Biotechnology



61. Tapping fungal diversity for drug discovery

Zhiqiang An. ChemGenics Pharmaceuticals Inc. Building 300 1 Kendall Square Cambridge, MA

Many of today's drugs are fungal natural products and most of these drugs have been isolated from species which can be easily cultured. However, culturable fungi constitute only a fraction of the fungal taxa. Due to technical limitations, unculturable and slow growing microorganisms have received little attention for drug discovery. While there is little doubt that many new agents will continue to be discovered from culturable fungi, there is a clear need to expand our potential drug source. Recent advances in our understanding of fungal genetics has opened a new avenue, known as combinatorial biology, to allow the expansion of the fungal drug source. Combinatorial biology comprises at least two approaches. The first captures genetic diversity from unculturable fungi through genetic engineering. The second approach directly manipulates gene(s) in specific classes of secondary metabolites, such as polyketides and non-ribosomal peptides. At ChemGenics Pharmaceuticals Inc., we are employing these approaches in our drug discovery efforts.

62. Identification of genes differentially expressed in biobleaching cultures of Trametes versicolor.

Kirk Bartholomew1, Tim Dumonceaux1, Trevor Charles1, Fred Archibald2. 1Department of Natural Resource Sciences,

Macdonald Campus, McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9. 2Pulp and

Paper Research Institute of Canada, 570 Saint Johns Blvd., Point Claire, Quebec, Canada, H9R 3J9.

Trametes versicolor has the ability to selectively bleach and delignify both hard and soft wood kraft pulps without significantly decreasing the quality or yield of the resulting fiber. Traditional biochemical techniques have identified one enzyme essential for this process(manganese peroxidase) and one probably essential (laccase). In order to identify other enzymes involved in the biobleaching and delignification of hard wood kraft pulp (HWKP) we performed a differential display polymerase chain reaction (DDPCR) screen for genes elevated in expression in the presence of HWKP. The primary screen of RNA isolated from cultures grown in the presence and absence of HWKP identified 74 messenger RNAs produced in increased abundance in the presence of HWKP. The differential expression of 12 of these messages has been confirmed by a secondary DDPCR screen of RNA isolated independently under identical conditions. We have cloned 6 of the cDNA fragments identified in the secondary screen for sequence and northern blot analysis to identify the gene products and confirm their elevated expression.



63. Trichoderma reesei EGIII expression and modification.

Ben Bower, Ed Larenas, Barbara Swanson and Mick Ward, Genencor International, Palo Alto, CA .

Trichoderma reesei is an important producer of cellulases for industrial uses. It has been reported to produce two cellobiohydrolases, CBHI, CBHII and four endoglucanases, EGI, EGII, EGIII and EGV. EGIII is a small 23 kDa enzyme that differs from the other Trichoderma cellulases by having a comparatively high pI and lacking a separate cellulose binding domain. We have overexpressed EGIII by placing the gene under control of the strong cbhI promoter. While there exist two potential glycosylation sites in the egIII gene, the protein has been thought to be unglycosylated. When EGIII is expressed under CBHI control, it is initially produced as a glycosylated protein of 28 kDa. It appears to be deglycosylated by an EndoH like activity post secretion. The glycosylated enzyme can be purified using ConA Sepharose. The glycosylation can be removed with Bacillus EndoH. Both enzymatic activity and heat stability appear to be unaffected by glycosylation.

64. Isolation and expression of the APS kinase (sD) gene of Aspergillus nidulans.

D.L.Clarke, R.W.Newbert, A. Ross and G. Turner, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield U.K.

As part of a study on supply of cysteine as a precursor for penicillin biosynthesis, we are investigating the sulphate assimilation pathway of Aspergillus nidulans, for which a number of structural and regulatory genes have now been isolated, Mutations in the sD (APS kinase) gene, which lead to a requirement for methionine, map approximately 3cM from benA. Cosmids known to contain benA were probed with the APS kinase gene of Saccharomyces cerevisiae, and a hybridizing cosmd selected. The sD gene was identified by transformation of an sD50 mutant and subsequently sequenced. Sequence comparison was made between a number of APS kinase genes, and the C-terminal region of the A. nidulans sC (ATP sulphurylase) gene, which resembles APS kinase, and may be involved in allosteric regulation of ATP sulphurylase by PAPS. Although the sulphate assimilation pathway is repressed by methionine, transcription of the sD gene is not noticeably affected. The sD gene has been expressed in A. nidulans under the control of the stronger alcA promoter, and the effect on pathway flux and penicillin titre will be examined.

65. Protein glycosylation in Aspergillus nidulans.

C. Joshua Eades and William E. Hintz, University of Victoria.

Glycosylation can have major effects on the structure and function of proteins, influencing the stability, antigenicity, and biological activity of the protein. We are currently cloning and characterizing the various enzymes of the N-glycosylation pathway of Aspergillus nidulans to allow production of heterologous glycoproteins having uniform carbohydrate moieties. The processing of mannose residues occurs early in the glycosylation pathway and can play a major role in the final carbohydrate structure of N-glycans. Historically, -mannosidase enzymes have been classified into two groups based on functional and sequence similarities. Certain -mannosidases, including the rat ER/cytosolic and yeast vacuolar enzymes, have been more difficult to classify and may consitute a third group. These cytosolic -mannosidases may play a catabolic role in the ER 'recycling' system and may facilitate glycoprotein breakdown in the ER by removing mannose residues from aberrant proteins. We have recently cloned a gene for an -mannosidase from the filamentous fungus Aspergillus nidulans which appears to belong to this third group of cytosolic -mannosidases. This gene has a similar length and shows considerable homology with both the rat and yeast 'cytosolic' -mannosidases. A phylogenetic comparison of all the cloned -mannosidases revealed three distinct groups. We suggest that cytosolic -mannosidases represent an evolutionarily independent group.

