Poster Category 8:


Fungal Biotechnology



Recombinant expression in Trichoderma reesei for improved hydrolysis of pretreated corn stover

Sandy Merino

Novozymes Inc.


The majority of commercial enzyme products for biomass hydrolysis are produced by the saprophytic mesophilic fungus Trichoderma reesei Trichoderma produces two cellobiohydrolases (CBHI and CBHII), five endoglucanases (EGs), and two β-glucosidases (BGs).  This mix of enzymes is relatively efficient at cellulose degradation and large quantities of these proteins are secreted from the fungus.  However, improvements in the total enzyme specific activity and secretion yield may improve this enzyme mix.  A primary factor in the high cost of enzymes for biomass hydrolysis is the amount of enzyme that must be applied for efficient cellulose conversion to glucose.  Compared with starch hydrolysis, 15-100fold more enzyme is required to produce an equivalent amount of ethanol, depending on specific process conditions.  It is well known that efficient cellulose hydrolysis requires a complex, interacting mix of cellulose degrading proteins.  To significantly reduce the enzyme loading required, one may replace Trichoderma components with more efficient candidates, or augment the enzyme system with additional components to improve the overall enzyme performance.  In this study, identification of new genes that improve specific performance in hydrolysis of pretreated corn stover, and their expression in Trichoderma will be discussed.  





Highly efficient gene replacements in ku70 disruption strain of filamentous fungus Penicillium decumbens

Tianhong Wang, Zhong-Hai Li, Chun-Mei Du,Yao-Hua Zhong

State Key Laboratory of Microbial Technology,Shandong University, P.R.China


Penicillium decumbens is an important industrial filamentous fungus and has been widely used in biorefinery for its high production of cellulase and hemicellulase. However, genetic engineering strategies are still rarely applied for strain improvement in P. decumbens. It has been proven that the frequency of targeted gene replacement in fungi can be increased by inactivation of the main components required for non-homologous end joining (NHEJ) pathway.

To improve gene targeting efficiency in P. decumbens, the putative pku70 encoding KU70 homolog involved in the NHEJ process was identified and deleted. The △pku70 strain displayed wild-type phenotypes regarding vegetative growth, conidiation and cellulase production, and appeared the similar sensitivity to hygromycin, EMS and H2O2 at different concentrations, compared with that of wild-type strain. The effect of the deletion of the pku70 on gene targeting was tested by disruption of the creA gene encoding carbon catabolite repressor and the xlnR gene encoding general xylanolytic activator. Efficiency of gene targeting for both genes was 100% in the △pku70 strain, compared with low efficiency (30%) in the wild-type strain.

Establishment of highly efficient gene targeting system opens the way to large-scale functional genomics analysis in P. decumbens and contributes to the study of the mechanism of lignocellulose degradation by P. decumbens.



Mutations in a beta-carotene oxygenase gene result in beta-carotene overaccumulation in the fungus Phycomyces blakesleeanus

Victor Garcia Tagua[1] H Ramirez-Medina[1] A Idnurm[2] C Sanz[3] AP Eslava[3] E Cerdá-Olmedo[1] LM Corrochano[1]

1Dep Genet, Univ Sevilla, 2School Biol Sci, Univ Missouri, 3Dep Genet Microbiol, Univ Salamanca


The biosynthesis of beta-carotene and its regulation by environmental factors, including blue light and sexual interaction, has been investigated in detail in the Zygomycete fungus Phycomyces blakesleeanus. Mutations in the regulatory genes carS, carF, or carD result in mycelia that accumulate beta-carotene.

The gene
carS is genetically linked to the structural genes carB and carRA. From the Phycomyces genome sequence we have identified several candidate genes in the vicinity of the  carRA/carB cluster. One of them encodes a putative protein similar to beta-carotene oxygenases, and we found that a carS strain had a single mutation that replaced a Ser at position 433 by Leu. We sequenced the gene strains carrying different carS mutations. We have sequenced 7 carS alleles and in all cases we have found single point mutations in the putative beta-carotene oxygenase gene. For this reason we now consider this putative beta-carotene oxygenase gene as the Phycomyces carS gene.

The gene carS encodes a 628 aminoacid protein. It contains two introns and the cDNA has been expressed in E. coli. The oxygenase CARS breaks beta-carotene in two molecules of 25 and 15 atoms of carbon. The carS gene is repressed by light and it is activated by sexual interaction.

The discovery that mutations in the gene of a beta-carotene oxygenase causes the acumulation of beta-carotene suggests that a derivate of beta-carotene acts as repressor of the biosynthesis or that the CARS enzyme itself could act as a repressor of the pathway.




BrlA and StuA are essential for conidiation in Penicillium chrysogenum

Claudia Sigl[2] Hubertus Haas[1] Heiko Eichhorn[2] Hubert Kürnsteiner[2] Ivo Zadra[2]

1Biocenter, Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria, 2Sandoz GmbH

Penicillium chrysogenum is the main industrial producer of the β-lactam antibiotic penicillin. Asexual conidiation is a key developmental process in this filamentous fungus. Evidence in filamentous fungi suggests that secondary metabolism might be linked with developmental processes such as conidiation.

We therefore generated deletion mutants of the transcription factors
brlA and stuA using a Pcku70 deletion strain with improved gene targeting efficiency. brlA is known as a central regulator of asexual sporulation and plays a crucial role in the development of conidiophores. The APSES domain transcription factor stuA influences conidiation by modulating the central developmental pathway that directs the differentiation of conidiophores and conidia from vegetative hyphae.

Both deletion mutants,
ΔbrlA and ΔstuA, showed drastic impairment in conidiation and morphology under light and electron microscope. Besides, micro array analysis disclosed altered regulation of various genes involved in differentiation in both mutants compared to the parental strain Pcku70. While conidiation was completely blocked in ΔbrlA, it was severely decreased in ΔstuA. Furthermore, HPLC-, qPCR- and array analysis revealed a dramatic regulation of penicillin biosynthesis in the ΔstuA mutant.


Development of a low-cellulase background Chrysosporium lucknowense C1 strain.

Hans Visser[1] Rob Joosten[1] Peter Punt[2] Sandra Hinz[1] Jan Wery[1]

1 Dyadic Nederland, Wageningen, Netherlands, 2 TNO Quality of Life, Zeist, Netherlands

Filamentous fungi, particularly species such as Aspergillus niger, A. oryzae, Trichoderma reesei and recently also Chrysosporium lucknowense  C1, have shown to produce large quantities of extracellular enzymes. As a result these fungi are used in industry as work horses for enzyme production. Usually, a mixture of enzymes is produced, which may be beneficial with regard to the degradation or modification of complex substrates such as plant cell wall polysaccharides. However, with respect to the production of a desired single target enzyme, the presence of large amounts of background proteins with non-relevant or even contra-productive enzyme activities is not desirable. A C1-strain almost devoid of background protein was obtained after random mutagenesis and extensive screening. The new C1 strain was shown no longer to produce extracellular cellulases. Furthermore, remaining undesired extracellular proteins were removed by targeted disruption of the corresponding genes. The new C1 strains proved in particular to be useful for the targeted production of specific single enzymes at high yields.





Development of a Chrysosporium lucknowense enzyme library

Rob Joosten, Sandra Hinz, Hans Visser, Jan Wery

Dyadic Nederland, Wageningen, Netherlands

Plant cell wall polysaccharides (PCWP) are large interconnected biopolymers that together form complex rigid structures.  For efficient breakdown of PCWP, e.g. in second generation biofuel production or in feed and food applications, a variety of enzymes is needed to efficiently hydrolyze the cellulosic and hemi-cellulosic fibers. The Chrysosporium lucknowense C1-genome contains over 115 genes that encode PCWP-degrading enzymes and each of these is being over-expressed in specially designed low protein background C1 strains. This has now resulted in an enzyme library of over 60 specific carbohydrate active enzymes. This growing C1-enzyme library is used  for mixing and matching experiments, e.g.  to establish limitations in current biofuel enzymes, as well as for specific enzyme characterization and application studies in the area of feed, food and pulp & paper. We continue to expand the enzyme library in C1, which now contains mainly cellulases, hemi-cellulases and other corresponding accessory enzymes.





Development of protease deficient Chrysosporium lucknowense strains

Vivi Joosten, Theo Verwoerd, Hans Visser, Jan Wery

Dyadic Nederland, Wageningen, Netherlands

Filamentous fungi have proven to produce and secrete large quantities of extracellular enzymes. Species such as Aspergillus niger, A. oryzae, Trichoderma reesei and recently also Chrysosporium lucknowense  C1 are used in industry as work horses for enzyme production. High yields of homologous enzymes or enzyme mixtures are readily obtained. The production of heterologous proteins, on the contrary, is often hampered by the presence of host proteases that partially or fully degrade the heterologous protein.  In order to significantly improve the production of heterologous proteins,  C1 strains are developed that have an as low as possible protease level without having their vital functions disturbed. To this end C1 was subjected to random mutagenesis and subsequent screening approaches.  Strains with greatly reduced overall protease activity levels were obtained . Additionally, the presence of specific proteases in C1 culture samples was shown by MS analyses. Some of these showed homology to notoriously harmful proteases known from other fungal systems. A protease gene disruption approach was used to knock-out the corresponding genes. This work was greatly facilitated by the use of strains defective in the non-homologous-end-joining of DNA. Thus a number of versatile low protease C1 hosts were developed for high level expression of heterologous proteins.





Vector development to simplify multiple gene expression in Aspergillus oryzae

Khomaizon Abdul Kadir Pahirulzaman, Colin Lazarus

University of Bristol

Vector pTAex3 was modified for use in characterising fungal polyketide synthases (PKS) and hybrid polyketide synthase-non-ribosomal peptide synthases (PKS-NRPS) in an arginine auxotroph of Aspergillus oryzae. In pTAex3GS the strong, starch-inducible amyB expression cassette was modified for GATEWAY LR recombination, and in further derivatives the argB selectable marker was replaced with basta- (bar) or bleomycin- (ble) resistance genes to allow co- or sequential transformation. Tailoring enzymes convert PKS/PKS-NRPS products to final secondary metabolites; tenellin production in Beauveria bassiana requires an enoyl reductase and two cytochrome P450s in addition to a PKS-NRPS encoded by tenS. We produced tenellin in A. oryzae by introducing all four genes in amyB expression cassettes on three separate plasmids. To simplify plasmid construction for whole-pathway expression pTAex3GS was converted to a yeast-E. coli shuttle vector. The A. nidulans argB gene functions well in A. oryzae, so promoters from three other arginine biosynthesis genes (carbomylphosphate synthase (P1), arginosuccinate synthase (P2) and arginosuccinate lyase (P3) were inserted by homologous recombination in yeast. The resultant pTAYAGSarg3P was further modified, replacing argB with bar and ble markers. Yeast recombination simultaneously placed the three tenellin tailoring genes downstream of the arg promoters, creating pTAYAGSarg3genes, and introduction of tenS by GATEWAY recombination reconstructed the whole tenellin synthesis pathway in pTAYAargTENELLIN, which was introduced into A. oryzae. While the easy construction principle has been proven, tenellin synthesis is not yet a likely outcome because in tests of the arg promoters only P2 gave an acceptable level of eGFP expression.





The role of the bZIP transcription factor MeaB in the nitrogen metabolite repression of Fusarium fujikuroi

Dominik Wagner[1] Anne Schmeinck[1] Magdalena Mos[2] Igor Y Morozov[2] Mark X Caddick[2] Bettina Tudzynski[1]

1Westfälische Wilhelms-Universität Münster, 2University of Liverpool

The rice pathogen Fusarium fujikuroi produces a broad spectrum of secondary metabolites. The synthesis of two of them, gibberellin and bikaverin, is subject to the nitrogen metabolite repression. One of the key elements of this complex regulatory network is the GATA transcription factor AreA which activates the expression of the gibberellin biosynthesis genes. In A. nidulans the main antagonist of AreA is NMR. The expression of NMR is positively controlled by the bZIP transcription factor MeaB which is therefore an indirect antagonist of AreA. We examined the role of MeaB in the nitrogen metabolite repression of Fusarium fujikuroi.

Knockout and overexpression mutants of meaB were created, and the impact on the expression of nitrogen repressed genes was studied by Northern analysis. It was shown that MeaB has also a mainly negative effect on the expression of these genes in F. fujikuroi, but that this effect is not as strong as in A. nidulans. Interestingly the repressing effect of MeaB is not mediated via the expression control of nmr as proved by promoter studies in the ∆meaB-background. Additionally, we identified two transcript sizes of meaB that are part of a complex regulation system. Western analysis indicated that only the large transcript is translated. This regulation system allows MeaB to migrate into the nucleus only under high nitrogen concentrations as shown by MeaB-GFP-Fusion. The results indicate the role of MeaB as a fine tuning regulator in the nitrogen regulation network and highlight differences between F. fujikuroi and A. nidulans concerning this role.




