| The conventional process for fuel and chemical production from cellulosic biomass involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Consolidating the process into fewer processing steps is one way to improve the overall economics. Utilizing lignocellulolytic microorganisms to directly hydrolyze the cellulose into reactive intermediates, such as sugar or sugar-like compounds, for subsequent conversion to fuels or chemicals is one such way to consolidate the process.;In the proposed biological process, Neurospora crassa, a cellulolytic fungus, is used as a model organism to convert cellulose to aldonic acids, primarily cellobionic acid. Efficient hydrolysis of cellulose requires several cellulase enzymes, including endoglucanases (EG, E.C. 3.2.1.4) and cellobiohydrolases (CBH, E.C. 3.2.1.91). EGs hydrolyze cellulose internally, while CBHs hydrolyze cellulose at the reducing and non-reducing ends. Cellobiose is the primary product of this hydrolysis, which can be further hydrolyzed by beta-glucosidase (BGL, E.C. 3.2.1.21) to form glucose. Alternatively, cellobiose can be oxidized by cellobiose dehydrogenase (CDH) to form cellobiono-delta-lactone, which spontaneously hydrolyzes to form cellobionic acid. A previously engineered strain of N. crassa (F5) with six out of seven bgl genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. The F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes cre-1 and ace-1 on cellulase production were investigated. The F5Delta ace-1 mutant showed no improvement over the wild type. The F5Delta cre-1 and F5Deltaace-1Deltacre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5Deltaace-1Delta cre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5Deltaace-1Delta cre-1DeltandvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, as compared to 18% by the strain F5Deltaace-1Deltacre-1.;Although the F5Deltaace-1Deltacre-1Delta ndvB showed improved yield of cellobiose and cellobionate from the hydrolyzed cellulose, 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. In the CDH-ABTS-laccase system, over 90% of the hydrolyzed cellulose can be converted to cellobionic acid.;While inhibition of cellulases by cellobiose has been extensively studied, cellobionic acid inhibition of cellulases has not received much attention, despite the discovery that oxidative enzymes produced by cellulolytic organisms generate aldonic acids from cellulosic substrates. Since our system converts cellulose to cellobionic acid, it became evermore important to understand the inhibitory effects of cellobionic acid on cellulases. To better understand the inhibition effects of cellobionate, we investigated the inhibition of Trichoderma reesei CBHI, one of the most extensively studied CBHs, and the N. crassa CDH. The well-studied cellobiose inhibitor was characterized in parallel for comparison. With the CBHI enzyme using p-nitrophenol-beta-D-lactopyranoside as a model substrate, both inhibitors showed a competitive inhibition mechanism based on Michaelis-Menten kinetic modeling. However, the Ki value calculated for cellobionate is approximately 23 times higher than that for cellobiose, indicating much weaker inhibition. The inhibition of CBHI by cellobiose or cellobionate using Avicel as a substrate was also evaluated, as well as the inhibition of Accelerase 1500, a commercial cellulase cocktail. In all cases, cellobionate showed a weaker inhibition effect compared to cellobiose. Contrastingly, cellobionate itself does not appear to inhibit CDH at concentrations up to 20 mM. However, below pH 6, aqueous solutions of cellobionate are in equilibrium with cellobiono-delta-lactone. Equilibrated solutions of cellobionate containing cellobiono-delta-lactone have significant inhibition of CDH, highlighting the importance of lactonases in whole cellulolytic systems. Our data show that the oxidation of cellobiose to cellobionate has significant potential to improve cellulose conversion rates through inhibition relief.;One of the main goals of this project is to utilize the hydrolyzed cellulose for biofuel production. Using a genetic engineering and fermentation optimization approach, we have developed a process for the biochemical conversion of cellulose to biofuel or chemical production. (Abstract shortened by UMI.). |