66. Overexpression of heme biosynthesis pathway genes results in increased heterologous hemoprotein production in Aspergillus oryzae.

Susan Elrod, Aubrey Jones and Joel Cherry. Novo Nordisk Biotech, Inc. 1445 Drew Ave., Davis, CA 95616.

Genes encoding the first two enzymes in the heme biosynthesis pathway were cloned from Aspergillus oryzae in order to investigate 1) the effects of their overexpression on heterologous hemoprotein production and 2) the regulation of heme biosynthesis in filamentous fungi. The first enzyme, 5-aminolevulinate synthase (ALAS), is a nuclear-encoded mitochondrial enzyme which catalyzes the condensation of glycine and succinyl-CoA to form 5-aminolevulinate. The second enzyme in the pathway, porphobilinogen synthase (PBGS), is a cytoplasmic enzyme which catalyzes the formation the monopyrrole, porphobilinogen, from two molecules of 5-aminolevulinate. ALAS is the rate-limiting enzyme in mammals while PBGS is thought to be the rate-limiting step in yeast. The A. oryzae genes encoding both enzymes, hemA (ALAS) and hemB (PBGS), were cloned and exhibit significant identity to their respective homologues from other organisms. Integration of multiple copies of the hemA gene, under transcriptional control of the TAKA promoter, resulted in a 2-fold increase in heterologous peroxidase production, while integration of a similar hemB construct had little effect. These data indicate that genetic manipulation of heme biosynthesis results in increased heterologous hemoprotein production.



67. Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains .

Robin J. Gouka, Peter J. Punt, Johanna (H.) G. M. Hessing and Cees A.M.J.J. van den Hondel. TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 5815, 2280 HV Rijswijk-, the Netherlands

Filamentous fungi, especially members of the genus Aspergillus, are able to secrete large amounts of homologous proteins into the medium which makes them attractive as a host for protein production. In contrast, heterologous proteins are very poorly produced and secreted.

The obective of our research is to obtain more insight in the parameters that influence heterologous protein production in Aspergillus awamori. To investigate this, a systematic analysis was carried out in which the expression levels of a number of different fungal and non-fungal genes were analyzed. This method is based on the single copy integration of different expression cassettes at the pyrG locus of A. awamori

Differences in expression mainly occured at the steady state mRNA level, varying from high mRNA levels for genes of fungal origin to low levels for genes of non-fungal origin. With one gene, encoding plant Cymamopsis tetragonoloba -galactosidase, no full length mRNA could be detected. With RT-PCR and nuclear run-on transcription assays it could be demonstrated that incorrect processing of full length mRNA was probably occurring, resulting in the lack of about 900 nt in the mRNA. By changing the DNA sequence of the gene improved levels of full length mRNA could be obtained.

In most cases the protein levels corresponded to the amount expected on basis of the mRNA levels. Only in the case of human interleukin-6, relatively high mRNA levels were obtained, whereas, only very low amounts of protein could be detected.

To further investigate the problems observed for plant -galactosidase and human IL6, gene fusions with the A. niger glucoamylase gene (glaA) were constructed. Data on improved mRNA and protein levels will be presented.

68. Studies on the production of single-chain antibody fragments by Aspergillus species.

Hanny (J.)G.M Hessing1, Leon G.J Frenken2, Marian van Miuijlwijk-Harteveld1, Wouter Musters2 and Cees A.M.J.J. van den Hondel1. 1Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food research, PO box 5815, 2280 HV Rijswijk, The Netherlands. 2Unilever Research Laboratorium, Vlaardingen, The Netherlands.

Filamentous fungi, such as Aspergillus niger and, Aspergillus awamori, are able to produce extracellularly significant amounts of homologous proteins. Furthermore, they have the capacity to secrete heterologous proteins although to a much lesser extent. We investigated their capacity for production of single-chain antibody (scFv-) fragments. These fragments comprise the variable fragment of an antibody heavy chain connected via a linker peptide to a variable fragment of a light chain. We studied a.o. the production of scFv-anti Hen egg white lysozyme [scFv-LYS; (1)] as a model. For expression of the scFv encoding sequence expression cassettes were made in which the gene to be expressed was flanked by efficient expression signals such as the A. niger glucoamylase promoter (2) or signals derived from the A. awamori 1,4--endoxylanase A gene (3). Transformants comprising multiple copies of the expression cassette produced up to 10 mg/l scFv-LYS in the presence of a signal sequence. However, when scFv-LYS was expressed as a fusion protein with glucoamylase - a protein which is well secreted by A. niger- production levels increased at least five-fold. To obtain mature scFv-LYS, a KEX2 cleavage site was inserted between the glucoamylase and the scFv-LYS sequences. More data on the production of scFv-LYS and on the production of other scfv-antibody fragments will be presented.

1. Ward, S. et al.,- Nature 341 (1989) 544-546; 2.Verdoes, J C et al.,- J of Biotechnology 36 (1994) 165-175; - 3. J. G.M. Hessing et al.,- Curr. Genet. 26 (1994) 228-232

69. Gene disruption as a method of fungicide target validation.

Kirsty Howard1, Stephen G. Foster2, R. Neil Cooley2 and Christopher E. Caten1. 1 University of Birmingham, UK

and 2AgrEvo UK Ltd., Saffron Walden, UK.

With increasing knowledge of the physiology and biochemistry of phytopathogenic fungi an expanding number of potential targets for biochemical fungicide design can be identified. To prevent wasted resources it is important to test at an early stage whether each putative target is in fact essential for infection and disease development, i.e. to validate the target. We are using Stagonospora (Septoria) nodorum to explore the use of gene disruption as a general method of fungicide target validation.