Formation of beta-fructofuranosidase by Aspergillus niger in submerged cultivations

Katina Andrea Kiep, Yvonne Göcke, Prof. Dr. -Ing. Dietmar C. Hempel, Prof. Dr. Rainer Krull

TU Braunschweig, Institute of Biochemical Engineering

Filamentous fungi like Aspergillus niger are used as versatile and efficient cell-factories for the production of lipases and proteases, exopolysaccharides, nutrition additives, and therapeutic agents in many industrial bioprocesses. Although A. niger shows a high production capacity and secretion efficiency, and is capable of carrying out post-translational modifications, obtainable yields of recombinant proteins are considerably lower than those of homologous proteins. Besides improvement of productivity due to genetically modified strains, current research is focussed on the optimisation of cultivation processes resulting in an increased, controlled and tailored formation of desired products while avoiding by-products.

The contribution displays the influence of pH-value, volumetric power input and inoculum concentration on the observed morphology and the formation of homologous recombinant β-fructofuranosidase under a constitutive promoter as model product. Batch cultivations are monitored starting with the germination of A. niger spores and ending with the stationary phase of growth. Every step of the protein formation path – transcription, translation and secretion – is shown: The expression of the β-fructofuranosidase gene as well as of genes, which show significant expression levels within the bioprocess, are quantified via real-time PCR. Intra- and extracellular enzyme activities are measured and related to gene expression levels and observed morphology, pellet size and concentration. Undesired by-products are analysed by HPLC.

In conclusion, the protein formation in batch processes is linked to defined cultivation conditions to reveal bottlenecks within the complex production path from gene to product. Therefore the shown results indicate targets for improving, optimising and controlling industrial bioprocesses.




A marker recycling system for filamentous fungi

Katarina Kopke, Birgit Hoff, Ulrich Kück

Christian Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany

To overcome the limited availability of antibiotic resistance markers in filamentous fungi we have established a system for marker recycling. For this purpose we have chosen the FLP/FRT recombinase system from the yeast Saccharomyces cerevisiae for application in the penicillin producer Penicillium chrysogenum. In a first step, we generated a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRTnat1 cassette) and ectopically integrated this construct into a P. chrysogenum recipient strain. In a second step the gene for the native yeast recombinase and in parallel a codon optimized Pcflp recombinase gene were transferred into the P. chrysogenum strain, carrying the FRTnat1 cassette. The corresponding transformants were analyzed by PCR, growth tests and sequencing to verify successful recombination events. Our analysis of several single and multicopy transformants showed that only with the codon-optimized recombinase a fully functional recombination system was achievable in P. chrysogenum. To further extend the application of the FLP/FRT recombinase system, we produced a ΔPcFRTku70PcFLP strain to establish a highly efficient homologous recombination system for the construction of marker free knockout strains. The applicability of the developed FLP/FRT recombinase system was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. In summary we have developed a optimized FLP/FRT recombinase system as a molecular tool for the genetic manipulation of filamentous fungi.





The Velvet-like complex In Penicillium chrysogenum – a novel target for industrial strain improvement

Birgit Hoff[1] Jens Kamerewerd[1] Sarah Milbredt[1] Ivo Zadra[2] Hubert Kürnsteiner[2] Ulrich Kück[3]

1Christian Doppler Laboratory for “Fungal Biotechnology“, Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, 2Sandoz GmbH, 6250 Kundl, Austria, 3Christian Doppler Laboratory for “Fungal Biotechnology“, Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany

The recent discovery of a velvet complex containing several regulators of secondary metabolism in the model fungus Aspergillus nidulans raises the question whether similar type complexes direct fungal development in genera other than Aspergillus. Penicillium chrysogenum is the industrial producer of the beta-lactam antibiotic penicillin, whose biosynthetic regulation is barely understood. Here we provide a functional analysis of major homologues of the velvet complex in P. chrysogenum. Data from northern hybridizations, HPLC quantifications of penicillin titres as well as detailed microscopic investigations clearly show that all regulators play not only a major role in penicillin biosynthesis but are also involved in different and distinct developmental processes. While for example deletion of the velvet homologue PcvelA leads to light-independent conidial formation, dichotomous branching of hyphae and pellet formation in shaking cultures, a ΔPclaeA strain shows a severe impairment in conidiophore formation in both the light and dark. Furthermore, detailed bimolecular fluorescence complementation assays together with yeast two-hybrid screens led not only to the identification for a velvet-like complex in P. chrysogenum but also to the detection of new components of this complex. Our results extend the current picture of regulatory networks controlling both fungal secondary metabolism and morphogenesis which is significant for the genetic manipulation of fungal metabolism as part of industrial strain improvement programs.


From chitin to a highly valuable, pharmaceutically relevant fine chemical: inserting a multi-step enzyme cascade into Trichoderma

Astrid R. Mach-Aigner, Matthias G. Steiger, Robert L. Mach

Gene Technology Group, Inst. of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166/5/2, A-1060 Vienna, Austria

Second to cellulose, chitin is a most abundant, renewable organic source in nature with an estimated annual biosynthesis of 109 to 1011 tons. The polymer is composed of ß-(1,4)-linked units of the amino sugar N-acetyl glucosamine (GlcNAc). For industrial applications this renewable resource is mainly extracted from crustacean shells. However, thitherto only a minute amount of this renewable resource is used in industrial and agricultural applications.

N-acetyl neuraminic acid (NeuNAc), a C9 mono-saccharide, is the most prevalent exponent of sialic acids. Currently, more than 50 derivatives of sialic acids are known to exist in nature. NeuNAc is believed to serve as a precursor of all these derivatives as all biochemical pathways precede via this substance.

In biological systems sialic acids are mostly terminal components of glycoproteins presented at the respective cell surface. Because of its exposed position in cellular systems they play an important role in infection cycles of viral diseases, e.g. influenza viruses A and B. Therefore, sialic acid derivatives are successfully applied in the therapy of such virus-born diseases. Preparations on the market are e.g. „Relenza“ (GlaxoSmithKline), functioning as neuraminidase inhibitor and produced from the precursor NeuNAc.

We will present an alternative strategy of NeuNAc production based on a genetically engineered Trichoderma strain producing and excreting NeuNAc. For the first time, this poses the introduction of a multi-step enzyme cascade into a filamentous fungus as heterologous host. This strategy involves the application of Trichoderma as a whole-cell catalyst for direct synthesis of NeuNAc from the cheap, renewable biopolymer chitin.




Development of a novel inducible expression system for the production of heterologous proteins in Aspergillus: successful production of lignolytic enzymes.
Sandra de Weert1, Nick van Biezen2, Dennis Lamers2, Jeroen Schouwenberg2, Kees van den Hondel1,2, Arthur Ram1 and Christien Lokman1,2
1 Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2HAN BioCentre, P.O. Box 6960, 6503 GL Nijmegen, the Netherlands

For industrial applications and (medical) scientific research efficient (heterologous) production of compounds like, enzymes, antibodies, viral epitopes, chemical compounds and antibiotics is of great importance. For these purposes Aspergillus functions as a suitable production host. Over the years all kinds of commercially available expression systems have been developed. A well established expression system is the one based on the protein Glucoamylase (GlaA). However, in the case of heterologous protein production the efficiency of these systems is still very depending on the protein to be produced. Recently, we identified the inuE gene, encoding for the exo-inulinase protein in A. niger, as the most strongly induced gene in the presence of inulin and sucrose (Yuan et al., 2006; Yuan et al., 2007).

Characteristics of the system were studied by placing gfp behind the inuE promoter. This reporter strain showed that the inuE gene is highly expressed when grown on inulin and sucrose. No expression was observed when grown on glucose, fructose and xylose indicating a tight control on different carbon sources. This tight control can be a benefit if the heterologous protein to be produced can be a disadvantage for fungal growth. Finally, different peroxidases and a laccase were successfully produced in Aspergillus using this novel inducible expression system.



Engineering an N-acetylneuraminic acid synthesis pathway into Trichoderma

Matthias Georg Steiger, Robert L. Mach, Astrid R. Mach-Aigner

Vienna University of Technology

N-acetylneuraminic acid (NeuNAc) belongs to the structural class of sialic acids and is an acidic α-keto sugar with a C9 backbone. Derivatives of NeuNAc are used as neuraminidase inhibitors to treat viral infections like influenza. Therefore, the pharmaceutical industry is interested in a cheap source for NeuNac, but its synthesis is costly (current market price: 3000 to 4000 US $/kg). Currently, NeuNac can be produced either by E. coli biosynthesis or enzymatic conversion. In both cases the substrate used is N-acetylglucosamine, which itself is costly.

Instead of N-acetylglucosamine, we use the renewable source chitin as a substrate. We developed a whole-cell-bio-catalysis process based on the fungus Trichoderma. Trichoderma is known for its high secretory capacity for hydrolytic enzymes. This filamentous fungus is able to utilize polysaccharides like cellulose or chitin which commonly occur in nature. We use this ability of the fungus to degrade chitin into its monomer N-acetylglucosamine. From N-acetylglucosamine we designed an intracellular enzyme-catalyzed pathway for the synthesis of NeuNac. Therefore, we heterologously expressed two bacterial enzymes, N-acetylglucosamine-2-epimerase and NeuNAc synthase, in Trichoderma. This is necessary because the wild-type of Trichoderma itself is not able to produce NeuNac. We will illustrate the properties of a recombinant Trichoderma strain, which expresses the two bacterial enzymes, and we will show the ability of this strain to form NeuNac. This work demonstrates the potential of using Trichoderma as a whole-cell catalytic system.





Transposon mutagenesis using a resident DNA transposon Crawler in Aspergillus oryzae industrial strains

Hironobu Ogasawara[1] Tsutomu Satoh[1] Hiroshi Konno[1] Yoji Hata[2] Saori Takahashi[3] Katsuya Gomi[4]

1Akita Konno Co. Ltd., Akita, Japan, 2Research Institute, Gekkeikan Sake Co. Ltd., Kyoto, Japan, 3Akita Res. Inst. Food and Brewing, Akita, Japan, 4Graduate School of Agricultural Science, Tohoku University, Sendai, Japan

An active DNA transposon Crawler isolated from the genome of industrially important fungus Aspergillus oryzae transposes under extreme stress conditions.  The DNA sequencing surveys revealed that the Crawler element is widely distributed among A. oryzae and A. sojae strains, which are commonly used in Japanese traditional fermentation manufacturing.  In the present study, we analyzed the relationship between various stress stimuli and inhibition of cryptic splicing of the Crawler mRNA by qRT-PCR to enhance the frequency of Crawler-mediated mutagenesis in an A. oryzae industrial strain, AOK139.  Under the optimized stress conditions, in which conidiospores were treated for 6 hr in 20 mM CuSO4 or at 52℃, various phenotypic mutants different from the parent strain were isolated. These mutants exhibited white color in conidiospores, less number of spores formed, shortened aerial hyphae, thin colony mat and so on.  DNA sequencing analysis of a white conidia mutant revealed that Crawler was newly inserted within a coding region of wA gene encoding polyketide synthase, which resulted in wA deficiency. The insertion occurred also at a TA site with duplication according to the manner of Crawler transposition.  These results suggested that transposon mutagenesis using active Crawler is potentially valuable to improve characteristics of A. oryzae industrial strains.




Bikaverin biosynthesis in Fusarium fujikuroi: Genes, their function and regulation

Bettina Tudzynski[1] Philipp Wiemann[1] Karin Kleigrewe[2] Marita Beyer[2] Hans-Ulrich Humpf[2] Bettina Tudzynski[3]

1Institut für Botanik / Institut für Lebensmittelchemie, WWU Münster, 2Institut für Lebensmittelchemie, WWU Münster, 3Institut für Botanik, WWU Münster

Fusarium secondary metabolites are structurally diverse, have a variety of activities, and their biosynthesis and regulation are generally poorly understood. The F. fujikuroi polyketide synthase gene bik1 was previously shown to be responsible for formation of the mycelial pigment bikaverin. Here we present the characterization of five genes adjacent to bik1 as encoding a putative FAD-dependent monooxygenase (bik2), an O‑methyltransferase (bik3), an NmrA‑like protein (bik4), a Zn(II)2Cys6 transcription factor (bik5), and an MFS transporter (bik6). Deletion of each gene resulted in total loss or significant reduction of bikaverin synthesis. Over-expression of genes involved in biosynthesis enabled us to identify at least one intermediate of the bikaverin pathway. Expression studies revealed that all bik genes are repressed by high amounts of nitrogen in an AreA‑independent manner and are subject to a time- and pH‑dependent regulation. Deletion of the pH regulatory gene pacC resulted in partial de-repression while complementation with a dominant active allele resulted in repression of bik genes at acidic ambient pH. Furthermore, the global regulator Velvet represses bik gene expression and product formation most likely by interconnection with the nitrogen regulation machinery. Thus, bikaverin synthesis is regulated by a complex regulatory network. Understanding how different factors influence the synthesis of this model secondary metabolite will aid understanding secondary metabolism in general.





