ECFG 9 Parallel Session 7 Fungal Biotechnology


Transcriptional regulation of Aspergillus oryzae genes and fermentation

Masayuki Machida1, Motoaki Sano2, Koichi Tamano1, Yasunobu Terabayashi1, Noriko Yamane1, Osamu Hatamoto3, Genryou Umitsuki3, Tadashi Takahashi3, Tomomi Toda1, Misao Sunagawa1, Hideaki Koike1, Keietsu Abe4, Yasuji Koyama3, Shinichi Ohashi2

1National Institute of Advanced Industrial Science and Technologies (AIST), Tsukuba, Ibaraki 305-8566, Japan, 2Kanazawa Institute of Technology, Hakusan, Ishikawa 924-0838, Japan, 3Noda Institute for Scientific Research, Noda, Chiba 278-0037, Japan, 4Tohoku University, Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

Aspergillus oryzae has been widely used in Japanese fermentation industries, Japanese alcohol beverage, soy bean paste, soy sauce and rice vinegar for longer than a thousand of years. High potential of secretory production of proteins has led A. oryzae to extensive use in modern biotechnology. The A. oryzae genome size is very close to those of A. flavus and A. niger, and 20-30% bigger than those of A. nidulans and A. fumigatus. Comparison of A. oryzae genome with those of the two species of smaller genome size revealed existence of non-syntenic blocks (NSBs) specific to the A. oryzae genome distributed in a mosaic manner throughout its genome. The NSBs are highly enriched with the genes concerning secondary metabolism and the A. oryzae-specific genes including those expanded in the A. oryzae genome. The transcriptional expression levels of the genes on NSBs were significantly lower than those on syntenic blocks (SBs) by the DNA microarray analysis. The genes on NSBs and SBs were globally down- and up-regulated, respectively, at heat shock. In contrast, the genes on NSBs appeared globally up-regulated in solid-state cultivation (SSC), which is widely used in the Japanese fermentation industries. There are several factors known to affect expression of CCS-specific genes including low water activity and physical barrier. Considering that most of the extra homologs are involved in metabolism, the enzymes encoded on the NSB-genes might enhance the potential of degrading raw materials. Interestingly, most of the extra homologs repressed at heat-shock in submerged culture were not repressed in membrane cultivation condition, which was a mimic of SSC. These results suggest that SSC is an ideal culture condition to take advantage of utilizing the genes uniquely existing in the A. oryzae genome.




Penicillium chrysogenum; what is the genomic secret behind penicillin production?

Marco van den Berg

DSM Anti-Infectives, Delft, Netherlands

In only 65 years the yearly consumption of world’s most important class of antibiotic, the B-lactams, has increased from 0 to 45000 tons. To coop with this high demand a tremendous effort in classical strain improvement of P. chrysogenum and a concomitant optimization of the industrial fermentation process lead to the current strains and production process. Although very successful this so-called classical black box approach has several limitations, one of them is that one does not know what has been changed. From literature only two features correlated to the increased productivity and induced by the decades of classical mutagenesis have been reported:

The multi-fold amplification of a 60-100 kb region of Penicillium chrysogenum, including the structural biosynthetic genes pcbAB, pcbC and penDE leading to increased mRNA levels and thus active enzyme (1,2).

A mutation in the first gene of the degradation pathway of the side chain precursor phenylacetic acid, leading a less active enzyme and more penicillinG (3)

With the advent of the ‘omics’ era new tools became available to study this unprecedented effort in strain improvement and learn more about what caused the increase in penicillin productivity. This lead to new insights, which will be reported.

1Barredo, J.L., Diez, B., Alvarez, E., Martín, J.F. (1989). Curr. Genet. 16, 453-459; 2 Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G. (1997) J. Ind. Microbiol. 19, 18-27; 3 Rodríguez-Sáiz M, Barredo JL, Moreno MA, Fernández-Cañón JM, Peñalva MA, Díez B. (2001) J Bacteriol. 183:5465-5471.




