 140 Intracoastal Pointe Drive, Suite 404, Jupiter, Florida 33477-5094, USA

Chrysosporium lucknowense, an ascomycetous cellulolytic fungus, is currently used for the commercial production of hemi(cellulases) and is being developed as a system for the expression of heterologous genes and gene discovery by high-throughput functional expression. Sequence analysis of peptides isolated from the major cellulase secreted by C. lucknowense showed similarity to published cellobiohydrolases. PCR amplification with primers based on these peptides resulted in the isolation of a DNA fragment homologous to cellobiohydrolases belonging to family 7 glycoside hydrolases, which includes the well-studied CBH I of Trichoderma reesei. The PCR fragment was used for the isolation of the corresponding gene, which we designated cbh1. The primary amino acid sequence of the C. lucknowense CBH I protein was deduced from the gene sequence. Two forms of C .lucknowense CBH I (Cel7A) were purified from the culture filtrate (52 kD and 65 kD). Their content makes up about 20% of the total extracellular protein. Analysis of the enzymatic properties of the two proteins showed that the 52 kD enzyme displayed much lower Avicel and cotton hydrolysis rates than the 65 kD enzyme, indicating the absence of a cellulose binding domain in the 52 kD protein. The thermostability of the 65 kD enzyme was significantly higher than that of the 52 kD protein, and better than that of the intact CBH I isolated from a commercial T. reesei preparation. Development of a genetic system for C. lucknowense allows selective overexpression of the C. lucknowense cbh1 and other cellulase genes.

414. Gene cloning and characterization of a novel glutaminase from Aspergillus sojae. Kotaro Ito¹, Kenichiro Matsushima¹, Genryou Umitsuki², Yasuji Koyama¹. ¹Research and Development Division, Kikkoman Corporation, ²Noda Institute for Scientific Research

Glutaminase is an enzyme that catalyzes the hydrolysis of L-glutamine to L-glutamatic acid. Glutaminase plays an important role to enhance umami taste in fermented food production. Using EST information of Aspergillus oryzae, we cloned a novel glutaminase-encoding gene, AsgahA, from Aspergillus sojae which was similar to a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis. The structural gene was 1929 bp long with no intron. This glutaminase protein, AsGahA, had an amidase motif, and showed 36% homology to that of C. nodaensis. Introduction of multiple copies of AsgahA intoA. oryzae RIB40 resulted in over expression of glutaminase activity. AsGahA was located at cell surface. AsGahA was subsequently purified from over expressing strain, and characterized. The molecular mass was estimated as 67 kDa by SDS-PAGE while it was estimated as 135kDa by gel filtration chromatography, which indicated that native form of AsGahA was a dimer. Its pH optimum was 9.5 and its temperature optimum was 50 degrees C. Analysis of substrate specificity revealed that AsGahA catalyzed not only L-glutamine but also L-asparagine, and was revealed as a glutaminase-asparaginase.

415. Agrobacterium tumefaciens-mediated transformation: an efficient tool for gene replacement in Aspergillus awamori. C.B. Michielse, A.H.A. van Dijk, M. Arentshorst, A.F.J. Ram, 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

Gene disruption or gene replacement is often used to generate precise deletion mutants in order to assess a possible function to the deleted gene. A. tumefaciens, a plant pathogen, which is being used for the transformation of plants, yeast and filamentous fungi, was shown to be an efficient tool for gene targeting in Kluveromyces lactis (Bundock et al., 1999). To determine the efficiency of gene replacement in Agrobacterium-mediated transformation of A. awamori, a systematic study was performed. The hygromycin selection marker was flanked with promoter and terminator sequence homologous to pyrG of varies sizes (1000 to 50 bp). Homologous recombination frequencies were determined and compared to frequencies obtained with the PEG/CaCl2 transformation method. Homologous recombination frequencies with the Agrobacterium-system increased 6-fold compared to the conventional method with 1000 bp flanks (30% versus 5%, respectively). Shortening the flanks to 500 and 250 bp led to a decrease in recombination frequencies to 5 and 1%, but these frequencies were again higher compared to the conventional method. By altering the length of the left and right flanking regions, it was shown that a long left flanking region increases the percentage of homologous recombination. Based on these data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the left border of the T-DNA plays an important role in homology search and might serve as a starting point for integration. Bundock et al. (1999), T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis, MGG 261(1): 115-21.

