Industrial Biology and Biotechnology
213. Identification and transcriptional regulation of new starch modifying enzymes in the Aspergillus niger genome. X. Yuan1, R. Van der Kaaij2, E. Martens3, C.A.M.J.J. van den Hondel1,4, and A.F.J. Ram1,4. 1 Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Leiden, The Netherlands. 2University of Groningen, Microbial Physiology Research Group, Haren, The Netherlands. 3 Wageningen University, Section Fungal Genomics, Wageningen, The Netherlands. 4 TNO-Nutrition and Food Research, Department of Microbiology, Zeist, The Netherlands.
Aspergillus niger is well known for its ability to secrete a wide variety of plant Carbohydrate Modifying Enzymes (CMEs). The secreted CMEs are able to degrade the plant cell wall materials as well as its storage carbohydrates, like starch. Based on its recently finished genomic sequence by Dutch life sciences company DSM, it is estimated that only a fraction of the potential of enzymes produced by A. niger is currently exploited.
Database mining of the A.niger genome resulted in the identification of twelve new enzymes, which contain motifs indicating that they might act on starch. In addition to the known starch degrading enzymes of A.niger, glucoamylase (glaA), two alpha-amylase (amyA and amyB), an acid amylase (aamA) and an alpha-glucosidase (aglU), six putative alpha-amylases, five putative alpha-glucosidases and one alpha1,4-glucan branching enzyme were identified. It is well established that the expression of starch modifying enzymes is coordinately regulated at the transcriptional level by a pathway specific transcription factor AmyR. AmyR binds to sequences (CGGN 8 CGG or CGGN 8AGG) in the promoter region of amylolytic genes. Inspection of the promoter regions of the newly identified amylolytic genes revealed that only five of them contain AmyR consensus binding sites. The expression of the newly identified genes in relation to the presence of starch and the role of AmyR during regulation has been examined using microarrays and will be presented.
214. The Fungal Hydrophobin RolA Bound on Hydrophobic Surfaces Recruits Esterase. Toru Takahashi1, Hiroshi Maeda2, Sachiyo Yoneda1, Shinsaku Ohtaki1, Yohei Yamagata1,2, Fumihiko Hasegawa2, Katsuya Gomi1,2, Tasuku Nakajima1,2, and Keietsu Abe1,2,*, 1Grad. Sch, of Agricul. Science, 2The New Indust. Creat.Hatchery Cent., Tohoku Univ., Sendai, Japan
When fungi grow on plant or insect surfaces coated with wax polyesters as protectants against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Hydrophobins, which are surface-active proteins found in fungi, coat aerial structures such as hyphae or conidiophores and are involved in adhesion to hydrophobic surfaces. When an industrial fungus Aspergillus oryzae was cultivated in liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the hydrophobin RolA and cutinase CutL1, which hydrolyzes PBSA, were simultaneously expressed. High levels of RolA and its localization on the cell surface in the presence of PBSA were confirmed by immunostaining. Preincubation of PBSA with RolA stimulated PBSA degradation by CutL1, suggesting that RolA bound to a PBSA surface was required for the stimulation. Immunostaining revealed that PBSA films coated with RolA specifically adsorbed CutL1. Quartz crystal microbalance analyses further demonstrated that RolA attached to a hydrophobic sensor chip specifically adsorbed CutL1. These results suggest that RolA adsorbed to the hydrophobic surface of PBSA recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis.
215. Heterologous Protein Expression in a Filamentous Fungus. Silke Alves and Frank Kempken. Abteilung für botanische Genetik und Molekularbiologie, Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany
In the last years filamentous fungi have become more important in expression of biotechnologically used proteins. They are capable to secrete a broad range of different proteins into the supernatant of the culture medium. Their ability to perform post-transcriptional modifications makes them an attractive host for the production of heterologous proteins. However, the amount of heterologous proteins rarely exceed more than a few milligrams per litre, due to problems during the secretion process and extracellular protease activity of the fungi. In this study, we constructed a set of different expression-vectors that enable us to exchange different genes, promoters and terminators without difficulties. To investigate the applicability of these expression-system, we tested the expression of eGFP and several other genes in filamentous fungi. Each expressed gene includes a specific fungal export-signal, which enables the secretion of the processed protein into the supernatant. Different promoters are being employed and tested under various physiological conditions.
