Current Projects
The saprotrophic fungus Trichoderma reesei is used for industry scale production of carbohydrate-active enzymes, which themselves are applied in many industrial processes. Unpleasantly, hyper-productive T. reesei strains sometimes spontaneously lose productivity during enzyme production fermentation. Such a spontaneous degeneration of microbial production strains is a widely observed phenomenon and poses a severe threat to the respective biotechnological process. To date strain degeneration cannot be prevented and is only poorly understood. Based on results obtained by the two applicants (TU Wien und Novozymes) this undesired phenomenon is due to cis epigenetic alterations going along with chromatin condensation as a result of changes in histone modifications. There is strong evidence that strain degeneration is a consequence of specific DNA methylation affecting targets upstream of the main transactivator of enzyme production. The alterations in physiology and gene expression that are implicated by the emergence of the degenerated strains are finally associated with proteins that mediate shifts in the epigenetic landscape of these strains. It is envisioned to identify these mediators and their targets followed by knowledge-based engineering and modification. This will lead to synthetic biology-based strategies to obtain stable production strains.
In addition to classical gene regulation by transcription factors, different non coding (nc) RNAs are nowadays seen as key regulatory elements in cells. Although there are good indications for the existence of ncRNAs in lower eukaryotes like industrially applied fungi, hardly any basic research or related applications have been reported up to now. The university partner has recently found experimental evidence for a long ncRNA (HAX1) functioning as an activator of enzyme production in T. reesei and that this effect was continuously reinforced during the random mutagenesis-based screening processes for industry strains. This evolution of HAX1 concerns an increase in length leading to structural changes. However, point mutations causing changes in secondary structure and/or HAX1 stability and changes in its expression have to be considered and investigated as well. Based on this, it is planed to develop optimized synthetic HAX1 versions derived by fusion of regions of differently evolved HAX1 versions or based on serial base permutation analysis. Finally, it is envisioned to design a set of HAX1 expression cassettes that can be directly introduced in any user-defined stain in order to improve its enzyme production capacity by a single-step genomic manipulation.
Information at Christian Doppler Gesellschaft, opens an external URL in a new window
Contact:
Senior Scientist Dipl.-Ing. Dr.techn. Astrid Mach-Aigner, opens an external URL in a new window
Fungi are responsible for the production of many natural products as a result of a wide variety of species and use of various biosynthetic pathways. Numerous of these products have been identified during the last decades and are nowadays widely exploited by the biotech and pharmaceutical industry. Although it is obvious that a plethora of these compounds still await discovery. Two main challenges limit the access to new compounds: i) the inability to cultivate diverse producers of natural products in the laboratory and, ii) the fact that a majority of secondary metabolite biosynthesis related genes and gene clusters are silent under standard laboratory conditions. To overcome latter obstacle several strategies including the manipulation of pleiotropic regulators of secondary metabolism are currently followed.
Hence, the applicants and their teams have recently isolated and preliminarily characterized a transcription factor functioning as a pleiotropic regulator of secondary metabolite biosynthesis. First results demonstrate its striking involvement in secondary metabolite production in the filamentous fungus Trichoderma reesei and its occurrence in other biotechnologically and agriculturally relevant fungal species. Based on these results the following investigations are proposed: i) Elucidating the regulon of this trans acting factor in T. reesei; ii) Analyze the regulatory function of it in the bio-control active fungus T. atroviride the plant pathogenic fungus Fusarium graminearum and F. fujikuroi and the pharmacologically exploited fungus Claviceps purpurea, iii) Investigate the molecular mechanisms how this factor exerts it regulatory function in T. reesei.
Funding:
FWF, project number P29556, opens an external URL in a new window
Contact:
Univ. Prof. Dr. Robert Mach, opens an external URL in a new window
Sekhmet’s gift is a novel methodology for the cost-efficient and sustainable production of drugs. For this purpose unused plant biomass is the favourable substrate, a fungus well established in industrial processed is the main tool, and the complete exploitation of all waste streams is envisioned.
Contact:
Senior Scientist Dipl.-Ing. Dr.techn. Astrid Mach-Aigner, opens an external URL in a new window
Second generation biofuel (“lignocellulosic bioethanol”) is a promising alternative to classic, fossil-based energy sources, and in contrast to first generation biofuel, it does not compete with human food or animal feed sources and does not demand for additional agricultural area. This is due to the fact that the raw material for its production is lignocellulosic biowaste (LCW).
Lignocellulose is the most abundant renewable resource on earth; this potentially cheap polymer is found as agricultural waste (wheat straw, corn stalks, soybean residues, sugar cane bagasse), industrial waste (pulp and paper industry), forestry residues, municipal solid waste, etc. It has been estimated that lignocellulose accounts for about 50% of the biomass in the world (10 – 50 billion tons). This is more than enough to cover the demand for the co-production of chemicals, materials, and fuel next to the demand for foods and feeds.
A commercial biofuel/biorefinery process implies several steps: i) the physical and/or chemical pre-treatment of LCW (for abscission of lignin and increase of water accessibility), ii) the enzymatic hydrolysis of LCW (for degradation of cellulose and xylan), and iii) the fermentation of the resulting monosacharides to ethanol.
