Enzyme production by T. reesei cultivated on lignocellulose

Cellulose is found in the plant cell walls. It is one of the most abundant sources of carbohydrates on earth, and has therefore great potential as raw material for the production of biofuels and chemicals. By using enzymes, the cellulose can be broken down to glucose and subsequently converted to a broad range of products. However, the commercial potential is limited by technical challenges, which are founded on the structure of the cellulose. The cellulose is organized into a complex structure that is resistant to degradation. Based on this rigid structure, a multitude of different enzymes is required to break down the cellulose effectively to glucose. Each type of enzyme thereby acts on a specific part of the cellulose. The resulting intertwined relationship between the cellulose structure and the various enzymes is not yet fully understood. This knowledge gap prevents optimization of enzyme cocktails, resulting in slow and incomplete break down of cellulose and a need for high amounts of enzymes. Neither is acceptable for economical production of bulk products from cellulose. The fungus Trichoderma reesei is able to produce all enzymes required to effectively break down cellulose in cellulosic materials. This enables it to use cellulose as a substrate to grow on. During growth, the fungus interacts closely with the substrate. It can adjust the composition of the produced enzymes and its form and shape (morphology) to the substrate. This allows it to break down a variety of cellulosic materials effectively. However, the underlying mechanisms of the fungus substrate sensing remain to be elucidated. This knowledge, however, could render production of optimized enzyme cocktails possible. The aim of the project is to analyze the relationship between the produced enzyme cocktail, the substrate structure, and the fungal morphology, when the fungus is grown on various different cellulosic materials. In dependence of the cultivation time and the utilized substrate the following will be analyzed: the change in the composition of the enzymes produced by T. reesei, the change of the substrate structure, and the adaptation of the fungus morphology. Altogether this study will provide fundamental understanding of a) how the cellulosic substrate and its structure influences enzyme production and the fungal morphology, b) how the fungus optimizes the composition of the produced enzymes to break down the cellulosic substrate effectively, and c) how the substrate structure changes in response to the produced enzymes. The gained knowledge is essential to improve the production of enzymes by T. reesei and the enzymatic breakdown of cellulose to glucose. The proposed project is an important contribution to advance the potential of the cellulosic biorefinery.

In this study we have created a method that allows us to quantitatively analyze how well a cellulose-degrading enzyme can access its binding site in the cellulose fiber, as well as the structural configuration of the cellulose itself. This novel method showed us that the cellulose is composed of distinct substructures, which interact with the enzymes to different extents, resulting in their faster or slower degradation. We further found that, depending on which enzymes are present in the applied enzyme cocktail, this degradation pattern varies, where the mot distinct difference was found when comparing commercial enzyme cocktails with the native enzymes secreted by the wood-decaying fungus Trichoderma reesei. In a second study, we found that the enzyme mixture secreted by this fungus varies in dependence of the substrate's chemical composition and its structural configuration. Together this indicates that the fungus has evolved to "sense" both the chemical composition and the physical structure of the lignocellulose it is growing on. From these results we can draw conclusion on how to tailor and optimize enzyme mixtures towards a biomass' chemical and structural characteristics, and we do so by learning from nature's own approach. This knowledge might help us to improve the efficiency of the lignocellulose saccharification step in the biorefinery process, designed to replace petrochemically-based chemicals and fuels with sustainable and environmentally benign lignocellulose-based alternatives.