The role of lignin in the conversion of wheat straw to cellobionic acid by Neurospora crassa HL10

An engineered Neurospora crassa strain (HL10) naturally produces cellulases, cellobiose dehydrogenase (CDH), and heterologously expresses laccase. It can convert Avicel (cellulose) to cellobionic acid (cellobionate) without any enzyme addition at high yields. However, the addition of a catalytic amount of artificial redox mediators was required to achieve a high yield conversion. When a lignocellulosic substrate like wheat straw was used as the carbon source, it was found that adding exogenous artificial redox mediators did not improve cellobionate yield. We hypothesized that lignin and lignin degradation products were able to serve as redox mediators for the CDH-laccase conversion system. Experiments were designed to prove the hypothesis. When N. crassa HL10 was grown on Avicel, the addition of enzyme hydrolyzed lignin (EHL) can achieve a similar level of cellobionate production as with ABTS addition. The addition of EHL can increase the cellobionate production rate in the CDH-laccase bienzyme system using cellobiose as the substrate. The formation of lignin radicals, and the quenching of lignin radicals by reduced CDH, were verified by an electron paramagnetic resonance (EPR) experiment, providing further evidence that lignin radicals can serve as the electron acceptor of reduced CDH. Hence, lignocellulosic biomass is a self-sufficient substrate for the production of cellobionate. The conversion of the lignocellulosic substrate to cellobionate using N. crassa HL10 can be achieved without any exogenous enzyme addition or redox mediator addition.