The role of the early genes of the mevalonate pathway, HMGR and FPPS, for gibberellin biosynthesis in Fusarium fujikuroi

Sabine Albermann,Bettina Tudzynski

Institute of Botany, Westf. Wilhelms-Universitaet Muenster


Each year about ten tons of gibberellins (GAs) are consumed by the agricultural industry as plant growth regulators. GAs function as phytohormones in higher plants and application of GAs induces flower bud formation and shoot elongation as well as an increased fruit size and yield of crops such as seedless grapes and corn.
The rice pathogen
Fusarium fujikuroi is known to produce high amounts of this secundary metabolite and constitutes a capable species for GA production by microorganisms. Therefore, we developed different strategies for strain improvement. By providing a higher amount of the GA precursor molecule geranyl-geranyl-pyrophosphate (GGPP) the yield of GAs could be enhanced and costs of the production would be reduced. For that reason expression of the key enzymes of the mevalonate pathway, 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMGR) and farnesyl-pyrophosphate-synthase (FPPS), should be increased.

In former studies truncation and overexpression of HMGR in Saccharomyces cerevisiae led to a higher accumulation of terpenoids. This is referred to the deletion of the transmembrane domains of HMGR where the enzyme is regulated by feedback inhibition.

Additionally, the ergosterol biosynthesis pathway, a branch of the mevalonate pathway, shall be downregulated as it competes with the GGPP synthase for FPP. Furthermore it would avoid formation of abnormal structures in the fungal membrane.

Finally, localization studies with GFP fusion proteins of HMGR and FPPS shall clearify subcellular organization of the mevalonate pathway and the role of the two key enzymes in F. fujikuroi.




Comparison of the polysaccharide degrading ability of 8 Aspergilli

Ronald de Vries[3] Isabelle Benoit[1] Pedro Coutinho[2] Hala Al-Bushari[1] Evy Battaglia[1] Birgit Gruben[1] Blanca Trejo-Aguilar[1] Guillermo Aguilar-Osorio[1] Ad Wiebenga[3] Bernard Henrissat[2]

1Microbiology, Utrecht University, 2Architecture et Fonction des Macromolecules Biologiques, UMR6098, CNRS, Univ. Aix-Marseille I & II 3CBS-KNAW Funngal Biodiversity Centre

Plant polysaccharides are among the most prominent carbon sources for fungi, which degrade these substrates through the production of diverse sets of extracellular enzymes. This topic has been best studied in the Aspergilli, in particular in A. niger and A. oryzae due to the many industrial application of plant polysaccharide degrading enzymes and the good fermentative properties of these fungi. The availability of genome sequences for several Aspergilli has allowed detailed comparisons between these species and enables us to identify differences in the strategies that they employ to release their carbon source from crude plant biomass.

CAZy-annotation of the genomes revealed significant differences in the sets of hydrolytic enzymes and growth profiling of these fungi demonstrated strong correlations between genome content and ability to degrade specific polysaccharides. In addition, analysis of the secreted enzymes demonstrated further differences that are likely caused by differences in transcriptional or post-transcriptional regulation. Highlights from these results will be presented.

These data will help with the further development of improved enzyme cocktails as they enable a link between the composition of the enzyme set and the efficiency with which different polysaccharides are degraded.





Targeted morphology engineering of Aspergillus niger for improved enzyme production

Habib Driouch, Becky Sommer and Christoph Wittmann

Institute of Biochemical Engineering, Technische Universität Braunschweig, Germany

Among filamentous fungi, Aspergillus niger is important as the major world source of citric acid and higher-value enzyme products including pectinases, proteases, amyloglucosidases, cellulases, hemicellulases and lipases. One of the outstanding and, unfortunately often problematic, characteristics of filamentous fungi is their complex morphology in submerged culture. Hereby, the productivity in biotechnological processes is often correlated with the morphological form.

                In the present work, supplementation with microparticles was used as novel approach to control the morphological development of A. niger [1]. With careful variation of size and concentration of the micro material added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium and short hyphal fragments could be reproducibly created. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Titres for glucoamylase and fructofuranosidase, were up to four fold higher. Moreover, accumulation of the undesired by-product oxalate was suppressed by up to 90 %. Using co-expression of glucoamylase with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production.
                The use of microparticles, allowing targeted engineering of cellular morphology opens novel possibilities for future design and optimization of recombinant enzyme production in
A. niger and probably also other fungi.

[1] Driouch H., Sommer B., Wittmann C (2009) Morphology engineering of
Aspergillus niger for improved enzyme production. Biotechnol Bioeng (in press).




Determination of viable spores in seeding cultures using fluorescent dyes

Becky Sommer, René Stellmacher, Rainer Krull, D. C. Hempel

 TU Braunschweig, Institute of biochemical engineering

In many biotechnological processes filamentous fungi like Aspergilli are used for the production of organic acids, enzymes and antibiotics. Furthermore, there is an increasing application of fungi as catalysts in biotransformations. Therefore spores are used in inoculum development or directly as starting culture in submerged cultivation processes. While the influence of process conditions during cultivation is well-known, the influence of the spore quality in seeding cultures has not been investigated in depth. However the spore viability in seeding culture is an important criterion for product quantity.

The contribution displays the investigation of the transferability of fluoresence based rapid screening assays, developed for bacterial cells, to study spore quality. The results of this method show the applicability to determine the proportion of viable and defect spores in seeding cultures of filamentous fungi. Additionally, good correlations to germination characteristics in submerged cultivation are demonstrated and allow fast, reliable and quantitative distinction between viable and dead spores. Furthermore a biochemical method, based on enzyme activity, has been verified to characterize the spore viability in seeding cultures. The assay allows the characterization of the growth stadium of the filamentous fungi during the sporulation process. Furthermore the enzyme based assay enables the determination of biomass activity in the early phase of submerged cultivation and reveals first information about the feasibility of the cultivation.

In conclusion the presented investigations display the possibility of assessing the ratio of viable to defect spores in seeding cultures, whereby a routine method prior to large scale cultivations could be applied.





A systems biology approach towards itaconic acid production in Aspergillus

An Li, Nicole van Luijk, Martien Caspers, Marloes ter Beek, Jan Jore, Mariet van der Werf, Peter J. Punt

 TNO Quality of life, Zeist, The Netherlands

The black filamentous fungi Aspergillus niger has a long tradition of safe use in the production of enzymes and organic acids, and is widely used in biotechnology as host for the production of food ingredients, pharmaceuticals and industrial enzymes. Besides, Aspergillus niger grows on a wide range of substrates under various environmental conditions. In our research we have addressed the production of one of the commercially interesting building-block organic acids, itaconic acid. To unambiguously identify the itaconic acid biosynthetic pathway several parallel approaches were taken using Aspergillus terreus as parental host strain. Using a combination of controlled fermentation design, reversed genetics and transcriptomics approaches the pathway specific cis-aconitate decarboxylase (CAD) encoding gene was identified. More specifically, data analysis from the transcriptomics study show that the cis-aconitate decarboxylase (CAD) gene and its clustered genes (Class I) are the most highly expressed ones related to itaconate production. Expression of the CAD gene in E.coli proved that this gene encodes cis-aconitate decarboxylase. Further more, expression of the CAD gene in Aspergillus niger resulted in the production of itaconate in the fermentation medium. Further genetic modifications of the itaconic acid metabolic pathway and fermentation medium improvement were initiated to improve itaconate levels.




Secrelection: a novel fungal expression system for selection of secreted enzymes

Robbert Damveld, Brenda Vonk, Cees Sagt, Hildegard Menke, Thibaut Wenzel

DSM Biotechnology Center

Modern biotechnology has generated an impressive set of molecular tools: for instance the ability to generate large sets of error prone mutant libraries or cDNA libraries. When these libraries are expressed in a host (e.g.
Aspergillus niger) not all strains produce a secreted protein. This is mainly dependent on the quality of the library. Here we describe a novel expression system that was developed by using genome expression profiling under different conditions. We were able to identify promoters that fit the required expression profile. These promoters were both up regulated during protein secretion and were not expressed during overexpression of intracellular proteins. By making use of transcriptomics for useful promoter identification, we were able to generate reporter construct(s) that allow us to easily select clones that secrete proteins. This technology can speed up novel protein discovery significantly. Additionally we have shown this approach is not limited to fungi but can also be applied to other production organisms.





Towards science-based process design in filamentous fungi

Andreas Posch, Christoph Herwig

Institute of Chemical Engineering, Division of Biochemical Engineering, Vienna University of Technology, Getreidemarkt 9/166, A-1060, Vienna, Austria.

Meeting QbD (Quality by Design) and PAT (Process Analytical Technology) guidelines launched by the U.S. Food and Drug Administration, biopharmaceutical manufacturers need to move away from traditional empirically based process development towards optimized and science-based process design and control.
Within recent years, numerous PAT compliant tools allowing effective, non-invasive in-situ monitoring of microbial production processes have been successfully introduced. Moreover, several approaches for online process characterization in bacterial and yeast systems have been suggested. Promising procedures include applying metabolic modeling and online evaluation of stoichiometric balances enabling to distinguish between different metabolic states of cellular production systems.

For cultivation processes of filamentous fungi however, implementation of such methods has been hampered by increased complexity of filamentous fungal systems and additional factors affecting process performance, e.g. growth morphology and extracellular proteolytic activity.

The aim of this work is to establish a methodology allowing generically characterizing fungal filamentous systems by accurately quantifying fungal response behavior to physiological stress under transient culturing conditions, linking metabolic regulations to morphological changes. Dynamic experiments are designed using DoE (Design of experiment) strategies, and carried out in fully instrumented bioreactors coupled to a process management system performing online data evaluation and reconciliation.

Combining process technology, morphological, and physiological analysis, allows accurately quantifying filamentous fungal process behavior. Our approach will thus facilitate process development and allow understanding biological variability and, in turn, control those complex processes.




Novel low-temperature performing cellulase for textile industry

Terhi Puranen[1] Leena Valtakari[2] Marika Alapuranen[1] Jarno Kallio[1] Jari Vehmaanperä[1]

1Roal Oy, Rajamäki, Finland, 2AB Enzymes Oy, Rajamäki, Finland

Cellulases are traditionally used in textile, detergent, pulp and paper, feed and food industries, and are currently also extensively studied for cellulosic biomass conversion to ethanol. Endoglucanases have been widely used in textile industry for biostoning and biofinishing of cellulosic fibers. There has been trend in the market, especially in Asia, to use lower washing temperatures for energy savings and environmental reasons. Here we describe molecular cloning of glycosyl hydrolase (GH) family 45 cellulase from the Geomyces pannorum as well as its heterologous expression in Trichoderma reesei. Geomyces pannorum Cel45A contains bimodular structure composed of a catalytic module linked to C-terminal carbohydrate-binding module (CBM), and the protein has low homology to the known Cel45 cellulases. Geomyces pannorum Cel45A enzyme shows exceptional broad application range from 20 ºC to 50 ºC with high biostoning performance and excellent depilling properties.





Secretome analysis of concentric zones of Aspergillus niger colonies

Pauline Krijgsheld[1] Jeffrey H. Ringrose[2] A. F. Maarten Altelaar[2] Albert J. R. Heck[2] Han A. B. Wösten[1]

1Microbiology and Kluyver Centre for Genomics of Industrial Fermantation, Utrecht University, Padualaan 8, 3584 CH Utrecht, 2Biomolecular Massspectrometry and Kluyver Centre for Genomics of Industrial Fermantation, Utrecht University, Padualaan 8, 3584 CH Utrecht

In nature fungi degrade organic matter such as wood. To this end, they secrete enzymes that convert the polymeric substrates into small molecules that can be taken up to serve as nutrients. Hyphae at the periphery of the colony are exposed to unexplored organic material, whereas the center of the colony experiences a (partly) utilized substrate. This suggests that the enzymes that are secreted by distinct zones in the colony are different. This hypothesis was tested using the industrial important fungus Aspergillus niger. Spatial protein secretion was monitored by growing A. niger colonies between two porous polycarbonate membranes that have been placed on top of agar medium. This mode of growth inhibits sporulation and forces the colony to grow two-dimensionally. After 6 days of growth, the colony was transferred to a ring plate. This plate contains 5 concentric wells that are filled with liquid medium. After growing the colony for 24 h on the ring plate the medium of the wells was collected. Proteins in each of the wells were identified by mass spectrometry. 70 and 50 different proteins were found to be secreted at the periphery and the centre of xylose-grown colonies, respectively. Of these, only one is predicted not to have a signal sequence for secretion. This indicates that lysis in the colony is minor, if present at all, even within the colony centre. Interestingly, six proteins were secreted in the central zone that were not found at the periphery of the colony. Thus heterogeneity of the colony is reflected in the spatial secretion pattern.  





