ECFG 9 Poster session 3

ECFG 9 Poster Session 3

Fungal Biotechnology, Hyphal Growth and Morphogenesis, Evolutionary and Population Biology

PR7.1

Identification and phytotoxical properties of fungi genus Trichoderma from ancient lands and antagonistic activity by Trichoderma koningii against Fusarium oxysporum in experiences in vitro

Hector Alejandro Cabrera Fuentes, Farida Alimova, Rezeda Tukhbatova, Elina Rafailova

Kazan State University, Kazan, Tatarstan, Russian Federation

Different species of the genus Trichoderma are employed in different areas of human activity. We acquired more than 400 stems of fungi from the genus Trichoderma, from burial grounds dating 800-600 BC, on the territory of the Republic of Tatarstan, Russia. The objective of this study was to look for stems, perspective for the manufacture of biological products and evaluate Trichoderma species as potential biocontrol agents to reduce the impact of the Fusarium oxysporum. In order to reach this goal we identified, classified and systematized allocated stems with the use of morphological and molecular-genetic methods and investigated their physiological-biochemical properties. Descriptions of the appearance of colonies and their morphology have been recorded in a before-stated work developed by identifiers Samuels, Chaverri, Bissett. Molecular-genetic research was carried out with use of method PCR (RAPD-analysis). On the basis of the received results investigated stems have been related to species: Tr. longibrachiatum, Tr. koningii, Tr. harzianum, Tr. asperillum, Tr. citrinoviride, Tr. atroviride, Tr. oblongisporum, Tr. spirale. Classification of these stems was done with the help of an earlier specially developed program. Also have been investigated antagonistic activities and phytotoxical properties, enzymatic activity of the complex exohydrolase (xylanase, protease and cellulase activity) of allocated stems.Morphological analysis revealed the 2 types of colony (II and VI). Their kinetic parameters were also determined: slow growth isolates and fast growth isolates were calculated as 0.01-0.07 mm/h and 0.1 mm/h. We have also investigated antagonistic activities. As a result of our work, selected stems of Trichoderma koningii with a positive influence on the growth of rye and wheat with prospects in biotechnology have been identified.

PR7.2

Weak acid preservative resistance in Aspergillus niger

Malcolm Stratford, Andrew Plumridge, David Archer

University of Nottingham, Nottingham, United Kingdom

Sorbic acid is a weak acid, added to foods and beverages as a preservative. Certain moulds and yeasts are highly resistant to sorbic acid and therefore contaminate food products and cause spoilage. The mechanism of resistance employed by Aspergillus niger, a common food contaminant, to sorbic acid is mediated by PadA1. PadA1 (phenylacrylic acid decarboxylase, encoded by padA1) has been shown to decarboxylate and, thereby detoxify sorbic acid producing the volatile compound 1,3-pentadine. A. niger D padA1 mutants have been shown to be unable to decarboxylate sorbic acid and as a result are more sensitive to this preservative compared to the wild-type. The genome of A. niger contains two other genes (padA2 and padA3) encoding putative phenylacrylic acid decarboxylases (PadA2 and PadA3, respectively). Gene disruption studies showed that the absence of padA2 or padA3 did not increase the sensitivity of these strains to sorbic acid, nor did they reduce the ability to decarboxylate this preservative. Putative PadA proteins have been identified in a number of moulds whose genome sequences have been published and their ability to decarboxylate sorbic acid has been confirmed. Certain yeasts are also able to decarboxylate sorbic acid but to a much lesser extent than moulds. Pad1p from Saccharomyces cerevisiae, a common spoilage yeast, has been shown to facilitate the decarboxylation of sorbic acid, producing 1,3-pentadiene. The growth rate of pad1 mutants was retarded during sorbic acid-stress but the minimium inhibitory concentration of the preservative required to cause complete growth arrest was identical for the mutant and the wild-type strains. Thus, Pad1p-mediated detoxification of sorbic acid, is a genuine resistance mechanism in moulds but not in yeast.

PR7.3

Splicing activity of an artificially engineered mini-intein from Penicillium chrysogenum

Skander Elleuche¹, Constantin Pelikan¹, Nicole Nolting², Kristin Döring², Stefanie Pöggeler²

1Ruhr-University, Bochum, Germany, ²Gerorg-August University, Göttingen, Germany

Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing. Thereby they religate the host protein with a peptide bond. In contrast to mini-inteins, large inteins also contain an endonuclease domain that is important in intein propagation, called homing. The first PRP8 intein was found within the prp8 gene of the basidiomycete Cryptococcus neoformans. The PRP8 protein occupies a central position within the catalytic core of the spliceosome.

In our study, we selected several members of the ascomycetous genera Penicillium and Eupenicillium and investigated them for the presence of PRP8 inteins. We were able to identify mini-inteins in P. expansum, P. vulpinum, P. chrysogenum, E. baarnense and E. crustaceum but not in P. thomii, E. terrenum and E. meridianum. All of the PRP8 inteins identified are mini-inteins and undergo autocatalytic protein splicing when heterologously expressed in a model host protein in E. coli.

In a second approach, we tested the importance of conserved domains inside the P. chrysogenum intein and were able to produce split intein versions capable of protein splicing in trans, when theintein sequences encoding the N- and C-terminal moiety of the intein are transcribed as polycistronic mRNAs. The insertion of split sites in two different domains restored splicing activity. By deleting non-conserved amino acids at two different sites Furthermore, we produced the smallest functional eukaryotic intein known so far. This indicates that the mini-intein of P. chrysogenum might have a more robust structure than do many other inteins and that it might be of practical importance to engineer this intein for various applications.

PR7.4

A single amino acid change in the L-arabitol dehydrogenase (LadA) from Aspergillus niger broadens its substrate specificity and increases its activity

Blanca Trejo-Aguilar¹, Lucy Rutten², Cecile Ribot³, Manuela Pail¹, Han A.B. Wösten¹, Ronald P. de Vries¹

1Microbiology, Utrecht University, Utrecht, Netherlands, ²Crystal and Structural Chemistry, Utrecht University, Utrecht, Netherlands, ³CNRS-UCB-INSA-Bayer CropScience, Lyon, France

L-arabitol dehydrogenase and xylitol dehydrogenase are part of the fungal pentose catabolic pathway. They are related to sorbitol dehydrogenases from higher eukaryotes.

Phylogenetic analysis of L-arabitol dehydrogenases (LAD), xylitol dehydrogenases (XDH) and sorbitol dehydrogenases (SDH) shows that XDHs are more similar to SDHs than LADs. By modeling A. niger LadA and XdhA on the structure of human SDH, we identified two residues in XdhA that were hypothesized to be important for the activity on D-sorbitol. Mutagenesis of one of these residues in LadA resulted in a nearly complete enzyme inactivation for reasons unknown. The other mutation resulted in increased affinity and activity for L-arabitol and sorbitol, demonstrating that this residue is not only important for activity on sorbitol, but also improves the general activity of the enzyme.

PR7.5

An improved & markerfreeÔ expression system for Aspergillus niger enzyme production

Ilse de Lange, Alrik Los, Janny Bakhuis, Noël van Peij, Herman Pel

DSM Food Specialties, Delft, Netherlands

For many decades, Aspergillus niger has been safely used in the commercial production of various food enzymes, such as glucose oxidase, pectinase, alpha-amylase and glucoamylase.

Genetically modified industrial strains of A. niger have been used as a host to over-express food and feed enzymes such as phytase and xylanase. Traditionally, the genes encoding these enzymes are integrated in a random fashion in the genome of the host organism.

New technological developments have enabled us to construct a new generation of A. niger CBS513-88 lineage strains according to a "design and build" concept, in which the genes of interest are targeted integrated in the host genome. A recombinant strain was derived from a glucoamylase production strain, in which the glucoamylase (glaA) genes were deleted, creating so-called ΔglaA loci. Each of these loci was designed in such a way that it individually can be detected by gel electrophoresis. Therefore, targeted integration of the gene of interest in all seven loci can be monitored, allowing selection of strains with multiple integrated expression units of the gene of interest. The homologous integration frequency was improved by disruption of the A. niger hdfA and/or hdfB genes, homologues of the human KU70 and KU80, that are essential for non-homologous end joining of DNA in double strand break repair. Deletion of hdf genes greatly reduces the frequency of non-homologous integration of transforming DNA fragments leading to dramatically improved gene targeting. The hdf knockout has contributed to a highly controlled strain construction process, with both increased targeting frequency and increased co-transformation percentage.

PR7.6

Automated screening for high-copy Aspergillus niger production strains

Ilse de Lange, Alrik Los, Janny Bakhuis, Noël van Peij, Herman Pel

DSM Food Specialties, P.O. Box 1, 2600MA Delft, Netherlands

The filamentous fungus Aspergillus niger has extensively been used for production of recombinant proteins. We have modified A. niger CBS513-88 lineage strains in order to enable highly controlled production strain construction, in which genes of interest are integrated at defined high expression loci. A recombinant strain was derived from a glucoamylase production strain, harboring 7 glucoamylase (glaA) genes. The glaA genes were deleted stepwise; the resulting Δ glaA-loci serve as tool for recombinant production strain construction according to the ‘design and build’ concept (see poster De Lange et al). Following integration of genes of interest at the target Δ glaA-locus, the gene copy number is subsequently amplified via gene conversion of the different Δ glaA-loci. This process can easily be monitored via PCR-screening and gel-electrophoresis. The throughput of this screening has significantly been increased by implementation of the LabChip 90 system (Caliper Life Sciences), thereby accelerating production strain construction.

PR7.7

Schizosaccharomyces pombe as host for high throughput screening of fungal proteins

Paulien Neefe, Lydia Dankmeyer, Pim van der Kley, Daan Meyer, Pauline Teunissen, Piet van Solingen

Genencor. A Danisco Division., Leiden, Netherlands

High Throughput Screening of libraries of enzymes from filamentous fungi cannot easily be carried out in the fungal host strains themselves for a number of reasons:

A limited number of stable transformants per transformation event

Instability of transformants because of multi nuclear character of mycelial protoplasts

Non-uniform growth at microtiterplate-scale leading to variable production yields

Difficulties in automated handling, e.g. inoculation and transfer of fungal colonies and cultures

High background of endogenous extracellular enzymes, co-expressed together with a protein of interest.

Other screening systems such as E.coli, Bacillus or Streptomyces are often not suitable either, as they are mainly limited to the expression of prokaryotic genes.

Therefore, the most obvious screening host is an unicellular yeast, for example Saccharomyces cerevisiae or Schizosaccharomyces pombe. One of the major advantages of S. pombe is high transformation efficiency and the absence of interfering background proteins. Additionally, it is easy to handle robotically.

It is rather unpredictable if enzymes from filamentous fungi can be expressed well in yeast host strains. S.pombe has proven to be an excellent host for cloning and expression of heterologous genes such as endoglucanase2 and cellobiohydrolase2 of Trichoderma reesei.

This poster describes the successful application of S.pombe as the screening host for HTS of protein engineered enzymes from the filamentous fungus T.reesei.

PR7.8

Intracellular protein production in Trichoderma reesei with the help of zera peptide technology

Ann Westerholm-Parvinen¹, Kevin O'Connor², Miriam Bastida², Blanca Llompart², Markku Saloheimo²

1VTT Technical Research Centre, Espoo, Finland, ²ERA Biotech, Barcelona, Spain

Several species of filamentous fungi, such as certain Aspergillus species and Trichoderma reesei, are utilised as cell factories for protein production, largely due to the superb secretion capacity of these organisms. However, for many heterologous proteins the production yields have been modest, due to various reasons like inefficient folding and transport along the secretory pathway and susceptibility to host proteases. In order to avoid these problems, we have developed an intracellular protein production system in T. reesei that utilises the Zera peptide technology commercialised by ERA Biotech. In this technology the foreign protein is fused with the Zera peptide, a fragment of the maize gamma-zein storage protein. In maize seeds this protein is localised into protein bodies that are derived from the ER membrane and work as protein storage organelles. When fused with a foreign protein of interest, the Zera peptide has been shown to recruit the foreign protein efficiently into protein bodies in plants, but also in mammalian and insect cells. The protein bodies have a high density and thus they are easy to isolate from the cells for product recovery.