In initial studies, nitrate reductase was chosen as a hypothetical target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistance and nitrate non-utilising). Three approaches to targeted disruption of the NIA1 gene have been tried: (1) cotransformation, (2) integrative gene disruption, (3) one step gene replacement; with each approach hygromycin resistance was used as the selectable marker. Around 2% of transformants from the cotransformation approach became chlorate resistant and Southern analysis confirmed disruption of the resident NIA1 gene. These disruptants retained full pathogenicity thereby invalidating nitrate reductase as a fungicide target. Transformants from the other two approaches are presently being screened for disruptants. To test this approach on a realistic target, we are cloning chitin synthase genes from S. nodorum. These clones will be used to disrupt resident chitin synthase genes and the disruptants assayed for pathogenicity. Reduction in pathogenicity would identify a major chitin synthase in this species that might be constitute a valid fungicide target.

70. Isolation of hazardous chemicals-induced mRNA in white-rot fungus Coriolus versicolor using differential display

Yosuke Iimura and Kenji Tatsumi, National Institute for Resources and Environment, AIST, MITI, 16-3 Onogawa, Tsukuba, Ibaraki 305, JAPAN

White-rot fungi such as Pheanerochaete chrysosporium and Coriolus versicolor, a ligniolytic basidiomycete, has been extensively studied for its ability to degrade hazardous chemicals. Hazardous chemicals degradation by the fungi involves complex families of secreted phenoloxidases. The transcriptional regulation of the genes in hazardous chemicals stress has been studied. In this study, we searched for hazardous chemicals specific genes using the mRNA differential display technique on C. versicolor exposed to pentachlorophenol (PCP). Eleven partial cDNA fragments were cloned and DNA sequences of the five fragments were further analyzed. Four of the clones represent novel genes that has not been identified previously. One of them show strong sequence homology to enolase and is up-regulated in PCP-treated C. versicolor.

71. Expression cloning and characterization of full-length cel45 cDNAs from Myceliophthora thermophila, Thielavia terrestris and Acremonium sp. reveals several conserved regions common to fungal family 45 cellulases.

Sakari Kauppinen, Soren F. Lassen, Lene N. Andersen and Henrik Dalboge. Screening Biotechnology, Novo Nordisk A/S, Novo Alle, bldg. 1BM1.05, DK-2880 Bagsvaerd, Denmark.

The glycosyl hydrolase family 45 is defined as a group of cellulases containing a highly conserved amino acid sequence at the NH2-terminus. To date, only one bacterial and four fungal endoglucanases belonging to this family have been reported. We have constructed directional cDNA libraries from cellulase induced mycelia of Myceliophthora thermophila, Thielavia terrestris and Acremonium sp. in the yeast expression vector pYES 2.0, and isolated 12 full-length family 45 cellulase (CEL45) cDNAs by functional expression in the yeast Saccharomyces cerevisiae. The positive clones were identified using 0. 1 % AZCLHE-cellulose as substrate in agar plates. Yeast colonies producing cellulose activity were surrounded by a blue halo. Nucleotide sequence analysis of the cDNAs classified the clones into four groups representing transcripts of four genes; cel45 of M. thermophila, cel45 of T. terrestris, and cel45A and cel45B of Acremonium sp. The T. terrestris and Acremonium endoglucanases are composed of a catalytic core domain (211, 212 and 213 residues, respectively), a linker and a cellulose binding domain (CBD). In contrast, the M. thermophila CEL45 endoglucanase contains a 209-residue catalytic core, but neither a linker nor a CBD. Comparison of the deduced amino acid sequences with the endoglucanase V from Humicola insolens and Kfaml - encoded endoglucanase V from Fusarium oxysporum revealed a sequence similarity of 68-81 %. Besides the consensus sequence, several additional, highly conserved regions can be found in the core region, which together with the conserved cysteines and glycines reflect the structural relatedness of the family 45 endoglucanases. By comparison, the Ser and Thr rich linker regions are less similar varying both in length and composition, while the CBDs are both similar to one another and to other fungal CBDS.

72. Purification and molecular cloning of acid stable xylanase from Penicillium sp.40.

Tetsuya Kimura, Takashi Makino, Hideki Kondo, Jun Ito, Kazuo Sakka and Kunio Ohmiya Faculty of Bioresources, Mie University, Tsu, Mie, 514 Japan.

We have isolated Penicillium sp.40 which can grow in acidic medium at pH2.0. This Penicillium sp. 40 produces a xylanase in the xylan medium as a carbon source.We purified this xylanase (XynA) by DEAE-Toyopearl column and Superdex200pg column. The optimum pH of XynA is 2.0 and XynA is stable at pH2-5, indicating that XynA is acid stable enzyme. The molecular mass of purified XynA was 25 kDa on SDS-PAGE gel. The expression of XynA is induced by xylan and b-methyl-D-xyloside and repressed by glucose. Also, the expression of XynA is dependent on pH of the culture medium, suggesting that expression of XynA is controlled by multiple factors. To know the expression mechanisms of XynA, we isolated the gene encoding XynA (xynA). A 4 kb of XbaI fragment was isolated and the nucleotide sequence of this fragment was determined. The xynA appeared to be 721bp long and was interrupted by a single intron of 58bp. The xynG1 encodes a polypeptide composed of 221 amino acids which contained a putative signal sequence composed of 32 amino acids in the N-terminal region. N-terminal amino acid sequence determined by purified XynA showed perfect match to the deduced amino acid sequence from nucleotide sequence. XynA showed 73% amino acid identity with the XynC of acidophilic Aspergillus kawachii and showed strong similarity to the other fungal xylanases classifed as family G.

73. Heterologous expression of human tumor necrosis factor in Aspergillus niger.

Nada Kragevec, National Institute of Chemistry, Slovenia.