416. A molecular approach for the development of a biosensor using Aspergillus nidulans. Milton Roque and Reinhard Fischer, Philipps-University of Marburg, Dept. of Microbiology and Max-Planck-Institute for Terrestrial Microbiology, Karl- von-Frisch-Str., D-35043 Marburg, Germany

The sensitive detection of heavy metals or pesticides in soil is a challenge for the development of biosensors. We are aiming to use the soil microorganism Aspergillus nidulans for this purpose. The strategy will be to isolate genes, which are highly expressed upon exposure of the living organism to the toxic compounds. The corresponding genes will be used to establish a reporter with the green fluorescent protein (GFP) as a tool. Genes which respond to general stress conditions can be used for a first evaluation of the contamination of a given soil and specific genes for a subsequent quantitative analysis. In order to develop a sensory system for general stress conditions, we have chosen superoxide dismutase (SOD). Northern blot analysis showed that sod-transcription is strongly induced in the presence of the herbicide diuron (100 ug/ml) and the primary metabolite 3,4 dichloroaniline (100 ug/ml), as well as the insecticide carbofuran (75 ug/ml) and the heavy metals cadmium (0.01, 0.2 and 1.0 ug/ml), mercury (2.5 ug/ml) and lead (5.0 ug/ml). In addition to transcript analyses, we fused 1.8 kb of the putative promoter region with GFP and introduced the construct in A. nidulans. In addition to the targeted approach, we constructed a differential library to isolate genes specifically induced upon diuron application. We used the SSH technique. We obtained fragments of genes with homology to glycosyltransferase, acetyl coenzyme A synthetase or multi drug resistance (MDR). The putative promoter region (~ 1.8 kb) from the MDR gene was transcriptionally fused to GFP. First results using the two reporter systems (SOD and MDR) will be presented.

417. Functional Analyses of Two Genes Encoding Catalytic Subunits of cAMP-Dependent Protein Kinase A (Pka) in Sclerotinia sclerotiorum. Wayne M. Jurick II¹, Martin B. Dickman², and Jeffrey A. Rollins¹ . ¹Department of Plant Pathology, University of Florida-Gainesville. ²Department of Plant Pathology, University of Nebraska-Lincoln

Cyclic AMP-dependent protein kinase A (Pka) is involved in regulating diverse biological processes in humans, mice, yeast, and filamentous fungi. Pka mutants in phytopathogenic fungi have been shown to affect appresorial formation, pathogenicity, and mycelial growth. Results of a previous study in Sclerotinia sclerotiorum indicated that compounds which increase intracellular levels of cAMP inhibited sclerotial formation and increased oxalic acid production. Pka was hypothesized to be involved in mediating these effects. To test this hypothesis, a genomic clone corresponding to the catalytic subunit of Pka (pkaS) was isolated and disrupted between subdomains II and III using a hygromycin cassette. Southern hybridization revealed that pkaS was a single copy gene and targeted disruption was confirmed by PCR and genomic DNA hybridization. Interestingly, pkaS mutants showed no morphological aberrations in sclerotial development, were pathogenic, and produced sclerotia in the presence of cAMP-amended medium. Multiple pka catalytic subunit genes have been characterized in Ustilago maydis (uka1 & adr1) and indentified in recently sequenced genomes of Neurospora crassa & Magnaporthe grisea. It is hypothesized that a additional pka gene encoding an additional catalytic subunit exists in S. sclerotiorum. Our current objective is to clone, disrupt and phenotypically evaluate a additional gene encoding a Pka catalytic subunit in S. sclerotiorum.

418. The Pc-mco1 gene of Phanerochaete chrysosporium encodes for a novel extracellular multicopper oxidase with ferroxidase activity. Luis F. Larrondo^1*, Loreto Salas¹, Francisco Melo¹, Dan Cullen², and Rafael Vicuña¹. ¹Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile and Millenium Institute for Fundamental and Applied Biology. ²USDA Forest Products Laboratory, Madison, Wisconsin 53705, USA