We have tested whether Zera technology functions in filamentous fungi by expressing the eGFP fluorescent protein as a fusion with the Zera peptide in T. reesei from the strong cbh1 promoter. The transformants carrying the Zera-eGFP expression construct were observed to contain strongly fluorescent bodies in their cytoplasm, whereas a control construct expressing the eGFP alone with an ER localisation signal produced much lower and diffuse fluorescence. Western analysis with GFP antibodies showed that a greatly higher level of Zera-eGFP than eGFP alone was produced in the cells. The transformant cells were disrupted and the cell content was fractionated by gradient ultracentrifugation. Good yields of relatively pure Zera-eGFP protein could be isolated from the cells with the centrifugation as the single purification step. Our study demonstrates that the Zera peptide is fully functional in formation of protein bodies in T. reesei, and that the Zera technology can be successfully used in protein production in this fungus.

PR7.9

Improvement of the biocontrol of Heterobasidion root rot with antagonist fungus
Phlebiopsis gigantea

Hui Sun¹, Kari Korhonen², Risto Kasanen¹, Jarkko Hantula², Fred Asiegbu¹

1Department of Forest Ecology, University of Helsinki, Helsinki, Finland, ²Finnish Forest Institute, Vantaa, Finland

Root and butt rot caused by Heterobasidion species is one of the most destructive diseases of conifers in the northern temperate regions of the world. Phlebiopsis gigantea (Fr.) Jül. is currently used as biocontrol agent. However, little is known about the physiological and molecular basis of interaction between P. gigantea and Heterobasidion spp.The hypothesis of our study is: the traits of P. gigantea related to biocontrol could be targets of breeding and could be used in developing the biocontrol abilities of P. gigantea. The aims of this investigation are: a) To study the importance of different traits (spore production ability, antagonism, and growth rate) related to biocontrol. b) To test whether the biocontrol abilities of P. gigantea can be enhanced with breeding of these traits. C) To study the differentially gene expression of P. gigantea to select candidate genes contributing to biocontrol.

64 Phlebiopsis gigantea isolates from different countries and hosts (Pinus sylvestris and Picea abies), including commercial Rotstop F and Rotstop S, were tested and P. gigantea strains with ‘good’ biocontrol ability were selected for homokaryon collection and breeding to produce heterokaryon progeny. The biocontrol ability of progeny was tested. Selected P. gigantea strains with ‘good’ and ‘bad’ biocontrol ability were studied for differentially gene expression using Macroarray method and quantitative reverse transcript Real-time PCR.

Preliminary result indicated that there was significant variation in each tested trait between different P. gigantea strains, but no correlation between three traits. P. gigantea progeny showed higher ability against H. annosum in three traits than that in parental P. gigantea. Moreover, there was significant difference between P. gigantea progeny and parental in mean efficacy with 88% and 78%, respectively. This indicated the biocontrol ability of P. gigantea against H. annosum could be enhanced through P. gigantea breeding. Future perspectives will be selecting candidate genes contributing to biocontrol by Macroarray and verifying candidate genes by quantitative RT-RT PCR.

PR7.10

Fast and efficient single step construction of replacement vectors by USER friendly cloning, for targeted gene replacement in fungi

Rasmus J.N. Frandsen, Jens A. Andersson, Henriette Giese

Institute for Ecology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark

Functional genetics in filamentous fungi have always been dependent on the isolation or construction of mutant strains. The genome sequencing of over 40 fungi genomes has increased the need for faster and more efficient methods to construct targeted replacement and overexpression mutants. To accommodate this we have developed a new vector system that allows single step construction of vectors for targeted gene replacement, thereby cutting vector construction time from ten to only three days and removing half of the required work load. The vector system is dependent on the Uracil-Specific Excision Reagent cloning technology (USER Friendly^TM), which in its commercial version offers high efficient directional cloning of a single PCR amplicon. However, our research shows that USER friendly^TM cloning technology also can be used for the simultaneous directional cloning of several PCR amplicons and vector fragments, with a cloning efficiency of 85 %, thus allowing single-step construction of replacement vectors. In addition to the increased speed and reduced workload, the single-step construction strategy also offers greater freedom of operation with respect to the placing of the homologous recombination flanks, as it is independent of restriction enzymes.

The new vector system includes vectors for targeted gene replacement (pRF-HU2), promoter exchange (pRF-HU2E), ectopic overexpression (pRF-HUE) and general purpose cloning (pRF-HU). All are compatible with both protoplast and Agrobacterium tumefaciens mediated transformation technologies. The system has been used to analyse putative polyketide gene clusters in Fusarium graminearum.

PR7.11

Enhancement of the Trichoderma reesei expression system

Shingo Miyauchi¹, Helena Nevalainen¹, Peter Bergquist¹, Junior Te'o²

1Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney / NSW, Australia, ²Applimex Systems Pty. Ltd, Sydney / NSW, Australia

Recent advances in fungal genomics and proteomics have facilitated further development of Trichoderma reesei as a production host. In most studies, the strongly inducible promoter of the cbh1 gene in T. reesei has been utilised for the expression of various gene products (1, 2) In addition to CBH1, T. reesei also produces significant amounts of other cellulases such as CBH2 and EG2, as shown by transcriptomic and proteomic studies (3). Therefore, the genes encoding these enzymes provide an alternative source of strong gene promoters.

We have constructed four expression vectors that feature either the cbh2 or egl2 promoter. The first pair of vectors, named pEG2/pCBH2-sigpro, feature a promoter sequence, secretion signal and pro-region derived from either the cbh2 or egl2 gene, whereas pEG2/pCBH2-cbmlin vectors contain a promoter sequence, secretion signal, pro-region, carbohydrate-binding module and a linker region. The xynB gene encoding xylanase B from the thermophilic bacterium Dictyoglomus thermophilum has been inserted into the vectors for heterologous expression. The codon usage of the xynB gene has been modified for the expression in T. reesei (4).

Expression of xynB from the pEG2-cbmlin and pCBH2-sigpro vectors was found to be greater than that using the EG2-sigpro and CBH2-cbmlin constructions based on zymogram analysis and liquid enzyme activity assays. In this presentation, we discuss the capability of the novel promoters to drive protein expression as influenced by the structural differences in the expression cassette motifs.

^{1 Ward et al (2004). Appl. Environ.Microbiol. 70: 2567–2576, ² Punt et al (2002). Trends Biotechnol. 20: 200–206, ³ Foreman et al (2003). J. Biol. Chem. 278: 31988-31997, ⁴ Te’o et al (2000). FEMS Microbiol Lett 190: 13-19}

PR7.12

Selection of microbial production hosts for real-life feedstock utilization

Karl Rumbold, Hugo van Buijsen, Johan van Groenestijn, Peter Punt, Mariët van der Werf

TNO Quality of Life, Zeist, Netherlands

Bulk chemicals produced by microbial production processes are becoming more competitive in comparison to those traditionally synthesized from crude oil. It is expected that products representing 10% of total sales in chemical industry by 2010 will originate from microbial production processes. Improvements in fermentation technology and down stream processing, as well as economies of scale, have significantly improved the competitiveness of bulk chemicals produced by biotechnology. Now, cost of goods, in particular substrate costs, has become a major parameter.

The use of lignocellulosic hydrolysates is gaining increasing attention as low-cost substrate feedstocks. These feedstocks contain, however, mixtures of different fermentable sugars, high salt concentrations as well as specific compounds that strongly inhibit microbial growth, depending on the feedstock pretreatment process applied. We have performed a benchmark study with six industrial microorganisms (i.e. Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia stipitis, Trichoderma reesei and Aspergillus niger) to evaluate their fermentation performance, versatility in feedstock use, and product producing capacities using ‘real-life’ feedstocks as the substrate. In this study, the performance of these microorganisms with respect to their substrate utilization potential and sensitivity towards inhibitors as well as extreme process conditions was evaluated. Moreover, these microorganisms were evaluated for their ability to ferment hydrolysates of "real-life" feedstocks, i.e. corn stover, wheat straw, sugar cane bagasse, willow wood and waste glycerol from biodiesel production.

Combining the data from the benchmarking test with existing knowledge, we were able to rank these industrial production hosts based on their suitability to utilize ‘real-life’ feedstocks for the production of bulk chemicals. A. niger and P. stipitis were found to perform the best. In general, all hydrolysates of "real-life" feedstocks were good substrates for fermentation.

The benchmark test developed in this study is a generic and can be applied to test the performance of other (industrial) production hosts and their ability to utilize different feedstocks as well their potential to produce specific chemicals.

PR7.13

EroA and oxidative protein folding in Aspergillus niger

Anna Harvey¹, Andrew Plumridge¹, Mick Ward², Huaming Wang², David Archer¹

1University of Nottingham, Nottingham, United Kingdom, ²Genencor Intl, Palo Alto, California, United States

Disulphide bonds are recognised as an integral feature within the structure of many folded proteins. In the yeast model organism Saccharomyces cerevisiae, the formation of protein disulphide bonds occurs via an enzyme pathway involving the oxidoreductase Ero1p, and the foldase Pdi1p, which are encoded by the ERO1 and PDI1 genes respectively. In this pathway, oxidising equivalents are transferred between Ero1p, Pdi1p, and substrate proteins requiring disulphide bonds. In the filamentous fungus, Aspergillus niger, the presence of a PDI1 homologue, PDIA, has been demonstrated. PDIA has been shown to catalyse the refolding of denatured and reduced RNase A, and to complement Pdi1p function in a pdi1 strain of S. cerevisiae. We describe an A. niger homologue of ERO1, eroA, and show that it is transcriptionally up-regulated in the presence of agents that cause endoplasmic reticulum stress and induction of the unfolded protein response. We demonstrate that eroA is likely to be essential for viability, and are further characterising the functions through down-regulation, over-expression, and complementation studies.

PR7.14

Development of a new fungal RNA-silencing system in Acremonium chrysogenum and Penicillium chrysogenum

Ricardo Vicente Ullán², Ramiro Pedro Godio², Fernando Teijeira¹, Inmaculada Vaca², Carlos García-Estrada², Juan Francisco Martín²

1Area of Microbiology, Faculty of Biological and Environmental Sciences, University of León, Leon, Spain, ²Institute of Biotechnology (INBIOTEC), Science Park of León, Leon, Spain

In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double stranded RNA expression cassette for gene silencing in the filamentous fungi Acremonium chrysogenum and Penicillium chrysogenum. Targeted gene disruption (knockout) in both microorganisms is too complicated, tedious and time-consuming due to the ectopic integration of exogenous DNA. As substitute, we propose a new system based in the RNA silencing machinery. We have tested this vector with two genes of the -lactam pathway; the pcbC gene for P. chrysogenum and the cefEF for A. chrysogenum. Antibiotic production and mRNA transcripts levels analysis showed gene knockdown in both pcbC and cefEF genes. This new RNAi vector is a new tool for exploring gene function into genomes of P. chrysogenum and A. chrysogenum.