216. Directed evolution of barley beta-D-glucan endohydrolases. Graham Eariss 1, Maria Hrmova 2, Geoffrey Fincher 2 & David E. A. Catcheside1. 1School of Biological Sciences, Flinders University, Adelaide, South Australia. 2Department of Plant Science, University of Adelaide, South Australia.
We are using an in vivo gene diversification technique developed in Neurospora crassa to investigate the functional divergence of a barley (1-3) and (1-3,1-4)-beta-D-glucanase, and to attempt to produce a (1-3,1-4)-beta-D-glucanase with enhanced thermostability. The primary sequences and three dimensional structures of the barley (1-3)-beta-D-glucan endohydrolase isozyme GII and (1-3,1-4)-beta-D-glucan endohydrolase isozyme EII imply a close relationship in evolutionary terms, yet they perform completely different functions. While the (1-3)-beta-D-glucanase capable of hydrolyzing the linear, substituted and branched (1-3)-beta-D-glucans often found in fungal cell walls appears to be involved in plant protection, the (1-3,1-4)-beta-D-glucanase is responsible for digestion of the starchy endosperm cell wall during germination of barley grains. Possibly in response to the hostile environment it encounters, the (1-3)-beta-D-glucanase has evolved to be significantly more thermostable than the (1-3,1-4)-beta-D-glucanase.
217. Multiplex PCR for the detection of ochratoxigenic fungi in coffee beans. Maria Helena Pelegrinelli Fungaro*; Daniele Sartori, Márcia Cristina Furlaneto.Centro de Ciências Biológicas, Universidade Estadual de Londrina, P.O.Box, 6001, 86051-990 Londrina-PR, Brazil.
Ochratoxin A (OA) is a nephrotoxic and carcinogenic mycotoxin that has been found in cereal and food commodities. This mycotoxin was originally described as a secondary metabolite of Aspergillus ochraceus strains, but in subsequent years other Aspergillus and Penicillium species were described as OA producers. Nowadays, Aspergillus carbonarius, A. niger and A. ochraceus have been recognized as culprit species of OA in coffee beans. This situation demonstrates the importance of using appropriate methods to control the mycological status of coffee beans. The use of DNA markers for easy and rapid detection of these species in the interior of coffee beans can be useful in order to substitute the conventional methods, which are based in fungal cultivation and requires an expertise in taxonomy. In this study, strains representing closely related black aspergilli species were analyzed by molecular markers aiming at the development of specific primers for detection of A. niger in coffee beans. The primer pair designed in this study and other two primers pair developed for A. carbonarius and A. ochraceus were used in the multiplex-PCR optimization. Our group has successfully used this method in order to assess the presence of these ochratoxigenic species in coffee beans samples.
Acknowledgement CAPES/CNPQ/Fundação Araucária/CPG-UEL
218. Expression and characterization of xylanases from Aspergillus fumigatus. Haiyan Ge, Janine Lin, Suzie Otani and Debbie S. Yaver. Novozymes Biotech, Inc, Davis CA.
Xylanase, which catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans, are used in the food industry, primarily for baking and in fruit and vegetable processing such as fruit juice production or wine making. Using bioinformatics, three putative GH10 xylanase genes, xyn1,xyn 2, and xyn3, were discovered in A. fumigatus genome. The alignment showed that the deduced amino acid sequence of xyn1 shared 72% identity to NCU08189.1 of the Neurospora crassa; the deduced amino acid sequence of xyn2 shared 54% identity to XynF1 of the Aspergillus oryzae, and the deduced amino acid sequence of xyn3 shared 67.8% identity to Xynia of the A. aculeatus. We successfully cloned and expressed the three putative xylanases in A. oryzae. Xylanase activities were assayed using AZCL-arabinoxylan as substrate. On SDS-PAGE gel, proteins with expected sizes bands were observed. Further characterization of the xylanses will be presented.
219. Directed Evolution of Human Growth Factors in Neurospora crassa. Steven T. Henderson1,2, Leah J. Cosgrove2, Colin W. Ward3 & David E. A. Catcheside1. 1Flinders University, Adelaide, SA 5042, Australia; 2CSIRO Division of Health Sciences and Nutrition, Adelaide, SA 5000, Australia; 3CSIRO Division of Health Sciences and Nutrition, Parkville, VIC 3052, Australia.