The human tumor necrosis factor (hTNF) monomer is a 17 kDa non-glycosylated protein. The active form of this important cytokine is a trimer with a wide range of biological activities. We decided to express hTNF in the GRAS filamentous fungus A. niger, because Aspergillus expression-secretion system has proved to be able to produce therapeutically important proteins as cheaply as industrial enzymes, even though it cost more time and effort than initially hoped. On the other hand also has to be taken in account, it has not yet been generalized to a wide range of heterologous protein products. For heterologous expression of hTNF in A. niger the same strategies were used, which were proved to be the most successful for several other non-fungal proteins, including (i) the use of protease deficient host strain AB1.13, (ii) the use of strong fungal transcription control regions and efficient secretion signals of the A. niger glucoamylase gene, (iii) the use of gene fusion with the A. niger glucoamylase GII form as a carrier-gene and (iv) the introduction of large number of gene copies due to the amdS selection marker.

74. Fungi from geothermal soils in Yellowstone national park.

Anastassia Litvintseva1, Regina S. Redman2, Joan Henson1 and Rusty J. Rodriguez2,3. 1Microbiology Department, Montana

State University, Bozeman, MT 59717; 2National Biological Service, NWBSC, Seattle, WA 98115; 3Botany Department,

University of Washington, Seattle, WA 98115.

Eighteen species of thermotolerant and thermophilic fungi representing nine genera were isolated from geothermal soil depths of 5 to 20 cm. The soils ranged in temperature from 16 to 91 C and pH from 2.58 to 5.79. Temperature and pH optima in vitro culture of these fungi ranged from 25-45 C and 3.0-6.0 respectively. Temperature growth ranges were 5-37 C for thermophilic fungi. Transect studies indicated that a higher number of fungi were obtained from soil core samples in and below the root zone of the tropical plant Dicanthelium lanuginosum. Extracellular enzyme analysis revealed that 8 of these fungal species were capable of secreting proteases and/or cellulases at 35 C. Chemical analysis of soil samples indicated that high levels of phosphorus, lead, iron and sulfur were present. Isolates representing 13 species were able to grow on media containing 1 mg/ml of iron and 200 ug/ml of lead.

75. Cloning of glycolytic genes and their 5' flanking region from Aspergillus oryzae.

Masayuki Machida1, Sumiko Kunihiro1, Zhi Song1, Susumu Eto2, Makoto Yasukawa3. 1Department of Molecular Biology, National Institute of Bioscience and Human- Technology, Tsukuba, Japan; 2Food Science and Technology Division, Oita Industrial Research Institute, Oita, Japan; 3Fukushima Technology Center, Kooriyama, Japan.

Aspergillus oryzae is considered a favorable host for heterologous protein production (Barbesgaard et al. 1992) owing to its ability to secrete large amounts of proteins (Christensen et al. 1988) and wide range of application in food industries. We have already cloned the genomic enolase gene (enoA) and corresponding cDNA from A. oryzae (Machida et al. 1996). The enoA gene was highly expressed (approximately 3% of total mRNA) and the 5'- flanking region of the enoA gene may be useful for foreign gene expression in A. oryzae.

In Saccharomyces cerevisiae, most of the glycolytic genes are regulated by the general transcription factor, Rap1p (Chambers et al. 1995). We examined the DNA-binding activity specific to Rap1p-binding sequence in A. oryzae crude extract using 36 bp DNA fragment containing S. cerevisiae ENO1- UAS. Several strong binding activities were detected, however, none of them was sequence specific. This suggests that the recognition sequence of the factor in A. oryzae glycolytic genes may be different from that of S. cerevisiae or that the general transcription factor, such as Rap1p, may not be present in A. oryzae.

We cloned several glycolytic genes from A. oryzae by the random sequence analysis of A. oryzae cDNA or by PCR using mixed primers which have the sequence homologous to the corresponding genes from several other organisms. We cloned the 5' flanking region of the genes from A. oryzae genomic DNA by PCR-based DNA walking using primers designed from their cDNA sequences. Sequencing is under progress.

76. The characterization of the second laccase gene (CVLG1) from white-rot basidiomycete Coriolus versicolor.

Junko Mikuni*, Masaya Nakamura** and Atsumi Nishida**. *Tokyo Univ. of Agriculture and Technology. Tokyo 183 **Forestry and Forest Products Research Institute. Ibaraki 305, Japan

A white-rot basidiomycete Coriolus versicolor secretes laccase during wood decay. Many studies show that laccase plays an essential role in fungal brakedown of lignin. The extracellular laccases from C. versicolor were fractionated as laccase I, II and III by using anion exchange chromatography. Laccase III which is the most abundant isozyme among them has been characterized biochemically and genetically. In this study, second gene (CVLG1) coding for laccase isozyme has been isolated from C. versicolor. CVLG1 is interrupted by eleven putative introns and exhibits high similarity (70%) with laccase III gene (CVL3). From the deduced amino acid sequence, CVLG1 product is predicted to be a preprotein of 526 residues with twelve N-glycosylation sites. Genomic southern analysis showed that CVLG1 and CVL3 are not allelic. From the CHEF analysis, CVLG1 and CVL3 were mapped on distinct chromosomes. It has been cleared that laccases of C. versicolor were encoded by a gene family.



77. An expression system based on the improved promoter containing multiple copies of the conserved sequence in the amylase genes of Aspergillus oryzae.

Toshitaka Minetokil, Kenji Ozekil, Chieko Kumagail, Katsuya Gomi2, and Yuzuru Iimura2. lGeneral Research Laboratory, Ozeki Corp., Nishinomiya 663, 2National Research Institute of Brewing, Higashi-Hiroshima 739, Japan.