PR7.15

Characterisation of the Trichoderma reesei proteasome

Liisa Kautto¹, Liisa Kautto², Jasmine Grinyer¹, Peter Bergquist¹, Peter Bergquist², Peter Bergquist³, V.S.Junior Teo¹, V.S.Junior Teo², V.S.Junior Teo³, K.M.Helena Nevalainen¹, K.M.Helena Nevalainen²

1Department of Chemistry and Biomolecular Sciences, Macquarie University, 2109 NSW Sydney, Australia, ²Macquarie University Biotechnology Research Institute, Macquarie University, 2109 NSW Sydney, Australia, ³Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand

The broad aim of this work is to advance understanding of the role of the proteasome in cellular protein quality control in Trichoderma reesei, which provides an easy and cost-effective factory for protein production. Some proteins are synthesised with high yields, but others, especially foreign proteins suffer from low yields. This effect could be partly because of misfolding of the foreign proteins in the fungal host resulting in their targeting to the fungal proteasome for degradation.

We have isolated and characterised the 26S proteasome from T. reesei. From the 2D reference map, 12 of the 14 20S proteasome subunits, five of 19S subunits and several interactive proteins that were co-purified with the 26S proteasome have been identified.

We are now exploring the function of the proteasome in ERADication of misfolded proteins from the secretory pathway by using mutant forms of the main cellulase of T. reesei, CBHI. We are examining the CBHI mutant proteins by confocal microscopy so as to trace their secretion and presumed degradation by the proteasome of T. reesei. Also, we are in the process of analysing the transcriptional response of the cells to the production of mutant CBHI proteins by the application of genome-wide CombiMatrix oligonucleotide array analysis.

PR7.16

Artificial generation of large chromosomal deletions in Aspergillus oryzae and Aspergillus sojae by using ku70 deficiency

Tadashi Takahashi, Feng Jie Jin, Yukio Senou, Yasuji Koyama

Noda Institute for Scientific Research, Noda City, Chiba, Japan

By using the combination of a ku70-deficient strain and bidirectional markers of the koji molds Aspergillus oryzae and Aspergillus sojae, we established a fundamental technology for efficiently generating large chromosomal deletions. Previously, we have reported that the genomic deletion of a 60-kb region including the aflatoxin gene cluster was efficiently carried out in an A. sojae strain (1). In this study, we investigated the limitations of the possible deletion range in one deletion cycle and the efficiency of 2 deletion methods, the loop-out (resolution-type recombination) and direct (replacement-type recombination) deletions. The large genomic deletions generated by a loop-out deletion mechanism enable us to make multiple deletions by using marker recycling. As a result, over 300-kb regions of chromosome 3 including the aflatoxin biosynthetic gene cluster and those of chromosome 4 have been completely deleted from the koji molds. Moreover, no additional sequence remained in the resultant deletion strains, which makes this technique suitable for the breeding of food-grade microorganisms.

In addition, we recently discovered that the limitation of deletion range in the replacement-type recombination method was highly expanded in ku70 mutant strains. Thus far, replacement-type recombination between 2 homologous regions separated by a large nonhomologous sequence has been practically impossible due to the low frequency of recombination in wild-type koji mold strains. In this study, we have reported the successful deletion of regions larger than 200 kb in chromosome 3, including the aflatoxin gene cluster, and 470 kb in chromosome 8 with high efficiency by using ku-deficient strains of the koji molds A. sojae and A. oryzae. The results obtained in this study indicate that the ku70 mutation elevated not only the conventional gene-targeting frequency but also the frequency of recombination between 2 distant sites.

The technology described here is applicable for genomic engineering and molecular breeding of industrially used filamentous fungi by artificially generating large-scale genomic deletions and marker-free strains.

^{1 Takahashi et al. (2006) In: Abstracts of ECFG8, PP272.

PR7.17
A trial of the minimization of the chromosome 7 by large-scale chromosomal deletion in A. oryzae RIB40
Feng Jie Jin¹, Toshi Furukido¹, Masafumi Tokuoka¹, Masahiro Ogawa¹, Tadashi Takahashi¹, Masayuki Machida², Yasuji Koyama¹
1Noda institute for scientific research, Noda city,Chiba pref., Japan, ²National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Chromosome engineering is a powerful technology with effectively allowing any chromosomal rearrangement to create desired strains by artificially introducing gene disruption and foreign gene insertion. In particular, large-scale DNA rearrangement is becoming an increasingly important method in this field. We have previously developed a method to efficiently construct large-scale deletion mutants by using a ku70-disrupted strain (1). In this study, using this method with pyrG-mediated transformation system, we attempted to construct a mutant with the minimum set of genes in chromosome 7, which is the smallest chromosome (2.93 Mb) in A. oryzae RIB40 strain. In this way, our final goal is to breed industrially favored strains by appropriately altering regulation of metabolite production.
We first constructed series of large-scale deletion mutants (16~150 kb) with a particular focus on the non-syntenic regions, which were characterized by the comparison of A. oryzae genome with those of other Aspergilli (2). Based on these results, we extrapolated that approximately a quarter of chromosome 7, at least 740 kb sequence, was non-essential and could be deleted. We then sequentially deleted these confirmed non-essential regions using the A. oryzae ku70 disruption strain. Until now, we have successfully constructed a mutant lacking as large as 685 kb genome sequence (23.4% of chromosome 7) by multiple deletions. In addition, we also observed and compared the growth phenotype of obtained large-scale deletion mutants and the parental strain. In one of deletion mutants, the number of formed conidia increased more than 1.6-fold compared with that of the parental strain. More interestingly, the number of metabolites of this mutant was markedly decreased. These results suggested that the large-scale deletion method not only could be useful to breed industrial strains, but also opened up a new avenue of research to analyze global genetic function on a large scale in chromosomes.

^{1 Takahashi et al. (2006) In: Abstracts of ECFG8, P. 272, ² Tamano et al. (2008) Fungal Genet Biol. 45, 139-151

PR7.18
Extracellular beta-glucosidase activities in Zygomycetes
Csaba Vagvolgyi¹, Miklos Tako¹, Elvira Farkas¹, Tamas Papp¹, Judit Krisch²
1Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary, ²Department of Food Engineering, Faculty of Engineering, University of Szeged, Szeged, Hungary

Introduction: Beta-glucosidases constitute a group of well characterized, biologically important enzymes that catalyze the transfer of the glycosyl group between oxygen nucleophiles. They play important roles in biology, including degradation of cellulose biomass by fungi and bacteria, degradation of glycolipids in mammalian lysosomes, and the cleavage of glycosylated flavonoids in plants. Several Zygomycetes fungi have biotechnological significance as producers of extracellular enzymes, e.g. proteases and lipases. However, this fungal group is poorly characterized from the aspect of beta-glucosidases. The aim of this work was to find new producer strains potentially applicable in further basic and biotechnological studies.
Methods: Detection of the extracellular beta-glucosidase activity of 71 strains was performed in minimal medium containing cellobiose as the sole carbon source. Ten isolates from the good producers were chosen for solid state fermentation studies, in which wheat or oat bran were used as carbon source. Enzyme activities were assayed spectrophotometrically by using p-nitrophenyl beta-D-glucopyranoside; isoenzyme analysis was also carried out with the tested strains. Temperature and pH optimum of some purified enzymes were determined also.
Results: Enzyme activities of the tested strains were found to be highly variable. On the basis of the liquid culture fermentation, ten isolates representing different Gilbertella, Rhizomucor and Rhizopus species showed high extracellular beta-glucosidase activity. In each case, the highest activity was detected on the sixth culturing day. In the solid state fermentation assay, an R. miehei isolate gave the highest activity, however culture filtrate from the same isolate growth in liquid medium showed the lowest activity among the selected producers. Extracellular enzyme activities of the other strains were also stronger in solid state fermentation than in liquid culture. Enzymes with different temperature optimum and stability could be isolated.

This research was supported by ETT grants (214/2006; 261/2006) and the J. Bolyai Research Scholarship of the Hungarian Academy of Sciences.

PR7.19
Carotenoid content of different species belonging to the class Zygomycetes
Arpad Csernetics¹, Tamas Papp¹, Andras Szekeres², Csaba Vagvolgyi¹
1Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary, ²Analytical Laboratory of Cereal Research Non-profit Company, Szeged, Hungary

Introduction: Three closely related Zygomycetes fungi, Phycomyces blakesleeanus, Blakeslea trispora and Mucor circinelloides have been traditionally involved in the studies on the fungal carotene biosynthesis. The main industrial carotenoid producer fungus is even now the B. trispora, where high production level has been achieved by the selection of mutants and the optimization of the growth conditions. On the basis of morphological observations, several other Zygomycetes seems to be promising producer, but their carotenoid content has not been analysed.
Methods: The main objective of this study was to reveal the carotenoid composition and to characterize the carotenoid production of different fungi belonging to the order Mucorales. Twenty isolates representing several species of the genera Mucor, Backusella, Rhizopus and Gilbertella were involved in the study. After cultivation under continuous light, carotenoid content of the strains was analysed by HPLC and TLC. The effects of different carbon sources and the growth temperature on the carotenoid production were also examined.
Results: Some Mucor species produced the valuable hydroxylated derivatives of beta-carotene, e.g. beta-cryptoxanthin and zeaxanthin, in considerable quantities. Some strains belonging to the species M. bainieri and M. hiemalis produced two fold more amount of carotenoids than M. circinelloides and the wild-type B. trispora, model organisms of the carotenogenic studies. G. persicaria produced significant amount of pigments only if it was plated as a mixture of two strains with the opposite mating types.

This research was supported by ETT grants (214/2006; 261/2006). T. P. is a grantee of the J. Bolyai Research Scholarship.

PR7.20
A defensin-like protein from Aspergillus nidulans is a potent inhibitor of fungal growth
Andrea Eigentler¹, István Pócsi², Florentine Marx¹
1Biocenter, Division of Molecular Biology, Innsbruck Medical University, Innsbruck, Austria, ²Department of Microbial Biotechnology and Cell Biology, Faculty of Science, University of Debrecen, Debrecen, Hungary

Antimicrobial proteins have great potential for the development of new therapeutic strategies. In the genome of Aspergillus nidulans we identified a small gene of 345 nt which we called defensin A (defA). The open reading frame of 225 nt encodes a preproprotein of 74 amino acids (aa). The mature protein has a molecular mass of 4 kDa and its primary sequence exhibits 51% identity and 83% homology with the aa sequence of the insect defensin AaDefA1 from the mosquito Aedes aegyptii. Northern blot analysis confirmed gene transcription and we found a strong induction of gene expression under stress conditions. By in silico analysis we identified four potential stress consensus sequences (STRE) in the promoter region of the defA gene, which correlates well with its expression pattern.
To study the putative antimicrobial activity of DefA we recombinantly expressed the protein by using the Pichia pastoris multicopy expression system (Invitrogen) to produce a high amount of protein for further analysis. The recombinant protein was purified from the supernatant of a 72 h culture by ion-exchange chromatography and the identity of the protein was confirmed by mass spectroscopy.
The purified protein showed potent growth inhibitory activity against filamentous fungi and yeasts as determined by growth inhibition assays. Further analyses of its species specificity, the MIC and its mode of action are currently in progress.

This work is in part supported by the D. Swarowski Forschungsfonds (FB2/06).

PR7.21
Prospecting of novel cold-active enzymes and their genes from Antarctic microfungi
Suja Mohammed¹, Suja Mohammed², V. S. Junior Te'o¹, V. S. Junior Te'o², Peter L. Bergquist¹, Peter L. Bergquist², Peter L. Bergquist³, K. M. Helena Nevalainen^1,2

1Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia, ²Applimex Systems Pty. Ltd., Sydney, NSW, Australia, ³Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand

Cold-active enzymes have a high specific activity at low and moderate temperatures and are thermolabile (1). Their cold activity and thermolability makes them attractive as industrial catalysts to facilitate biotechnological processes at low temperatures and also as potential research tools. The aim of this study was to identify and isolate genes responsible for the synthesis of cold-active enzymes of interest from Antarctic microfungi. We have selected six Antarctic fungal strains to investigate the secretion of lipases, proteases and cellulases, which have major applications in food and detergent industry.
The initial study was carried out by plate assays with appropriate substrates for each enzyme. The fungal strains were grown in liquid culture and the enzyme activities in the culture supernatants were analyzed. Out of the six Antarctic strains, Geomyces pannorum and Phoma sp. were found to exhibit the highest activities for lipase and protease respectively, when compared to the other strains. These enzyme activities were confirmed by zymogram activity analysis. We have carried out modifications of the culture medium to improve enzyme production in the best strains. The pH and temperature profile of the enzyme activities were studied since these factors contribute to the use of enzymes for particular industrial purposes. Our results indicate the presence of a cold-active lipase and protease from the Antarctic strains Geomyces pannorum and Phoma sp. respectively.

