Poster Abstracts, Extracellular Enzymes

A Reporter Gene System for Analysis of Promoter Functions in the Basidiomycete Fungus Phanerochaete chrysosporium

P.R.J. Birch1, P. F. G. Sims2 and P. Broda2. 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK, 2 Dpt of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, Manchester M60 I QD, UK.

Lignocellulose degradation by Phanerochaete chrysosporium involves families of genes with differential expression. To establish a system for functional analysis of gene promoters involved in the process, we made a DNA construct comprising the minimal promoter region of the cbhI.1 gene of P. chrysosporium fused to the phlR gene of Streptoallotiechus hindustanus. This was used to transform P. chrysosporium to phleomycin resitance.

Southern blot analysis revealed that, in phleomycin-resistant transformants, the transforming DNA was maintained extrachromosomally. The incoming donor DNA was also methylated by the fungus. Inhibition of such methylation elevated expression of the phlR gene. RT-PCR analysis of gene expression revealed that the cbhl.1 promoter/phlR gene construct was transcribed, demonstrating the successful use of this vector for a reporter system. However, an intron within the cbhl.1 gene promoter, which was excised from transcripts of the cbhI.1 gene, was not excised from transcripts of the transforming DNA construct.

Purification and Partial Characterisation of an Glucosidase (GLUA) from the Entomopathogenic Fungus Metarhizium anisopliae

B.D. Cobb, J.M. Clarkson and A.K. Charnley. School of Biology and Biochemistry, University of Bath, Bath, BA 2 7A Y, UK.

Efficient mechanisms for carbon uptake by entomopathogenic fungi are likely to be important factors in disease development. The predominant carbon source in the hemocoel of many insects is the glucose disaccharide trehalose. We describe the purification and characterisation of an extracellular -glucosidase (GLUA) produced by M. anisopliae which shares a number of characteristics with -glucosidases from other sources.

Purification was achieved using ammonium sulphate precipitation, preparative isoelectric focusing and fast-flow anion exchange chromatography. This resulted in a 109-fold purification of the enzyme. SDS-PAGE resolved a major protein of 37kDa. GLUA hydrolysed a range of -linked disaccharides but showed no detectable activity toward any -linked disaccharides tested. GLUA was inhibited by EDTA however no metal ion requirement was determined. GLUA displayed transglycosylation activity which resulted in non-linear MchaelisMenton kinetics. Transglycosylation products were resolved using thinlayer chromatography. Progress toward the characterisation and cloning of this gene will be presented.

Expression of the Starch-binding Domain of Glucoamylase from Aspergillus niger and Determination of its Structure

Marie-Francoise Le Gal-Coeffet1,2, David Archer1, Kay Sorimach1, Amanda Jacks2 and Mike Williamson2. 1Institute of Food Research, Norwich Research Park, Norwich, UK. 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.

The starch-binding domain (SBD) of glucoamylase 1 (GI) of Aspergillus niger is a separate domain required for the binding of glucoamylase to granular starch. SBD has been produced by proteolysis of GI but the yields were poor and the method was time and labour consuming. An expression vector, using the glucoamylase sequence (residues 1-498) as carrier in a fusion construct has been used to produce from Aspergillus niger the starchbinding domain on its own. The endoproteolytic cleavage recognition site, Lys-Arg, introduced between G498 and the SBD sequences, was correctly processed in vivo. Yields of secreted SBD in different culture media have been studied and up to 300mg/l achieved in shake flasks. Biochemical and physical analysis showed that the engineered SBD is functionally active and 2 mol cyclodextrin/mol protein are bound with an affinity very similar to the proteolytically-derived SBD. The threonine residues 510, 511 and 513 are glycosylated with an average of three mannose residue per SBD molecule. 15N labelled SBD has been successfully produced at 20-40 mg/l with a specific activity of >99% for high resolution NMR analysis. The labelled protein was used to determine NMR ambiguities. The secondary structure has been resolved and it consists of one parallel and five antiparallel pairs of B-strands forming two B-sheets. The three dimensional structure of the SBD has been also completed. In order to study the molecular basis of starch binding, mutagenesis of specific amino acid residues of the SBD, which are thought to be involved in the binding of polysaccharides, has been undertaken. Binding affinity studies with the mutated SBDs are underway.

Sequence Analysis of Esterases from the Rumen Anaerobic Fungus Neocallimastix patriciarum: Members of a New Family of Hydrolases

Brian P. Dalrymple, Daisy H. Cybinski and Ingrid Layton. Commonwealth Scientific and Industrial Research Organisation, Division of Tropical Animal Production, Private Bag No. 3., P.O. Indooroopilly, QLD., 4068, Australia.

A Neocallimastix patriciarum cDNA library was screened for esterase activity and four different esterase-encoding genes (bnaI, bnaII, bnaIII and bnaIV) were isolated. Two of the esterases bnaI and bnaIII were shown to possess acetyl xylan esterase activity.