Lignin degradation by the white rot basidiomycete P. chrysosporium involves an array of extracellular oxidative enzymes, including lignin peroxidase, manganese peroxide and glyoxal oxidase. Recent studies suggest that laccases may also be produced, although this has been a controversial issue. A search of the P. chrysosporium genome database revealed four sequences distantly related to laccases and ferroxidases (Fet3). One of these multicopper oxidase sequences, Pc-mco1, is interrupted by 19 introns and is transcribed in defined media and in colonized wood. The cDNA is predicted to encode a mature MCO protein of 543 aa with a 16 aa secretion signal. Structural analysis and multiple alignments led to the identification of residues common to both laccases and Fet3. The recombinant MCO, expressed in Aspergillus nidulans, has a molecular weight of 78 kDa and the UV-visible spectrum confirms the presence of a copper I type center. The purified enzyme oxidizes a variety of compounds such as ABTS and aromatic amines, although it poorly oxidizes phenols. Its best substrate is Fe²⁺ (Km ~ 2 uM). Because only ceruloplasmin and Fet3 are known to oxidize Fe²⁺, we also searched the P. chrysosporium database for Fet3-like sequences. The P. chrysosporium Fet3 gene and corresponding cDNA were cloned and characterized. This is the first report of a fungal extracellular MCO capable of oxidizing Fe, but distinct from Fet3. Our results also firmly establish the view that P. chrysosporium lacks conventional laccase. This work was financed by grants 8990004 and 2000076 from FONDECYT-Chile, by the MIFAB-Chile, and by the U.S. Dept. of Energy grant DE-FG02-87ER13712. * Fellow from Fundacion Andes.

419. Fungal Genomics and Tools. Marco van den Berg, Remco Klasen, Herman Pel, Han de Winde and Roel Bovenberg DSM Life Science Products, DSM Gist (624-0270), PO Box 425, 2600 AK, Delft, The Netherlands

Fungi as Pencillium chrysogenum, Saccharomyces cerevisiae and Aspergillus niger are the most important fungi used for a the production of a range of products within DSM Life Science Products. In order to rationalize the ongoing improvement of production strains and processes, as well as to speed up the identification of potential new products, genome seqeunces and tools are essential to embarke onto 'omics'-type of R&D. Analyzing the genomic sequences of our favourite fungi resulted not only in a wealth of new genes, but more importantly it resulted in much more insight in the organisms themselves. To be able to study these, several new and old tools were adapted tofunction in the easy 'fungus' Saccharomyces, as well as in the more difficult fungus Penicillium. Within the penicillin gene cluster of Pencillium chrysogenum unexpected genes were found to be amplified in higher producing strains. Using a combination of molecular and biochemical technologies we identified and demonstrated the function of several proteins in relation to penicillin production. In the enzyme producing fungus Aspergillus niger over 14000 genes were identified, using a trained gene-prediction algorythm and all were cheked manually. At this moment relevant fermentations are being sampled and studied using Affymetrix gene chips, 2D proteome gels, cDNA sequencing, etc. WWW5.DSM.COM

420. Improvement of foreign protein production by constitutive UPR induction inAspergillus niger var. awamori. Mari Valkonen¹, Markku Saloheimo¹, Michael Ward² and Merja Penttilä^{1 1}VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland ²Genencor International, Inc., 625 Page Mill Rd., CA 94304-1013, USA

A signalling pathway from ER to nucleus called the unfolded protein response (UPR) controls the expression of genes for several endoplasmic reticulum-resident (ER) chaperones and foldases. When the unfolded protein response pathway is active, a signal is transduced from the ER lumen to the transcription machinery in the nucleus. It has been shown that the yeast transcription factor mediating the UPR induction of the chaperone and foldase genes is Hac1p. The UPR-induced form of the A. niger var. awamori hacA cDNA was expressed in A. niger var. awamori strain producing Trametes versicolor laccase. For the overexpression of hacA, the induced form of the A. niger var.awamori hacA cDNA was first created by deleting the 20 bp intron and truncating the 5' flanking region by about 150 bp, which omitted the upstream open reading frame. The laccase activity measurements made from the supernatants show that all the transformants produce more laccase than the parental strain. The production levels of the transformants were in the fifth day samples 3 to 7.6 fold higher than in the parental strain. On the seventh day of cultivation the transformants produced 2 to 5.4 fold more laccase than the parental strain. This indicates that the overexpression of HACA protein induces the production of heterologous proteins in A. niger.