^{1 Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J., Claverie, P., Collins, T., D’Amico, S., Dumont, J., Garsoux, G., Georlette, D., Hoyoux, A., Lonhienne, T., Meuwis, M. and Feller, G. (2000). Trends Biotechnol. 18: 103-107.

PR7.22
Improved dominant selection markers for Coprinus cinereus: re-evaluation of hygromycin & phleomycin resistance vectors and a "new" carboxin resistance system
Sreedhar Kilaru, Catherine Collins, Amanda Hartley, Gary Foster, Andy Bailey
University of Bristol, Bristol, United Kingdom

Dominant selection systems are beneficial for transformation of many fungi. In this study, we used Coprinus cinereus as a model organism to develop vectors containing dominant selectable marker genes based on antibiotic selection. Plasmids previously used to establish hygromycin (hph) transformation systems of several basidiomycete species failed to give rise to hygromycin-resistant transformants of C. cinereus. Sequence analysis of these constructs showed that they had all been modified removing the two lysine residues adjacent to the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two residues in C. cinereus. Phleomycin-resistant (ble) transformants were also obtained but only with the intron-containing construct pblei004, not with intron-less construct pble004, showing that an intron is necessary for ble expression in C. cinereus. A carboxin (cbx) resistant allele of the C. cinereus sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the conserved histidine block. This modified gene was used to successfully confer carboxin resistance upon transformation of C. cinereus protoplasts. Based on the total number of transformants obtained, C. cinereus protoplasts were more efficiently transformed to carboxin resistance when compared to hygromycin and phleomycin resistance highlighting the potential use of this homologous marker gene. In order to investigate the expression of two different dominant selectable marker genes in a single transformation experiment, vectors pYES-hph-cbxgene and pYES-hph-icbxgene with dual marker genes were constructed with hph and sdi^R cassettes in yeast through in vivo recombination. The unique restriction sites present in the sdi^R and hph genes allow the replacement of one of the selectable marker genes with a gene of interest and consequently avoid the requirements for co-transformations. We replaced the sdi^R gene in these vectors with the fluorescent marker gene Discosoma red (DsRed). Expression of DsRed was only observed in transformants containing vector with intron. These vectors should allow several transformations of same strain of C. cinereus and be of use in establishing transformation systems of other basidiomycete species.

PR7.23
Breeding of the hyper-producing strains for heterologous proteins by double proteinase disruption and mutagenesis in Aspergillus oryzae

Jun-Ichi Maruyama, Taisuke Watanabe, Takashi Nemoto, Katsuhiko Kitamoto
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Aspergillus oryzae has drawn much attention as one of the most excellent hosts for protein production due to its ability to secrete vast amounts of proteins and the safety guaranteed by the use in fermentative food industry. While the production level of homologous (fungal) proteins by A. oryzae can reach gram-per-liter scale, the production of heterologous (mammalian and plant) proteins yields only milligram-per-liter scale. Although hyper-producing strains are required for higher-level production of heterologous proteins, only a few attempts have been reported for breeding of an excellent production host in A. oryzae.
We previously reported that the double disruption of the proteinase genes (tppA and pepE) increased the production of human lysozyme (HLY) by 1.63-fold (15.6 mg/l to 25.4 mg/l).¹⁾ Moreover, by using the double proteinase disruptant, the production yield of bovine chymosin (CHY) was 65.1 mg/l, which was 1.93-fold increment from the control strain (33.8 mg/l). High productivity of the two heterologous proteins by the double disruptant raised the possibility that the strain showing a higher-level production of HLY might also produce other heterologous proteins in larger amounts. In order to test this hypothesis, hyper-producing mutants of HLY were screened from the double disruptant. For this purpose we developed an efficient screening system by employing halo assay with bacterial cells, the HLY substrate. Among 22 hyper-producing mutants obtained from ~80,000 colonies, the highest production level of HLY was 50.8 mg/l, which was 2.00-fold increment compared to that of parental strain (the double disruptant). Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection using chlorate, which is metabolized into a cytotoxic compound by the nitrate assimilation pathway. The 8 strains obtained by the curing were named as AUT1~8 (A. oryzae hyper-producing strains bred in The University of Tokyo). The maximum production yield of CHY by the AUT strains was 107.9 mg/l, which was 1.66-fold increase from the double disruptant. In conclusion, we successfully increased heterologous protein production (3.26- and 3.19-folds for HLY and CHY, respectively) by double proteinase disruption and mutagenesis in A. oryzae. The AUT strains are expected as powerful hosts for higher-level production for many heterologous proteins.

^{1 Jin et al., Appl Microbiol Biotechnol. 2007; 76: 1059-1068.

PR7.24
Characterisation of novel oxalate decarboxylase from the white rot fungus Dichomitus squalens

Miia Mäkelä, Kristiina Hildén, Annele Hatakka, Taina Lundell
Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland

We have cloned a novel oxalate decarboxylase (OxDC) -encoding gene and partially characterised an OxDC protein from a basidiomycetous white rot fungus Dichomitus squalens. Certain bacteria and fungi produce OxDC (EC 4.1.1.2) enzyme which catalyses the decarboxylation of oxalic acid into stoichiometric quantities of formic acid and carbon dioxide in a highly specific reaction. The regulation of organic acid production is an essential part of effective lignin degradation caused by white rot fungi which makes the specific oxalate decomposing enzymes, such as OxDC, important to study.
So far, only four sequences of OxDC-encoding genes from basidiomycetes are available: Flammulina (Collybia) velutipes, Flammulina sp., Phanerochaete chrysosporium, and Trametes versicolor (only cDNA). Recently, P. chrysosporium has been shown to produce the OxDC protein under growth conditions that promote wood degradation.
Because of their high specificity to oxalate, OxDCs have been used in clinical and food assays for the determination of oxalate. Transgenic crop plants expressing an oxdc-gene have been generated in order to increase the resistance of host plant to fungal infections. The use of OxDC for the degradation of oxalate deposits in breweries has been patented (pat.no US4652452). The potential use of oxalate-degrading enzymes to prevent the formation of calcium oxalate deposits during the bleaching of pulp in paper making processes has also been noticed.
D. squalens produced oxalic acid during the growth in liquid cultures and on natural solid wood substrate. The fungus showed high intracellular OxDC activity after induction with oxalic acid. In order to find out the properties of D. squalens OxDC, the gene was cloned and the protein was partially characterised. The amino acid composition of the predicted Ds-OxDC has the highest pair wise identity of 56.9 % with P. chrysosporium OxDC. Two putative His-containing cupin motifs and four N-glycosylation sites are found in the translated Ds-OxDC aa-sequence. Ds-OxDC has a molecular weight of 72 kDa and in chromatofocusing it shows an isoelectric point of 4.2.

PR7.25
Transformation of Aspergillus oryzae RIB40 with the bleomycin acetyltransferase-encoding gene as a selection marker
Satoshi Suzuki¹, Mari Fukuoka¹, Hiroko Taketani¹, Sawaki Tada¹, Mayumi Matsushita¹, Ken-Ichi Kusumoto¹, Yutaka Kashiwagi¹, Masanori Sugiyama²
1National Food Research Institute, Tsukuba, Ibaraki, Japan, ²Hiroshima University, Hiroshima, Hiroshima, Japan

Introduction: Aspergillus oryzae is one of the most important fungi in Japanese traditional fermentation industry. Since this microorganism also produces many-food processing enzymes, it is significant to establish a host-vector system for A. oryzae. In various filamentous fungi, the genes, which confer resistance to hygromycin B, aureobasidin, and G418, have been used as genetic markers for the gene manipulation. However, A. oryzae is resistant to these antibiotics. We notice that high concentration of bleomycin inhibits the growth of A. oryzae RIB40 which is known as a genome reading strain. We have recently found that some agents, such as tritonX100, increase the bleomycin-susceptibility of A. oryzae (data not shown). In the present study, we developed a transformation system for A. oryzae RIB40 by using bleomycin-resistance gene as a selective marker.
Methods: The codon of the bleomycin N-acetyltransferase gene from Streptomyces verticillus were optimized according to the codon usage of the fungus and inserted into the plasmid pPTRI under control of the histone H2B promoter. The resulting chimeric plasmid was introduced into the protoplasts from A. oryzae RIB40 by the polyethylene glycol method. The transformants were selected on the maltextract-peptone medium containing 27 microgram/ml of bleomycin and bleomycin potentiation agents. The integration of the plasmid into genomic DNA of transformants was confirmed by the colony PCR method using FTA card and the Southern blot analysis.

Results: We successfully selected the transformants, which exhibit bleomycin resistance, on the agar plate. The background outgrowth of non-transformants was clearly suppressed by bleomycin. The transformants were inoculated on the non-selective PDA plate and allowed to grow. The colony PCR analysis shows that the growing tip of colony inoculated on non-selective medium retains the DNA fragment carrying the bleomycin N-acetyltransferase gene.
Discussion: In the present study, we established the transformation system for A. oryzae by using the Streptomyces bleomycin N-acetyltransferase gene. We expect that the established system contribute to many research for A. oryzae.

PR7.26
Efficient production and partial characterization of one of metalloproteases, aspartyl aminopeptidase from Aspergillus oryzae

Ken-Ichi Kusumoto¹, Mayumi Matsushita¹, Ikuyo Furukawa¹, Satoshi Suzuki¹, Youhei Yamataga², Yoshinao Koide³, Hiroki Ishida⁴, Michio Takeuchi⁵, Yutaka Kashiwagi¹
1National Food Research Institute, Tsukuba, Ibaraki, Japan, ²Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan, ³Gifu R & D Center, Amano Enzyme Inc., Kagamihara, Gifu, Japan, ⁴Research Institute, Gekkeikan Sake Company Ltd., Fushimi-ku, Kyoto, Japan, ⁵Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan

Aspartyl aminopeptidase from Aspergillus oryzae has high substrate specificity, degrading only amino-terminal acidic amino acids from peptides, and may be suitable for processing physiological peptides. However, little is known about the biochemical properties of this enzyme and its efficient production in Aspergillus oryzae.
The gene encoding aspartyl aminopeptidase was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a cobalt-containing medium. The enzyme was extracted from mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high affinity to peptide substrate rather than synthetic substrates. Thus, recombinant A. oryzae aspartyl aminopeptidase was purified to homogeneity with an increased specific activity, when cultivated in a cobalt ion-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help increase specific activity of other metalloproteases and their functional analysis.