The complete nucleotide sequences of bnaI, and bnaIII and a partial sequenceof bnaII have been determined and encode proteins with several distinct domains. BnaI and BnaIII each contain a putative signal sequence followed by the catalytic domain, a threonine-rich linker and two copies of a cysteine-rich repeat at the carboxy-terminus. The carboxy terminal repeats of BnaI and III are homologous to the carboxy terminal domains of the CelB and the XynA proteins from N. patriciarum. These domains may constitute a novel cellulose binding domain or alternatively be involved in cellulosome assembly. From the bnaII cDNA clone (only a partial clone) the complete nucleotide sequence of the catalytic domain only has been determined. The catalytic domains of BnaI,II and III are members of a new family of serine active site hydrolases. Candidate active site aspartic acid and histidine residues have been identified as part of a consensus sequence for the new family of enzymes. The amino acid sequence of BnaII was found to exhibit homology with a domain of the Clostridium thermocellum CelE enzyme, while the amino acid sequence of BnaIII exhibits extensive amino acid sequence identity with a domain from XynB from Ruminococcus flavefaciens and a lower level of identity with a domain of the C. thermocellum CelX enzyme. BnaI, possibly the major extracehular esterase of N. patriciarum is much less similar to any of the members of the family than BnaII or BnalIl and appears to represent the first member of a new branch of the family. The partial sequence of BnaIV is related to a group of esterases that includes lipases, carboxyl esterases and a para-nitrobenzyl esterase from Bacillus subtilis.

Isolation and Characterization of Pectinolytic Enzymes and Encoding Genes from Fusarium oxysporum

A. Di Pietro, M. C. Ruiz-Roldan, M. D. Huertas-Gonzalez and M. 1. G. Roncero. Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, Avda. San Alberto Magno sln, E-14071 Cordoba, Spain.

Pectinolytic enzymes, produced by the vascular tomato wilt pathogen Fusarium oxysporum f.sp. lycopersici have been proposed to be important pathogenicity factors. We have purified and characterized an endopolygalacturonase (PG1), an exopolygalacturonase (PG2), and an endopectate lyase (PL1) produced by this pathogen on tomato vascular tissue. The three enzymes were N-glycosylated and their secretion was inhibited by tunicamycin, indicating a role of glycosylation in the secretary pathway. Sequencing of N-termini showed high homology of PGl and PL1 with a F. moniliforme endopolygalacturonase (Caprari et al., Mycol. Res. 97:497-505) and a F. solani pectate lyase (Gonzalez-Candelas and Kolattukudy, J. Bacteriol. 174:6343-6349), respectively. The N-terminal sequence of PG2 showed no homology with other proteins in the databases. cDNAs of the F moniliforme and the F solani genes were used as heterologous probes to screen a -EMBL3 genomic library of F. oxysporum f.sp. lycopersici, race 2. Hybridization patterns of four phage clones hybridizing to the F. moniliforme gene indicated that they all encompassed the same genomic region. A 2.2 kb Hind III fragment containing the putative PGl gene was subcloned into pUC118. On the other hand, a 2.5 kb Sal I fragment from a phage clone hybridizing to the F solani gene was subcloned and is being characterized.

Molecular Characterization of the Endoxylanolytic System of the Rumen Anaerobic Fungus Neocallimasix frontalis

Durand, R., Rascle, C., Fevre, M. Laboratoire de Biologie Cellulaire Fongique, Centre de Genetique Moleculaire et Cellulaire, CNRS UMR 106, Bdt 405, Universite Claude Bernard Lyon I, 69622 Villeurbanne Cedex France

Anaerobic fungi belonging to the class Chytridiomycetes are potent producers of enzymes degrading the various components of plant cell wall material in the rumen of herbivorous animals. The secreted xylanolytic system of Neocallimastix frontalis is characterized by a multiplicity of activities as revealed by zymogram analysis. In order to know if these polypeptides are the products of different xylanase genes or the result of post-traductional modifications of the products of a limited number of genes, we started upon the molecular characterization of the xylanolytic system. Different cDNAs designated xyn3 and xyn4 were isolated from an expression library of the anaerobic rumen fungus N. frontalis. Xyn3 was further characterized and was shown to contain a single open reading frame of 1821 bp coding for a protein XYN3 of Mr 66000. The predicted primary structure of XYN3 consisted of two large N-terminal reiterated regions of 223 amino acids with high homology (88.3%). Each domain of XYN3, XYN3A and XYN3B showed significant homology with fungal and bacterial xylanases belonging to endoxylanase family G. Xyn3 and xyn3A were cloned in a bacterial expression plasmid harbouring a 6His-C terminal tag and the recombinant proteins XYN3 and XYN3A were purified from Escherichia coli. The recombinant proteins had a Mr of 66800 and 34000 respectively and hydrolysed xylan to xylo-oligosaccharides. Analysis of truncated forms of XYN3 confirmed that the full length protein contained two catalytic domains which displayed similar substrate specificity. Western blot analysis using antiserum raised against XYN3 showed that the N. frontalis xylanase was not extensively glycosylated. XYN3 antiserum recognized similar polypeptides in the culture medium of two other rumen fungi Piromyces rhizinflata and Caecomyces communism Xylanases have potential industrial applications in the feed industry, food processing and paper industry. The availability of recombinant fungal xylanases offers a potentially attractive source of xylanase for industrial applications. Experiments are in progress to obtain expression of the N. frontalis xylanases in species of aerobic fungi that are more amenable to genetic manipulations.

