SBIR Phase II: One-step Production of Lactic Acid from Lignocellulosic Biomass by Recombinant Cellulolytic Bacillus subtilis

This Small Business Innovation Research Phase II project will further develop new proprietary cellulolytic Bacillus subtilis strains that can produce high-titer, optically- pure L-lactate in high yields from pretreated lignocellulosic biomass through consolidated bioprocessing (CBP) technology. Lactate, or equivalently, lactic acid, is the precursor of the biodegradable plastic polylactic acid (PLA). The following Phase I goals were achieved: (i) the creation of a cellulolytic B. subtilis strain with an enhanced cellulolytic ability, (ii) the demonstration of lactate production from pretreated biomass without the use of cellulases, and (iii) the secretion of large-size heterologous proteins in B. subtilis. This Phase II project will further engineer strains with enhanced cellulolytic ability, and will seek to increase product yield, productivity (i.e., its space time yield), titer, and purity using systems biology and synthetic biology tools. At completion of this project, the goal is to have industrially-ready CBP strains that can hydrolyze pretreated lignocellulosic biomass efficiently, with product yields of>90% based on mixed biomass sugars and>95% based on glucose, a titer of ~150 g/L, and a productivity of ~1 g/L/h. Such Bacillus strains will be ready for large-scale fermentation as a continuing commercialization phase. The broader impact/commercial potential of this project is the production of a key building-block chemical from biomass. New proprietary recombinant cellulolytic B. subtilis strainsdeveloped in this project will provide an ultra-low-cost platform for producing L-lactate from the non-food biomass, with many advantages over other developing CBP microorganisms. Large-scale production of L-lactate from pretreated lignocellulosic biomass will enable the development of other CBP microorganisms that could produce PLA, biochemicals (e.g., succinate) and advanced drop-in biofuels (e.g., isobutanol, jet fuel) in the future.