PR7.27
Alternative oxidase is responsible for the oxidative defense in Aspergillus fumigatus

Taisa Magnani, Frederico Soriani, Anna Policarpo, Carlos Curti, Sergio Uyemura
Sao Paulo University, Ribeirão Preto/ São Paulo, Brazil

A. fumigatus is a human pathogenic fungus capable of inducing a range of disease states in immunocompromised patients. Fungal respiration exhibits peculiar features and involves an alternative oxidase pathway not yet fully elucidated. In this sense, we have silenced an aox gene in A. fumigatus (Afaox) by RNA interference. Inverted repeats of Afaox coding sequence were cloned in the pALB1 plasmid, which contain an alb1 gene cloned. In xylose medium, the Afaox/alb1 and alb1 transformants had the same phenotype, a green color, as the wild strain, indicating that the interference was not induced. However, in maltose medium the Afaox/alb1 transformant grew in white color indicating an entire silence and a Real-time PCR revealed a decrease of 95% in RNAm levels. It has been suggested that AOX may be important for fungal survival in macrophages, based on accumulating evidence for its antioxidant defense. ROS production was monitored using fluorescent probe CM-H₂DCFDA. In Afaox/alb1 transformants, ROS production increased 12.5 and 2-fold when compared with wild-strain and alb1 transformants, respectively, suggesting that AOX can prevent ROS formation. The survival rate of silenced strain in macrophages was measured by MTT assay. Afaox, double silenced strain, revealed a highest survival when compared with the other strains. Conidia were incubated with different ROS donors and conidia viability was measured by flow cytometria with FUN-1 and PI uptakes.
These results indicate that the alternative oxidase has an important role in oxidative defense in Aspergillus fumigatus.

Supported by: FAPESP and CNPq

PR7.28
Overexpression of an E. coli phytase mutant in Trichoderma reesei and characterization of the enzyme
Dietrich Löbel, Tatiana Schwarz, Silvia Paladino, Anja Lingner, Gerhard Leiss, Oliver Schmidt, Astrid Pfeifer, Bruno Winter, Jari Vehmaanperä, Khanh Nguyen
AB Enzymes GmbH, Feldbergstrasse 78, 64293 Darmstadt, Germany

Phytases catalyze the sequential hydrolysis of phytic acid (IP6) to obtain less phosphory-lated myo-inositol derivatives plus inorganic phosphate. About 70% of phosphate in grain used as livestock feed exists in the form of phytic acid. However, monogastric animals such as pig, poultry and fish are unable to digest phytic acid efficiently, resulting in low phosphate availability. Phytases added to the diets of monogastric animals enhance the bioavailability of organic phosphate and reduce the need for inorganic phosphate supplementation.
Phytases belong to the family of histidine acid phosphatases and are found in several microorganisms and plants. E. coli phytase (EC 3.1.3.26) is known to have a particularly high specific activity, paired with adequate gastric performance at low pH and strong pepsin resistance (Konietzny & Greiner, Int.J.Food Sci.Technol., 2002, 37:791). When expressed in Trichoderma reesei the E.coli phytase retains its favourable molecular characteristics and results in commercially feasible yields.
The aim of this study was to enhance E.coli phytase production through site-directed mutagenesis. The E. coli phytase gene used in this investigation is based on the amino acid sequence of acid phosphatase appA (Dassa et al., J.Bacteriol., 1990, 172(9):5497). The E.coli phytase encoding gene was designed and synthesized with the codon usage of T.reesei. The gene was placed under control of the T.reesei cbhI promoter and cbhI-terminator and the plasmid containing the WT-phytase sequence designated pKDa4. In another plasmid pKDa2 the amino acid Val²⁰⁰ (GTG) of the phytase gene was changed to Tyr²⁰⁰ (TAC).
The E.coli phytase product (spent medium) reached the highest activity after submerged fermentation in industrial medium for 6 days. The phytase mutant V200Y exhibited a 2.2-times higher expression level than the wild type.
The E.coli phytase produced in T. reesei is non-uniformly glycosylated and therefore present in 3 main bands in SDS-PAGE. Treatment of the phytase with PNGase F led to a shift in molecular weight towards a single band of 44.5 kDa. The pH dependence of the enzyme was investigated between pH 3.0 and pH 7.0 and the temperature activity profile of the purified enzyme was determined between 30°C and 70°C. The thermostability of the purified phytase was analyzed by differential scanning calorimetry (DSC).

PR7.29
Pcr-based early detection of Penicillium expansum in apple
Valentina Tolaini, Massimo Reverberi, Federico Punelli, Corrado Fanelli
Università La Sapienza, Roma, Italy

Penicillium expansum is one of the major post-harvest pathogens on apple fruit and its infection is associated with patulin production, a secondary metabolite with immunological, neurological and gastrointestinal toxic effects. Use of mouldy apples contaminated with P. expansum greatly increases the risk of patulin contamination in fruit juices. Early detection of this pathogen on apples is important for ensuring quality and safety of fruits and fruit juices. The objective of this study was to develop and optimize a PCR-based assay for rapid and specific detection of P. expansum in apple.
In order to discriminate P. expansum from other natural apple-contaminating fungi by PCR, specie-specific primers have been designed on the basis of the consensus conserved sequence of the Pepg1 gene of P. expansum, which encodes for the enzyme responsible of fruit tissue rot. Another set of primers was designed on the basis of the sequence of a PCR fragment of P. expansum obtained by amplification with M13 minisatellite primer. These primers were used for the amplification of DNA extracted by different fungi isolated from apples (cv Annurca and cv Golden) and grown in vitro on potato dextrose agar (PDA) and from apples contaminated and not contaminated with P. expansum.

The use of Pepg and Pem13 primers in optimal PCR conditions shows their ability to amplify a specific sequence of P. expansum DNA. None of the other species have been detected. These results indicate the specificity of this PCR system for P. expansum. Preliminary results obtained in contaminated apples confirm the specificity and sensitivity of the PCR-based assay.
Nucleic acid-based method as PCR could be used as a rapid tool for detecting P. expansum in apple. This assay can be a good alternative to the traditional diagnostic methods as cultural method.

PR7.30
Modified hydrolase profiles in Hypocrea jecorina by changing the expression of the general activator protein Xylanase regulator 1 (Xyr1)
Astrid R Stricker, Matthias G Steiger, Marion E Pucher, Robert L Mach
Vienna University of Technology, Vienna, Austria

The filamentous fungus Hypocrea jecorina (Trichoderma reesei) for a long time has been used for different important industrial applications in the areas food and feed, textile, as well as pulp and paper production. Its fundamental role in fermentation technologies remains unaltered, not only but in particular due to its high secretory capacity. Systematic strain improvement resulted in production of levels of extracellular protein of over 100 g/L. However, molecular approaches to change and/or improve the hydrolase cocktail composition remained rare. Here we present that the modification of the expression level of the major transcriptional activator Xyr1 significantly changes the expression profile of several genes coding for the hydrolase enzyme system of this fungus. Interestingly, an increase in the expression of xyr1 does not consequently increase the transcription of all hydrolytic enzyme-encoding genes subject to regulation by this factor, but changes the ratio of the respective mRNA levels. In addition, we can prove that a constitutive expression of xyr1 does not lead to the induction of the hydrolase expression, since the presence of an inducer molecule is strictly required. Finally, we provide evidence that a posttranslational modification, most probably a phosphorylation, is responsible for the conversion of Xyr1 into its active form.

PR7.31
PCR and spectral imaging based assays for the early detection of aflatoxigenic fungi on maize kernels
Antonella Del Fiore¹, Silvia Serranti², Alessandra Ricelli³, Massimo Reverberi², Annadele Fabbri², Giuseppe Bonifazi², Corrado Fanelli²
1ENEA CR Casaccia, Roma, Italy, ²Univesità La Sapienza, Roma, Italy, ³CNR, Roma, Italy

Fungi are a real issue for the cereal industry. Some fungal species, such as Aspergillus flavus and A. parasiticus, can produce under suitable conditions, aflatoxins, secondary metabolites which are toxic for human and animals. Many methods have been utilized to measure fungal contamination on cereals. The aim of this work is to use a PCR based method and a spectral imaging based method to detect aflatoxigenic fungi on maize. In the experiments were used two aflatoxigenic strains, A. parasiticus NRRL 2999, A. flavus NRRL 3357, and one non aflatoxigenic strain of A. parasiticus NRRL 11096. An A. Niger strain and other strains of Aspergillus spp., Penicillium sp. and Fusarium sp. isolated from maize were also analyzed. Maize hybrids employed in food industry, were used in the PCR-based assay. Genomic DNA was extracted from pure fungal strains, not inoculated and inoculated maize kernels. A primer pair was designed on the coding portion of omt-1 gene which encodes for the enzyme O-methyltransferase II necessary for the last step of aflatoxin biosynthesis. The DNA extracted was used as template for PCR amplification with the primer pair selected. PCR amplification generate a fragment of 1254 bp length in A. flavus and A. parasiticus. Amplification operative conditions were optimized on pure fungal strains and confirmed on maize. DNA amplification was achieved only with DNA from fungal strains of A. parasiticus and A. flavus and from maize inoculated with A. flavus or A. parasiticus, never with DNA of other fungal strains. Specificity was confirmed with DNA extracted from different fungal strains. These results indicate that this PCR-based method could be able to discriminate maize kernels infected with A. flavus and A. parasiticus from un-infected ones; this method could be used like early detection system, for aflatoxigenic strains in maize. Several studies have tried non-destructive, spectral methods to detect fungal contamination on cereals. At this concern we present a method, based on the spectral imaging, to detect fungal contamination on maize. A desktop spectral imaging system, ImSpectorTM, V10 were used in the work; two aflatoxigenic species were used, A. parasiticus, and A. flavus. They are inoculated on maize and were imaged on day 7 of growth. Changes in reflectance of maize were observed during fungal growth. This approach could be a rapid method of detecting the aflatoxigenic fungi on cereals.

PR7.32
A molecular early detection method of Aspergillus carbonarius on grape and a novel analysis for Ochratoxin A in wine
Patrizia De Rossi¹, Massimo Reverberi², Alessandra Ricelli³, Domenico Caputo², Giampiero De Cesare², Augusto Nascetti², Riccardo Scipinotti², Corrado Fanelli²
1ENEA CR Casaccia, Roma, Italy, ²Università La Sapienza, Roma, Italy, ³CNR, Roma, Italy

Aspergillus carbonarius is an important ochratoxin A (OTA) producing fungus which is responsible for toxin contamination of grapes and wine. OTA is a secondary metabolite produced by fungi belonging to Aspergillus and Penicillium genera which has been shown to be nephrotoxic, nephrocarcinogenic, teratogenic and immunosuppressive.
The objective of this research was to investigate the presence of A. carbonarius in grapes and of OTA in wine.
Two PCR-based assays have been developed to detect the presence of A. carbonarius in grapes by designing specie-specific primers on the basis of Internal Transcribe Spacers of rDNA units (Ac-ITS) and of the polyketide synthases (Ac-PKS) sequences. These specific primers were used for A. carbonarius detection in grapes. For early detection of Ochratoxin A the performance of a system based on hydrogenated amorphous silicon photosensors was investigated. This device is based on the measurement of the photocurrent induced in a hydrogenated amorphous silicon (a-Si:H) photodiode by the fluorescence of the mycotoxin excited by a UV radiation.
The results of PCR using DNA extracted from various grape-contaminating fungi show that only A. carbonarius DNA was amplified with Ac-ITS and Ac-PKS primers. None of the other species gave a positive result with this PCR primers set used. Concerning OTA detection in wine the photocurrent measured by the sensor is due to the mycotoxin and the value is proportional to its quantity.
PCR-based methods that target DNA are considered a good alternative with respect traditional diagnostic methods for early detection because of their high specificity and sensitivity. This PCR analysis was also successfully employed to detect A. carbonarius in grape. Currently, the most of the methods available for the determination of OTA in wine are based on an extraction step, a clean-up passage and determination by High Performance Liquid Chromatografy (HPLC) with fluorescence detection. The sensor we used could contribute to early and rapid detection of potential presence of OTA in wine samples.