Electrotransformation of the obligate biotrophic cucurbit pathogen Podosphaera fusca

DAVID VELA, Antonio de Vicente, Alejandro Pérez


Powdery mildew fungi (Erysiphales) are probably the largest group of plant pathogens that remain uncharacterized from the genetic and molecular points of view. Their lifestyle as obligate biotrophic parasites and consequent inability to grow on culture media has significantly hampered research. Appropriate disease management requires a good understanding of the biology of the responsible pathogen. Regarding powdery mildews research, a technologically important unsolved issue is their genetic manipulation. Since the first reports of DNA-mediated transformation in model filamentous fungi such as Neurospora crassa and Emericella nidulans, many commercially and agriculturally important fungal species have been successfully transformed. However, the obligate biotrophs have, hitherto, proved recalcitrant to this process.Most common methods such as protoplast fusion or Agrobacterium-mediated transformation are not suitable for such organisms like the first, or show important methodological limitations like the second. In fact, to date particle bombardment has been reported as the only valid method to transform this type of parasites, although this technique has shown certain lack of reproducibility.

We report here on progress in developing a reliable and stable transformation protocol for
Podosphaera fusca, the causal agent of cucurbit powdery mildew, by the highly versatile electrotransformation method. Two selectable markers have been used:the hygromycin B resistance gene (hph) and the enhanced green fluorescent protein gene (egfp). These markers were tested under the control of different promoter and terminator sequences such as those of the gpd and trpC genes of E. nidulans, the isl gene of N. crassa, and the promoters of the b-tubulin and CYP51 genes of P. fusca. Transformants were obtained with most of the constructs used. Under selective conditions the hygromycin B resistant transformants grew very slow. Similarly, weak GFP signals were associated to fluorescent hyphae of transformants. In all cases we detected sequences of marker genes by PCR and found copies of the transforming plasmids integrated in the fungal genome. To our knowledge this is the first case of genetic transformation of a powdery mildew fungus by electroporation of conidia. An update of the results obtained so far will be presented.




The regulation of ergot alkaloid biosynthesis in Claviceps purpurea

Barbara Oportus, Nicole Lorenz, Anna Koppe, Sabine Giesbert, Paul Tudzynski

Institut für Botanik, Westfälische Wilhelms-Universitaet, Schlossgarten 3, D-48149 Muenster,Germany

Ergot alkaloids (EA) are secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. The altogether 14 genes encoding the specific enzymes for the biosynthesis of EA are clustered. To date the molecular mechanisms of cluster regulation in C. purpurea are unknown. No transcription factor gene has been found within the cluster region involved in the synthesis of EA. It is only known to date that the EA in C. purpurea wild-type are produced during the ripening of the sclerotium and not in axenic cultures. Mutant strains producing alkaloids in submersed cultures require under specific conditions: (a) tryptophan as inducer and precursor, (b) a high osmotic value, (c) a low phosphate level. The alkaloid biosynthesis was speculated to be regulated by changes in the chromatin organization, a hypothesis checked by the cultivation of C. purpurea in the presence of either inhibitors of histone deacetylases (HDACis) or histone acetyltransferases (HATis). The production strain P1 of C. purpurea was cultivated under both alkaloid producing (T25N medium) and non-producing (BII medium) conditions. Unexpectedly, the alkaloid biosynthesis is repressed instead of induced by the addition of HDACis (euchromatin formation) and vice versa, indicating a complex regulation system (1). Another global regulatory system of secondary metabolism and development was discovered in Aspergillus nidulans, velvet (2). We are investigating if in C. purpurea the biosynthesis of ergot alkaloids could be regulated through velvet. We have identified and sequenced a velvet homologue in C. purpurea (cpvel1) and started a functional analysis. (1) Lorenz et al. (2009) Phytochemistry. 70:1822-1832. (2) Kim et al. (2002) Fungal Genet Biol. 37:72-80.





Disruption of a vacuolar protein sorting receptor gene, Aovps10, enhances production level of heterologous protein by the filamentous fungus Aspergillus oryzae

Jaewoo Yoon, Tuerxun Aishan, Jun-ichi Maruyama, Katsuhiko Kitamoto

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Filamentous fungi have received attention as a host for heterologous protein production because of their high secretion capability. However, a bottleneck in post-transcriptional processing in the secretory pathway limits protein production yields. The vacuolar protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar proteins. Although it can also target several recombinant and aberrant proteins for the vacuolar degradation, there is limited knowledge of the effect of its disruption on heterologous protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was isolated. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent protein showed that the fusion protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of AoCpyA to vacuoles in A. oryzae. To investigate the extracellular production level of heterologous protein, we constructed an ΔAovps10 mutant expressing bovine chymosin (CHY). Interestingly, ΔAovps10 increased the maximum extracellular production by three fold. Western blot analysis of extracellular CHY also demonstrated an improvement in productivity. These results suggest that the AoVps10 plays a role in the regulation of heterologous protein secretion in A. oryzae, and may be involved in vacuolar protein degradation through the Golgi apparatus. This is the first report demonstrating that disruption of a vacuolar protein sorting gene in filamentous fungi leads to enhanced production levels of heterologous protein.




Cloning, expression, and characterization of beta-glucosidases from a black Aspergillus spp.

Sorensen Annette, Peter S. Lubeck, Mette Lubeck, Birgitte K Ahring

Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University

Through a broad cellulolytic activity screening of fungi isolated from various locations, a very promising fungus, identified as a black Aspergillus, was selected for further studies. An enzyme extract of this fungus was obtained through solid state fermentation and tested to profile its beta-glucosidase activity. Four beta-glucosidase genes from this fungus have been cloned through pcr with degenerate primers designed from conserved motif regions of known beta-glucosidase genes from Aspergilli followed by genome walking strategies. The goal is to have these genes expressed in Trichoderma reesei QM6a ΔpyrG. An expression system was constructed with the constitutive ribosomal promoter RP27 for expression of the his-tagged beta-glucosidase genes and with the pyrG gene to complement the mutation. Analysis of the different transformants is currently on-going and subsequently the different beta-glucosidase proteins will be purified for detailed analysis of specific activity, Km, sugar tolerance, thermostability as well as their ability to break down shorter chained oligosaccharides.



Laccases from the biopulping fungus Physisporinus rivulosus: molecular characterization of thermotolerant oxidoreductases and reactions with mediator compounds

Kristiina Hilden[4] Taina Lundell[1] Miia Mäkelä[1] Johanna Rytioja[1] Paula Nousiainen[2] Jussi Sipilä[2] Alexey Chernykh[3] Ludmila Golovleva[3] David Archer[4] Annele Hatakka[1]

1Department of Applied Chemistry and Microbiology, University of Helsinki, Finland
2Department of Chemistry, University of Helsinki, Finland
3G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Puschino, Russia
4School of Biology, University of Nottingham, UK

The white rot, softwood biopulping basidiomycetous fungus Physisporinus rivulosus isolate T241i produces two laccase isozymes with novel catalytic properties such as thermotolerance and atypically low pH optima (pH 3.0-3.5) for the methoxylated phenolic laccase substrate compounds syringaldazine, 2,6-dimethoxyphenol and guaiacol (1). The fungus was previously distinguished in a large screening study as the most selective and efficient lignin decomposer of spruce (Picea abies) wood (2, 3), and is shown to produce several manganese peroxidase and laccase isozymes as well as oxalic acid when growing on spruce wood chips (4). Recently, we have cloned and characterised at molecular level two P. rivulosus laccase genes. The translated mature Priv-lac1 and Priv-lac2 enzymes demonstrate structural similarity with Trametes versicolor and Lentinus tigrinus laccases, respectively, as deduced by protein 3-D homology modelling. The Priv-lac1 and Priv-lac2 coding sequences are interrupted by 11 and 12 introns and they host the four conserved His-containing Cu-binding regions typical for laccases. The protein architectures are viewed at the modelled substrate-binding pocket to elucidate the atypical pH optima for the phenolic compounds as electron donors. When P. rivulosus T241i is cultivated in spruce sawdust-charcoal containing liquid medium, higher levels of laccase activity is produced. The activity is separated within over five isoforms, and purified P. rivulosus laccases display catalytic efficiency for oxidation of the higher-redox potential substrates Reactive Black 5 and veratryl alcohol when the reactions are supplied with natural-type of oxidation-mediator compounds. These properties well support the P. rivulosus laccases to be applied as robust biocatalysts for e.g. bleaching of dye compounds.

(1) Hildén K., Hakala T.K., Maijala P., Lundell T.K., Hatakka A. (2007) Appl Microbiol Biotechnol 77:301-309
(2) Hakala T., Maijala P., Konn J., Hatakka A. (2004) Enzyme Microb Technol 34:255-263
(3) Hatakka A., Maijala P., Hakala T.K., Hauhio L., Ellmén J. (2003) Int pat appl WO03/080812
(4) Hakala T., Lundell T., Galkin S., Maijala P., Kalkkinen N., Hatakka A. (2005) Enzyme Microb Technol 36:461-468


Improving A. niger asparaginase via directed evolution

Ilse de Lange, Jan-Metske van der Laan, Richard Kerkman, Wim Bijleveld, Mark Stor

DSM Biotechnology Center , P.O. Box 1, 2600 MA Delft, The Netherlands,

The filamentous fungus Aspergillus niger has extensively been used for recombinant protein production. The sequencing of the A. niger CBS513-88 genome, an ancestor of our current enzyme production strains, yielded easy access to numerous protein encoding genes. Some of these genes encode valuable new food enzymes. One of those new products is A. niger asparaginase, an enzyme effective in mitigation of acrylamide formation in food products. To improve efficiency of A. niger asparaginase in application, we applied directed evolution to optimize the pH-activity profile of the enzyme. Several new variants were identified, which show improved performance both in vitro and in small scale application tests.





The secreted yields of heterologous proteins from Saccharomyces cerevisiae and Pichia pastoris are dependent on protein stability and the UPR

Graham Whyteside, Rohana Mat Nor, Marcos Alcocer, David Archer

University of Nottingham

S. cerevisiae and P. pastoris are hosts for the production of heterologous proteins. The secreted yield of a heterologous protein varies according to the protein being expressed and we aim to provide predictability based on the stability of protein folded states. Evidence was gained from the secretion of human lysozyme variants that have single amino acid changes. A direct relationship between decreasing protein stability and secreted yield was demonstrated in P. pastoris (Kumita et al. 2006 FEBS J 273, 711-720) and in S. cerevisiae (unpublished). The reasons underlying the impact of stability on secreted yields were investigated and the unfolded protein response (UPR) was the focus in terms of its kinetics of induction and its severity. We showed that genes encoding both BiP (chaperone) and PDI (foldase) were transcriptionally up-regulated more quickly and to higher levels in response to secretion of less-stable lysozymes. The transcript level of HAC1 mRNA is not altered substantially in S. cerevisiae by ER stress but the transcript level of HAC1 from P. pastoris was enhanced markedly and increasingly so with the more unstable variant lysozymes. We conclude that the folded state of a protein is detected and responded to in the ER lumen by the quality control system and the response is a measured one and proportionate to the folded state stability of a secreted protein. This analysis is being extended to other proteins, e.g. antibody fragments, and the differences in the UPR between S. cerevisiae and P. pastoris are being explored further.





Engineering intracellular metabolism by altering gene expression of Aspergillus oryzae

Junichiro Marui[1] Sumiko Ohashi[1] Marie Nishimura[2] Hideaki Koike[1] Masayuki Machida[1]

1National Institute of Advanced Industrial Science and Technology (AIST), 2Natl. Inst. Agrobiol. Sci.

Aspergillus oryzae is one of the most important organisms in Japanese fermented food industry. Although it hardly produces secondary metabolites, related organisms are producers of diverse metabolites. A major objective of our project is to develop a system using A. oryzae to generate diverse metabolites. To reveal regulation of metabolic pathways in A. oryzae under various conditions, gene expression profiles under variety of conditions and change depending on time were analyzed by DNA micro array and so on. The factors affecting expression level of metabolic genes are being studied. To develop the system, novel vectors and host strains of A. oryzae have been constructed. We have replaced promoter regions of some metabolic genes and successfully altered the level of some metabolite productions. Although it is well known that A. oryzae does not produce toxic metabolites, less attention has been paid to its non-toxic secondary metabolites. Genomic analysis revealed that A. oryzae possessed the orthologous gene cluster for penicillin production. The penicillin production was positively regulated by a global gene regulator required for transcriptional expression of the penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production. Transcriptional repression of a wide range of secondary metabolism genes in A. oryzae is a valuable characteristic for the production of a particular secondary metabolite with higher purity and safety. It appears that genetically engineered A. oryzae should be extremely useful as a cell factory for industrial production of beneficial secondary metabolites.