Cellular responses to protein production in the filamentous fungus Trichoderma reesei

Tiina Pakula, Mikko Arvas, Jari Rautio, Bart Smit, Marilyn Weibe, Heini Koivistoinen, Markku Saloheimo, Merja Penttila

VTT, Espoo, Finland

The filamentous fungus Trichoderma reesei is known as an efficient producer of a variety of cellulases and hemicellulases, and it is used as an industrial host organism for production of the fungal enzymes as well as for heterologous proteins. Production of secreted proteins in large quantities, or production of heterologous proteins originating from distantly related organisms, are known to provoke stress responses in the cells. The known stress responses include e.g. activation of the unfolded protein response (UPR) pathway resulting in induction of a number of genes needed in enhancement of protein folding and transport. The fungal cells have also a feed-back mechanism to reduce the load in the secretory pathway by negative transcriptional regulation of genes encoding the major secreted proteins.

The availability of the genome sequence information has made it possible to apply genome-wide approaches in studies of the cellular responses to protein production under different conditions and to obtain further information on protein production and factors influencing it at different physiological conditions. We have applied transcriptome and proteome analysis to study the effects of production of heterologous proteins in T. reesei, as well as to analyse different protein production conditions in batch bioreactor and chemostat cultures. Specifically, we have analysed protein production and cellular responses in carbon-limited chemostat cultures at different specific growth rates and cell densities.




Production of oxygenated beta-carotene derivatives by modified Mucor circinelloides strains

Tamas Papp1, Enrique Iturriaga2, Arpad Csernetics1, Andras Szekeres3, Arturo Eslava2, Csaba Vagvolgyi1

1Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary, 2Área de Genética, Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain, 3Analytical Laboratory of Cereal Research Non-profit Company, Szeged, Hungary

Introduction: Carotenoids are yellow to orange-red natural pigments with a broad range of biological functions. Recently, they are attracting an increasing attention, due to their beneficial effects on health. Mucor circinelloides is a beta-carotene-accumulating Zygomycetes fungus, which has been used to study the fungal carotene biosynthesis. In a previous study (1), heterologous expression vectors based on the Agrobacterium aurantiacum b-carotene ketolase gene (crtW) were constructed to achieve the production of xanthophylls in Mucor. In the present work, transformations of a M. circinelloides strain have been performed with combinations of these heterologous vectors and vectors containing Mucor genes encoding the rate-limiting steps of the isoprenoid pathway.

Methods: The genes of isopentenyl pyrophosphate (IPP) isomerase; farnesyl pyrophosphate (FPP) synthase and geranylgeranyl pyrophosphate (GGPP) synthase were introduced via PEG mediated protoplast transformation into M. circinelloides in combinations with the crtW gene. Carotenoid production of the transformants was analysed by TLC and HPLC.

Results: Increased carotenoid production was observed in the new transformants: they produced astaxanthin and canthaxanthin in higher amounts than the strain transformed just with the crtW. In general, co-transformants produced about two times more carotenoids than the original strain.

Conclusion: Strains able to produce oxygenated beta-carotene derivatives were constructed by heterologous genetic transformation. Carotenoid synthesis also could be increased by the over-expression of the early isoprenoid steps via the gene doses effect. The highest carotenoid content was observed in the transformant harbouring crtW together with extra copies of carG. The product of this gene determines the last isoprenoid step before the carotenoid-specific biosynthetic steps.

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

1Papp, Velayos, Bartók, Eslava, Vágvölgyi, Iturriaga 2006. Applied Microbiol. Biotechnol. 69: 526-531.




The contribution of the Aspergillus niger genome sequence and annotation to the discovery of novel products.

