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We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of  Neurospora crassa  (F5∆ ace-1 ∆ cre-1 ∆ ndvB ) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically,  N .  crassa  produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native  N .  crassa  laccase in such a conversion system was evaluated against the exogenous  Pleurotus ostreatus  laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

Production of Cellobionate from Cellulose Using an Engineered Neurospora crassa Strain with Laccase and Redox Mediator Addition