Overproduction and characterization of prolyl aminopeptidase from Aspergillus oryzae

Ken-Ichi Kusumoto, Satoshi Suzuki

National Food Research Institute

Overproduction and characterization of prolyl aminopeptidase from Aspergillus oryzae Ken-Ichi Kusumoto1, Mayumi Matsushita-Morita1, Ikuyo Furukawa1, Youhei Yamagata2, Yoshinao Koide3, Hiroki Ishida4, Michio Takeuchi2, Yutaka Kashiwagi1, Satoshi Suzuki1 1National Food Research Institute, Tsukuba, Ibaraki, Japan; 2Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 3Gifu R & D Center, Amano Enzyme Inc., Kagamihara, Gifu, Japan; 4Research Institute, Gekkeikan Sake Company Ltd., Fushimi-ku, Kyoto, Japan  


Introduction: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides.  The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts.  Therefore, we present here an efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties.

Methods and Results: The gene encoding PAP was overexpressed as a His-tag fusion protein under a amyB promoter with a limited expressing condition in A. oryzae.  The PAP activity in the mycelia grown in rich media containing glucose (repressing condition) was twice that in starch (inducing condition).  The enzyme prepared as cell-free extract was partially purified through two-step column chromatography.  A. oryzae PAP was purified 1800-folds.  The PAP was estimated to be a homo hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l-1.

Discussion: Overproduction of PAP under promoter inducing conditions led to an increase of inactive PAP, possibly because of irregular folding.  PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods. 


The Trichoderma harzianum pH regulation factor Pac1 is involved in osmotic and saline stress resistance

ANA MARÍA RINCON ROMERO[3] Moreno-Mateos[1] Espeso, E.A.[2] Codón, A.C.[3] Benítez, T.[3]

1Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Av. Américo Vespucio, s/n, 41092 Sevilla, Spain
2Centro de Investigaciones Biológicas CSIC. Ramiro de Maeztu, 9, 28040 Madrid, Spain
3Universidad de Sevilla, Departamento de Genética, Facultad de Biología, Avda.
Reina Mercedes, s/n, 41012 Sevilla, Spain

Trichoderma spp. is a filamentous fungus used in biocontrol. Many attemps were carried out to improve its antagonistic ability. However, Trichoderma adaptability to different soils might be affected under environmental stress conditions, and finding resistance to adverse conditions would be a desirable feature for the use of Trichoderma in agriculture. Recently, implication of Pac1, the homologue of the pH transcriptional regulator PacC, in Trichoderma harzianum parasitic relationships has been demonstrated. As its homologous PacC proteins in other filamentous fungi, Pac1 is expected to be directly involved in fungal stress response. Two mutants of T. harzianum CECT2413 have been obtained in Pac1: P2.32 expresses a constitutively active Pac1, and R13 contains a null allele by gene silencing. Strain P2.32 shows better growth than wild type in NaCl-, KCl- or sorbitol-containing media, while strain R13 shows higher sensitivity in the same conditions. Phenotypes of P2.32 and R13 mutants in the presence of Lithium are opposed but contrary to those described for PacC/Rim mutants in yeasts and other filamentous fungi. Transcriptional regulation of pac1 is altered under salt/osmotic stress. Pac1 mRNA levels are higher and the protein produced able to bind DNA. However, Pac1 DNA binding ability did not correlate to those levels of pac1 expression in non stressing media. These results indicated that Pac1 is a key factor for saline and osmotic stress resistance and might be subjected to a complex combination of traductional and posttranslational regulation in T. harzianum.




Applications of genetic transformation of Aspergillus oryzae with bleomycin resistant selection

Satoshi Suzuki[1] Sawaki Tada[1] Mari Fukuoka[1] Yoshiki Tsukakoshi[1] Mayumi Matsushita-Morita[1] Yutaka Kashiwagi[1] Masanori Sugiyama[2] Ken-Ichi Kusumoto[1]

1NFRI, 2Hiroshima Univ.

Introduction: Since Aspergillus oryzae produces large quantity of enzymes, it is significant to establish a host-vector system to use this microorganism as a host for heterologous protein expression.  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 have recently developed a transformation system for A. oryzae RIB40 by using bleomycin-resistance gene as a selective marker. In the present study, we generated ligD knock out strain, by using bleomycin resistance selection. And we generated exogenous or endogenous enzyme over-expresser by transformation with expression vector carrying bleomycin resistant gene. Methods: The ligD disruption cassette consisted of bleomycin-resistance expression cassette and 2kb of 5’ and 3’ franking sequence of ligD, the β-glucuronidase encoding gene uidA from E. coli over-expression vector and a polygalacturonase encoding gene pgaB from A. oryzae over-expression vector were introduced into the protoplasts from A. oryzae RIB40 by the polyethylene glycol method, respectively.  Results: The disruption of the ligD locus in genomic DNA of transformants was confirmed by colony PCR and the Southern blot analysis. Activity of each enzyme was detected from cell free extract or culture filtrate of the each transformant selected by bleomycin resistance. Discussion: In the present study, we successfully generated a gene disruptant and over-expresser of two kinds of enzymes by using bleomycin resistance transformant selection system of A. oryzae.



Genetic chemotyping in Fusarium species predicts toxin content in wheat

Matias Pasquali, Frederic Giraud, Emmanuelle Cocco, Lucien Hoffmann, Torsten Bohn

CRP Gabriel Lippmann

Fusariotoxins present in wheat and barley are a major cause of economic loss, especially for alimentary industries and breweries. Many predictive models based on climatic and agronomic practices have been implemented in order to estimate toxin content in grains. Here we show that genetic chemotyping of the Fusarium population within a field may improve significantly the prediction of toxin content on wheat. We analysed nivalenol (NIV, a trichothecene with higher toxicity when compared to deoxynivalenol) content in wheat by LC/MS-MS analysis from 17 different wheat fields sampled for two consecutive years. We determined species and chemotype by using multiplex PCRs on a total of 1084 isolates. A significant correlation (R= 0.75, p<0.00001) was found between the quantity of Fusarium culmorum isolates per 100 seeds with the NIV chemotype and the amount of NIV in grains. A threshold, based on the amount of NIV isolates per 100 seeds for detecting NIV in grains has been tested successfully on 9 independent additional fields (P<0.001). Moreover we showed that the development of a real time PCR method for the detection of F. culmorum with NIV chemotype from grains further improved the accuracy and speed of the test. Here we demonstrated the reliability of Fusarium genetic chemotyping for toxin prediction in the field. Furthermore, we verified the importance of targeting genes linked to function (in this case trichothecene synthetic genes) in PCR based screening for mycological application.





Development of a dual marker selection system for repeated gene deletions in Fusarium venenatum and the resulting strain harboring five gene deletions

Jeffrey Shasky, Wendy Yoder, Alfredo Lopez, Ron Kloberdantz, Michael McCulloch, Brian Gore-Clancy, Dawn Lysaght

Novozymes Inc

The industrially important QuornTM fungus Fusarium venenatum has previously been used as a host for heterologous protein expression.  Since the wild-type organimsm can grow on the toxic analogue 5-fluoroorotic acid (FOA) despite having an intact pyrG gene, an alternative couter-selectable marker was needed for sequential gene deletions to improve the host.  A system was developed using two separate gene cassettes, each controlled by a different heterologous promoter and terminator, flanked by direct repeats.  The E. coli hygromycin phosphotransferase gene (hph), conferring resistance to the herbicide hygromycin, was used for positive selection and deletion of the gene of interest.  The herpes simplex virus thymidine kinase (tk) gene was used for counterselection on the uridine analogue 5-flourodeoxyuridine (FdU), allowing selection for spontaneous excision of the region containing both markers.   Deletion constructs incorporating the two markers listed above, flanking direct repeats, as well as flanking sequence for the gene of interest were generated for two mycotoxin genes (trichodiene synthase gene (tri5) and enniatin synthase gene (dps1)), the orotidine- 5- phosphate decarboxylase gene (pyrG), an alpha amylase gene (amyA) and an alkaline protease (alpA) gene.

Using deletion constructs above genes were sequentially deleted in Fusarium venenatum strain A3/5 in the following order:  tri5, pyrG, amyA, alpA, dps1.  The uridine auxotrophy caused by the pyrG deletion could be rescued by transformation with a pyrG fragment, allowing its use as a marker for heterologous protein expression. The resulting strain also showed desired host characteristics as a result of the gene deletions. 




Enzymatic hydrolysis of lignocelluloses: identification of novel cellulases genes from filamentous fungi

Marta Kolasa, Birgitte K. Ahring, Peter S. Lübeck, Mette Lübeck

Copenhagen Institut of Technology, Section for Sustainable Biotechnology

Lignocellulosic materials form a huge part of the plant biomass from agricultural and forestry wastes. They consist of three major components: cellulose, hemicellulose and lignin. Cellulose, the main constituent of plant cell wall, is a polymer of D-glucopyranose units linked by β-1,4-glucosidic bonds. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production of a variety of cellulolytic enzymes. The aim of this work is to discover new thermostable and robust cellulolytic enzymes for improved enzymatic hydrolysis of biomass. For this purpose two screening methods are applied in different fungal strains with high cellulolytic activities: an expression-based method using suppression subtractive hybridization and a targeted genomic screening approach using degenerate PCR. Suppression subtractive hybridization facilitates identification of genes encoding cellulolytic enzymes that are expressed when cultivating a fungal strain in medium with cellulose as the carbon source. By means of degenerate PCR, specific genes, homologous to the genes of previously classified glycoside hydrolases from CAZY database, are searched for in selected strains of Aspergillus sp., Trichoderma sp. and Penicillium sp. Both methods are anticipated to facilitate identification of target genes which subsequently will be cloned and expressed in a relevant fungal host for further characterization of the expressed enzymes. The goal is to introduce new enzymes to industrial processes.




Colony picker as screening-tool in fungal biotechnology

Thomas Haarmann, Ute Knirsch, Jasmin Hack

AB Enzymes GmbH

The aim of this project is to establish the QPix2 colony picker (Genetix) to pick colonies from different filamentous fungi (e.g. Aspergillus, Trichoderma) in a fast and efficient way. This is important because classical mutagenesis and screening projects require huge amounts of mutants to be picked and analysed which is highly time consuming if done manually. One additional benefit is that all the work is carried out in an enclosed chamber which is easily kept sterile by exposure to UV-light.

Since the robot was originally designed for picking bacteria or yeast we had to overcome different obstacles regarding e.g. the composition of the culture media, the design of the picking-pins or the speed of movement of the picking head. Other critical parameters are the density of plated spores, age of cultures and size of the colonies. Several experiments will be shown in which we try to define the optimal values for each of these parameters.

Additionally, the colony picker will be used to carry out simple plate assays to measure e.g. enzymatic activities of generated mutants semi-automatically.

All of these improvements will substantially enhance the speed of classical strain development by mutagenesis and screening approaches.




Identification and regulation of gene fusA responsible for fusarin production in Fusarium fujikuroi

Violeta Díaz-Sánchez, Javier Avalos, M. Carmen Limón

Universidad de Sevilla

Fusarium fujikuroi is a rice pathogen which produces different secondary metabolites. Some of them are mycotoxins, i.e. bikaverins and fusarins, and others have biotechnological applications, i.e., gibberellins and carotenoids. Fusarins are near UV-absorbing polyketides that, accumulated at high concentration, provide a yellowish pigmentation to F. fujikuroi mycelia. Little information is available on the regulation of fusarin biosynthesis by this fungus. As formerly described, the synthesis was enhanced at high temperature (30°C vs 22°C). However, in contrast to the nitrogen repression described for the synthesis of other metabolites, such as gibberellins and bikaverins, we found that fusarin production is repressed by nitrogen starvation. As other fungal polyketides, the synthesis of fusarins starts through the activity of a multidomain enzyme, termed type I polyketide synthase (PKS). The PKS gene responsible for fusarin biosynthesis was formerly indentified in Fusarium graminearum and other Fusarium species. High sequence conservation between Fusarium genomes has allowed us to clone the orthologous gene of F. fujikuroi, which we named fusA. Deletion mutants for this gene showed a total absence of fusarin production, confirming the role of  PKS FusA in the biosynthesis of this metabolite. Real-time RT-PCR analyses of fusA mRNA were achieved using a 3’ gene segment, absent in the other known Fusarium PKS genes. The results showed that the two regulatory conditions tested, temperature and nitrogen availability, act at least partially on fusA gene expression.





