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

这一小型企业创新研究二期项目将进一步开发新的专利枯草芽孢杆菌菌株，该菌株可以通过联合生物加工技术从经过预处理的木质纤维生物质中高产率地生产高滴度、光学纯的L-乳酸。乳酸，或相当于乳酸，是可生物降解塑料聚乳酸(PLA)的前体。实现了以下第一阶段的目标：(I)创造一株具有更强的纤维分解能力的枯草杆菌菌株，(Ii)在不使用纤维素酶的情况下从经过预处理的生物质中产生乳酸，以及(Iii)在枯草杆菌中分泌大尺寸的异源蛋白。这个第二阶段的项目将进一步设计具有增强的纤维素分解能力的菌株，并将利用系统生物学和合成生物学工具寻求提高产品产量、生产力(即其时空产量)、效价和纯度。在该项目完成后，目标是获得能够有效地水解经过预处理的木质纤维生物质的工业化菌株，以混合生物质糖为基础的产品产率为90%，以葡萄糖为基础的产品产率为95%，效价为~150g/L，生产能力为~1g/L/h。这些芽孢杆菌菌株将作为继续商业化的阶段进行大规模发酵。该项目更广泛的影响/商业潜力是从生物质中生产一种关键的积木化学品。本项目开发的新型专利重组纤维素分解菌枯草芽孢杆菌将为利用非食用生物质生产L乳酸提供一个超低成本的平台，与其他正在开发的CBP微生物相比具有许多优势。从经过处理的木质纤维生物质中大规模生产L乳酸，将有助于开发其他CBP微生物，在未来生产聚乳酸、生物化学品(如琥珀酸)和先进的插入式生物燃料(如异丁醇、喷气燃料)。

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.