A synthetic expression platform for the T. reesei Xyr1 regulon (SYNEX)

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.

During the course of the research project SYNEX a novel biotechnological method for the sustainable production of proteins was successfully developed. The microorganism that was used for this purpose is Trichoderma reesei, which is a saprotrophic fungus. This means that in nature, this fungus degrades plant biomass, which mainly consists of lignocellulose. In this way, the fungus secures its own growth and covers its demand for energy. Lignocellulose is the world`s most abundant renewable resource and is a main side-product in agriculture (e.g. grain and corn straw, residues of soy beans), industry (e.g. paper industry), forestry and wood processing, and it is occurring in municipal waste. Consequently, it is a cheap, renewable, and sustainable resource that should not be unused considering the growing demand of mankind for energy, resources and goods. Hence, the above-mentioned fungus is an ideal candidate to be used in biotechnological production processes. During this project, some artificial genetic switches were introduced in this fungus, which enables the fungus to produce a certain protein of interest in high amounts, regardless on which nutrient it grows. This production can be turned on by the addition of tiny amounts of the human oestrogen hormone. The spectrum of proteins that can be produced range from enzymes, which themselves contribute to more ecological production processes (e.g. by reducing or even replacing chemicals), to proteins applied for medical purposes.