Effects of CBM insertion on the properties of Trichoderma reesei α-Amylase

Taija Leinonen[1] Kari Juntunen[1] Leena Valtakari[2] Tapani Suortti[3] Sanni Leinonen[3] Kristiina Kruus[3] Marja Paloheimo[1]

1Roal Oy, 2AB Enzymes Oy, 3VTT

The carbohydrate binding modules (CBMs) are non-catalytic domains of carbohydrate-acting enzymes, and are known to promote the association of enzyme with its insoluble substrates. We studied the effects of CBM attachment on the properties of Trichoderma reesei α-amylase (ID 105956 in This amylase does not, in its native form, contain a CBM. A sequence encoding a family 20 CBM from another fungal α-amylase gene was fused 3’ to the T. reesei amylase gene.  The T. reesei amylase gene and amylase-CBM fusion construction were overexpressed in T. reesei using the strong cbh1 (cel7A) promoter. The two enzymes were purified from the culture supernatants, and their physicochemical properties and performance in stain removal were determined. The amylase-CBM fusion protein was stable in T. reesei culture supernatant. Its temperature and pH profiles were similar to those of the native T. reesei amylase. However, its specific activity on Azurine-crosslinked amylase (AZCL-Amylose) was over three times higher when compared to the native amylase. Addition of the CBM20 was shown to have a positive effect on starch hydrolysis rate but the hydrolysis product profiles were similar with the two enzymes. The hydrolysis products consisted mainly of glucose and maltose during the whole time-course of the reaction, and only low amounts of maltotriose, -tetraose and –pentaose were detected.  Insertion of the CBM had no positive effect on stain removal performance on standard starch stain, possibly due to attachment of the amylase-CBM form to the stain and/or on fabric surface.



Using spore autofluorescence to measure viability of fungal inocula

Matthias Gehder, Jan-Hendrik Sachs, Dietmar Hempel, Rainer Krull

TU Braunschweig

Filamentous fungi such as Aspergilli are widely used in numerous biotechnological applications. Their use ranges from the large scale production of citric acid to the expression of highly complex catalytic and therapeutic proteins. Furthermore, they are used in biotransformations both, inside and outside the bioreactor. The widespread industrial usage of filamentous fungi has consequently triggered tremendous interest in the research and optimization of the cultivation process which is now well understood. However, what has been left out so far is the influence of spore quality. This is partially due to the lack of adequate methods for spore quality assessments and hinders the interpretation of cultivation outcomes.

The contribution shows the applicability of spore autofluorescence as an indicator for spore quality. Autofluorescence allows high throughput, non-invasive investigations of important cell compounds and can be correlated to key metabolic substrates also present in spores. The results of the autofluorescence assay show a good correlation to different standard viability assays.

The findings of these investigations show the possibility of assessing spore quality as a routine measure prior to large scale cultivations, thereby improving the cultivation outcome and minimizing bioprocesses with filamentous fungi with inferior yields. The presented results also offer a new and fast tool for the optimization of sporulation media.





Biochemical producing fungi

Lena Nilsson, Birgitte K. Ahring, Mette Lübeck, Peter S. Lübeck

Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University

We are in the process of developing a biorefinery concept for the use of selected plant biomasses for production of high value biochemicals aiming at replacing chemicals produced from fossil fuels. One aspect will be the engineering of efficient biomass converting fungal strains with the ability to produce high amounts of specific organic acids. Aspergillus niger producing citric acid is a classical example of industrial application of fermentative processes in a filamentous fungal strain. We would like to utilise both the large potential for secretion of hydrolytic enzymes and the organic acid producing machinery of the filamentous fungi for further genetic engineering.  We anticipate to initiate the engineering by manipulating central pathways in carbon metabolism i.e. glycolysis to increase the funnelling of sugars to acid production. By initial screening of a large collection of fungal strains isolated from natural habitats we have identified isolates with high production and excretion of organic acids. Among these are several Aspergillus species and one Penicillium sp. with higher excretion of TCA-cycle intermediates, and these strains have been chosen for genetic engineering.





2-DE analysis of the Penicillium chrysogenum extracellular proteome: An impressive number of isoforms

Mohammad Saeid Jami Foroushani[1] Carlos García-Estrada[1] Carlos Barreiro[1] Rebeca Domínguez-Santos[2] Zahra Salehi-Nagafabadi[2] Ricardo V. Ullán[1] Fernando Teijeira[1] Marta Fernández-Aguado[2] Juan-Francisco Martín[3]

1INBIOTEC, Instituto de Biotecnología de León, Avda. Real nº. 1, Parque Científico de León, 24006 León, Spain
2Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain
3Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain. INBIOTEC, Instituto de Biotecnología de León, Avda. Real nº. 1, Parque Cient&a

Penicillium chrysogenum is well-known by its ability to synthesize penicillin and related b-lactam antibiotics as well as several other secondary metabolites. In addition, P. chrysogenum might be consideered, like other filamentous fungi, as an excellent host for secretion of extracellular proteins due to the high capacity of their protein secretion machinery. In this work, we have analysed by 2-DE the extracellular proteome reference map for P. chrysogenum Wisconsin 54-1255. IPG strips with a pH range from 4 to 7 were used and visualization of spots was achieved by “Blue Silver” staining. This method allowed the correct identification of 279 spots by peptide mass fingerprinting (PMF) and tandem MS. The 279 spots represented 328 correctly identified proteins (with an average error of 6.47 ppm), since some spots contained more than one protein. Almost all the significant MASCOT searches provided an exact match with proteins inferred from the P. chrysogenum Wisconsin 54-1255 genome, which indicates high accuracy of the genome annotation process. From the 328 proteins identified in the reference map, 62 proteins showed a total of 259 isoforms. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, glucan 1,3-beta-glucosidase), extracellular acid phosphatases, 6-hydroxy-D-nicotine oxidase and catalase R. These two proteins showed a high number of isoforms (20 and 18, respectively). The deep analysis of the P. chrysogenum extracellular proteome may be of relevant interest for white biotechnology.





Transcriptional analysis of Trichoderma reesei cultivated in the presence of different lignocellulose substrates

Mari Häkkinen, Markku Saloheimo, Merja Penttilä, Tiina Pakula

VTT Technical Research Centre of Finland

Transcriptional analysis of Trichoderma reesei cultivated in the presence of different lignocellulose substrates   Mari Häkkinen , Markku Saloheimo, Merja Penttilä and Tiina Pakula VTT Technical Research Centre, P.O.Box 1000 (Tietotie 2, Espoo), Fin-02044 VTT, Finland;, tel. +358 20 722 111, fax. +358 20 722 7071   Trichoderma reesei is a soft rot Ascomycete fungus able to secrete enzymes extremely efficiently and which is therefore used for industrial production of cellulolytic and hemicellulolytic enzymes and heterologous proteins for applications in pulp and paper, detergent, food, textile and feed industries and in biorefinery applications. Production strains have been traditionally improved by classical mutagenesis as well as by specific genetic modifications. The availability of the complete genome sequence of T. reesei has made it possible to utilise genome wide analysis methods to study physiology and protein production by the fungus at different conditions and to utilise the information obtained to develop new strains with better enzyme production qualities, such as capability for enhanced protein production or production of modified enzyme mixtures for degradation of specific types of lignocellulose materials.   In this study Trichoderma reesei Rut-C30 was cultivated in the presence of different lignocellulose substrates in order to study the hydrolytic system of T. reesei in the presence of the substrates. The substrates included e.g. sophorose, cellulose, pretreated wheat straw, pretreated spruce, xylan and bagasse. For bagasse, different pre-treatments were used in order to get different combinations of the inducing substances. The cultures were subjected to transcriptional profiling using oligonucleotide microarrays. Differentially expressed genes were indentified from the data by comparing the signal intensities between the induced samples and un-induced controls, and expression profiles of glycoside hydrolase genes and other genes encoding lignocellulase degrading enzymes were compared in the presence of the different substrates to identify co-regulated groups of genes





Production of a CBH1-EG1 fusion enzyme in Trichoderma reesei and evaluation of its activity on lignocellulosic substrates

Senta Heiss-Blanquet[1] Nicolas Lopes Ferreira[1] Gaëlle Brien[1] Sarah Morais[2] Ed Bayer[2] Henri-Pierre Fierobe[3] Antoine Margeot[1]

1IFP, 2Weizmann Institute, 3CNRS

The enzymatic cocktail of Trichoderma reesei is widely used for the hydrolysis of lignocellulosic substrates to fermentable sugars in the bioethanol production processes. Its efficiency has still to be increased, however, to lower the cost of the hydrolysis step. In order to increase the specific activity of the enzyme mixture, a fusion protein comprising the entire CBH1 enzyme linked to the catalytic domain of EG1 was constructed. The construct was transformed into a T. reesei strain lacking the cbh1 gene (CL847Dcbh1) where it was expressed under the control of the cbh1 promoter. Two stable clones secreting the fusion protein were isolated and one of them was cultivated in a larger scale for purification of the recombinant protein by FPLC. A partially pure protein could be obtained and its activity on cellulosic substrates determined. The initial hydrolysis rate and final sugar yield of a T. reesei enzyme cocktail supplemented with the fusion enzyme were measured on pretreated wheat straw and softwood and compared to the hydrolytic activity of the native mixture. In addition, the degree of synergy was evaluated by comparing the activity of the fusion protein with an equimolar mixture of CBH1 and EG1.




Real time qPCR-based assay for the early detection of aflatoxigenic fungi on maize kernels

Antonella Del Fiore[1] Massimo Reverberi[2] Patrizia De Rossi[3] Valentina Tolaini[2] Corrado Fanelli[2]

1ENEA, Centro Ricerche Casaccia, Roma, Italy, 2Dipartimento di Biologia Vegetale, Università “La Sapienza”, Roma, Italy, 3ENEA, Centro Ricerche Casaccia, Roma, Italy

The aim of this work is to set up a SYBR-green real time qPCR method,  based on the use of specie-specific primers for the early detection and quantification of potential aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus on whole maize kernels. A primer pair was used for amplifying a 352 bp fragment of aflR, gene regulator of the aflatoxin biosynthesis gene cluster. DNA amplification was achieved only with DNA extracted from fungal strains of A. parasiticus and A. flavus and from maize kernels inoculated with A. flavus or A. parasiticus, never with DNA of the other fungal species. Amplification was evident in maize artificially inoculated with A. flavus 3357 starting from 6 hours of incubation after inoculation, when mycelium was not visible by stereomicroscope analysis as yet. This real time qPCR method could be a real, effective method for the early detection and quantification of the most important aflatoxin-producing fungi in food commodities. The method proposed in this work represents a useful tool to evaluate the quality of raw material at different critical points of the food chain. It could be used to predict potential risk for the presence of potentially aflatoxigenic strains. The combination of this approach with the more expensive and laborious conventional chemical analysis of toxin, could be a real, effective alternative respect to traditional diagnostic methods for the early detection and quantification of aflatoxigenic fungi in food commodities.





Applications of Trichoderma harzianum in postharvest fungicidal biocontrol

Jesús Fierro Risco, Rincón, A.M., Codón, A.C., Benítez, T.

Universidad De Sevilla, Departamento de Genética, Facultad de Biología, Avda. Reina Mercedes, s/n, 41012 Sevilla, Spain

Although chemicals are usually used to preserve postharvest fruit quality, the acceptability of these treatments by consumers is becoming increasingly limited. Alternatively, biocontrol of postharvest fruit decay using antagonistic microorganisms has been considered a desirable method. Trichoderma is a filamentous fungus widely used as a biocontrol agent against phytopathogenic fungi. One of the mechanisms employed by Trichoderma during antagonism is the production of cell wall degrading enzymes (CWDEs) and antibiotics (e.g. pyrones). In this study, treatments with conidia of Trichoderma in sterile distilled water were conducted to assess the effect on Penicillium expansum inoculated on apple and pear fruits. Application of T. harzianum CECT 2413 on apples was able to reduce to 60% the number of infected fruits. However, treatment of pears with T. harzianum PF1 –a derived mutant overproducing CWDEs and secondary metabolites- was more effective in disease reduction compared to the treatment with the wild type. According to this, effect of enzymes and antibiotics excreted by Trichoderma was tested. Combinations of supernatants of PF1 growing either in hydrolase-inducing (fungal mycelia) or pyrone-inducing media significantly inhibited the growth in vitro of P. expansum. Moreover, the treatment with these combinations of supernatants drastically reduced the number of apple fruits infected with P. expansum. These results indicated the importance of T. harzianum enzymes and antibiotics in protecting apple fruits from blue mold infection and the suitability of these components in postharvest biocontrol.




Production of meso-galactarate (mucic acid) in fungi

Dominik Mojzita, Marilyn Wiebe, Satu Hilditch, Harry Boer, Merja Penttilä, Peter Richard

VTT - Technical Research Centre of Finland

Mucic acid is a dicarboxylic 6-carbons-containing organic acid, which has applications in food and pharmaceutical industries and can also be used as a platform chemical. Nowadays, mucic acid is produced either by oxidation of D-galactose with nitric acid or by electrolytic oxidation of D-galacturonate. These industrial processes are energy demanding and produce a significant amount of toxic waste. Here we present an alternative route of synthesis of mucic acid by means of biotechnology in Aspergillus niger and Hypocrea jecorina.