This project aims to generate novel human growth factors (hGF) by utilising the in vivo gene diversification system developed for the filamentous fungus, Neurospora crassa. Directed evolution of hGFs in Neurospora will utilise the high frequency of meiotic recombination initiated at the cogL recombination hotspot to diversify hGF DNA sequences juxtaposed to cogL during a sexual cross. Specifically, targeting vectors will be used to transplace a functional hGF gene between his-3 and cogL. Repeat-Induced Point mutation (RIP) is a natural phenomena occurring in the pre-meiotic phase of Neurospora's sexual stage that results in G:C to A:T transition mutations in duplicated sequences. Thus, an additional non-functional hGF gene will be ectopically transformed into Neurospora to induce low frequencies of RIP, generating hGF alleles in vivo. Additional hGF variation is created by subsequent meiotic recombination shuffling the hGF alleles created by RIP. This has the additional advantage of potentially separating deleterious mutations. Progeny from the cross will be screened to identify novel hGF variants.
220. Aspergillus nidulans uvsC and genetic transformation. Yasuo Itoh. Shinshu University, Nagano, Japan
Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Aspergillus nidulans uvsC is an ortholog of budding yeast RAD51 that plays central roles in recombination-based repair and meiotic recombination. Occurrence of gene targeting at argB locus was under a detection limit in a null mutant of uvsC (less than 5% of control strains) when randomly chosen transformants were examined for their integration profiles. Other differences were also observed in the integration profiles during genetic transformation that were consistent with the expected functions of uvsC. Transcription of uvsC was elevated using the 5f sequences of the glyceraldehyde-3-phosphate dehydrogenase (Pgpd) and Taka-amylase A (Ptaa) genes from A. nidulans and A. oryzea, respectively. Maximum of 5-fold increase in the efficiency of targeting was observed at argB, yA, and wA loci when uvsC was under control of Pgpd. Higher level of transcription was achieved with Ptaa, but at the inducible condition with maltose, mycelial growth was significantly suppressed. These results demonstrate that uvsC is involved in integration of extra-cellular DNA integration into chromosomes and is a possible rate-limiting factor for gene targeting. However the increased efficiency of gene targeting is hindered by a deleterious effect of increased transcription on cell proliferation.
221. Targeted disruption of fungal genes based on a dual selection method. Chang Hyun Khang1, Sook-Young Park1, Hee-Sool Rho2, Yong-Hwan Lee2, and Seogchan Kang1. 1Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA; 2School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
The creation of gene knock-out (KO) mutants through homologous recombination is the most direct method to determine gene function. In filamentous fungi, however, this method is often hampered by the low efficiency of homologous recombination, which requires molecular analysis for a large number of transformants to identify gene KO mutants. We have developed an efficient gene KO method, which is based on Agrobacterium tumefaciens-mediated transformation with a binary vector containing a mutant allele of the target gene flanked by a negative selection marker, the herpes simplex virus thymidine kinase (tk) gene. Ectopic transformants are eliminated through a negative selection resulting from the expression of the tk gene, thus significantly enriching for gene KO mutants. The efficiency of gene KO by this method was evaluated using Magnaporthe grisea and Fusarium oxysporum. Although a significant enrichment of gene KO mutants was achieved in both fungi, false positives occurred mainly due to deletion of the tk gene on the T-DNA. An array of gene KO vectors were developed and tested for their efficiency in gene KO.
222. Overexpression of basidiomycetous enzymes in Coprinopsis cinerea. S. Kilaru, M. Rühl*, A. Saathoff, R. C. Dwivedi, M. Zomorrodi, K. Lange, A. Majcherczyk, P. Hoegger and U. Kües. Georg-August-University of Göttingen, Institute of Forest Botany and NHN*, 37077 Göttingen, Germany
Basidiomycetous fungi are the source of many biotechnologically interesting enzymes, e.g. laccases, peroxidases, and others. When such enzymes are attempted to be overexpressed in ascomycetous fungi, they often show low yields and have altered properties, most probably due to improper posttranslational modifications. A commonly observed problem is the overglycosylation of the proteins. Therefore, we developed an efficient expression system in Coprinopsis cinerea as a basidiomycetous host. The laccase gene lcc1 of C. cinerea served as a tool to establish the best activities among various homologous and heterologous promoters. Enzymes produced under control of foreign promoters exhibit natural extents of glycosylation. Various laccases with different activities and specificities have now been obtained from recombinant C. cinerea. With our expression system, we were able to produce much higher amounts of enzyme than obtained from natural inductive promoters in this and other white rot fungi. The purification, biochemical characterization and comparison of the different overexpressed laccases is now in process.
