This project will provide a low-cost route for high-yield fumarate production from pretreated non-food cellulosic biomass mediated by recombinant cellulolytic Bacillus subtilis. The cellulose fraction of biomass feedstock contains more than one half oxygen by weight, making it a good starting material for the production of oxygen-containing polymeric monomers. Fumaric acid or fumarate, among the DOEs top building block chemicals is a precursor of succinic acid, malic acid, L-aspartic acid, and biodegradable polymers. B. subtilis strains have numerous advantages in industrial chemical production, such as their extensive usage as industrially-safe microorganisms, an inherent ability to use monomeric and oligomeric C5 and C6 sugars, tolerance to organic acids, low medium nutrient needs, and fast growth rates. The goal of this project is to develop an industrially-safe recombinant cellulolytic Bacillus subtilis strain that can produce high-yield fumarate from cellulosic materials in a two- step fermentation. Aerobic fermentation is used first, for fast cell mass synthesis and cellulase production, followed by anaerobic fermentation, which produces two moles of fumarate from two moles of CO2 and one mole of glucose (released from cellulose), with a theoretical yield of 1.29 g fumarate per g glucose. Compared to other dicarboxylic acids (e.g., succinic acid), the production of fumarate from cellulosic materials has balanced cofactors between the substrate and desired product, along with potentially high product yields, easy regulation of complex metabolic pathways, and low separation costs. Different from other consolidated bioprocessing (CBP) microorganisms, such as Gram- positive Clostridium spp., Gram-negative Escherichia coli, and yeast, B. subtilis strains have lots of advantages for industrial fermentation. Furthermore, we have developed several new techniques so that we can modify B. subtilis strains rapidly and easily, such as rapid scar-free chromosome knockout or knockin, directed evolution for enhanced activity secretory cellulases, and creation of the first generation recombinant cellulolytic B. subtilis. To produce high-yield fumarate from cellulosic materials and simplify product separation, the specific objectives are to (i) redirect the carbon flux to a fumarate-producing pathway, by knocking out other side-product pathways, (ii) over-express key enzymes, such as phosphoenolpyruvate carboxykinase, and (iii) co-express family 5, 9 and 48 cellulase genes for fast microbial cellulose utilization. This project provides a new route for generating high-yield fumarate from biomass with an industrially-safe B. subtilis strain, without the addition of costly fungal cellulase or expensive medium nutrients.