Robbert Damveld, Noël van Peij, Hein Stam, Herman Pel

DSM Food Specialties, Delft, Netherlands

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of extracellular enzymes and organic acids, particularly citric acid. We sequenced 33.9 megabases of the genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains1. The genome sequencing and annotation enabled on one side the construction of a metabolic model, the development and application of proteomics and transcriptomics2. On the other side the sequencing yielded easy access to numerous (14,165) protein encoding genes. Some of these genes encode for valuable industrial proteins. Using our STIPT technology3, production strains were generated expressing numerous extracellular proteins. The sequencing and annotation of the A. niger CBS 513.88 genome contributed to the rapid discovery of several interesting enzyme products, some of which examples will be shown.

1Pel et al., Nat Biotechnol. 2007 25(2):221-31 2 see poster van de Vondervoort et al; 3 see poster de Lange et al




Cellular responses to the expression of aberrant secretory proteins in Aspergillus oryzae

Takahiro Shintani, Daisuke Shiro, Jun-ichi Yokota, Katsuya Gomi

Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan

Filamentous fungi have been used as hosts that produce useful proteins because of their ability to secrete proteins at a very high level. Despite a great success in producing the proteins derived from eukaryotic microbes, the efficiency of secretion of higher eukaryotic proteins was quite poor in many cases. One of the reasons for the decreased production could be the instability of heterologously expressed proteins in the secretory pathway. In this research, we used Aspergillus oryzae as a host strain to analyze the cellular response to the overexpression of an aberrant secretory protein.

We expressed the mutant version of 1,2-alpha-mannosidase from Aspergillus saitoi, whose cystein residue at the position 443 was substitute to phenylalanine (C443F MsdS). It has been reported that this amino acid residue is crucial to maintain the proper folding of MsdS and this mutation therefore causes the decreased production of this protein in A. oryzae. We integrated the mutant msdS allele in a niaD locus as well as the wild type allele whose expressions were controlled under a moderate enoA promoter or a strong No.8142 promoter. When expressed from the enoA promoter, the mutant MsdS was not secreted whereas the wild type was detected in a medium fraction. The decreased production of the mutant was not due to the defect in its secretion, because it was not seen even in a cellular fraction. Moreover, it was stabilized in the hrdA deletion strain, suggesting that the mutant MsdS was degraded via the ER-associated degradation pathway. When expressed from the No.8142 promoter, although the mutant MsdS was secreted at a level comparable to the wild type, it was hyperglycosylated, which is rarely seen in Aspergillus. Since the mutant MsdS in the intracellular fraction did not receive hyperglycosylation, which occurs in Golgi apparatus, the proteins might be accumulated in the ER. DNA microarray analysis revealed that the transcriptions of ER chaperones and components of the ER-Golgi transport were specifically upregulated in the strain overexpressing the mutant MsdS, suggesting that the accumulated mutant MsdS induced the unfolded protein response. These findings demonstrate that aberrant secretory proteins are primarily degraded by the ERAD but also the ER-Golgi transport and the hyperglycosylation might relieve the stresses caused by their accumulation in the secretory pathway.




Deceiving Aspergillus niger to illuminate new putative antifungal compounds from natural extracts and their targets

Benjamin M Nitsche1, Vera Meyer2, Cees AMJJ Hondel van den1,3, Arthur FJ Ram1

Leiden University, Leiden, Netherlands, 2 Berlin University of Technology, Berlin, Germany, 3 Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands

Increased resistance to currently used antifungal compounds and the fact that these agents are often harmful to man and environment have resulted in a growing demand for new antifungals, which selectively act on cellular processes that are unique to fungi. To meet this demand, we have established a luciferin/luciferase based reporter system for high-throughput screening of extracts obtained from natural sources. This system allows us to identify compounds that specifically target fungal cell wall biosynthesis. It has been well established in the yeast Saccharomyces cerevisiae that cell wall synthesis is a highly dynamic process, which is orchestrated by the cell wall integrity (CWI) pathway. Such CWI pathway is also present in the filamentous fungus Aspergillus niger and probably widespread among fungi. We have previously shown that transcription of the A. niger agsA gene, encoding an alpha-1,3-glucan synthase, is strongly and specifically up-regulated in response to cell wall stress . We have also shown that the induced expression of the agsA gene is mediated via the RlmA transcription factor and its putative RlmA binding site within the agsA promoter . We are here presenting results for a new cell wall stress responsive A. niger reporter strain obtained by cloning a modified agsA promoter containing two additional RlmA binding motifs in front of a codon optimized luciferase gene . The performance of the system was verified by screening several antifungal compounds with a known mode of action. The specific response dynamics of the new reporter system will allow us to identify new putative antifungal compounds from natural extracts.