Aspergillus oryzae produces a copious amount of enzymes that are important in Japanese fermented food production such as sake, shoyu and miso manufacturing. Among them, enzymes involving in the degradation of starch are most important. These include -amylase, glucoamylase, and -glucosidase, which are known to be produced in the presence of starch or malto-oligosaccharides such as maltose, maltotriose, and isomaltose, but not of glucose. We found that there exist three highly conserved sequences, designated Region I, II, and III, in the promoter regions of the genes encoding -amylase (amyB), glucoamylase (glaA), and glucosidase (agdA) of A. oryzae. Deletion and insertion analyses of the promoter regions were carried out and indicated that Region III is mainly involved in high-level expression and maltose induction of the amylase genes (Minetoki et al., Curr. Genet., 30, 432-438, 1996). To enhance the promoter activity, multiple (6-12) copies of the fragment comprising Region III were inserted into the agdA or glaA promoter. Promoter activities were assayed by GUS expressed under control of the improved promoters and were shown to be elevated by 3-5 fold higher than those of the intrinsic promoters. Using the improved agdA promoter, we could achieve a remarkably high yield of native proteins, of which secreted level is very low by nature in wild type of A. oryzae, and observed concomitantly a significant decrease in -amylase and glucoamylase yield in the transformants. Northern blot analysis showed that transcriptional levels of amyB and glaA in the transformants were extremely reduced, suggesting that titration of a common regulatory protein(s) involved in high-level expression and maltose induction of the amylase genes may occur. In addition, advantages of conventional solid-state culture system for protein production will be presented.

78. An Aspergillus fumigatus extracellular phytase with high activity.

E. J. Mullaney, C. B. Daly and A.H.J. Ullah. Southern Regional Research Center, ARS, USDA, New Orleans, LA.

In 1968, an isolate of Aspergillus niger (ficuum), NRRL 3135 was identified as the producer of the most active extracellular phytase. It produces 12.5 nkat/ml of enzyme in the crude culture filtrate. Since then, the gene for this enzyme has been cloned in NRRL 3135, over expressed, and marketed commercially. In this period of almost 30 years, no other extracellular phytase has been reported to equal the yield of phytase produced by this isolate. The reason for its unique high activity level remains unknown. Recently, in a survey of other Aspergillus species, an isolate of A. fumigatus was identified as another high producer of phytase. When this isolate was grown under the identical conditions as NRRL 3135, it produced 11.0 nkat/ml of phytase in the crude culture filtrate. By employing PCR technology we are cloning the phytase gene from this fungal isolate and examining it for domains it shares with NRRL 3135 that may be associated with high levels of enzyme activity.

79. Characterization of a heat tolerant Aspergillus terreus phytase.

E. J. Mullaney, A.H.J. Ullah, and C. B. Daly. Southern Regional Research Center, ARS, USDA, New Orleans, LA.

The animal feed industry is seeking a heat tolerant phytase for use as a feed additive. Phytic acid, the major storage form for phosphorus in soybean seeds, is undigestible to monogastric animals. Phytase makes the phytin phosphorus in soybean meal available and thus reduces phosphorus contamination of the environment from animal waste. A heat tolerant phytase would survive the brief period of elevated temperature necessary for animal feed production, such a phytase would be more cost effective to use with current animal feed production methods. We have identified several isolates of Aspergillus terreus that secrete phytase with a higher thermal stability than found in the previously reported A. niger (ficuum) NRRL 3135 phytase. The heat tolerant phytase from one of these isolates is being characterized and its gene cloned. We are determining the molecular basis of this heat stability, i.e., primary structure, disulfide bonds, etc., in order to engineer an enzyme with both high phytase activity and heat tolerance.

80. Expression of -lactam biosynthesis genes of Acremonium chrysogenum.

Jorg Nosek, Renate Radzio & Ulrich Kuck, Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat Bochum, D-44780 Bochum.

The filamentous fungus Acremonium chrysogenum is the most important producer of the -lactam antibiotic cephalosporin C. Many attempts have therefore been made to achieve a detailed understanding of the biosynthesis of this metabolite. For several -lactam genes a clustered organization has been shown. The pcbAB and pcbC genes as well as the cefEF and cefG genes are transcribed from a bidirectional promoter region (for review, see [1]; [21). We have constructed chimeric reporter genes to investigate the effect of potential controlling sequences of the promotor regions. For this purpose non translated regions of both pairs of genes have transcriptionally been fused to the lacZ reporter gene derived from plasmid pSI8.8 [3]. Further on we show that regulation of cefEF/cefG gene expression is different to the expression of genes, which are specific for penicillin biosynthesis in other filamentous fungi.

[1 ] Martin JF et al. (1 994) Antonie van Leeuwenhoek 65: 227-243

[2] Brakhage AA, Turner G (1995) In: Kuck (ed) The MycotaII. Springer Verlag, Berlin pp 263-285

[3] Menne S, Walz M & Kuck U (1994) Appl Microbiol Biotechnol 42: 57-66

81. Isolation of a -tubulin gene conferring resistance to carbendazim in the phytopathogen Septoria tritici.

Andrew Payne, IACR-Long Ashton, Department of Agricultural Sciences, University ofbristol, Long Ashton, Bristol, BS18 9AF.

Infection of wheat by the fungal pathogen S. tritici has not been well studied at the molecular level. A transformation system is an essential tool in the molecular analysis of pathogenesis, especially in systems recalcitrant to genetic analysis. In our laboratory and elsewhere (Pnini-Coben et al 1996) S. tritici has been transformed to hygromycin B resistance. Transformation based on the homologous MBC-resistant allele of the -tubulin gene has been established in a number of fungal systems. This selectable marker has the advantage of being of potential use as a reporter gene in planta.

The -tubulin gene of a carbendazim resistant (MIC > 10 ug ml-1) isolate of S. tritici (ST16) was isolated by screening an EMBL3 genomic DNA library with a homologous PCRgenerated probe. An 8.1 kb Sall fragment common to a number of positive clones, and which hybridised to the PCR probe, was subcloned into pUC18 (pST-BT.1). Sequence analysis of pSTBT. 1 indicated the presence of the whole of the -tubulin gene with at least 400 bp of sequence upstream of the start codon. pST-BT.1 was transformed into the carbendazim sensitive (MIC = 0. 1 ug ml-1) S. tritici strain 12-3B.8 by PEG treatment of protoplasts. Transformants were directly selected on 1 ug ml-1 carbendazim and arose at a frequency of between 0.6 and 2.2 per ug DNA. Results of studies of carbendazim sensitivity of the transformants, and the nature of the integration events, are presented.