Molecular Characterization of the Xylanolytic System from the Fungal Pathogen Claviceps purpurea

Sabine Giesbert and Paul Tudzinski. Westfalische Wilheims-Universitat, Institut fur Botanik, Schlossgarten 3, 48149 Munster, Germany

Calviceps purpurea is a phytopathogenic fungus which is a parasite on grasses and cereals. Cell wall degrading enzymes probably play a role during colonization of Secale cereals ovary by C pupurea. As the primary cell walls of monocotyledonous plants consist of approximately 40% arabinoxylan xylan-degrading enzymes might be an important factor in pathogenicity of the fungus. In axenic culture C purpurea secretes at least two different xylanases of which one has been purified by ion exchange chromatography and isoelectric focusing.

Using the XYL I -gene from Cochlioboliis carbonum as a probe a xylanase encoding gene (XYL 1) was isolated from a genomic library of C. purpurea and subsequently sequenced. The XYL I -gene consists of 83 1 bp, the open reading frame is interrupted bv an 182 bp intron. The sequence exihibits great homology on both nucleic acid (up to 66%) and amino acid (up to 67%) level to other xylanases and belongs to family G xylanases according to the classification of Gilkes et al. (1991). Southern hybridizations indicate the presence of at least one more gene with homology to XYL I from C. purpurea. A further genomic region shows homology to Magnaporthe grisea xylanase gene Xyn33 belonging to family F xylanases.

The expression and regulation of XYL I in axenic culture and in planta is currently being examined by Northern and Western hybridization and RTPCR, respectively.

A disrupted copy of Xyl I was used to create a xylanase lacking mutant by transformation mediated gene replacement. Integration of the vector into the homologous genomic Xyl I region in the chromosome was confirmed bv Southern analyses. Disruption of Xyl I results in reduced total xylanase activity. Western analyses using monoclonal antibodies raised against Trichodernia reesei Xylanase Xyn I as a probe show the disappearance of one out of three crossreacting bands wrih a MG of 30.000 Da strongly indicating that this is the XYL I gene product.

In order to elucidate the role of Xyl I for pathogenicity the Xyl I -Mutant is currently being examined in pathogenicity tests.

References: Gilkes, N.R., Henrissat, B., Kilbourn,D.G., Miller R. C. and Waffen R.A.J. (1991), Microbiol. Rev. 55, 303-315

Kinetics of Endo- and Exocellular Production of Glucose Oxidase by Recombinant A. niger

Karsten Hellmuth, Stefanie Pluschkell and Ursula Rinas. GBF National Research Center for Biotechnology,

Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany

The kinetics of endo- and exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. Cells were grown in batch culture on glucose minimal medium supplemented with yeast extract. An improved method for cell disruption was developed that subsequently allowed to monitor quantitatively the time dependence of endocellular GOD activity and concentration during bioreactor cultivations. During fast growth in the first part of the cultivation, rapid endocellular accumulation of GOD up to 40% of soluble cell protein was observed followed by a slow release of the enzyme into the culture fluid. A second, weaker phase of GOD synthesis that did not cause a significant increase in endogenous enzyme activity occurred concomitantly with a second phase of slower growth. At the end of the cultivation, about 97% of total GOD recovered were detected in the culture supernatant. Two dimensional gel electrophoresis analysis provided evidence that endo- and exocellular GOD are indistinguishable clearly demonstrating identical posttranslational modifications (e.g. signal sequence cleavage, glycosylation pattern). These results also show that the initial steps of the secretary pathway are fast and that the release of the enzyme into the culture fluid is most likely delayed due to cell-wall association.

Molecular Genetics of Xylanase Production in Aspergillus nidulans

Perez-Gonzalez, JA., MacCabe, AP., De Graaff, LH., Visser, J., Fernandes-Espinar, M.T. and Ramon, D.

Aspergillus nidulans produces at least three xylanases of molecular masses 22, 24 and 34 kDa (X22, X24 and X34). These enzymes have been purified and biochemically characterized. The genes xlnA, xlnB and xlnC encoding the three enzymes have been isolated and sequenced. xlnA and xlnB were cloned by cross-hybridisation with the xlnB gene of A. tubingensis and share a high degree of homology. Each ORF is interrupted by a single intron. Both genes have been overexpressed in A. nidulans multicopy transformants and their cDNAs expressed in yeast. xlnC which encodes X34 was cloned by heterologous hybridisation with an A. kawachii xynA gene probe,xlnC contains nine introns, one of which has atypical donor/acceptor sites. Overexpression of xlnC in A.nidulans multicopy transformants resulted in elevated levels Of X34 but no apparent overexpression of xlnA and xlnB.

