A Synthetic Expression System for protein production in Trichoderma reesei

Trichoderma reesei is a filamentous fungus that is used world-wide as a host for industrial enzyme production. The enzymes produced are used, for example, in pulp and paper production, in the food and feed industries, and in the textile industry. T. reesei enzymes are also increasingly important because of their ability to turn lignocellulosic biomass into sugars that can be used to produce biofuels and chemicals. The strongest promoters used in this system typically require the use of inducing molecules, which create limitations and generate extra cost in the process. To simplify and improve the production process we have employed a novel orthogonal expression system based upon a synthetic transcription factor (sTF) that regulates expression of the target gene via a sTF-dependent promoter. The great benefit is that new system allows for high level, constitutive expression in diverse growth conditions and growth stages. Multiple sTF-binding sites can be designed to enable selection of a wide range of target gene expression levels, from very low to extremely high. We have taken advantage of this unique expression system to improve native and heterologous protein production in the industrial microbe T. reesei. We have been able to produce high levels of secreted proteins without the use of inducing molecules, which are typically used for this production organism. For instance, with T. reesei we could produce the native CBH1 cellulase, Candida antarctica CALB lipase, and bovine beta-lactoglobulin at high levels and high purity when grown in glucose medium. The new universal expression system allows for simpler media and bioprocesses to be used for industrial protein production.