Engineering Anaerobic Gut Fungi for Lignocellulose Breakdown

Currently bio-fuel is an attractive and promising energy source to secure our nation’s energy future. Anaerobic gut fungi the live in the digestive tract of large herbivores are among the most efficient degraders of lignocellulose, which is the most abundant raw materials on earth for the production of bio-fuel. The major contributor to fungi enzymatic efficiency is the large macro molecular structure called cellulosome complex which is consist of four major components: functional enzyme, non-catalytic dockerin, and dockerin’s binding partner cohesin and scaffoldin.  While the bacterial cellulosome is well-characterized, the fungal cellulosome complex’s molecular architecture remains elusive. Since the interaction between dockerin and cohesin is essential to the construction of cellulosome complex, it is crucial to identify dockerin’s binding partner in order for us to be able to decipher fungal cellulosome architecture. In an effort to identify dockerin-binding proteins, DNA encoding a dockerin sequence was amplified using polymerase chain reaction and inserted it into an expression vector for high-level expression in E. coli. Then the isolated and purified dockerin proteins were put together with the cellulosome complex to measure binding activity to the cellulosome complex. It was revealed that multiple proteins bind to the dockerin, indicating the presence of more than one scaffoldin protein.