Our laboratory is supported by the Deutsche Bundesstiftung Umwelt (DBU), the NHN by the European Regional Development Fund and the country of Lower Saxony.
223. Characterization of the telomere-attached vectors and their transformants of Aspergillus oryzae. Ken-Ichi Kusumoto, Ikuyo Furukawa, Tae Kimura, Mayumi Matsushita, Satoshi Suzuki, and Yutaka Kashiwagi (National Food Research Institute, JAPAN)
The authors isolated the telomere sequences at the chromosomal terminal regions of Aspergillus oryzae, and showed that their structure was a repeated sequence constituted of 12bp repeat unit (TTAGGGTCAACA) (AMB61: 247, 2003). As telomere is participated in the replication of the chromosomal DNA and its stabilization of their ends, development of new type of the replicating vectors is expected. The replicating function of the telomeres of A. oryzae and the conservation of the chromosomal ends by them are not investigated yet. In this study, we constructed the two types of the telomere-attached vectors to examine their function. The vector I was constituted of single copy of TEL134, a clone of A. oryzae telomere (TTAGGGTCAACA)10 with its associated sequence, inserted into a vector of a integrated type, pPTRI (ptrA as a marker gene, TaKaRa Bio, JAPAN). The vector II was constituted of two copies of TEL134 inserted into pPTRI, where the two TEL134 were inserted as inverted repeat. A 0.8 kb DNA fragment derived from a vector for Escherichia coli was put as a spacer sequence between those TEL134 DNA. Namely, the spacer sequence was positioned as vector-(TTAGGGTCAACA)10-spacer-(TGTTGACCCTAA)10-vector. A. oryzae NFRI1599 were transformed with those telomere-attached vectors and pPTRI as a control plasmid. We are going to characterize the structure of the introduced vectors and the transformants.
225. A transfer of key primary metabolism genes of Aspergillus niger into Aspergillus terreus. G. Tev1, M. Benèina1, A. Kern2, A. Glieder2, M. Legiša1. 1National Institute of Chemistry, Hajdrihova 19, Si-1001 Ljubljana, Slovenia. 2Institute for Molecular Biotechnology, Technische Universitaet Graz, A-8010, Graz, Austria
In Aspergillus niger, one of the most important industrial micro-organisms, strong anaplerotic reactions were found to be responsible for high productivity of primary metabolites. Two enzymes, 6-phosphofructo-1-kinase (PFK), a key regulatory enzyme of glycolytic pathway, and alternative oxidase, a membrane protein that uncouples NADH re-oxidation from ATP formation, have been recognised to play a key role in replenishing citric acid cycle intermediates. After a transfer of genes coding for both enzymes into Aspergillus terreus cells, a similar effect on the level of tricarboxylyc acid cycle intermediates was expected to be achieved and concomitantly increased productivity and/or yield of specific A. terreus bio-products. The genes pfkA coding for 6-phosphofructo-1-kinase and aox1 carrying the information for synthesis of alternative oxidase were isolated from A. niger gene library by the aid of the known sequence. Namely, both genes have been previously isolated, cloned and sequenced by other laboratories. The genes together with their native promoter and terminator regions have been integrated into appropriate vectors. Heterologous pfkA gene was introduced into A. terreus protoplasts by co-transformation with a plasmid carrying hygB gene as a marker for hygromycine resistance, while double transformants were isolated after additional co-insertion of a plasmids carrying aox1 and amdS gene. The integration of genes was confirmed by standard recombinant DNA techniques. In cell free extract of the modified strain higher specific activity of PFK was detected by comparing to the parental strain and significantly higher sensitivity of double transformant was recorded toward SHAM, a specific inhibitor of alternative oxidase. The obtained results indicate that A. niger genes could be efficiently expressed in other Aspergillus species, while their effect on primary metabolism will have to await further studies.
226. Purification, characterization, and expression of AP025 protease from Thermoascus aurantiacus. Janine Lin, Wenping Wu, Jan Lehmbeck, and Debbie Yaver. Novozymes Biotech, Inc, Davis CA.