In nature, mucic acid is the first intermediate in the D-galacturonate catabolic pathway in some bacteria, such as Agrobacterium or Pseudomonas. The reaction is catalyzed by D-galacturonate dehydrogenase (udh), which is an NAD-dependent enzyme. D-galacturonate is a main component of pectin, abundant and cheap raw material derived from agricultural production. The bacterial udh gene was introduced into the fungal strains with disrupted intrinsic D-galacturonate metabolism in order to direct metabolic flux towards mucic acid. Both modified strains converted D-galacturonate to mucic acid. H. jecorina produced mucic acid in high yield. A. niger regained the ability to grow on D-galacturonate, which implies the existence of yet undiscovered pathway for mucic acid degradation in this organism.





The master regulator of the Unfolded Protein Response revisited

N.D.S.P. Carvalho, T.R. Jørgensen, M. Arentshorst, C.A.M.J.J van den Hondel, A.F.J. Ram

Institute of Biology Leiden

The Unfolded Protein Response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). In Aspergillus niger the beta-zip transcription factor HacA plays a central role in the activation of genes involved in this pathway. Activation of HacA is mediated via an unconventional splicing event of 20 nucleotides in its mRNA. In the present study we engineered an A. niger strain that expresses only the activated form of HacA and we show that the removal of the 20 nucleotides results in a constitutive activation of the well established UPR target genes. The wild type strain (HacAWT) and the strain expressing the constitutive active form of HacA (HacACA) were cultured in glucose-limited batch cultures using bioreactors. Initially, HacA grew with the same specific growth rate (≈0.22 h-1) as the wild type. However, halfway into the growth phase HacA shifted to linear growth. RNA from the batch cultures was isolated for transcriptomic analysis of the effect of expressing a constitutive activated HacA. Statistical analysis defined 1119 genes as differentially expressed (significance p<0.005) relative to the wt. Not surprisingly, genes described as UPR direct targets - pdiA, bipA, clxA, prpA - are found among the most differentially expressed ones. GO enrichment analysis revealed that the expression of several secretion and protein modification related genes was up-regulated in the HacACA mutant. Biological processes overrepresented in the down-regulated genes include several metabolic pathways, and terms related to transcription and translation. A comprehensive overview of the transcriptional response in the HacACA mutant will be presented.





Genome mining of secondary metabolites in Aspergillus

Clay Wang
University of Southern California

Aspergillus species are known to produce high value secondary metabolites such as lovastain. Recently the genomes of several Aspergillus species have been sequenced. One of the most important findings from these genome efforts is the realization that these organisms have the potential to produce far more secondary metabolites than have ever been isolated and identified. I will present our recent efforts in identifying new secondary metabolites and their corresponding biosynthesis pathways from several different Aspergillus species. Finally we will present approaches to use natural products we have isolated from Aspergillus as starting point for further drug discovery.





Characterization of the Aspergillus nidulans biotin biosynthesis gene cluster: the bifunctional biDA gene as a new transformation marker in Aspergilli

Michel Flipphi[3] Pasqualina Magliano[1] Dominique Sanglard[2] Yves Poirier[1]

1Department of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland
2Department of Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland

3Co nsejo Superior de Investigaciones Cientificas (CSIC)

Biotin (vitamin H) is an essential cofactor for some (trans)carboxylation reactions in carbon metabolism. It also plays roles in cell signaling, epigenetic regulation, and chromatin structure. In fungi, biotin biosynthesis genes are clustered. In Aspergilli, the central gene we termed biDA, encodes a protein that bares similarity to both Escherichia coli dethiobiotin synthetase (BioD) and 7,8-diaminopelargonic acid (DAPA) aminotransferase (BioA). The divergently transcribed biF gene codes for a protein similar to 7-keto-8-aminopelargonic acid synthase (BioF) while the biB gene, situated downstream of biDA, is similar to biotin synthase (BioB).  E. coli mutants deleted for either of the structural genes bioF, bioA, bioD or bioB, could be complemented by the expression of A. nidulans biF, biDA or biB cDNAs. This confirmed that while seperate genes encode DAPA aminotransferase and dethiobiotin synthetase in bacteria and yeast, both these activities are performed by a single, bifunctional protein in A. nidulans. Three classical A. nidulans (biA1-3) biotin-auxotroph mutants were found to have distinct mutations in the part of the biDA gene that specifies the DAPA aminotransferase domain. Such mutants could be complemented by transformation with the functional biDA gene from either A. nidulans or Aspergillus fumigatus. Approximately ten biotin autotroph colonies per 106 protoplasts per microgram of plasmid, were routinely obtained. Co-transformation of biDA with a plasmid carrying sGFP under A. nidulans transcriptional control yielded co-transformants stably expressing sGFP over several generations. These results showed that biDA orthologs can serve as the basis for a robust and convenient transformation system in Aspergilli.




Transposon Vader: From a mobile element towards a molecular tool

Frank Kempken, ElkBir Hihlal

Botanisches Institut, Christian-Albrechts-Universität, Olshausenstraße 40, 24098 Kiel, Germany

Transposons are mobile DNA sequences, which are found in all eukaryotic genomes (1-3). We have analyzed the transposon content in two fungal genomes, Aspergillus niger and Penicillium chryosogenum. One non-autonomous element, the A. niger transposon Vader, was shown to be active during strain development (4). Vader mobility could also be shown in a transposon trap experiment. Due to its obvious activity and to its ability to insert into genes Vader appears to be suitable as a gene tagging tool. A vector was constructed which carries a Vader element between the promoter and open reading frame of the hygromycin resistance gene. The Vader element was modified by adding a unique oligonucleotide binding sequence. Conidiospores were plated on hygromycin B containing media which allows for selection of excision of transposon Vader. Reintegration sites were determined using TAIL-PCR. Vader transposition shows a high insertion rate into genes which appears very promising. At current we analyse a large number of integration sites to determine any target site preferences. In addition we will introduce Vader into A. nidulans and other heterologous hosts.


  1. Kempken F, Kück U (1998) BioEssays 20:652-659
  2. Kempken F (2003) In: Arora DK, Khachatourians GG (eds) Applied Mycology and Biotechnology, Vol. 3 Fungal Genomics, Elsevier Science Annual Review Series, pp83-99
  3. Pöggeler S, Kempken F (2004) In: Kück U (ed) The Mycota II, Genetics and Biotechnology, 2nd edition, Springer Verlag, Heidelberg, New York, Tokyo, pp165-198
  4. Braumann I, van den Berg M, Kempken F (2007) Fungal Genet Biol 44:1399-1414





Array comparative genomic hybridization analysis of Trichoderma reesei high cellulase producing strains

Marika Vitikainen, Mikko Arvas, Tiina Pakula, Merja Oja, Merja Penttilä, Markku Saloheimo

VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland

Trichoderma reesei is the main industrial producer of cellulases and hemicellulases used to depolymerize biomass in many biotechnical applications. Many production strains in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in hyperproducing mutants of T. reesei by high-resolution comparative genomic hybridisation tiling array (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms that underlie the efficient cellulase production and enable targeted genetic engineering for improved production of proteins in general. Results: We carried out aCGH analysis of four hyperproducing strains (QM9123, QM9414, NG14 and RutC-30) using QM6a genome as a reference. In QM9123 and QM9414 we detected altogether 44 previously undocumented mutation sites including deletions, chromosomal breakpoints and single nucleotide mutations. In NG14 and RutC-30 we detected 126 mutation sites of which 17 were new mutation sites not documented previously. Among these new sites there are first chromosomal breakpoints found in NG14 and RutC-30. We studied the effect of two deletions identified in RutC-30 (a deletion of 85 Kb in the scaffold 15 and a deletion in the gene 72076 encoding a fungal transcription factor) on cellulase production by constructing knock-out constructs in the QM6a background. Neither of the deletions affected cellulase production.  Conclusions: ArrayCGH analysis identified dozens of mutation sites in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a useful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially if large set of interesting strains is to be analyzed.


Comparative analysis of koji mold's genomes

Masayuki Machida[1] Hideaki Koike[1] Morimi Teruya[2] Masatoshi Tsukahara[3] Yumi Imada[3] Youji Wachi[4] Yuki Satou[4] Yukino Miwa[3] Shuichi Yano[3] Yutaka Kawarabayasi[1] Osamu Yamada[5] Koji Jinno[6] Akira Hosoyama[6] Takasumi Hattori[7] Motoaki Sano[8] Koichi Tamano[9] Kazurou Fukuda[10] Takaomi Yasuhara[10] Kenichi Higa[11] Shinichi Ohashi[5] Kotaro Kirimura[12] Masanori Arita[13] Kiyoshi Asai[13] Keietsu Abe[14] Katsuya Gomi[14] Shigeaki Mikami[5] Takeaki Ishikawa[15] Kaoru Nakasone[16] Nobuyuki Fujita[6] Takashi Hirano[17] Ikuya Kikuzato[4] Kazuhiro E. Fujimori[17] Hiroshi Horikawa[6]

1Natl. Inst. Advanced Inst. Sci. Technol, Okinawa Cutting-edge Genome Project, 2Okinawa Iind. Technol. Center, Okinawa Cutting-edge Genome Project, 3Tropical Technol. Center, Okinawa Cutting-edge Genome Project, 4Okinawa Sci. Technol. Promotion Center, Okinawa Cutting-edge Genome Project, 5Natl. Res. Inst. Brewing, 6Natl. Inst. Technol. Eval., 7Waseda U., 8Kanazawa Inst. Technol., 9Natl. Inst. Advanced Inst. Sci. Technol., 10Asahi Breweries, 11Okinawa Iind. Technol. Center, 12 Tropical Technol. Center, 13U. Tokyo, 14Tohoku U., 15Brewing Soc. Japan, 16Kinki U., 17Natl. Inst. Advanced Inst. Sci. Technol., Okinawa Cutting-edge Genome Project

Koji mold is the traditional name of Aspergillus species that are used for Japanese fermentation industries. Aspergillus oryzae has been widely used in Japanese fermentation industries, Japanese alcohol beverage, soy sauce and so on for longer than a thousand of years. Comparison of A. oryzae genome with those of other
Aspergillus species of smaller genome size revealed existence of non-syntenic blocks (NSBs) specific to the A. oryzae genome. Aspergillus awamori is another industrial filamentous fungus, widely used for brewing Japanese traditional spirits, Awamori, in Okinawa prefecture. We have sequenced A. awamori NBRC 4314 (RIB2604). The total length of non-redundant sequences reached 34.7 Mb consisting of contigs fallen into 44 major linkage groups. High potential of secretory production of proteins has led A. Oryzae and A. awamori to extensive use also in modern biotechnology.  A. awamori is genetically very close to Aspergillus niger and close to A. oryzae. Like A. niger, A. Awamori vigorously produces citric acid, lowering pH of the product. A. awamori is genetically very close to Aspergillus niger. However, mapping of short reads from A. awamori by SOLiD revealed that the species have remarkable difference. Comparison between the genomes of A. awamori and A. oryzae showed higher diversity of genes located on the non-syntenic blocks of the A. oryzae genome. We are currently sequencing various strains of A. awamori by SOLiD. Analysis of the relationship between genetical and phenotypical differences among the strains should provide important information for gene function.




Single-use bioreactors for the heterologous production of a peroxidase by Aspergillus niger. An alternative for stirred tank reactors?

Nick van Biezen[1] Jeroen E. Schouwenberg[1] Nico M.G. Oosterhuis[2] Anton Tromper[2] Christien Lokman[1]

1HAN BioCentre, Nijmegen, The Netherlands, 2CELLution Biotech BV, Assen, The Netherlands

The application of single-use equipment becomes more and more common in the biopharmaceutical industry.  Disposable filters, bags for medium and product storage are more and more used and the application of single-use process equipment becomes more common nowadays. Compared to the traditional glass or stainless steel stirred tanks, the single-use bioreactor offers clear advantages: a shorter turnaround time (no SIP or CIP required), resulting in a cost reduction; minimal utilities required; greatly reduced potential of cross contamination (safety); greater operational flexibility; reduced validation requirements.

Due to its two-dimensional movement profile, the CELL-tainer® single-use bioreactor creates a superior gas-liquid mass-transfer compared to other single-use bioreactors presently available. This offers the opportunity to reach higher cell-densities and therefore higher volumetric productivities. With the CELL-tainer kla values of 300 h-1 and above are measured, making the system suitable for high density mammalian cell cultures, but especially also for microbial, yeast, and mycelia cultures. Measurement of the heat exchange capacity (cooling capacity) indicates that accurate temperature control of such exothermic type of cultures is possible as well.

A comparison is made between the CELL-tainer® and the standard stirred bioreactor during the heterologous production of a peroxidase in Aspergillus niger under the control of the inuE expression signals.