421. Preliminary results of using Aspergillus nidulans microarrays to monitor gene expression in recombinant protein producing strains. Andrew Sims¹, Manda E. Gent¹, Geoffrey Robson¹, Nigel Dunn-Coleman², Rolf Prade³, Hugh Russell³, Stephen G. Oliver¹. ¹School of Biological Sciences, Stopford Building, University of Manchester, Manchester, UK.,²Genencor International Inc, 925 Page Mill Road, Palo Alto, CA 94304, USA.,³Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA

The genus of filamentous fungi, Aspergillus has a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However the secretory pathway, which is key to the production of extracellular proteins is rather poorly characterised. The use of microarrays in the analysis of gene expression is becoming widespread for many organisms. Although a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy for these organisms. Aspergillus nidulans microarrays have been produced and validated in our laboratory. Work has now begun using these cDNA arrays with a range of different approaches to profile the effects of recombinant protein secretion on gene expression levels. Batch shake flask cultures have shown that chaperones such as binding protein (bipA) and protein disulphide isomerase (pdiA) are over expressed during production of the industrially and economically relevant recombinant protein, bovine chymosin. Chemostat fermentations have also been performed to compare gene expression in recombinant protein producing and wildtype strains during steady-state growth.

422. Cellular responses to secretion stress in Trichoderma reesei. Markku Saloheimo, Tiina Pakula, Mikko Arvas, Mari Valkonen and Merja Penttilä VTT Biotechnology, P.O Box 1500, 02044 VTT, Espoo, Finland

Trichoderma reesei is known for its extremely high capacity of protein secretion. High loads of protein, and especially foreign protein, in the secretory pathway form a challenge to the production organism and expose it to secretion stress. Unfolded protein response (UPR) denotes the induction mechanism of genes encoding ER-resident chaperones and foldases and numerous other genes involved in protein secretion. This induction is triggered when unfolded proteins accumulate into the ER. We have cloned the transcription factor involved in UPR induction, HACI, from T. reesei. Our results indicate that the hac1 gene is activated by a dual mechanism operational at the mRNA level. This mechanism includes a splicing event of an unconventional intron of only 20 nt in length and a truncation of the mRNA at the 5' flanking region. This truncation removes an upstream open reading frame from the mRNA, and we have shown that these uORFs are involved in translational control of the HAC1 protein formation.

We have observed that concurrently with the induction of the UPR pathway, the genes encoding secreted proteins are rapidly down-regulated in Trichoderma reesei. This type of regulation can be caused by different secretion inhibitors and by foreign protein expression. The down-regulation is dependent on the promoter of the affected gene, suggesting that it is functional at the transcriptional level. The down-regulation of genes encoding secreted proteins during secretion stress has not been reported before from any other experimental system and thus it could be unique for filamentous fungi.

423. Genomic Organization, Transcript Analysis and Heterologous Expression of a Gene Family Encoding Cu-radical Oxidases (CRO) in the White Rot Basidiomycete Phanerochaete chrysosporium. Amber Vanden Wymelenberg ¹, Philip Kersten ², Robert Blanchette ³ and Daniel Cullen ^{1,2 1} Department of Bacteriology, University of Wisconsin-Madison, USA, ²USDA Forest Service, Forest Products Laboratory, Madison, Wisconsin, and³Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota.

The white rot basidiomycete Phanerochaete chrysosporium is able to efficiently degrade all major components of wood: cellulose, lignin and hemicellulose. The oxidative enzymes thought to be involved in lignin degradation by this model system include lignin peroxidases (LiP), manganese peroxidases (MnP), and the peroxide-generating enzyme glyoxal oxidase (GLOX). Recently, a draft P. chrysosporium genome sequence has been made publicly available (http://www.jgi.doe.gov/programs/whiterot.htm). Blast searches of this database unexpectedly revealed at least five additional GLOX-like sequences. Transcripts were detected in various defined culture media and from P. chrysosporium-colonized wood. Full length cDNAs were cloned and sequenced. Comparisons of these sequences to GLOX and to related Cu-related oxidases showed close structural similarities around the active site. Interestingly, three highly conserved CRO sequences are located within a LiP gene cluster. Designated cro3, cro4, and cro5, all three feature a conserved N-terminal putative carbohydrate binding domain. Active enzyme corresponding to cro3 has been produced in Aspergillus nidulans under the control of the A. niger glucoamylase promoter. Substrate specificity of the cro3 protein is under investigation.