Synthesis of the xylanases is induced by xylan and xylose and, with the exception Of X24, is subjected to carbon catabolite repression by glucose. Regulation of xylanase gene expression is currently being studied at the transcriptional level. Expression of xlnA and xlnC in wild type is i) repressed by glucose (2%), ii) commences approximately nine hours after

transfer to xylan medium and iii) is temporally advanced in the carbon catabolite repression mutant creAd30. xlnA and xlnC' transcription remains repressed in the presence of 2% glucose in the creAd30 strain.

Analysis of the sequences upstream of the xylanase genes has revealed the presence of both putative CREA and PACC binding sites. Nucleic acid protein binding assays and promoter deletion experiments are being performed in order to determine the functional significance of these motifs.

This work was supported by grants from DG XII of the European Commission (BIOTECH BIO2-CT93-0174) and the Spanish Government (CICYT AL193-0809).

A CCAAT-box Mediates Sophorose-induction of CBH2 (Cellobiohydrolase II- Encoding)-gene Expression in the Filamentous Fungus Trichoderma reesei

B. Seiboth, R Rauscher, H. Stangl, F Gruber, R.L. Mach, and C.P. Kubicek. Abteilung fur Mikrobielle Biochemie, Institut fur Biochemische Technologie und Mikrobiologie, TU Wien, A-1060 Wien, Austria

The cellulose system of the filamentous fungus Trichoderma reesei consists of several cellobiohydrolases, endoglucanases and -glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disacharide sophorose. In order to identify nucleotide-motifs involved in the induction of cellulases, we prepared protein extracts from induced and noninduced mycelia of T. reesei, and used them for electrophoretic mobility shift assavs (EMSA) with a 615-bp fragment of the cbh2 promoter. This assay detected a specific protein-DNA complex present only in sophorose-induced mycelia. Using various overlapping fragments and competitive oligonucleotides, the DNA target motif for this protein complex was shown to be a CCAAT-box. CCAAT-motifs were also shown to be present in the 5'-noncoding region of other sophorose-inducible cellulase(cbh1, egl1, egl2) and xylanase (xyn2) genes. To study the in vivo role of the CCAAT-box in cellulose induction, T. reesei strains were constructed, which carried a copy of the cbh2 gene in which this CCAAT-motif was mutated to CCTTT. Sophorose did not induce cbh2 transcription in these strains. Protein extracts were also prepared from a mutant strain of T. reesei, in which cellulose formation could not be induced by cellulose or sophorose, but which formed a complete cellulose system upon cultivation under carbon catabolite derepression. These protein extracts did not form the sophorose induced CCAAT binding protein complex. It is proposed that binding of a protein complex, of which at least one component is induced by sophorose, to a CCAAT-box mediates the triggering of expression of cbh2 (and probably also that of the other cellulose genes) by sophorose in T. reesei.

Isolation and Characterization of a Novel Arabinofuranosidase from Aspergillus niger

Susan M. Madrid, Preben Rasmussen, Ida Hilden, Anita Baruch, Clive P. Walter, Jette Rasmussen, and Jom D. Mikkelsen. Danisco Biotechnology, Langebrogade 1, PO Box 17, DK-1001 Copenhagen, Denmark.

Arabinofuranosidases are enzymes that split off arabinose substituents from arabinose-containing hemicelluloses such as arabinoxylans, arabinans and arabinogalactans. We have purified and partially sequenced an arabinofuranosidase from the culture medium of Aspergillus niger grown on sugar beet pulp/wheat bran. The purified arabinofuranosidase has a molecular mass of 33,270 D + 50 D, determined by laser desorption mass spectrometry and a pI of 3.7. The purified protein has been shown to specifically cleave arabinose side chains from arabinoxylans, branched arabinan and sugar beet pectin. This protein in combination with Xylanase A, synergistically breaks down the xylan backbone.

Degenerate oligonucleotides designed from the partial amino acid sequence were used to amplify a 90 bp genomic DNA fragment. The PCR product obtained was used to screen an Aspergillus niger genomic library. A genomic clone was isolated, and the structural part of the gene encoded a 26 amino acids long hydrophobic signal sequence and a mature protein of 270 amino acids. The deduced polypeptide has 35% amino acid sequence identity to the catalytic domain of a 59 kDa Xylanase C from Pseudomonas fluorescens subsp. cellulose.

Purification and Characterization of a -galactosidase from Schizophyllum commune

Mankel, A. and Kothe, E. Dept. of Genetics, Philipps-University, Karl -von- Frisch Str., 35032 Marburg, Germany

Schizophyllum commune secretes extracellular -glycosidases, which possess activity for the substrate o-nitrophenyl-galactoside. The enzyme with the highest activity was purified and characterized.

Purification to apparent homogenety was achieved by affinity chromatogaphy, gel filtration and ion exchange chromatography.

A highly active -galactosidase was induced in the presence of lactose in the medium, lowest activity was observed when a medium with glucose as carbon source was used.

Denaturing gelelectrophoresis of this preparation showed a single band of an apparent molecular mass of about 66 kDa.

We intend to use the N-terminal sequence of the purified enzyme to identify the corresponding gene. This may be used as a homologous reporter gene to study gene expression in Schizophyllum commune.