Pnini-Cohen, S. et al (1 996). Phytopathology. 86: S40.

82. Characterization of superoxide dismutases from Trichoderma harzianum.

June I. Pounder* and Anne J. Anderson, *University of California, Santa Cruz, CA; Utah State University, Logan.

UT Superoxide dismutases, which transform the toxic superoxide radical to hydrogen peroxide and divalent oxygen, produced by two biocontrol strains of Trichoderma harzianum were characterized. The metal prosthetic groups of the three common types of superoxide dismutases (SODs); copper-zinc (CuZnSOD), iron (FESOD) and manganese (MnSOD) were differentiated by selective chemical inactivation. CuZnSODs were inactivated by treatment with cyanide, hydrogen peroxide or diethyldithiocarbamate. MnSODs were inhibited by azide and FeSODs, if present were sensitive to azide and hydrogen peroxide treatment. The strains studied were T. harzianum from soil suppresive to Rhizoctonia solani plant disease and its'rhizosphere competent mutant generated by nitrosoguanadine (T95). Both strains produced CuZnSOD and MnSOD isozymes. Strain T95 produced an additional CuZnSOD isozyme. Exposure of mycelia in liquid cultures to paraquat, increasing intracellular superoxide concentrations, resulted in increased SOD activity. The intensity of the MnSOD isozyme increased with paraquat exposure. PCR of genomic DNA with primers specific for conserved sites in MnSOD amplified two bands, 294 bp and 378 bp in size, that appear to be different gene products. The PCR products had homology to MnSOD genes from yeast, corn, tobacco and a thermophilic bacterium.

83. Analysis of the -amylase production in a transformed Aspergillus oryzae strain.

Anne L. Santerre, Lone B. Petersen, Jure Piskur and Jens Nielsen, Danish Technical University, DK-2800 LYNGBY, Denmark.

For industrial application of recombinant strains genetic stability is a very important aspect especially related to continuous cultivations. In this study we investigated the production of - amylase in chemostat cultures of a transformant of a wild-type strain of A. oryzae containing additional copies of the -amylase gene. To quantify strain instability a parameter has been introduced which is characteristic of the rate of instability and has different values depending on the nitrogen source during the cultivations. From the cultivations several mutants have been isolated and further characterised with regards to their -amylase production, to their resistance to the marker used for transformation, and to the number of copies of the -amylase gene.

84. Expression of Aspergillus niger Glucoamylase in Fusarium graminearum.

Jeffrey R. Shuster and Donna L. Moyer, Novo Nordisk Biotech, 1445 Drew Avenue, Davis, CA 95616.

The filamentous fungus, Fusarium graminearum, has been shown to be an excellent host for the production of heterologous proteins. Transcriptional control elements derived from a Fusarium oxysporum trypsin gene have been utilized to achieve the expression of many different proteins. Transcription systems that can be regulated by simple media changes are often useful in a number of protein expression regimes. Therefore, the regulation of the trypsin promoter in F. graminearum was investigated. The A. niger glucoamylase (GLA) gene was utilized as a reporter gene in transformants of F. graminearum. Functional glucoamylase protein was produced in these transformants. Northern analysis of RNA extracted from cultures grown under different conditions indicated that considerable levels of steady state RNA were observed when nitrate was used as the nitrogen source and no, or only a low level of RNA, was observed when ammonium salts were used. GLA RNA levels from cultures growth in glucose/nitrate medium were similar to those from succinate/nitrate medium suggesting no strong repression of the promoter by glucose. The GLA RNA present in wild type A. niger is found in two different forms as a result of differential splicing of intron E. The GLA RNA produced in F. graminearum was also observed to be present in two forms consistent with this differential splicing.



85. Extrachromosomal Replication of Transforming DNA by a Filamentous Fungus.

Eric D. Smidansky, Gary A. Strobel, and David M. Long, Department of Plant Pathology,Montana State Univ Bozeman, MT 59717.

Effective molecular genetic engineering of filamentous fungi to increase their utility and to further our understanding of their natural roles has been, as compared to bacterial and yeast studies, difficult. Among the factors causing this are: 1) many filamentous fungi can not be transformed in an efficient manner, and 2) there are almost no broad host range plasmids with the properties required by molecular genetic methods. Any significant improvements in these, as applied to molecular engineering of filamentous fungi, would be expected to open vast opportunities in terms of our understanding and exploitation of these organisms. We have discovered a filamentous fungus, Pestalotiopsis microspora, that can be transformed with an unusually high efficiency using methods which give much lower efficiencies, or sometimes fail completely, with other filamentous fungi. A combination of Southern blotting and phenotypic stability experiments has established that P. microspora transformants replicate transforming DNA in an extrachromosomal mode, rather than by the integration route used by nearly all other filamentous fungi. To accomplish this, P. microspora recombines transforming DNA, which does not contain a replication signal recognizable by filamentous fungi, with its own to yield autonomously replicating plasmids. These plasmids display remarkable structural stability and the ability to replicate over extended periods of growth. Thus, P. microspora is an unusual filamentous fungus in that it readily accepts exogenous DNA and, in effect, synthesizes from this, its own autonomously replicating plasmids. These are precisely the type of DNAs that are, arguably, required to advance fungal molecular genetics.

86. Expression and secretion of a functional, humanized, IgG monoclonal antibody from a N. crassa heterokaryon culture. W. Dorsey Stuart, Douglas C. Vann , Elie Kato, Charles A. Long, Glenn M. Magyar, Doreen Morris, Faye Nagano, Stephen Buczynski, Gordon Edlin and Helen Tu. Neugenesis Corporation, Honolulu, Hawaii.