424. Role of the bga1-encoded extracellular beta-galactosidase of Hypocrea jecorina in lactose metabolism and cellulase induction. Bernhard Seiboth¹, Lukas Hartl¹, Noora Salovuori², Jari Vehmaanperä², Merja E. Penttilä² and Christian P. Kubicek¹. ¹ Institute of Chemical Engineering, TU Wien, Wien, Austria. ² VTT Biotechnology, Espoo, Finland

Lactose is the only economic carbon source for protein production under the control of cellulase promoters by the ascomycete H. jecorina (anamorph: Trichoderma reesei). However, the mechanism by which lactose triggers cellulase formation is not understood. We have investigated the role of beta-galactosidase in lactose metabolism and cellulase induction in H. jecorina. A genomic copy of the bga1 gene predicting a protein with a MW of 111 kDa (incl. a signal sequence) was cloned using degenerate primers. The Bga1 belongs to the Glycosyl hydrolases family 35. Transcriptional analysis of the bga1 expression shows that it is highly expressed on L-arabinose, D-galactose and lower on lactose. Deletion of the gene showed that it is not essential for growth on lactose but that knock-out strains grew slower and produced about half the biomass of the wt strain, whereas amplification of the bga1 gene under the pki1 promoter resulted in faster growth with a reduced lag phase. Bga1 is not necessary for lactose-dependent induction of cbh1 gene expression in H. jecorina but its overexpression impairs lactose-induction of cbh1 expression. Analysis of the deletion strain further showed that bga1 encodes the major extracellular beta-galactosidase which is also partially cell-wall bound. An additional cell wall-bound beta-galactosidase activity could be shown but no evidence for an intracellular beta-galactosidase was obtained.

425. Mutants of Neurospora crassa deficient in secreted protease activity. Edward B. Cambareri, Juan P. Montufar and W. Dorsey Stuart. Neugenesis Corporation, San Carlos, CA 94070-3389

Heterologous proteins produced by secretion in filamentous fungi are often quickly degraded by endogenous extracellular proteases. As part of our strain-improvement program we have induced, by UV as well as by integrative mutagenesis, a number of mutants that show decreased extracellular protease activity. Mutants were isolated by screening for colonies with decreased halo size on plates containing casein and gelatin. Chromatographic analysis demonstrated distinct loss of some species of media proteins that normally bind to a bacitracin protease affinity column. As reported by others, the genetic marker associated with the DNA mutagen does not always co-segregate with the mutant phenotype. Results of gene-disruption of a putative transcriptional regulator that may control secreted protease activities, related to the prt-13 factor of A. niger, also will be presented.

426. Cytochrome P450 system genes in the white rot fungus Phanerochaete chrysosporium: cloning, differential regulation, and expression. Jagjit S. Yadav, P. Mishra, M. Safaie, V. Subramanian, and H. Doddapaneni Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0056, USA

 P. chrysosporium has been widely studied for its unique ability to biodegrade the plant polymer lignin and a broad range of recalcitrant organic pollutants, with a focus on its exo-oxidases. Lately, cytochrome P450 enzyme systems have been reported to be critical in several of these biotransformations. Characterization of the P450 systems will help understand overall metabolic pathways of biodegradation in this and other white rot fungi and their potential use in environmental or industrial applications. Our efforts have included cloning of the first three complete P450 monooxygenase genes, PC-1, PC-2, and PC-3 and their 2 splice variants (Fungal Genet. Biol.) and the P450 reductase gene, POR (Curr. Genet. 2000). Subsequently, recent whole genome sequencing has led to the identification of over 150 cytochrome P450 genes in this organism, the highest among lower eukaryotes. Real-time quantitative RT-PCR analysis showed that PC-1 is differentially regulated by nutrient levels; the transcript level was several fold higher in nitrogen-limited cultures than in N-rich or N- and C-rich cultures. POR expression did not seem to be as tightly regulated by the N & C conditions. Temporal analysis showed that the cloned genes are expressed in both primary and secondary metabolic phases of growth. PC-1 transcription was shown to be induced in presence of alkyl-substituted aromatics. The POR gene was heterologously expressed in active form in E. coli and the recombinant enzyme (82 kDa) was purified. Transcriptional analysis also showed its expressibility in S. cerevisiae. In an initial microarray analysis, about 30 of the selected 86 P450 monooxygenase genes were shown to be transcriptionally expressed.