A metalloprotease AP025 was purified from culture broth of a thermophilic fungus Thermoascus aurantiacus. The AP025 protease has high optimal temperature (70̊C), and is most active at low pH (pH 6). The enzyme is most stable at pH 4, but is also stable from pH 4-10. The AP025 protease may have potential in industrial applications. The gene encoding the protease AP25 was cloned and sequence analysis showed that this protease is a metalloprotease. The cDNA of the AP025 gene was successfully expressed under two variants of Aspergillus niger amylase promoter in Aspergillus oryzae and in Fusarium venenatum under the control of the F. venenatum glucoamylase promoter. Expression of AP025 under a stronger promoter in A. oryzae resulted in the highest yield of AP025.
227. Genetic stability of direct and inverted chromosomal repeats in Aspergillus nidulans. Michael Lynge Nielsen, Line Albertsen & Uffe Mortensen, Center for Microbial Biotechnology, BioCentrum-DTU, DK-2800 Kgs. Lyngby, Denmark
Homologous recombination facilitates error free repair of DNA double strand breaks because it uses a homologous sequence as a template for the repair. However, most genomes contain repetitive sequences such as transposable elements, rDNA, centromeric repeats, etc, which in some cases cause inadvertent homologous recombination leading to chromosomal translocations or genomic instabilities. In many filamentous fungi, including Aspergillus nidulans, transformation of recombinant DNA often leads to the integration of multiple copies of this DNA either as inverted or tandem repeats. For many industrial applications transformants with multiple gene copies are obtained from screening because such strains often have high gene expression levels, but consequently, the repeated sequences result in genetic instability. For filamentous fungi there are no accurate methods for assessing this type of genetic instability. Therefore, we have developed a sensitive conidiospore color assay in A. nidulans that allows the detection of as little as a single spontaneous or induced recombination event within a fungal colony and also determines whether the event proceeded as a loop-out or a gene conversion event. The assay provides a unique way to measure the recombinogenic effects of specific direct or inverted repeats, and can effectively be used to measure the genotoxicity of compounds or the effect of various gene mutations on recombination.
228. Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production. Tiina Nakari-Setälä1, Marja Paloheimo2, Jarno Kallio2, Jari Vehmaanperä2, Merja Penttilä1, and Markku Saloheimo1. 1VTT Biotechnology, Espoo, Finland and 2Roal Oy, Rajamäki, Finland.
The cre1 gene mediates the carbon catabolite repression in Trichoderma reesei in the presence of glucose. The CREI protein has been shown to bind to specific sequences in the promoter of the major cellulase gene cbh1. CREI target sequences have also been identified in the promoter regions of other cellulase and hemicellulase genes such as cbh2 and xyn1. A truncated form of CREI (CREI-I), present in the hypercellulolytic T. reesei strain Rut-C30, has been shown to be responsible for derepression of cellulase and hemicellulase gene expression on glucose-containing media. We modified the carbon catabolite repression of T. reesei by deleting the wild type cre1 gene from the T. reesei strains QM6a and VTT-D-80133, a hypercellulolytic mutant from the strain QM9414. In addition, QM6a transformants were constructed in which the cre1 gene was replaced by the “Rut-C30 -type” truncated cre1-1 gene. Bioreactor cultivations were carried out with both types of cre1 mutant strains. Increases in the levels of protein and cellulase production were detected compared to the parents, both when the strains were grown on glucose and on lactose based complex medium. Higher levels of increase compared to parent were detected from the QM6a transformants. The results obtained from the cultivations and the expression analysis will be described.
229. Fungal biosensors based on novel luminous and fluorescent probes. Emma Perfect and Patrick Hickey. LUX Biotechnology
Luminescent and fluorescent proteins form an increasingly important component of the biologist's tool kit. These non-invasive probes are proving to be invaluable for investigations into the dynamics and localisation of cellular components and have aided drug discovery programmes, the development of biosensors and helped understand plant-fungal interactions.
In addition to being a distributor of fluorescent and luminescent genes, proteins and reagents, LUX is using its expertise in fungal molecular biology and imaging to develop fungal biosensors for environmental testing, drug discovery and R+D. These biosensors harness the power of bioluminescence and fluorescence to generate strains whose light output allow a monitoring of fungal health as well as indicating the presence of specific molecules in the environment. The characteristics of a variety of LUX biosensors, which incorporate a range of fluorescent, luminescent and calcium regulated photoproteins are presented.