Heterologous protein production and purification of a His-tagged peroxidase in Aspergillus niger

Christien Lokman, Dennis Lamers, Nick van Biezen, Andrea Thiele, Theo Goosen, Kees van den Hondel

HAN BioCentre, Nijmegen, The Netherlands

Filamentous fungi have a very efficient protein-production capacity which make them suitable host organisms for the overproduction of commercially interesting homologous and heterologous proteins. The overall efficiency of an enzyme production process is influenced by the production yield (fermentation) and purification yield (Down Stream Processing). Unfortunately, since every protein is different, in many cases production and purification protocols and strategies must be developed for each individual protein. In E. coli the fusion of proteins to oligohistidine tags followed by affinity chromatography is a very common protein purification strategy. As far as we know, the use of His-tags in the extracellular production and purification of heterologous proteins in Aspergillus has not yet been demonstrated.

Recently, we successfully produced Arthromyces ramososus peroxidase (ARP) in Aspergillus niger under control of the inuE (exo-inulinase) expression signals. To allow fast and easy purification we introduced N- and C-terminal His6-tags, respectively. Extracellular peroxidase activity could be measured and was obtained with both, N- and C-terminal His6-tagged ARP. The ability to purify the different His6-tagged proteins by affinity chromatography is under investigation.




Transcriptome and proteome analysis of an antibody Fab fragment producing Trichoderma reesei strain and its parental strain at different cultivation temperatures

Tiina Pakula, Lise Bernard-Granger, Jaana Toikkanen, Merja Oja, Marilyn Wiebe, Markku Saloheimo, Merja Penttilä

VTT Technical Research Centre, P.O.Box 1000 (Tietotie 2, Espoo), Fin-02044 VTT, Finland

The work is part of a EUROSCOPE programme project Genophys, in which the aim is to compare the physiology of heterologous protein production in different microbial production hosts using a common model protein and applying genome-wide analysis methods to study the cellular events at a selected set of cultivation conditions.
We have carried out transcriptome and proteome analysis of a
Trichoderma reesei strain producing antibody 3H6 Fab-fragment and a control strain expressing only the selection marker gene amdS, and analysed the effects of production of the hetereologous protein as well as cultivation temperature on the cellular responses.  For the analyses, the strains were cultivated in lactose-limited chemostats at 21.5°C, 24°C and 28°C. The dilution rate used in the cultivations, 0.03/h, has been previously shown to be optimal for protein production in similar type of chemostat cultures of T. reesei. The lower temperatures used in the study, favoured protein production by both strains. In the Fab producing strain, both the total protein production into the culture medium as well as Fab production were the highest at 21.5°C, the values obtained at 24°C being close to the ones obtained at 21.5°C. In the control strain the highest protein production was at 24°C. Comparison of differentially expressed genes between the strains showed relatively few genes affected by the heterologous gene expression, whereas comparison of the cultures at different temperatures revealed a large number of genes with altered expression level.  However, the proteome analysis of the cultures using the 2D DIGE method indicated also stress responses to the heterologous protein production. The analysis showed differences especially in the intensity of protein spots corresponding to different type of heat shock proteins (ER, mitochondrial, cytoplasmic, ribosome-associated HSPs), actin and tubulin assembly factors, and proteins related to ER-associated protein degradation.




Transient disruption of the kusA gene in a citric acid producing strain of Aspergillus niger facilitates high throughput gene deletion

Kenneth Bruno, Kristen M. Meyer, Scott E. Baker

Pacific Northwest National Lab. Richland, WA, 99352. USA

Mutation of the kusA gene in several fungi, including Aspergillus niger, has proven to greatly increase the rate of homologous integration. The kusA gene is required for non homologous end joining and is required  for repair of double strand breaks in genomic DNA.  A transient kusA- mutant was created in A. niger strain ATCC 11414 by inserting pyrG, a counter-selectable marker, at the kusA locus. Screening of targeted gene deletion transformants in this background reveals the rate of homologous recombination was significantly higher (95%) than in the non mutant parent (20%).  Restoration of KusA activity is important for maintaining strain stability in A. niger, an ascomycete with no established sexual crossing procedures. Spores from the wildtype, kusA, and reverted kusA+ strains were exposed to gamma radiation ranging from 50 to 400 Gy. Our analysis confirms that the ability to recover from radiation-induced mutagenesis was restored in the kusA revertant strains.  High throughput genesis of gene deletion constructs can be achieved by utilizing yeast GAP repair and has been used to great effect in other large scale genomic analyses.  We have adapted these tools to A. niger. Large numbers of gene deletions are underway in order to analyze the metabolic and morphological changes associated with highly efficient citric acid production.  This capability will rapidly advance the analysis of organic acid production and morphology in this organism.



Functional characterization of the CPCR1 ortholog (regulatory factor PcRFX) in Penicillium chrysogenum

Rebeca Domínguez-Santos1[1] Carlos García-Estrada[2] Mohammad-Saeid Jami[1] Ricardo V. Ullán[2] Fernando Teijeira[2] Marta Fernández-Aguado[3] Juan-Francisco Martín[4]

1Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain
2INBIOTEC, Instituto de Biotecnología de León, Avda. Real nº. 1, Parque Científico de León, 24006 León, Spain
3Area de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain
4Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain INBIOTEC, Instituto de Biotecnología de León, Avda.
Real nº. 1, Parque Cient&

Beta-lactam biosynthesis is subjected to complex regulatory processes. One of the transcriptional factors recently identified in Acremonium chrysogenum is CPCR1, which acts as a regulator of the cephalosporin C biosynthesis and shows a high similarity to transcriptional factors from the human family of “winged helix” regulatory X factors (RFX).

The CPCR1 ortholog was characterized in Penicillium chrysogenum. The gene encoding this transcription factor (Pcrfx) was identified in the P. chrysogenum genome (Pc20g01690). The protein encoded by this gene (PcRFX) shared a 47% homology and 30% identity with the A. chrysogenum CPCR1 factor. Analysis of the promoter region of the penicillin biosynthetic genes (pcbAB, pcbC and penDE), revealed the presence of one binding site (X-box) for this factor in each promoter region. Gene silencing of Pcrfx gave rise to a decrease in the production of isopenicillin N and penicillin G in the knock-down mutant after 48 and 72 h of culture. In this mutant, the steady-state levels of the penicillin biosynthetic genes transcripts were diminished, thus confirming the regulatory role of PcRFX in penicillin biosynthesis.



Efficient gene targeting in Aspergillus niger using a transiently disrupted ku70 gene

M. Arentshorst, A.F.J. Ram and V. Meyer

Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands.


Homologous recombination frequencies in filamentous fungi, including Aspergillus niger, are low. Several reports over the last years have shown that mutants defective in the Non-Homologous-End Joining (NHEJ) pathway display increased homologous integration efficiencies up to 80 to 100%. Recently, we described that deletion of the A. niger kusA gene, encoding the orthologue of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency. However, deletion of kusA also causes increased sensitivity of A. niger towards UV and X-ray and the consequences of loss of kusA in relation to DNA repair and genome stability are currently unknown.

To avoid any potential side effects of a kusA loss-of-function mutation on growth and viability of A. niger, we transiently disrupted kusA. We made use of the counter selectable amdS marker, flanked by 300 bp direct repeats of the kusA gene. Disruption of the kusA gene resulted in similar homologous recombination frequencies compared to the ΔkusA strain. After completion of the gene targeting approach, we re-established an intact kusA copy using counter selection on fluoroacetamide, proven by sequencing of the kusA locus. In A. niger, which lacks an sexual reproduction cycle, the transient disruption system is especially important as the NHEJ pathway cannot be restored by performing a sexual cross. 





Improved tools for fungal strain development

Margreet Heerikhuisen, Cora van Zeijl, Peter Jan Punt

TNO Quality of Life, Dept. Microbiology, P.O. Box 360, 3700 AJ Zeist, the Netherlands


Already from the very start of the molecular era for filamentous fungi, TNO has been involved in developing molecular tools for gene expression and protein secretion for filamentous fungi. In this poster we present a number of improved tools for fungal strain development, like novel promoters and versatile selection markers.

From the development of fungal gene transfer systems, the isolation of efficient expression signals is one of the research lines to obtain efficient protein production. As a result of gene expression studies we have discovered a new and very strong promoter from A. niger. Furthermore, we have optimized the existing very strong A. niger gpdA promoter by modification of its promoter sequence.


Versatile genetic modification of filamentous fungi requires the possibility to modify, disrupt and express a number of different genes in a single fungal strain. Therefore, the availability of a (series) of different selection markers is essential. However, the possibility of repeated use of the same marker in subsequent experiments can circumvent the requirement for multiple selection markers. The usage of counter selectable markers like pyrG and amdS is very suitable for this purpose, because these allow selection of the mutant and the transformant. Also a synthetic amdS selection marker was constructed which is devoid of most restriction sites that are present in the native amdS gene.




Single cell analysis as a way to study hyphal heterogeneity

Charissa de Bekker, Arman Vinck, Han A.B. Wösten

Microbiology and Kluyver Centre for Genomics of Industrial Fermentations, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands


Mycelial fungi use the hyphae growing at their apices to colonize a substrate. These hyphae secrete enzymes that convert complex polymers into small molecules that can be taken up by the fungus to serve as nutrients. Using GFP as a reporter it has been shown that exploring hyphae of Aspergillus niger are heterogenic with respect to enzyme secretion; some hyphae strongly express the glucoamylase gene glaA, while others express this gene lowly. This was a surprising finding considering the fact that all hyphae were exposed to the same nutritional conditions. Apparently, a vegetative mycelium is more complex than generally assumed.               

We here demonstrate that the expression of other genes encoding secretion enzymes in A. niger is also heterogenic. Co-expression studies, using GFP and dTomato as reporters, showed that hyphae that highly express one of these genes also highly express other genes encoding secreted proteins

To unravel the mechanisms underlying heterogeneity we aim to perform a genome-wide expression analysis of highly and lowly expressing hyphae. To this end, protocols have been set up to collect individual hyphae using laser micro-dissection and pressure catapulting (LMPC) and to isolate RNA from these samples. QPCR has shown that we are able to extract RNA from a single hypha only. 


This research is supported by the Dutch Technology Foundation STW, applied science division of NWO and the Technology Program of the Ministry of Economic Affairs.




Oligosaccharide transport by Neurospora crassa during cellulolytic growth

Jonathan M. Galazka1, Chaoguang Tian2, William T. Beeson1, N. Louise Glass1 and Jamie H. D. Cate1

1University of California Berkeley  2Tianjin Institute of Industrial Biotechnology


The filamentous fungus, Neurospora crassa, is capable of depolymerizing and metabolizing plant cell walls, but this process is not understood in great detail.  Our group recently reported a systematic study of plant cell wall degradation by N. crassa, utilizing transcriptomics, secretome analysis, and the phenotypic characterization of gene knockouts (1).  Whole genome microarrays identified 114 genes that show overlapping expression differences when N. crassa is grown on either ground Miscanthus stems, or pure cellulose as carbon sources.  Within this set were 10 predicted major facilitator superfamily transporters.  We hypothesized that at least one of these proteins may transport oligosaccharides, an activity that has been reported in H. jecorina (2), but never attributed to a particular gene.  Mutations in three of these transporters significantly affected growth of N. crassa on crystalline cellulose.  We developed a method by which to rapidly assay the substrates of these transporters, and report the identity of three oligosaccharide transporters in N. crassa.  We believe these transporters may be useful in downstream processing of depolymerized plant cell walls.


1. Tian & Beeson et al. (2009) PNAS 2009 106: 22157-22162.

2. Kubicek et al. (1993) J. Biol. Chem. 268: 19364-19368.





Genome-wide analysis of the transportome of the industrial filamentous fungal strain Penicillium chrysogenum

Stefan Weber1 , Jeroen G. Nijland1, Andriy Kovalchuk1, Marco van den Berg2, Roel Bovenberg2 and Arnold J.M.  Driessen1

1University of Groningen, Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and the Kluyver Centre for Genomics of Industrial Fermentation, Kerklaan 30, 9751 NN Haren, The Netherlands, and

2DSM anti-infectives, PO Box 425, 2600 AK Delft, The Netherlands


Industrial production of β-lactam antibiotics using the filamentous fungus Penicillium chrysogenum is based on successive microbial strain improvement cycles. Analysis of these strains has led to the identification of several important mutations in high-producing strains. These are amongst others the amplification of the penicillin biosynthesis genes, the elevated transcription of genes involved in biosynthesis of the amino acid precursors, and genes encoding microbody proteins. However, many of the key (intra)cellular transport processes have remained obscure.


To identify ABC-transporters involved in β-lactam production, P. chrysogenum was grown in the presence and the absence of phenylacetic acid (PAA), a side chain precursor of penicillin G. Expression of all ABC transporters was determined by Quantitative Real Time PCR. This revealed a significantly increased expression level of ABC40 when grown with PAA. In another experiment, expression of all ABC transporters was determined upon a penicillin G challenge, resulting in elevated levels of the expression of ABC30, ABC42 & ABC48. Independent knock-outs strains of ABC30, ABC40, ABC42 and ABC48 in a high producing strain were constructed, but none of these strains showed a detectable phenotype. In another approach, the same genes were inactivated in an early (low) production strain. The further analysis of these strains is in progress.




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