427. Production of full-length antibodies and antibody fragments in Aspergillus niger. H. Wang, C Lin, D. Victoria, B. Fox, B. Fryksdale, D. Wong, H. Meerman, J. Pucci, M. Heng, X. Wang, R. Fong, and M Ward. Genencor International, Palo Alto, CA

Genencor International has previously developed methods for efficient secretion of foreign proteins in filamentous fungi, including Aspergillus niger. We have now demonstrated production of full-length humanized IgG1 and Fab' in A. niger. Both light chain and heavy chain were fused to the catalytic domain of the glucoamylase in separate plasmids and co-transformed into A. niger strains that were deleted for the glucoamylase gene. Antibody was subsequently cleaved off from the glucoamylase throught the engineered kex2 process sites. Yields of humanized IgG1 are competitive with those in mammalian cell systems and much higher than previously reported for other microbial hosts. The composition of the N-linked glycan added to the Fc region of human IgG1 by A. niger is of the high-mannose type and thus differs from the complex glycan structure of mammalian cells. We believe that A. niger will provide an attractive alternative for manufacturing some therapeutic antibodies.

428. Analysis of heterologous expression from Aspergillus nidulans gpdA andalcA promoters. David Lubertozzi and Jay D. Keasling, Dept. of Chemical Engineering, Univ. of Ca. Berkeley

Random integration of transforming DNA is common is Aspergillus, resulting in multiple copies throughout the genome. To study the effects of position, copy number, and promoter character on expression, A. nidulans strains were constructed with single- and multi-copy integrations of a plasmid bearing an expression cassette consisting of an Aspergillus alcA (inducible), or gpdA (constitutive) promoter; the E. coli lacZ gene; and the Aspergillus trpC terminator. Homologous integration to the target loci was selected for by transformation with a truncated or mutated version of the wild-type genes, such that ectopic integration results in a null phenotype.

Integration of the lacZ gene was confirmed by PCR amplification of a fragment oflacZ and of the argB gene as control. Genomic locus and copy number were determined by Southern blotting of genomic restriction digests and probing with a³²P-labeled lacZ fragment. A rapid method to quantify transgene dosage utilizing real-time PCR was developed. A single-copy native gene (trpC) and lacZ were simultaneously amplified from genomic template from the transformants; by comparing the product curves to a series of standards, the amount of starting template DNA and hence gene copy number was determined. A tentative linear relation appeared for beta-galactosidase expression levels versus lacZ copy numbers, excepting a group of high expressing clones at ~10 copies.

429. Structural and expression analyses of retrotransposons from Aspergillus oryzae. Katsuya Gomi and Motohiro Sato. Agriculture, Tohoku University, Sendai, Japan.

We found an EST clone of Aspergillus oryzae highly homologous to the gene encoding reverse transcriptase (RT) of the LINE-like DNA element in a fungus, Ascobolus immersus. This EST clone seemed to be transcribed from the gene in an A. oryzae LINE-like element, and to be involved in its transposition through an RNA intermediate. Southern blot analysis showed that A. oryzae RIB40 has two copies of the DNA fragment hybridizing to the EST clone. Screening of the genomic library with the EST clone as a probe resulted in isolation of two positive phage clones, which are derived from different chromosome loci. Sequencing analysis of the DNA fragments inserted in the isolated phage clones revealed the existence of two different types of the LINE-like element, designated Aoret1 and Aoret2. One phage clone contained whole putative retrotransposon, Aoret1, and the other had Aoret2 about 6-kb downstream of Aoret1. Both putative retrotransposons contained two long ORFs encoded on the strand of the same direction. First ORF (ORF1) encoded a putative protein containing cysteine-rich motifs near the C-terminal region commonly found in gag-like proteins. The second ORF (ORF2) encoded a protein homologous to the RT. Northern blot analysis using a respective ORF2 of both retrotransposons as a probe revealed that major transcripts of approximately 5.5-kb in length were transcribed from the both retrotransposons. The transcripts were somewhat larger than the expected size for ORF2, but coincided with the full-length transcripts used as a LINEs RNA transposition intermediate. Thus if two proteins, gag-like protein and RT protein, would be produced, they might be translated from a single full-length transcript.