230. Proteolysis and protein processing in filamentous fungi. Peter J. Punt, Margreet Heerikhuisen, Cora M.J. van Zeijl, B. Christien Lokman and Cees A. M. J. J. van den Hondel. TNO Quality of Life, Department of Microbiology, P.O.Box 360, 3700 AJ Zeist, The Netherlands
Filamentous fungi are being as production organisms for fungal and, more recently, non-fungal proteins. Our research is focused on three different areas of research related to efficient protein production:(i) protease mutants,(ii) protein processing in the secretion pathway,(iii) vacuolar proteases
(i). Already early on, protease production was addressed as a bottleneck in achieving high levels of secreted protein. Several approaches to isolate strains with reduced protease production, including a new direct selection approach and options for functional genomics and HTS-type of approaches will be discussed
(ii). A "secretion carrier" approach results in increased levels of secreted heterologous protein. To analyze the role of the KEX2/furin like protein processing protease in this approach, we have isolated the corresponiding fungal kexB/pclA genes and analyzed the effect of gene-disruption on protein secretion. The resulting mutant strains show significantly increased levels of several fusion proteins. Detailed analysis of some of the secreted fusion proteins revealed the presence of alternative, PclA- independent, protein processing pathways.
(iii) . Also protein targeting to the vacuole and release of vacuolar proteases is identified as possible reasons for low levels of secreted proteins. To study this we have isolated vacuolar protease mutant strains to study proteolysis and protein processing in vacuolar protease mutants.
231. ThPTR2 , A PEPTIDE TRANSPORTER GENE FROM T. harzianum CECT 2413. Vizcaíno, J. A1., Cardoza, R. E2., Hermosa, R1., Rey-Barrera, M3., Gutiérrez, S2. and Monte, E1. 1Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca. Edificio Departamental. Lab 208. Plaza Doctores de la Reina s/n. 37007. Salamanca, Spain. 2Microbiology Area. University of León. Campus of Ponferrada. Ctra. Astorga s/n, 24400. Ponferrada. 987-442000 Fax. 987-442070. 3Newbiotechnic, S.A. (NBT) Parque Industrial de Bollullos A-49 (PIBO). 41110 Bollullos de la Mitación. Sevilla. Spain.
Some Trichoderma strains are used as biological control agents (BCAs) of important plant-pathogenic fungi. Due to the interest of Trichoderma species, a functional genomic project ("TrichoEST") was undertaken by an International Consortium comprised of academic institutions and enterprises. The aims were to identify genes and gene products from Trichoderma spp. with biotechnology value.
The PTR (peptide transport) family transporters are found in both prokaryotes and eukaryotes (fungi, plants and animals). Members of this family have been shown to transport a wide range of nitrogen-containing substrates, including amino acids, peptides and nitrate . PTR2 from S. cerevisiae has been the only fungal PTR gene studied so far .
We decided to study the EST 6935, obtained from an EST library that was made from conditions related to biocontrol, due to its homology to PTR genes. Here, we report the cloning and characterization of the gene ThPTR2. This gene was 1,738 bp long and contained one 64-bp intron. In silico study of the codified protein (557 aa) predicted a 12 transmembrane domain transporter as expected. A Northern analysis of ThPTR2 expression was performed and at last, using a protoplast-mediated transformation strategy, we overexpressed ThPTR2 in T. harzianum CECT 2413 (homologous) and T. longibrachiatum T52 (heterelogous expression).
 Stacey, G., Koh, S., Granger, C. and Becker, J. M. (2002) Trends Plant Sci, 7, 257-263.
 Perry, J. R., Basrai, M. A., Steiner, H. Y., Naider, F. and Becker, J. M. (1994) Mol Cell Biol, 14, 104-115.