PR7.33
Production of full length antibody chains in Chrysosporium

Peter Jan Punt³, Margreet Heerikhuisen³, Eddy Motshagen³, Marian van Muijlwijk³, Cora van Zeijl³, Rich Burlingame², Martijn Meens¹, Jan Verdoes¹
1Dyadic Nederland B.V, Wageningen, Netherlands, ²Dyadic Intl, Jupiter, Florida, United States, ³TNO Quality of Life, Zeist, Netherlands

Filamentous fungi are widely used for enzyme production for a wide variety of uses – for example food, feed, textile, paper and pulp, fuels and chemicals, detergents - due to the development of extremely productive strains and production processes. Levels in the range of 100 grams protein per liter have been reported, repeatedly. Therefore, these organisms have also been considered for the production of pharmaceutical proteins. In the pharmaceutical industry, the main production platforms are E. coli and mammalian cell lines. However, for those pharmaceutical proteins for which high yields and low production costs are important, filamentous fungi could provide a viable alternative.
We have explored the use of a highly productive fungal production platform (Chrysosporium C1) for the production of a very versatile class of pharmaceutical proteins, i.e., antibody molecules. Antibody molecules and molecules carrying antibody domains are currently the largest and fastest-growing class of biopharmaceuticals. Production of functional full-length human monoclonal antibodies has been accomplished using highly productive low protease mutant Chrysosporium host strains. High level expression was achieved using a glucoamylase-carrier approach, and recombinant strains expressing both heavy and light chains were obtained. Heterodimeric antibody molecules were formed efficiently, allowing simple purification of the protein from the culture fluid using protein A. Cell-based bio-assays performed on the culture supernatant and the purified samples revealed almost complete bioactivity.

PR7.34
Bioconversion of ginsenocides in red ginseng by fermentation with Monascus

Jee-Hwan Oh, Sung Yun Hong, Inhyung Lee

Kookmin University, Seoul, Republic of Korea

Red ginseng contains ginsenosides that have various healthy effects. Some of ginsenosides are transformed from its glycone to aglycone forms during processing of red ginseng, but lots of them still remain as glycone form. Because alycone ginsenosides are superior in bioavailability and some aglycone ginsenocides such as Rh1, Rh2 and Rg3, have excellent antitumor activities, it is important to transform glycone to aglycone gincenosides to benefit more healthy function of red ginseng. Here we converted glycones to aglycone ginsenosides in red ginseng by fermentation with Monascus. Because Monascus produces several bioactive compounds, Monascus fermented red ginseng may have additional healthy functions. Especially monacolin K, one of metabolites produced by Monascus, has effects of reducing cholesterol in blood. We isolated three Monascus sp., RC02, RK03 and RU03 that have high activities of β-D-glucosidase, α-L-rhamnosidase, and α-L-rhamnosidase with high monacolin K and without apparent citrinin production in 10% red ginseng medium. We scaled up to 50 L fermentation with 13% red ginseng, 2% glucose using two fermentation conditions of RC02, RK03, and RU03 mixed culture and RK03 single culture. Lyophilized powder of fermented product using mixed and RK03 cultures contained 693.04 mg/kg and 3,089 mg/kg of monacolin K, respectively. The total amount of ginsenosides, Rh1, Rh2, and Rg3 was 838.7 mg/kg in red ginseng and that increased up to 4,016 mg/kg and 4,117 mg/kg after fermentation with mixed culture and RK03 culture, respectively. In any case, citrinin was not detected. In addition total gensenocides was almost the same as in red ginseng after fermentation, indicating that fermentation with Monascus did not affect total amount of ginsenosides. The newly developed fermented red ginseng using Monascus would have not only better healthy effects of red ginseng but also additional functions by Monascus fermentation.

PR7.35
Approaching zero growth in retentostat cultures of Aspergillus niger

Thomas R. Jørgensen, Cees A.M.J.J. van den Hondel, Arthur F.J. Ram
^{1Institute of Biology Leiden University, Leiden, Netherlands, ²Kluyver Centre, Delft, Netherlands}Micro-organisms are frequently exposed to suboptimal growth conditions, in nature as well as in culture. The ideal industrial production culture is characterized by high product yield and minimal biomass formation. We study how cellular processes are affected by starvation in energy-source maintained cultures. For this purpose, the enzyme and organic acid producing fungus, Aspergillus niger, was cultivated in maltose-limited retentostat cultures. A newly developed filter, in stainless-steel, allowed efficient and durable retention of mycelia. The filtration efficiency exceeded 95% at any time. Retentostat cultivation was initiated in end-exponential growth phase of batch cultures and lasted for about 11-12 days. Cultivation was ended at this time, because of massive foaming.
Carbon- and energy-source starvation induced morphological and metabolic differentiation, which became apparent after 5-6 days. Simple asexual reproductive structures, resembling phialides, and reduced conidiophores were formed at tips of leading and branching hyphae. Concomitantly, the culture broth turned dark brown reflecting melanisation of mycelial structures and formation of conidia. Effects of maintenance energy requirement (1, 2) were apparent from biomass accumulation over time, although the growth rate never reached zero. Analyses of major cellular components revealed that accumulation of protein and lipid was time-proportional, and that the apparent maintenance effect reflected a decreasing content of carbohydrate. Changes in cell composition and biomass accumulation appeared to coincide with asexual differentiation and changes in hyphal dimensions. Product formation, in terms of secreted protein and glucoamylase activity, will be assessed for different strains in retentostat cultures. Thorough characterisation of A. niger N402 in retentostat cultures will be used for basal description of starvation-induced changes in cellular processes and to identify target pathways/elements for genetic engineering. In addition, these cultures will serve as reference for comparison of a glucoamylase hyper-producing strain and other engineered strains.

^{1 Pirt, S.J., Proc. Roy. Soc. B 163, 224-231. 1965, ² van Verseveld, H.W., Metwally, M., el Sayed, M., Schrickx, J.M., A.H. Stouthamer, Antonie van Leeuwenhoek 60, 313-323. 1991.

PR7.36
The N-terminal region of Aspergillus oryzae hydrophobin RolA is important for RolA-cutinae CutL1 interaction
Toru Takahashi¹, Kenji Uehara², Yohei Yamagata², Katsuya Gomi², Fumihiko Hasegawa¹, Keietsu Abe¹
1New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan, ²Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae is cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding a Type I hydrophobin RolA is highly transcribed. High levels of RolA are localized on the cell surface and also secreted into the liquid medium. Under these conditions, A. oryzae simultaneously produces the cutinase CutL1, which hydrolyzes PBSA. RolA adsorbed to the hydrophobic surface of PBSA particles in the medium recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis (1, 2). The ability that RolA attached to the PBSA surfaces recruits esterase CutL1 is a newly discovered function in hydrophobin (1). In the present study, we studied amino acid residues involved in the RolA-CutL1 interaction by means of site-directed mutagenesis and chemical modification of RolA. Teflon particles were coated with RolA and its derivatives. The Teflon particles coated with RolA or its derivatives were incubated with soluble CutL1 for recruitment, and then the supernatant and particles were separated by centrifugation. Recruited CutL1 was extracted from the centrifuged particles with SDS and quantitatively measured by SDS-PAGE analyses. We found that the N-terminal region of RolA is important for RolA-CutL1 interaction. We discuss amino acid residues involved in the interaction.

^{1 Takahashi T. et al. Mol. Microbiol. 57:1780-1798 (2005), ² Ohtaki S. et al. Appl. Environ. Microbiol. 72:2407-2413 (2006)

PR7.37
Improvement of homologous recombination in Penicillium chrysogenum: impact of the hdfA mutation on fitness and genome wide transcriptional response.
Ishtar Snoek¹, Zita van der Krogt¹, Hesselien Touw², R Kerkman², Jack Pronk¹, Roel Bovenberg², Marco van den Berg², Jean-Marc Daran¹
1TU Delft, Delft, Netherlands, ²DSM, Delft, Netherlands

Targeting a transformed DNA fragment to a specific site in the chromosome of an organism makes use of homologous recombination repair mechanisms. While integration of exogenous DNA occurs readily in Saccharomyces cerevisiae, this mechanism of DNA repair is not predominant in other fungi. However, the targeting efficiency can be increased by disabling the non homologous end joining (NHEJ) pathway. The deletion of the gene homologous to the human KU70 which encodes a protein that functions in the NHEJ pathway has been shown to improve homologous recombination successfully in many filamentous fungi, including Neurospora crassa, Kluyveromyces lactis and several Aspergilli.
Comparable to what was observed in other filamentous fungi, the deletion of hdfA, the Penicillium chrysogenum KU70 homolog, resulted in a significant improvement of gene targeting through homologous recombination; from 0.1% in WT to 50% in the mutant (using 1.8 kb flanking regions). Furthermore, the potential of such a high recombinant P. chrysogenum strain to serve as platform for gene targeting was studied. In order to characterize the impact of the hdfA deletion, the mutant was compared to its isogenic reference using chemostat cultures, transcriptome analysis and quantitative fitness profiling. Whereas little to no marked difference was recorded at both physiological and transcriptome levels, in a direct competition experiment, the isogenic reference strain had a clear advantage over the hfdA strain.

PR7.38
Functional analysis of essential genes in A. fumigatus by co-precipitation of associated proteins using an S-tag fusion peptide
Gethin Allen¹, Mike Bromley², Mike Birch², Geoff Turner¹
1The University Of Sheffield, Department of Molecular Biology and Biotechnology,, Sheffield, United Kingdom, ²F2G Ltd., Manchester, United Kingdom

In the opportunistic human pathogen A. fumigatus essential gene products are potential drug targets, however, because A. fumigatus is an asexual haploid organism traditional techniques used to identify and characterise genes are not always feasible. Essential genes can be identified using the MycoBank® screening method where genes are disrupted in a diploid background and essentiality of the disrupted gene is determined by loss of a marker when the diploids are broken down in to haploids.
Bioinformatics can sometimes indicate the function of genes by identifying previously characterised homologues or domains. This is not always fruitful as the homologues may be uncharacterised genes. In these cases functional analysis is required.
Genes can be characterised by analysing the phenotypes of mutants. Disruption of an essential gene is lethal so mutant strains are difficult to generate and the phenotypes are often uninformative.
Identifying previously characterised proteins that interact with uncharacterised proteins can provide clues to the function of the uncharacterised proteins. We used the S-tag fusion peptide as an affinity tag for co-precipitation experiments to identify proteins that interact with a selection of putative essential proteins identified by MycoBank® screening and analysis of clusters of orthologous genes.
The S-tag system was used because the purification procedure is simple and the tag is very small so that it will not significantly disrupt the structure or function of the tagged protein. The tagged protein must be functional otherwise strains containing S-tagged essential genes will be inviable. A 5X glycine-alanine polylinker was used to attach the tag to the target proteins to prevent the tag disrupting the native structure of the protein.
This approach has identified a novel component of the SWI/SNF ATP-dependent chromatin remodelling complex that is unique to fungi and conserved across many fungal species. This data correlates with data obtained from other approaches; GFP localisation had previously shown this protein to be present in the nucleus and probably closely associated with DNA.