Acetyl Xylan Esterase from Trichoderma reesei Contains an Active Site Serine and a Cellulose Binding Domain

EmilioMargolles-Clark, Maija Tenkanen and Merja Petittild. V7T Biotechnology and Food Research P.O. Box 1500, FIN-02044 VTT, Espoo, Finland

The hemicellulose gene, axel, encoding acetyl xylan esterase, was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axeI codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axeI encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases, but has a clear similarity especially in the active site with fungal cutinases, which are serine esterases. Similarly to serine esterases AXEI was inactivated with PMSF. At its C-terminus AXEI carries a cellulose binding domain (CBD) of fungal type, which is separated from the catalytic domain by a serine, glycine, threonine and proline-rich region. 'ne CBD can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Cloning of Hemicellulase Genes Encoding Alpha-galactosidase and Alpha-arabinofuranosidase Activities from Trichoderma reesei by Expression in Yeast

Emilio Margolles-Clark, Tiina Nakari-Setala, Elina Luonteri, Maija Tenkanen and Merja Penttila. VTT Biotechnology and Food Research P.O. Box 1500, FIN-02044 VTT, Espoo, Finland.

In order to clone novel genes encoding hydrolytic enzymes without no need of pre-existing data, a cDNA expression library of T. reesei RutC-30 has been constructed in the yeast S. cerevisiae. We have previously reported isolation of new -glucanase genes with this strategy. Here we report the cloning of three different -galactosidase genes and two -L-arabinofuranosidase genes by screening the expression bank in the presence of the substrates pNP- -galactopyranoside and pNP- -arabinofuranoside, respectively. The -galactosidase genes, agl1, agl2 and agl3, encode for 444, 746 and 624 amino acids respectively. AGLI and AGLIII show similarity between them and with the -galactosidases of the family 27 of glycosyl hydrolases. AGLII shows high similarity with the -galactosidases of family 36. The -L-arabinofuranosidase genes, abf1 and abf2, encode for proteins of 500 and 758 amino acids, respectively. ABFI displays high similarity with the aarabinofuranosidase B (ABF B) of A. niger. ABFII shows similarity with the -glucosidases of family 3. The yeast produced enzymes were tested for enzymatic activity against different substrates. The three -galactosidases showed different specificities. AGLI was able to liberate galactose from small galactose containing saccharides and polymeric galacto(gluco)mannan. AGLII was almost inactive towards polymeric substrates. AGLIII was active against all substrates tested but at a very low extend compared with the other enzymes. AGLI acted synergistically with the -mannanase, and AGLII and AGLIII showed synergism with - mannanase and -mannosidase. The -arabinofuranosidase ABFI released .arabinose from pNP- -arabinofuranoside and arabinoxylan. ABFII did not acted on arabinoxylan and has both -arabinofuranosidase and -xylosidase activity, the second activity being higher.

Improved Production of Trichoderma harzianum Endochitinase in T harzianum and T reesei.

Emilio Margolles-Clark1, Christopher K. Hayes2, Gary E. Harman 2 and Merja Penttila1. 1 VTT Biotechnology and Food Research, P. O. Box 1500, FIN-02044 V7T, Espoo, Finland. 2 Departments of Horticultural Sciences and Plant Pathology, Cornell University, Geneva, NY 14456, USA

The mycoparasite fungus T harzianum P1 has been used as a biocontrol agent against plant pathogenic fungi. The endochitinase secreted by T. harzianum has been suggested to play an important role in the biocontrol capacity, alone or acting in synergy with hydrolases produced by the fungus. In order to obtain improved biocontrol strains or enzyme preparates increased in endochitinase, the chromosomal endochitinase gene (ThEn-42) of T. harzianum P1 was isolated and overexpressed in T. harzianum P1 and T. reesei RutC-30, respectively, under the control of the promoter of the major cellulose gene cbh I of T. reesei.

T. reesei RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T. harzianum endochitinase was correctly processed in T. reesei. On the other hand, no differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg/l of active enzyme which is about 20 fold increase to the endochitinase activity produced by the non transformed T. harzianum. The presence of multiple copies of the expression cassette in the transformant lead to limitation in transcription and/or regulation factors needed for full activity of the cbhl promoter, although this was not the major limiting factor for even higher production of endochitinase. The T. reesei strain RutC-30 appeared to be tolerant to the endochitinase and can be used as a production host for this enzyme.

The transformed strain of T. harzianum P1 cultivated in shake flask with cellulose inducing medium increased the production of extracellular endochitinase in 4 to 5 fold compared with the non-transformed T. harziatium. However, the extracellular activity towards colloidal chitin increased approximately 10 fold, most likely because of the synergistic action of the endochitinase with other chitinases produced by the fungus. The expression of the T. reesei cbhl promoter was regulated in T. harzianum in a similar manner than in T. reesei. The endogenous endochitinase gene of T. harzianum was expressed at a low basal level on glucose or sucrose containing media. No specific induction of the endogenous gene by chitin could be observed.