Genes coding for the kappa and gamma chains of a humanized monoclonal antibody (type IgG1) were each inserted separately into an expression vector and cotransformed into Neurospora host cells with mutations at the his-3 and trp-1 loci. The cotransformations were designed such that kappa chain transformants would grow on tryptophan only and the gamma chain transformants would grow on histidine only. Putative kappa chain transformants were further screened by ELISA assay of media for production and secretion of the humanized kappa chain protein. A stable producer of kappa chain was then fused in Vogel's minimal medium in a microtiterplate format with putative gamma chain transformants. The cells readily formed heterokaryons and their media was screened by ELISA for production of IgG. One strain which secreted both kappa and gamma chains was chosen for further study. Media from this strain produced intact, assembled IgG1 as demonstrated by Western blot analysis of reduced and native media proteins. The IgG1 was bound by protein A and showed functionality by antigen specific binding. Additional characterization of the IgG1 molecule is underway.

87. Development of a novel fungal expression system for combinatorial biology.

Kelly J. Thibault, Christopher J. Silva, and Lyndon M. Foster, ChromaXome Corporation *, San Diego, CA 92121 * a company of Houghten Pharmaceuticals, Inc.

ChromaXome Corporation (CXC) is conducting pioneering research into combinatorial biology, which combines the power of molecular genetics and biodiversity to produce novel chemistries for drug discovery. Our collaboration with Bristol-Myers Squibb which employs terrestrial actinomycetes and CXC's combinatorial biology technology has proven to be extremely successful. As a result, we have embarked on a program to extend this technology to fungi.

Fungi are widely recognized as valuable sources of chemically diverse secondary metabolites. Furthermore, heterologous gene expression among the major fungal groups, particularly those of the Ascomycetes and Deuteromycetes, is well documented, and a wide variety of genes have been cloned via heterologous complementation. Therefore, heterologous expression of secondary metabolite genes with the appropriate fungal host should be possible, and thus provide access to novel natural products for pharmaceutical development. We report here on a fungal expression system designed to exploit these aspects of fungal biology. This system utilizes a cosmid-based expression vector, PombeCOS-U, and the fission yeast, Schizosaccharomyces pombe as a host. To demonstrate the potential utility of this system, we have examined the expression of the polyketide 6-methylsalacylic acid derived from Pencillium patulum source DNA.

88. Expression of the GFP protein in Aspergillus oryzae.

Sheryl A. Thompson, Beth A. Nelson, Novo Nordisk Biotech, Davis.

The Green fluorescent protein (GFP) isolated from the marine cnidarian Aequorea victoria, was subcloned into a fungal expression vector placing it under the control of the TAKA amylase promoter and the AMG terminator. This vector when used to transform Aspergillus oryzae failed to direct expression of the GFP protein. Northern blots revealed that GFP specific mRNA was present but that a substantial portion of the mRNA population was not of the expected size. 3- Prime RACE analysis was performed on mRNA from an A. oryzae transformant which contained the GFP expression vector. DNA sequencing of the resulting RACE clones revealed deletions within the coding sequence. The deleted regions contained fungal consensus intron sequences at their 5-prime and 3-prime ends. In order to correct this aberrant splicing, a new GFP allele was constructed in which the consensus intron sequences were removed and the GC content of the coding sequence increased. Transformants which contained this new expression vector produced functional GFP, as determined by fluorescence spectra of intracellular protein extracts.

89. Expression of cellulases genes of Phanerochaete chrysosporium in Aspen wood chips.

Marcelo A. Valliml, Bernard Janse 2, and Dan Cullen3 IESALQ/USP, Depto. de Genetica Cx. Postal 83, 13400-970-

Piracicaba-SP, Brazil; 2 Department of Microbiology, University of Stellenbosh, 7600 Stellenbosh, South Africa,

and 3 Forest Products Laboratory, One Gifford Pinchot Dr., Madison, WI 53705, USA.

The white-rot basidiomycete Phanerochaete chrysosporium has been intensively studied for its ability to degrade lignocellulose. Genes encoding cellulolytic enzymes characterized to date include six closely related cellobiohydrolase I clones (cbh1s), a cellobiohydrolase 11 (cbh2), and a cellobiose dehydrogenase (cdh). Owing to the inherent complexities of 'natural' substrates (e.g. woody tissue), studies of transcriptional regulation of these genes have been limited to submerged cultures with synthetic media. We describe a generally applicable methodology for the quantitative assessment of fungal mRNAs in wood. In brief, the approach involves magnetic capture of PolyA RNA followed by competitive RTPCR. The techniques were applied to analyses of cbh and cdh transcripts in P. chrysosporium colonized Aspen wood chips. Transcript patterns in wood were substantially different from those previously observed in submerged cultures. Results provide a foundation for directed strain improvement.

90. The xylanolytic transcriptional activator xlnR of Aspergillus niger.

Noel N.M.E. van Peij, Jaap Visser, and Leo H. de Graaff, Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands.

Little is known about pathway-specific induction of extracellular enzyme systems due to the inability to select for, and complement in, mutants with a negative phenotype. A system has been developed that allows both the isolation of regulatory mutants, as well as the cloning of these regulatory functions by mutant complementation. A xylan induction-responsive element of the endo-xylanase gene xlnA gene of A. tubingensis has been used to isolate A. niger mutants lacking xylanolytic expression. The cloning of the xylanolytic transcriptional activator xlnR was done by complementation and subsequent recovery of the introduced wild-type copy of the mutant regulation allele. A. niger XYLR will be discussed on the basis of mutant characterisation and the primary structure. A model for the regulation of the A. niger xylanolytic sytem will be presented.

91. -Xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of xylan by Aspergillus niger, but not for induction of the xylanolytic enzyme spectrum.

Noel N.M.E van Peij, Joep Brinkmann, Maria Vrsanska1, Jaap Visser, Leo H. de Graaff, Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands, 1Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.