233. Characterization of the chitinolytic system of Trichoderma: development of a novel screening method to monitor gene expression on multiple carbon sources. V. Seidl, B. Huemer, B. Seiboth, I. Druzininha and C.P. Kubicek
The mycoparasitic fungus Trichoderma atroviride antagonizes the growth of several plant pathogenic fungi. Different hydrolytic enzymes such as chitinases and glucanases are involved in the cell wall degradation of the host. Biomining of the sequenced genome of T. reesei, which is industrially used to produce cellulolytic and hemicellulolytic enzymes, revealed a total of 19 genes encoding putative chitinases. 11 of these genes encode novel, so far not characterized chitinases. Based on these chitinase genes from T. reesei, 5 new chitinase encoding genes from T. atroviride P1 were isolated and sequenced. Origin and diversity of the novel chitinases from T. atroviride were asessed by phylogenetic analysis. Transcription of the 5 new chitinases differs from previously characterized chitinases: only low basal transcription levels, but no induction upon growth on Rhizoctonia cell walls and chitin were found. In order to identify novel, potent inducers of chitinase gene expression, a new method based on phenotype microarrays (containing 96 different carbon sources), which were developed for studying the global carbon utilization pattern of Trichoderma strains, was applied. Expression patterns of exo- and endochitinases of the wild-type and Gox-reporter strains of the two major chitinases of T. atroviride were monitored on a variety of carbon sources. The data show that phenotype microarrays in combination with reporter strains could be a useful tool to characterize the expression of novel enzymes.
234. REMI mutagenesis and identification of infection defective mutants in Wheat Scab Fungus Fusarium graminearum. Kye-Yong Seong1, Jiqiang Yao1, H.Corby. Kistler2, Jin-Rong Xu1. 1Dept of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907. 2USDA-ARS, Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108.
Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Wheat scab (head blight), caused primarily by F. graminearum in North America, poses a major threat to wheat production. To better understand the molecular mechanisms of plant infection and virulence of F. graminearum, we used the REMI (Restriction-Enzyme Mediated Integration) approach to generate random insertional mutants. Eleven pathogenicity mutants were identified by screening 6,500 hygromycin-resistant transformants. In mutant M8, the transforming plasmid was integrated 110-bp upstream from the start codon of the cystathionine beta-lyase gene (CBL1). Gene replacement mutants deleted for CBL1 was also obtained. The cbl1 mutants were methionine autotrophic and significantly reduced in virulence, indicating that the methionine synthesis pathway is important for pathogenesis in F. graminearum. We also have identified genes disrupted by the transforming DNA in three other REMI mutants. In mutants M68, the transforming vectors were inserted in the NADH: ubiquinone oxidoreductase. The putative b-ZIP transcription factor gene ZIF1 and the transducin beta-subunit-like gene TBL1 disrupted in mutants M7 and M75, respectively, had no known homologs in filamentous fungi and were likely to be novel fungal virulence factors. Further characterization of ZIF1 and TBL1 genes are under the way.
235. Efficient degradation of dichlorodibenzo-p-dioxins by recombinant basidiomycete Coriolus hirsutus strains. K. Shishido1, K. Orihara1, H. Kawashima1, Y. Okajima1, T. Yamazaki1, T. Sakaki2, A. Tsukamoto3 and J. Sugiura3. 1)Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan, 2)Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan, 3)Advanced Technology Research Laboratory, Oji Paper Co. Ltd., Shinonome, Tokyo, Japan.
Basidiomycete extracellular lignin-degrading enzymes catalyze degradation of polychlorodibenzo-p-dioxins (PCDDs). The plasmid MIp30-lip containing the expression cassette of C. hirsutus lignin peroxidase (LiP) gene (lip) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078, obtaining two good transformants ChTF3-1(Ch.LiP) and ChTF3-2(Ch.LiP) which carry several copies of the lip expression cassette on their chromosomes. Their culture supernatants showed the 2,7-DCDD-degrading activities 2-3 times higher than control strain. Rat cytochrome P450, CYP1A1 plays an important role in metabolism of PCDDs. The plasmid MIp5-(cyp1a1+arg1) containing the rat CYP1A1-expression cassette was introduced into protoplasts of OJ1078, obtaining three good transformants ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1) which carry 9, 6, and 7 copies of the expression cassette on their chromosomes respectively. Their microsomal fractions contained similar amounts of rat CYP1A1. The three transformants degraded 2,7/2,8(1:1)-DCDDs six times more efficiently than OJ1078.