Regulation of Cellulase and Hemicellulase Genes of Trichoderma reesei

Emilio Margolles-Clark and Merja Penttila. VTT Biotechnology and Food Research P.O. Box 1500, FIN-02044 VTT, Espoo, Finland

The filamentous fungus Trichoderma reesei uses plant polysaccharides to survive in nature producing a great array of cellulases and hemicellulases that act synergistically to degrade complex substrates. A large number of the T reesei genes encoding cellulases and hemicellulases have been isolated recently. This allows expression of several genes to be compared simultaneously in the same conditions and possible coregulation mechanisms to be studied. Here we present a study on the expression patterns of three cellulose and ten hemicellulase genes of T reesei. The analysis was carried out at the level of mRNA produced by the fungus cultivated in media containing different complex plant polysaccharides, oligosaccharides or monosaccharides.

Phytases from Thermotolerant Fungi

Luis Pasamontes, Monika Haiker, Denis Hug, Maria Huecas, David B. Mitchell and Adolphus P.G.M. van Loon.

Biotechnology Section, Vitamins and Fine Chemicals Division, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland

We screened a number of thermotolerant fungi for phytase production and cloned the complete phytase genes from 2 candidates (Aspergillus fumigatus, Talaromyces thermophilus), using degenerate oligonucleotides based on known phytase and histidine acid phosphatase sequences. Sequencing reveals that the presumed translation products are 465 and 466 aa, respectively, both containing a single short intron. Comparison of the sequences to each other and to published phytases show amino acid identity levels between 47-88%. The identity with known acid phosphatases are between 21-29%. Overexpression of these phytases in Aspergilli are under way and will facilitate the analysis of the enzymatic activities of both enzymes (e.g. pH activity profile with phytic acid and 4-nitophenylphosphate, temperature stability) as well as the testing of their performance in "in vivo" feed trials.

Cloning and Characterisation of an -amylase gene from the thermophilic fungus Thermomyces lanuginosus

Preben Rasmussen and Birgit Michelsen. DANISCO Biotechnology, DK-1001 Copenhagen K, Denmark

Fungal -amylases have been used for many years in food industry, especially in starch and bakery industry. Fungal amylases have traditionally been fermented from Aspergillus oryzae. However this amylase is not very heat stable and have little if any activity during starch gelatinasation. Several thermophilic fungi, among these T lanuginosus, produces -amylases (1) with significant higher heat stability, unfortunately in rather low amounts.

In order to study the -amylase from a thermophilic fungus we have cloned and characterised the gene encoding the -amylase from T lanuginosus . A PCR fragment of the gene was isolated by designing primers specific to conserved regions of fungal -amylases. The cloned fragment was used to screen a genomic library of T lanuginosus and 5 independent clones were found. The clones contained an ORF encoding a 493 residues long protein with a putative signal sequence of 18 residues. The ORF was interrupted by eight introns. Two peptides of purified -amylase was sequenced which confirmed the isolated gene encodes the correct enzyme.

This ORF was expressed in Aspergillus niger resulting in high expression of -amylase in the transformants. The heterologous protein was purified and characterised biochemically, which showed that it was substantially identical to the native protein. In contrast to the heterologous protein the native protein was N-terminally blocked.

The N-terminal sequence of the mature protein confirmed the putative identified signal sequence cleavage site.

The cloning and expression of this gene in A. niger have given a sufficient and reliable source of T lanuginosus -mylase for characterisation and application tests.

(1) Jensen, B. and Olsen J. (1992) Enzyme Microb. Technol. 14: 112-116

Hyperhydrolytic Mutant of Trichoderma harzianum with Improved Antifungal Activity

Jesus Delgado-Jarana, Manuel Rey, Jose A. Pintor-Toro*, Antonio Llobell**, Tahia Benitez. Departamento de Genetica. Universidad de Sevilla. *IRNA and ** IBFV, CSIC, Sevilla.

Strains of the filamentous fungus Trichodermia harzianum have been described as biological control agents against plant fungal pathogens. The cell wall degradation and further assimilation of phytopathogenic fungi have been proposed as a major mechanisms accounting for the antagonistic activity of the Trichoderma strains. It is therefore expected that strains with increased lytic activity will be more efficient biological control agents.

After nitrosoguanidine treatment of a mycoparasitic strain of T. harzianum mutant strains were isolated which produced halli of lysis in media with pustulan, a -1,6-glucan polymer, longer than that of the wild type. One of the mutant characterized, PFI, had higher levels of - 1,3-glucanase and chitinase, in addition to -1,6-glucanase, under both basal and induction conditions. Extracellular protein separation and deten-nination indicated each individual protein to be more abundant in PF1 than in the wild type, what seems to indicate that the mutant is hypersecretor. Preliminary results in experiments carried out in vitro in dual cultures with Rhizoctonia solani indicated that PFI possessed higher antifungal activity than the wild type. Finally, the mutant grows less than the wild type and sporulates sooner. Experiments in progress will allow us to establish whether this behavior reflects a higher sensitivity of the mutant to the lytic enzymes and/or a pleiotropic effect of the altered permeability, i. e. a higher energy consumption.