PR7.39
Evaluation of different protein extraction protocols for the comparative analysis of Monilinia spp proteomes
Olja Bregar, Stanislav Mandelc, Tjasa Gril, Branka Javornik
Biotechnical Faculty, University of ljubljana, SI-1000 ljubljana, Slovenia

The brown rot fungi Monilinia laxa (Aderh. & Ruhl.) Honey, Monilinia fructigena (Aderh. & Ruhl.) Honey and Monilinia fructicola (Wint.) Honey are known as an important plant pathogens in stone and pome fruits. The detection and identification of these species is complex, so new target sequences or proteins could contribute to the development of efficient diagnostic markers. In order to achieve this goal we have started proteomic analysis of fungi and, as a first step, have evaluated protein extraction protocols in terms of protein yield, 2-D image quality and reproducibility. Sample preparation is one of the most crucial steps in proteom analysis and we therefore tested various extraction protocols. Three different lysis buffers were tested (a combination of CHAPS and Tris-HCl pH 8,0; 2-D lysis buffer with thiourea/urea and acidic extraction), followed by grinding, precipitation with TCA/acetone and purification. Fungal cell wall is very resistant to fragmentation, so special attention was devoted to thorough grinding and the removal of interfering compounds. Two isolates of M. laxa, one from apple and the other from apricot, were grown in malt extract medium and sampled for protein extraction after 3 and 7 days growth. All different isolations of total proteins were analyzed with 2-D electrophoresis (300µg load, pH 3-10 NL, 12,5% T), stained with CBB, and 2-D patterns were analyzed with ImageMaster Platinum software. Image comparison and statistical analysis showed that the three methods differ in the number of protein spots detected. The most effective was acidic protein extraction. A 2-D protein reference map of M. laxa was established and we are currently performing comparative analysis of M.laxa isolate proteomes showing host specificity.

PR7.40
Highly improved gene targeting in Penicillium chrysogenum

Paulo de Boer¹, Hesselien Touw², Jeroen Bastiaans¹, Richard Kerkman², Karolina Dukik¹, Marco van den Berg², Remko Offringa¹
1Add2X Biosciences BV, Leiden, Netherlands, ²DSM Anti Infectives, Delft, Netherlands

Introduction: In most eukaryotes, including filamentous fungi, the predominant mode of DNA integration is via non-homologous recombination (NHR). For functional genomics studies and various other applications however, DNA integration via homologous recombination leading to gene targeting (GT) is the preferred pathway. The recent identification of key components of the NHR pathway, such as Ku70 and Ku80, has provided new tools for improving GT efficiencies. Knocking out the NHR pathway has resulted in efficient GT in a variety of eukaryotes, including several Aspergillus species. In the present study, we studied the effect of deletion of the Ku70 or Ku80 genes GT in the b
-lactam producer Penicillium chrysogenum.
Methods: Deletion constructs consisting of 2.5 kb up- and downstream flanks of the target genes (ku70 and ku80), interrupted by the amdS marker driven by the gpdA-promoter were assembled using the Multisite Gateway system. These constructs were then used for transformation of protoplasts. Stable deletion mutants were subsequently analysed for GT at the niaD locus in comparison to the wildtype.
Results: Deletion of the ku70 or ku80 homologues of P. chrysogenum resulted in stable transformants. After the initial transformations, removing the marker over the direct repeat occurred spontaneously. Subesequent analysis of the D
k70 mutant revealed a highly increased GT efficiency at the niaD locus compared to the wildtype (50% vs. 1% resp).
Discussion: GT efficiencies are highly improved in P. chrysogenum D
ku70 or D
ku80 mutants compared to the wildtype. The use of these deletion mutants opens up possibilities for efficient reverse genetics in this industrially important fungus. Recently, additional methods have been developed which enable GT efficiencies up to 100%.

PR7.41
Generation and characterisation of camelid antibodies against fungal melanin
Jeannette Schmaler¹, Gernot Habicht², Uwe Horn¹, Axel Brakhage¹
1Leibniz Institute for Natural Product Research and Infection Biology (HKI), Friedrich Schiller University, Jena, Germany, ²Darmstadt University of Technology, Darmstadt, Germany

Invasive aspergillosis is a leading cause of death in immunosuppressed patients. This is due to late diagnosis and limited therapies of the disease. Previously, we showed that Aspergillus fumigatus produces a specific melanin designated dihydroxynaphthalene (DHN)-melanin. This DHN-melanin might be produced during infection to protect the fungus from the host immune system. Our aim is to study melanisation as pathogenicity mechanism and to detect the fungus in tissue sections with the help of a melanin specific antibody.
To select antibodies against natural occuring DHN-melanin we chose conidia as target since they expose on their surface DHN-melanin. A competitive selection with conidia of a pksP mutant strain which lack DHN-melanin was performed to minimise the selection of unspecific antibodies. A recombinant library of camelid VHH-domains served as antibody pool. Selection was performed via a phage-display method. The antibody was produced as fusion to alkaline phosphatase and characterised in ELISA studies. Furthermore, we applied the biotinylated antibody to demonstrate the melanisation of conida and hyphae via an immunofluorescent staining method with streptavidin-Cy3 conjugate as detecting agent.
We successfully selected phages, that were presenting antibody domains, against melanised conidia. The camelid antibody domain was recombinantly produced as fusion to alkaline phosphatase in Escherichia coli via high cell density fermentation. The purified antibody possesses low molecular mass, good stability and binds with high affinity to melanised conidia of different Aspergillus species. A less stringent binding to synthetic DOPA-melanin and sepia-melanin was observed. The antibody also detects melanised hyphae and conidia but not pigmentless hyphae in in vitro grown cultures.
In future, we want to use this tool to study the pathogenicity mechanisms of A. fumigatus with regard to melanisation. We also intend to test the suitability of the antibody to detect the fungus in infected tissues.

PR7.42
Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19: a novel corticioid species re-organized under the genus Phlebia?
Kristiina Hilden¹, Ralf Bortfeldt³, Martin Hofrichter², Annele Hatakka¹, Taina Lundell¹
1University of Helsinki, Department of Applied Chemistry and Microbiology, Helsinki, Finland, ²International Graduate School Zittau (IHI Zittau), Zittau, Germany, ³Friedrich-Schiller University Jena, Jena, Germany

The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is adopted for degradation of natural polymers such as wood lignin, soil humic acids and brown coal components. Basidiomycete MNPs are among the most promising metalloenzymes to be used as biocatalysts in industrial biotechnology (so called white chemistry). The N. frowardii MNP has also shown strength in conversion of xenobiotic compounds like polycyclic hydrocarbons (PAHs) and trinitrotoluene (TNT). Despite all of its potential, this biotechnologically interesting fungus has not been extensively studied at molecular biology level before.
According to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3, three LIP (lignin peroxidase) and two laccase encoding genes, and the ribosomal ssu RNA encoding 18S gene, we show here that the fungus reveals close phylogenetic relationship to the white-rot in wood causing corticioid basidiomycete Phlebia radiata (Fr.).
Comparison of the 390-amino acid long primary structure of Nf b19 MNP2 reveals 96% aa-identity to the MNP2 of Phlebia radiata 79 (gene Pr mnp2) with differences only within 15-aa that are apparently not involved in enzyme catalysis or heme coordination. Nf b19 MNP2 belongs to the classical long MNPs which we have designated the phylogenetic group B within the superfamily of fungal class II secreted heme peroxidases (see also the abstract of Hildén K. et al. for this meeting).
Phylogenetic analysis on the ITS sequence (ITS1+5.8S+ITS2) more precisely re-orders the fungus as a possible representative of a novel species under the genus Phlebia, nearest to the P. acerina and P. radiata clades. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified to the family Strophariaceae under the order Agaricales. Our results thereby point to a need for traditional systematic re-identification and classification of the previously named N. frowardii isolate b19.

PR7.43
Evaluation of Coprinopsis cinerea genome annotations through proteomic analysis of the secretome
Andrzej Majcherczyk, Dorothea Fragner, Mojtaba Zomorrodi, Ursula Kües
Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University, Göttingen, Germany

The genome of the basidiomycete Coprinopsis cinerea has been established in 2003 and an annotation been published in the NCBI database by the Broad Institute. Other annotations - Twinscan, Snap, Glean, GleanMax300 and Augustus - were established by J. Stajich and M. Stanke with the help of EST libraries provided by A. Gathman, W. Lilly and co-workers. We combined the protein predictions of these six different annotations in a Mascot database used for searches of MS-data obtained from mass spectrometry analysis of C. cinerea proteins. C. cinerea secretes large number of proteins freely into its culture medium. Secreted proteins, beginning from the early exponential growth phase up to the starvation conditions, were separated by 2D-gel electrophoresis and ESI-LC-MS/MS analysis of proteins was either performed on single protein spots or by application of a shot-gun method on complex protein mixtures. Over 90% of single spots gave specific hits to one or to several predictions of protein sequence. The significance of protein identification and sequence coverage were compared with respect to the different annotation algorithms. Combining the annotations increased significantly the number of confidently identified proteins and demonstrated also an incompleteness of any single automated gene annotation method. Combining the Mascot results from repeated shot-gun analysis into SQL-databases increased further the number of confidently identified proteins. Several glucanases, proteins with sugar binding domains, various peptidases, redox-enzymes and some esterases/lipases have been identified. In addition, several low molecular weight proteins without known function and with low homology to known proteins from the NCBI database were detected. The secretome of C. cinerea changed significantly during growth and the highest complexity of the extracellular proteins was observed in the stationary growth phase. High amount of fungal polysaccharides and metabolites produced in the exponential growth phase hampered protein analysis by 2D-gel electrophoresis. However, this problem is solved by application of shot-gun proteomic methods.
We kindly appreciate sequencing and discussion inputs from members of C. cinerea annotation consortium.

This work is supported in frame of a Common Lower-Saxony-Israel Project (ZN 2043) by the Ministry of Science and Culture in Hannover, Germany.

PR7.44
Structurally and phylogenetically divergent manganese peroxidases from two lignin-degrading basidiomycetes
Kristiina Hildén, Miia Mäkelä, Terhi Hakala, Pekka Maijala, Annele Hatakka, Taina Lundell
University of Helsinki, Applied chemiatry and Microbiology, Helsinki, Finland

Class II extracellular heme peroxidases of filamentous fungi show diverse functions as biocatalysts (degradation of lignin, conversion of xenobiotics, bleaching of coloured compounds and polymeric dyes) but minor differences in protein structure. The lignin, manganese and versatile peroxidases (LiPs, MnPs and VPs) are involved in lignin brakedown thereby being described the lignin modifying enzymes.
Recently, we described structurally and phylogenetically divergent MnPs from two biotechnologically promising white-rot basidiomycetes, Phlebia radiata and Physisporinus rivulosus. Both species have two types of MnP that are functionally similar but differ significantly in protein and gene sequence and length, and gene intron-exon structure. The 3-D molecular models of the two divergent P. radiata MnPs confirm this diversity. MnP2 with a long C-terminal tail has the highest structural similarity with the crystal structure of Phanerochaete chrysosporium MnP, whereas the shorter MnP3 is structurally the most related to P. chrysosporium LiP.
Both P. radiata and P. rivulosus express the two divergent MnP isozymes when they are cultivated on milled wood. Expression levels of the two mnp genes of P. rivulosus were studied on defined media supplemented with Mn²⁺ions and veratryl alcohol (VA), which is a non-phenolic aromatic compound biosynthesized and converted by some white rot fungi. Expression of the two mnp genes in agitated liquid cultures implicated differential regulation in response to the effectors. Transcription of mnpA gene was induced by the addition of VA but not by Mn²⁺. Induction of mnpA expression was noticeably observed in the cultures supplemented with milled wood. In contrast, the transcription of mnpB was induced in both cases, that is by addition of either VA or Mn²⁺. Our data on the two basidiomycetous species indicate that differential regulation of the two divergent MnP enzymes may in turn reflect their different functions for fungal wood and lignin degradation.