Lack of aldose 1-epimerase in Hypocrea jecorina is a key to cellulase gene expression on lactose

Erzsébet Fekete1, Bernhard Seiboth2, Christian P. Kubicek2, Attila Szentirmai1, Levente Karaffa1

1Department of Genetics and Applied Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary, 2Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Vienna, Austria

The heterodisaccharide lactose (1,4-O-β-d-galactopyranosyl-d-glucose) induces cellulase formation in the ascomyete Hypocrea jecorina (=Trichoderma reesei). Lactose is thereby assimilated by extracellular hydrolysis to d-glucose and β-d-galactose, and subsequent uptake of the monomers. Lactose assimilation is slow and the assimilation of the β-d-galactose depends mainly on the operation of a recently described reductive pathway and is less dependent on the Leloir pathway of d-galactose catabolism which accepts only α-d-galactose. We therefore reasoned whether a galactomutarotase (aldose 1-epimerase) activity may limit lactose assimilation and thus influence cellulase formation. We identified three putative aldose 1-epimerase genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular (AEP1-2), and one an extracellular protein (AEP3). Although all three were found to be transcribed, only the extracellular aep3 did it on lactose, and only during the early but not the later stages of growth. Consistent with this, no mutarotase activity was detected in growing mycelia. Therefore, the effect of a galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal part of the S. cerevisiae Gal10 which is responsible for galactose mutarotase activity. These strains showed increased growth on lactose in a gene copy-number dependent manner, although their formation of extracellular β-galactosidase activity, and transcription of the genes encoding the first steps in the Leloir pathway and the alternative pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose was dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the β-anomer of d-galactose, and reveals the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.




Quantitative linkage mapping of lignin-degrading enzymatic activities in Pleurotus ostreatus

Francisco Santoyo, Cristina García, Aldo E. González, María C. Terrón, Lucía Ramírez, Antonio G. Pisabarro

1Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Navarre, Spain, 2Centro de Investigaciones Biológicas CIB, CSIC, 28040, Madrid, Spain

Pleurotus ostreatus and Phanerochaete chrysosporium are two model lignin-degrading basidiomycetes. The genome of P. chrysosporium has been released and that of P. ostreatus will be available soon. The lignin degrading strategies of these two fungi are, however, different as P. ostreatus lacks lignin peroxidases (LiP) whereas P. chrysosporium lacks phenol oxidases (Pox). Both fungi, in addition, contain genes coding for Mn-oxidizing peroxidases [manganese (MnP) and versatile (VP) peroxidases in P. ostreatus (mnp genes), and manganese peroxidases in P. chrysosporium]. We have mapped genetically the genes coding for different ligninolytic enzymes in P. ostreatus and we have found them linked to chromosomes IV (mnp1, VP activity), V (mnp3, MnP activity) and VI (mnp2, VP activity; pox1 and poxC, Pox activity). If the enzymatic activities are mapped as quantitative traits instead of mapping the structural genes, the genome regions containing regulators of these activities will be detected in addition to the structural genes. We have used this approach for the Pox and MnP/VP activities and we have identified various genomic regions that control them and that map to linkage groups different from those where the corresponding structural genes had been previously mapped to. These new sites could code for regulatory genes. Using a combination of this information and classic genetic techniques, we have selected new P. ostreatus strains enriched in specific activities while maintaining a low biomass production in submerged cultures.