Isolation and Characterisation of Genes Encoding Cell Wall Degrading Enzymes of the Pathogenic Fungus Botrytis cinerea

Wietse Mulder1, Arjen terl Have2, Jan A.L. van Kan2 en Jaap Visser1. 1 Section Molecular Genetics of Industrial Micro-organisms, WAU, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. 2 Department of Phytopathology, Wageningen Agricultural University Binnenhaven 9, 6709 PD Wageningen, The Netherlands.

The pathogenic fungus B. cinerea causes major problems in diverse agricultural crops. At this moment control of B. cinerea infection is hampered for several reasons including high genetic variation and the various infection strategies employed by this organism. Fungal extracellular hydrolases presumably play an important role during infection by degrading the plant cell wall.

In order to develop an effective control strategy, the ftmgus is extensively studied, however B. cinerea is still poorly characterised at the molecular level. Therefore, we recently started a molecular genetic study in order to identify and characterise extracellular hydrolases of the fungus and to elucidate the roles the various enzymes play in the process of pathogenicity.

A genomic DNA library was constructed from B. cinerea strain SAS56. Via heterologous hybridisation, using DNA-probes derived from several Aspergilli species, genes coding for pectinolytic enzymes were isolated. Amino acid sequence alignments showed that the Bcpgal gene encodes a polygalacturonase, which is highly homologous to the PG genes of Sclerotinia scierotiorum. Interestingly, as in S. sclerotiorum the ORF is not interrupted by introns. The Bcpel1 gene encodes a pectin lyase and shows significant homology with pelA of A. niger. Compared to the A. niger pelA gene, only one intron is conserved both in respect to position and consensus intron sequences, whereas the other three introns are absent in Bcpel1.

To manipulate the fungus genetically several methods have been modified and developed. Disruption of chromosomal copies of the Bcpga1 and Bcpel1 genes, using a haploid strain, derived from SAS56, is currently in progress.

The effect of gene disruption on the level of pathogenicity will be tested in a well defined bioassay using tomato leaves. Gene expression, enzyme activities and the effect on the process of maceration will be determined. Obtained results and prospects will be discussed.

Organisation and Expression of Two Laccase Genes in the Cultivated Mushroom Agaricus bisporus

M. Smith 1, A. Shnyreva2, D.A. Wood3 and C.F. Thurston1. Division of Life Sciences, King's College London, London

W8 7AH, U.K. 2Department of Mycology and Algology, Moscow Lomonosov University, Moscow 1198799, Russia.

3Horticulture Research International, Wellesbourne, Warwick CV35 9EF, U.K.

Laccase encoding sequences from the cultivated mushroom Agaricits bisporus were first isolated as cDNA. Two non-allelic sequences were found, one of which was also sequenced from genomic DNA (Perry et al., 1993. J. Gen. Microbiol. 139, pp 12091218). Cosmid clones containing laccase GDNA sequence all gave a comparable southern blot hybridisation pattern to total A. bisporus genomic DNA suggesting that the two genes were not segregating to independent clones. Restriction fragment mapping of cosmid clone LA16E2 was not completely unambiguous, but was consistent with both laccase genes residing within a few thousand base pairs of each other. PCR reactions gave a product of ca. 1.5 kb when specific primers were used directing synthesis (5' - 3) from the 3'-end of lcc 1 and (3' - 5') from the 5'-end of lcc2. The 1.3 kb sequence between the 3'-end of lcc1 and the 5'-end of lcc2 has been analysed and will be presented, together with studies of laccase gene expression by northern blot and competitive RT-PCR assays.

Fermentation Studies on the Overproduction of Glucoamylase in Aspergillus niger

G.H. van Geel-Schutten1, J. ten Veen1, P.J. Punt2 and A.J. van den Wijngaard1. 1 Department of Bioprocessing and Biomonitoring, TNO Nutrition and Food Research Institute, P.O. Box 306, 3700 AJ Zeist, The Netherlands. 2Department of Molecular Genetics and Gene-technology, TNO Nutrition and Food Research Institute, P.O. Box 5815, 2280 HV Rijswijk, The Netherlands.

Batch fermentation studies with a wild type A. niger and strain B36, derived from the wild type, containing multiple copies of the A. niger glucoamylase gene (glaA) were carried out in pH controlled fermentors with a working volume of 1.5 liters. In both strains the expression of the glaA genes is driven by the wild type glucoamylase promoter and glucoamylase production was induced with maltodextrin. For both strains, the production of glucoamylase was 2-3 times higher in the fermentor than the production in erlenmeyers. In addition, production in fermentors was much faster than in erlenmeyers. Strain B36 produced 5-10 times more enzyme than the wild type strain and there was low production of aspergillopepsin in the fermentor. The glucoamylase production in strain B36 was pH dependent. At pH 4 the glucoamylase production was higher than at pH 4.5 and pH 5.5 The addition of extra carbon source to the medium resulted in a higher biomass and a higher glucoamylase production. In this way, we were able to produce almost 4 g/l glucoamylase within 70 hours. Further studies will focus on fed-batch cultures with strain B36.