PR7.45
The fungal path for D-galacturonate catabolism
Satu Hilditch¹, Suvi Berghäll², Janis Liepins³, Merja Penttilä¹, Peter Richard¹
1VTT Technical Research Centre of Finland, Espoo, Finland, ²University of Helsinki, Department of Applied Chemistry and Microbiology, Helsinki, Finland, ³University of Latvia, Institute of Microbiology and Biotechnology, Riga, Latvia

Pectin is a major component of the primary cell wall of plants and mainly consists of D-galacturonate. D-galacturonate is an important carbon source for microorganisms living on decaying plant material. Bacterial catabolic pathways have been described but a eukaryotic pathway has remained unknown. We show that the D-galacturonate pathway in the mold Hypocrea jecorina (Trichoderma reesei) is different than the pathways in bacteria.
In the fungal catabolic pathway D-galacturonate is first reduced to L-galactonate by an NADPH-utilizing D-galacturonate reductase (GAR1). Then a water molecule is removed from L-galactonate by an L-galactonate dehydratase (LGD1) and L-threo-3-deoxy-hexulosonate is produced. Subsequently an L-threo-3-deoxy-hexulosonate aldolase (LGA1) splits L-threo-3-deoxy-hexulosonate to L-glyceraldehyde and pyruvate, and an NADPH-utilizing L-glyceraldehyde reductase (GLD1) converts L-glyceraldehyde to glycerol.
The activity of GAR1, LGD1 and LGA1 was induced when H. jecorina mycelia were grown on D-galacturonate but not when grown on other carbon sources, GLD1 was equally present on all the tested carbon sources. Deletion of the gar1, lgd1 or lga1 gene in H. jecorina resulted in a strain unable to grow on D-galacturonate. All the four genes of the pathway were identified, cloned from H. jecorina cDNA library, functionally expressed in the heterologous host S. cerevisiae, and the kinetic properties of the enzymes were determined.

PR7.46
Inhibitory effect of bamboo vinegar and wood vinegar towards sapstaining fungal growth
Natarajan Velmurugan¹, Sang-Sub Han², Dong-Min Sa³, Yang-Soo Lee¹
1Department of Forest Science and Technology, Institute of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, ²Institute of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, ³College of Agriculture, Life & Environments Sciences, Chungbuk National Universsity, Chunbuk 361-763, Republic of Korea

The antifungal activity of bamboo vinegar and wood vinegar were investigated by radial mycelial growth measurement technique and characterisation of both extracts were performed my GC-MS analysis. Both bamboo and wood vinegar are natural resources and acidic dark brown liquid by-products of bamboo and broad leaved trees charcoal burner. Two percentage MEA plates were prepared amended with 0.001, 0.01, 0.1, 0.5 and 1.0 % of both bamboo vinegar and wood vinegar. Six mm plugs from the plates growing Ophiostoma flexuosum, Ophiostoma tetropii, Ophiostoma narcissi and Ceratocystis ips were transferred to 2 % MEA plates contain different concentrations of both extracts. The results revealed that both extracts were highly active at minimum concentrations (0.1-1.0 %) used. GC-MS analysis was performed to search for compounds with antifungal activity. The chloroform fractions of compounds were identified as 2,6-dimethoxy phenol, dehydroacetic acid, 2,3,5-trimethoxy toluene and 5-hydroxy-3-oxo-2-propionyl-1gamma-1-acetone-4-heptnoic acid in bamboo vinegar and 2-methoxy phenol, 2-methoxy-4-methyl phenol, 2,6-dimethoxy phenol, dehydroacetic acid and 2,3,5-trimethoxytoluene in wood vinegar by MS analysis. These phenolic compounds have powerful antifungal activity. Organic acids comprises 21.89 % in bamboo vinegar and 18.60 % in wood vinegar, the total phenolic content was 58.06 % in wood vinegar and 62.64 % in bamboo vinegar. The antifungal activity of bamboo vinegar and wood vinegar may arise from its phenolic content and pH. These extracts are easily available, inexpensive and non-toxic to environment, hence the applications of these extracts in wood industries are beneficial and uncomplicated.

PR7.47
Differential responses to cadmium in the ectomycorrhizal fungus Suillus luteus, a proteomic approach
Karen Verstraelen, Jan Colpaert, Jaco Vangronsveld
Center for Environmental Sciences, Environmental Biology Group, Hasselt University, Diepenbeek, Belgium

Heavy metals are important environmental pollutants and can cause serious problems to organisms. Still, some micro-organisms show a genetic adaptation to heavy metals and could be useful in the revegetation of contaminated sites. A promising organism is the ectomycorrhizal fungus Suillus luteus, a common root symbiont of Pinus species. S. luteus populations thriving in pioneer forests that colonize sites severely contaminated by Zn smelters in NE-Belgium, have undergone a genetic adaptation to toxic cadmium (Cd) concentrations. However, the exact mechanisms of metal tolerance in ectomycorrhizal fungi are largely unknown. The aim of this study was therefore to identify fungal proteins involved in the mechanism sustaining the adaptive cadmium tolerance in S. luteus. Using the high-tech 2D-DIGE technology, a comparison of the proteome under Cd stress was performed for two fungal strains, one sensitive and one tolerant strain. In this time-response experiment (0-48hrs), fungal proteins were extracted after Cd treatment, labelled with fluorescent CyDyes and analysed by two-dimensional gel electrophoresis. Proteins that showed statistically significant changes between both isolates will further be analysed with massaspectrometry.

PR7.48
Gene replacement by mutant versions without co-insertion of a selectable marker in Podospora anserina

Evelyne Coppin¹, Riyad El-Khoury², Carole, H. Sellem², Antoine Boivin², Marc Maas², Annie Sainsard-Chanet², Robert Debuchy¹
1Univ Paris-Sud, Institut de Génétique et Microbiologie, CNRS UMR8621, Orsay, France, ²CNRS, Centre de Génétique Moléculaire, UPR2167, Gif sur Yvette, France

Testing the effect of targeted mutations, point mutations, deletions or promoter exchange, is a fundamental tool for the analysis of gene function. In particular, fusions to promoters that induce heterochronic expression of the gene of interest have proved highly valuable in investigating the function of developmental genes. However, in most cases, transgenes integrate in different ectopic positions, resulting in variations in their expression level from one transformant to another, and in possible and unpredictable alteration of the metabolism due to the structural change of the integration locus. These variations are a cause of concern with the expanding development of microarray analyses of transgenic strains, as this technology requires highly similar genetic context. It has been shown that the deletion of the KU70 orthologs in fungi is an efficient strategy to improve homologous recombination. By using a delta PaKU70 strain of P. anserina, we demonstrated that it is possible to reintroduce a mutated gene without co-insertion of a selectable marker and without any other modification of the target locus. Replacement of a gene of interest by versions in which it is fused to homologous or heterologous promoter sequences in a delta PaKU70 strain is in progress.

This work is funded by the Association Française contre les Myopathies, the European Commission (integrated Project MiMage LSHM –CT-2004-512020) and National Research Agency contract n° ANR-05-BLAN-0385-01

PR7.49
Cell wall-associated proteins of the basidiomycete Coprinopsis cinerea

Dorothea Fragner, Mojtaba Zomorrodi, Ursula Kües, Andrzej Majcherczyk
Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University, Göttingen, Germany

The fungal cell wall is a complex structure composed of a network of glucans, chitin and glycoproteins. To perform a comprehensive study on the largely unknown cell wall proteome in higher basidiomycetes, we use the model organism Coprinopsis cinerea grown in liquid medium to the early exponential phase. Secreted proteins were fractionated from the mycelium according to the nature of their binding to the fungal cell wall: proteins adsorbed to the outer hyphal sheath, extractable cell wall-associated proteins and non-extractable cell wall-bound proteins. 2D-gel-electrophoresis revealed a high diversity between the hyphal sheath proteins, the extractable cell wall-associated proteins and the free proteins in the culture supernatant, respectively. There is little overlap between the different protein fractions. By a mass spectrometry approach, we identified so far 62 proteins from the hyphal sheath, 86 extractable cell wall proteins and 41 proteins in the supernatant, adding up to a total of 141 different proteins. 10% of amounts of mycelium-isolated proteins are from the hyphal sheath and 90% from the cell walls. The amount of soluble proteins in the supernatant is equal to the total amount of extractable proteins from the mycelium. The protein complexity in the supernatant is thus comparably low with only 19% of all identified proteins, many of which are oxidases, glucanases (e.g. glyoxal and glucose oxidases) as well as numerous proteolytic enzymes. The protein variety with 53 % is much higher in the cell wall fraction and with 28% of the identified proteins highest in the hyphal sheath. Many of these proteins are new with so far no defined function.

PR7.50
The purification and characterisation of a low molecular weight xylanase from the hypercellulolytic filamentous fungus Trichoderma reesei

Mark Gaffney, Richard Murphy, Ronan Power
Alltech Biotechnology, Meath, Ireland

It has been well documented that arbinoxylans have anti-nutritional effects on monogastric animals. These arbinoxylans are a contributing factor to the solubility of non-starch polysaccharides (NSPs), which are found in the endosperm of cereal cell walls. NSPs attract large amounts of water, increasing the viscosity of the ingesta. The resulting effects are manifested as poor feed conversion, depressed weight gain and wet droppings. Incorporation of exogenous xylanase has been successfully proven to reduce gut viscosity and improve nutrient adsorption. The filamentous fungus Trichoderma reesei is not only an efficient producer of multiple xylanolytic enzymes, but also of cellulolytic and other hemicellulolytic enzymes when cultivated by solid state fermentation (SSF). The aim of this study was to produce, purify and characterise a low molecular weight endo-b
-1,4-xylanase from a crude extract of T. reesei cultivated by SSF. One such xylanase was purified using a combination of ultrafiltration, cation exchange and gel filtration chromatographies. Purification was to a factor of 8.9 and resulted in a 2% final yield. When analysed by SDS-PAGE, the enzyme migrated as a 21 kDa band which appeared free of any contaminating proteins. The activity of the purified enzyme was strongly inhibited by SDS and by the divalent cations Fe²⁺ and Hg²⁺. Activity was largely unaffected by agents often found in animal feeds, such as calcium, manganese, potassium, sodium and zinc. The purified xylanase retained relatively high activity over a broad pH range, 3-10. The enzyme was also active when incubated up to 50^°C, however a near complete loss in activity was observed when treated at 60^°C and above. The enzyme was exclusively xylanolytic, displaying little specificity for other polysaccharide substrates including cellulose, glucan or dextran. These physiochemical traits are advantageous when considering the potential utilisation of the enzyme as an additive in animal feeds but also in other areas such as the bleaching of kraft pulps, the quality improvement of baked products and the recovery of fermentable sugars from hemicelluloses.

PR7.51
Enzymes of basidiomycetes in wood and plant litter degradation
Andrzej Majcherczyk¹, Ravi Chandra Dwivedi¹, Dorothea Fragner¹, Mojtaba Zomorrodi¹, Yitzhak Hadar², Ursula Kües¹
1Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University, Göttingen, Germany, ²Laboratory of Fungal Physiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel

Higher basidiomycetes secrete numerous different proteins for the extracellular degradation of complex high molecular weight substrates. However, only few known enzymes of the complex fungal secretomes can be studied by direct detection of their activities and changes in substrate composition. To better understand and to follow the degradation processes, we developed special protocols for high-resolution separation of proteins from basidiomycetes by 2D-gel electrophoresis and in-gel-staining of oxidative enzymes. Various oxidative and hydrolytic enzymes from the white rots Pleurotus ostreatus and Trametes versicolor and the litter-degrading saprotroph Coprinopsis cinerea were identified by analyzing tryptic digests of separated proteins by mass spectrometry (ESI-LC-MS) and Mascot searching engine using a database with known protein sequences, annotated genomic sequences and ESTs. Secretion conditions and isoenzyme compositions were studied in different culture conditions with a focus on peroxidases and laccases. We are isolating selected proteins from culture supernatants and fungal cell walls for detailed biochemical characterization.

This work is supported in frame of a Common Lower-Saxony-Israel Project (ZN 2043) by the Ministry of Science and Culture in Hannover.

PR7.52
Sudies on the morphology of the filamentous basidiomycete Coprinopsis cinerea grown in liquid cultures
Martin Rühl, Max Richter, Sreedhar Kilaru, Karin Lang}}}}}}