Two proteins exhibiting -D-xylosidase activity were identified upon fractionation and purification of a culture filtrate of an arabinoxylan-grown Aspergillus niger. A single band of 110 kDa by SDS-PAGE was obtained in both cases and these were active on xylo-oligosaccharides but not on xylan. Partial xlnD cDNA clones were immunochemically identified and isolated from a cDNA expression library. Sequence analysis showed that all cDNA clones correspond to a single gene. A genomic clone was isolated and overexpressed in A. niger and A. nidulans. The xlnD gene has an ORF of 2412 nucleotides, encodes a protein of 804 amino acids and contains a potential signal peptide of 26 amino acids. This results in a mature protein of 778 amino acids with a predicted molecular mass of 85 kDa and an isoelectric point of 4.5. The protein is N-glycosylated and contains 15 potential N-glycosylation sites. Sequence similarity is found with -D-glucosidases both of bacterial and fungal origin. Both -xylosidase proteins purified have high activity on theartificial substrate para-nitro-phenyl- -D-xylopyranoside (XylNp) and a side activity on para-nitrophenyl--L-arabinofuranoside (AraNp) and para-nitro-phenyl- -D-glucopyranoside (GluNp). A. niger strains, in which the xlnD gene was disrupted, accumulate mainly xylobiose and xylotriose when grown on xylan and have no significant -xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular -xylosidase.

92. Coordinate regulation of expression of a cytochrome P450 enzyme system in Aspergillus niger.

Hans (J.).M van den Brink, Cees.A.MJ.1. van den Hondel and Robert FM van Gorcom. TNO Nutrition and Food Research, Department of Molecular Genetics and Gene technology, P.O. box 5815, 2280 HV Rijswijk, the Netherlands.

Cytochrome P450 enzyme systems comprise two elements; cytochrome P450 reductase (CPR), a generally acting electron donor and the reaction specific cytochrome P450 enzyme. In previous work we have identified the A.niger cytochrome P450 gene encoding benzoate para-hydroxylase (bpha) and the gene encoding cytochrome P450 reductase (cprA). Expression of both genes was shown to be regulated at the transcriptional level by benzoate. However, some indications were obtained that regulation also might occur at post-transcriptional level. To study the exact mechanism underlying the regulation of gene expression of both genes, the gene control region of both genes were fused to a reporter gene followed by generation of progressive deletions. Using this strategy we were able to identify regions (Benzoate Responsive Region = BRR) in both gene control regions involved in benzoate dependent induction of gene. Cloning of this DNA region upstream from a minimal promoter conferred benzoate inducibility on this promoter. Another mechanism involved in regulation of the BPH enzyme system is the use of different promoters1). Clear differences in mRNA size was observed between cprA and bpha mRNA obtained from induced and from non-induced mycelium. Using 5'-RACE we were able to determine the different transcription start points.

1) Promoter is defined as the part of the gene expression control region where the general transcription factors and the RNA polymerase assemble.

93. Heterologous protein expression system using a lipase gene, mdlA, from Penicillium camembertii.

S. Yamaguchi, H. Amano, K. Washizu, and A. Matsuura. Tsukuba Research Labs., Amano Pharmaceutical Co. Ltd., Tsukuba, Japan.

The mono- and diacylglycerol lipase (MDGL)-encoding gene, mdlA, from Penicillium camembertii (Pc) has advantageous characteristics for utilizing to heterologous protein expression. First, mdlA promoter functons efficiently in Aspergillus oryzae (Ao) as well as Pc, and is also functional in Saccharomyces cerevisiae (Sc). Secondly, the MDGL Pro-form is processed at a Lys-Arg site in the C-terminal region in both Pc and Ao. These findings led us to develop a novel heterologous protein expression system using the mdlA gene. In this system, a target protein can be expressed in three microorganisms, Pc, Ao and Sc by the introduction of a same expression cassette. The host organism is selected according to the purpose and is introduced with the cassette by an appropriate vector. The S. cerevisiae host is suitable for the plate colony-screening of large numbers of randomly mutagenezed target proteins. As a model protein, we have examined the expression of two filamentous fungal proteins, Myrothecium verrucaria bilirubin oxidase (BO) and Eupenicillium brefeldianum ascorbate oxidase (ASO) and an anti-bacterial protein, sapecin, from insect, Sarcophaa peregrina. The BO cDNA was efficiently expressed in Pc (1.0 g/L) and Ao (0.3 g/L), and Sc transformant could be conducted to plate-colony assay. The ASO gene was also expressed efficiently in this system. The expression and secretion of sapecin was examined by the use of a fusion gene, in which mature sapecin cDNA was connected at the Lys-Arg site of mdlA. The Pc transformant having the fusion gene secreted correctly processed recombinant sapecin with a yield of 7 mg/L and a normal biological activity.

94. A palindromic CCAAT-motif is involved in the regulation of the Trichoderma reesei cbh2 (cellobiohydrolaseII-encoding) gene.

Susanne Zeilinger, Robert L. Mach, Roman Rauscher and Christian P. Kubicek, Technical University of Vienna, IBTM 172/5, Getreidemarkt 9, A - 1060 Wien.

The cellulase system of the filamentous fungus Trichoderma reesei consists of several cellobiohydrolases, endoglucanases and B-glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disaccharide sophorose. In order to identify nucleotide-motifs involved in the induction of cellulases we prepared protein extracts from induced and non induced mycelia of Trichoderma reesei QM 9414, and used them for electrophoretic mobility shift assays (EMSA) with a 90 bp fragment of the cbh2 promoter. This assay detected different, specific protein-DNA complexes present when using cell-free extracts from glucose, lactose or sophorose grown cultures. Using various overlapping fragments and competitive oligonucleotides, the DNA target motif for these protein complexes was shown to be a palindromic CCAAT-box. A similiar motif is also present in the 5' noncoding region of another sophorose inducible gene (xyn2). To study the DNA-protein complex formation more in detail, the binding motif was elucidated by EMSA using native and mutated oligonucleotides and by in vitro DNA-footprinting with crude extracts.