Cutinase of Botrytis cinerea Is Not Essential for Infection of Gerbera Flowers and Tomato Fruits

Cecile van der Vlugt-Bergmans, Lia Wagemakers, John van 't Klooster, Dianka Dees, Jan van Kan. Dept. of Phytopathology, Wageningen Agricultural University, PO Box 8025, 6700 EE Wageningen, The Netherlands.

Cutinase was proposed to play an early role in the infection of host tissues by Botrytis cinerea. To investigate this role, the enzyme was purified, characterized and amino acid sequences of the purified cutinase were used to design primers for PCR based gene cloning from strain SAS56; the protein sequence shows significant homology to known fungal cutinases.

Expression of the cutinase gene during the infection of gerbera flowers and tomato fruits was studied, using a cutinase promoter-GUS construct, transformed into B. cinerea. During germination and penetration of host tissue, fungal structures demonstrated high GUS activity, indicating that the cutinase promoter is active in planta. During in vitro growth on water agar, GUS staining was observed, indicating that substrate induction is not essential. The addition of a cutin monomer to the agar resulted in a much more intense GUS staining. In vitro, cutinase expression is subject to catabolite repression.

To study the necessity of cutinase for penetration of host tissue by B. cinerea, cutinase deficient mutants were obtained by means of gene disruption. These mutants were tested on gerbera flowers and tomato fruits for their pathogenicity. The ability of these mutants to penetrate and cause infection was not altered. Microscopically, infection structures produced by the mutant did not differ morphologically from the wild type. Therefore we conclude that cutinase in B. cinerea is not an essential pathogenicity factor.

Novel Fungal Phytase Genes and Enzyme Activities

David B. Mitchell, Kurt Vogel, Luis Pasamontes and Adolphus P.G.M. van Loon. Biotechnology Section, Vitamins and Fine Chemicals Division, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland

We isolated and characterised the complete genes encoding novel fungal phytases from Aspergillus terreus and Myceliophthora thermophila and PCR fragments of the phytase genes from 4 other fungi. The encoded proteins show 48-60% identity compared to the phytase of Aspergillus niger and have 21-29% identity when compared with known acid phosphatases. The phytases form a separate subclass of the histidine acid phosphatase family. Phytase genes of additional fungal species were identified by cross-hybridization experiments. Interestingly, no cross-hybridization with the Aspergillus niger phytase gene was seen. The phytases of Aspergillus terreus and Myceliophthora thermophila showed novel enzyme activity profiles and have a higher preference for phytic acid as substrate compared to 4-nitrophenyl phosphate, than the A. niger phytase. Further characterization of the genes and enzymes is underway.

The SC3 Hydrophobin Is among the Most Surface Active Molecules

Han Wosten1, Teun Ruardy2, Wiesje van de Vegt2, Hennie van der Mei2, Henk Busscher2 and Joseph Wessels1. 1 Groningen Biotechnology and Biomolecular Sciences Institute, Department of Plant Biology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. 2 Materia Technica, University of Groningen, Bloetwingel 10, 9712 ICZ Groningen, The Netherlands.

Hydrophobins are small secreted proteins involved in emergent growth of fungi. These proteins are characterized by the conserved presence of 8 cysteine residues and similar hydropathy plots. Although more than 20 hydrophobin genes have been cloned, SC3 of Schizophyllum commune is the only hydrophobin that has been partially characterized. SC3 self assembles at a hydrophilic/hydrophobic interface into an SDS-insoluble amphipathic protein membrane. Self-assembly of this hydrophobin is solely determined by the degree of hydrophobicity and not by other chemical or physical properties at the interface. When an aqueous solution of SC3 is dried on a hydrophilic solid surface SC3 assembles at the water/air interface exposing its hydrophobic side (water contact angles 110 ) that is characterized by a mosaic of parallel rodlets. On the other hand, when a hydrophobic surface, e.g. Teflon, is incubated in an aqueous solution of SC3, the hydrophobin assembles at the water/Teflon interface making the Teflon wettable. The hydrophilic side of the SC3 membrane (water contact angles 48 ) appears smooth. The amphipathicity of assembled SC3 is accompanied by a different atomic composition at different sides of the protein membrane. Self-assembly of SC3 was also studied in situ by Axisymmetric Drop Shape Analysis by Profile (ADSAP). ADSAP allows simultaneous analysis of self-assembly of SC3 at the water/air and water/solid interface in a water droplet placed on a Teflon surface. The interfacial tension at the water/air interface decreased sharply from 70 to 28 mJ.m-1 at 35, which shows that SC3 is among the most surface-active molecules identified. This decrease was mainly caused by a conformational change and was not limited by difftision of SC3 monomer to the interface. At the water/Teflon interface, however, the contact angle did not decrease but increased from 110 (bare Teflon) to 122 (coated Teflon) after 30 min. of incubation. This contrasts to the low water contact angle measured after dipping Teflon in an aqueous solution of SC3. A model explaining this paradox will be presented.

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