The amount of hydrolytic enzymes required for the efficient lignocellulose hydrolysis is very high and obviously a crucial factor in the overall biofuel/biorefinery production costs. One of the most prominent enzyme producers applied on the industrial level for this purpose is the filamentous fungus Trichoderma reesei. To date, the common production approach requires the addition of expensive, inducing carbohydrates to the T. reesei fermentation medium in order to activate enzyme production. These inducers have to be produced in an additional process that significantly increases costs. Furthermore, glucose and high concentrations of D-xylose, the predominant end products of the lignocellulose hydrolysis, act as repressors of enzyme production, even when Cre1-mediated carbon catabolite repression is eliminated. To overcome these drawbacks and bottlenecks the applicant proposes a synthetic biology approach, i.e. the construction of chemically inducible systems based on the tansactivator Xyr1 from T. reesei, the human estrogen receptor, and the LexA operator from Escherichia coli. Obtained strains bearing the synthetic transactivator and a synthetic signalling cascades will lead to: i) the usage of an efficient and cheap inducer molecule that is readily commercially available, ii) thereby making the additional inducer production obsolete, iii) the complete release from glucose or high D-xylose repression of LCW-hydrolyzing enzyme expression, iv) the usage of alternative, low-cost carbon sources (i.e. lignocellulose hydrolysates comprising glucose, D-xylose, and L-arabinose) in LCW-hydrolyzing enzyme production.
Funding:
FWF, project number P26733, opens an external URL in a new window
Contact:
Senior Scientist Dipl.-Ing. Dr.techn. Astrid Mach-Aigner, opens an external URL in a new window
Recently, the spectrum of classical functions of so-called non-coding RNAs (ncRNAs) has been extended to additional aspects in cell biology. This is in particular due to intense investigations on this topic in the field of molecular biology applied in human medical research. In addition to classical gene regulation by transcription factors, a number of different ncRNA groups are nowadays seen as key regulatory factors in cells, drawing a bow from their involvement in embryonic development to tumor genesis. Although there are good indications for their existence in eukaryotes like filamentous fungi, only negligible research is carried out in biotechnological relevant microorganisms and no corresponding applications for e.g. industrially used fungi have been reported up to now.
In this project the long ncRNA HAX1, that is the hypothetical activator of Xyr1 (Xylanase regulator 1), which was recently isolated and preliminarily characterized by the team of the project initiator, is the main objective of research. Its structure, its molecular mechanistic mode of action, and its regulatory impact on gene expression will be determined. In particular, the involvement of HAX1 in plant cell wall degrading enzyme production in Trichoderma reesei will be investigated. Obtained results are expected to provide novel insights in fundamental regulatory circuits of gene expression in lower eukaryotes on the one hand and to reveal a new manipulation level of for targeted strain improvement of industrially important filamentous fungi on the other hand.
Funding:
FWF, project number P26618, opens an external URL in a new window
Contact:
Ao.Univ.Prof. Mag. Dr.rer.nat. Robert Mach, opens an external URL in a new window
Trichoderma reesei is widely used in industry as a source of cellulases and hemicellulases for the hydrolysis of plant cell wall polysaccharides. Today, these enzymes are employed for the production of renewable biofuels and other fine chemicals from plant biomass. These second generation biofuels derived from agricultural crop residues, grasses, wood and municipal solid waste would have important advantages over first generation biofuels produced from food crops as feedstock since they do not directly or indirectly compete with food production. Lignocellulosic biomass is however notoriously difficult to convert into fermentable sugars and one of the major obstacles that must be overcome are the high costs for hydrolyzing the insoluble and crystalline cellulose by cellulases. To make biofuel production cost-effective a number of technological breakthroughs in the area of enzymes, pre-treatment and fermentation are needed. New studies to understand and improve cellulase efficiency and productivity are therefore at the forefront of biomass research. Academic and industrial research programs have over the past decades produced different T. reesei strains by random mutagenesis whose production of cellulases exceeds 100 grams per liter. In contrasts, reports on successful strain improvement by direct targeted genetic engineering are rare and cellulase yields of these strains cannot compete with the producer strains generated by classical mutagenesis. Molecular manipulations would, however, make strain development more straightforward and would also eliminate undesired deleterious mutations which accumulate during random mutagenesis programs. With the development of genome-wide tools including next-generation DNA sequencing whole genome comparisons of improved producer strains to their ancestors are now feasible. However, these genome comparisons alone are not sufficient to interpret the observed genomic changes and further transcriptomic and phenotypic information are required to interpret the changes in the genomes and transcriptomes that accompanied the improvement of cellulase production. The obtained information will in turn be used to create more efficient cellulase producing strains through targeted molecular genetic manipulation rather than through random mutagenesis that leads to collateral and deleterious genomic damages.
Funding:
FWF, project number P24219, opens an external URL in a new window
Contact:
Piv. Doz. Dr. Bernhard Seiboth, opens an external URL in a new window
Trichoderma reesei is an important producer of industrial applied enzymes. Conventional strain improvement by mutagenesis and selection has led to strains that secrete more than 100 g/l of homologous proteins. The reasons for this protein hyperproduction are largely unknown and production levels of heterologous proteins are usually much lower. In addition these highly mutated strains have accumulated numerous negative mutations affecting their overall fitness. To design new recombinant strains which exceed the current production levels for proteins and show a better overall performance, different industrially relevant strain lines will be compared to their ancestors by state of the art systems biological approaches including genomics, transcriptomics and proteomics.
In combination with advanced genetic engineering techniques we will design yield improved strains for efficient protein production.
Partner:
THE AUSTRIAN CENTRE OF INDUSTRIAL BIOTECHNOLOGY, opens an external URL in a new window
Contact:
Piv. Doz. Dr. Bernhard Seiboth, opens an external URL in a new window
This project is a Community Science Program (CSP) sponsored by the Department of Energy Joint Genome Institute at Joint Genome Institute. The project compares the transcriptional and enzymatic response of the three different fungi Podospora anserina, Aspergillus niger and T. reesei to the presence of various plant feedstocks to provide insights in the diversity of plant biomass degradation strategies.
Partner:
Adrian Tsang (Concordia University)
Contact:
Priv. Doz. Dr. Bernhard Seiboth, opens an external URL in a new window