236. Characterization of a Fungal Gene Provides Technologies for Transgenic Bioremediation and Biosensing of Environmental Pollutants. Kristin M. Skinner and Lynda M. Ciuffetti, Department of Botany and Plant Pathology, Oregon State University
Graphium, a filamentous ascomycete, grows on n-alkanes. In Graphium, these unlikely growth substrates are rendered more palatable through an initial oxidation reaction, which produces the corresponding alcohol. The alcohol is shuttled through conventional metabolic pathways to provide energy and carbon for growth. After exposure to alkanes, Graphium cultures cometabolically oxidize a broad range of xenobiotic compounds including fuel oxygenates, chlorinated solvents, and aromatic hydrocarbons. Because these compounds do not support growth, subsurface conditions and biological limitations may impede Graphium's ability to metabolize xenobiotics in situ. However, transfer of the metabolic capabilities of the fungus into a different organism may result in new technologies for the remediation of polluted groundwater. Biochemical characterization of alkane metabolism in Graphium will facilitate this technology. Competition and inhibition assays suggest that both direct and cometabolic oxidation activity are catalyzed by an inducible cytochrome P-450 alkane monooxygenase (Alk1). A putative gene encoding Alk1, Grsp Alk1, has been identified and cloned. Although sequence analysis and expression data suggest that Grsp Alk1 encodes Alk1, heterologous expression and targeted gene disruption will ultimately assign Alk1 function. The inducible promoter region directly upstream of the putative Grsp Alk1 gene was also characterized. Four upstream regions were cloned and used to drive expression of Green Fluorescent Protein (GFP) in Graphium and in Verticillium dahliae. Fluorescence was quantified in the presence of different substrates to determine if low concentrations of xenobiotics are sufficient to drive GFP expression. These results may lead to the development of strains that can directly measure the presence or absence of toxic compounds in environmental samples.
237. Expression of fish antibodies in the filamentous fungus Trichoderma reesei. Valentino S. Jr. Te'o 1,2, Gemma C. MacDonald 1, Peter L. Bergquist 1,2,3, Stewart Nuttall 4, Helena K.M. Nevalainen 1,2. 1 Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia. 2 Macquarie University Biotechnology Research Institute, Sydney, NSW 2109, Australia. 3 Department of Molecular Medicine & Pathology, Auckland University Medical School, Auckland, New Zealand. 4 CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria, Australia
The increasing interest on recombinant low volume-high value products of pharmaceutical importance, typically of mammalian origin, has strengthened the attraction in using filamentous fungi as expression hosts. For example, the shark immunoglobulin new antigen receptor (IgNAR) antibodies which possess single variable domains (VNAR), lack the external hydrophobic region present in traditional antibodies (1). These small VNAR molecules have enormous potential in therapeutics because they can penetrate dense tissues to access unique epitopes (1).
Functional and correctly folded shark IgNAR variable domain (VNAR) antibodies have been produced in E. coli at levels of 3-5 milligrams per litre. We present here our results from the expression of the hydrophilic IgNAR molecules (VNAR 12F-11 and 14M-15) in the protease deficient T. reesei strain RutC30, using different fusions to the highly secreted CBHI protein.
(1) Nuttall SD, Krishnan UV, Hattarki M, De Gori R, Irving RA, Hudson PJ (2001). Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol Immunol 38 : 313-326.
238. Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation. David White, Tony Chen, and Weidong Chen. Agriculture Research Service/Washington State Univ.
The conditions for efficient transformation of Ascochyta rabiei using Agrobacterium tumefaciens-Mediated Transformation produce random integration events have been determined. Conidiaspores of an A. rabiei pathotype II isolate were co-cultivated with A. tumefaciens carrying a virulence plasmid to determine the efficiency of T-DNA insertion into the fungal genome. Pre- and co-cultivation conditions were varied to determine the optimal transfer efficiency. Randomness and frequency of T-DNA integration were measured and related to co-cultivation conditions. T-DNA insertions were stably maintained in the absence of selective pressure and during asexual development. The efficiency of T-DNA transfer of a hygromycin resistance cassette driven by the plant-derived CaMV35s promoter was found to be less efficient than an identical T-DNA molecule carrying an Aspergillus nidulans trpC promoter driving expression of hph. Increased co-cultivation times of A. tumefaciens cells with A. rabiei conidiaspores resulted in an increased rate of transformation into the A. rabiei genome without a significant increase in the number of insertions per genome. The growth phenotype of transformants was media-dependent in the presence of Hygromycin. A subset of A. rabiei transformants were screened for the loss of pathogenicity on chickpea cultivars using a minidome assay. Two transformants unable to infect chickpea were isolated for further analysis.