This Small Business Technology Transfer (STTR) Phase I project proposes to develop a new low-cost platform for the production of methyl ethyl ketone directly from pretreated cellulosic biomass in a single step using a novel recombinant cellulolytic Bacillus subtilis strain. Methyl ethyl ketone (MEK), also referred to as 2-butanone, is the second most important commercial ketone after acetone. MEK is currently only produced by the oxidation of 2-butanol. However, this industrial synthesis process uses starting materials derived from petrochemicals and is generally expensive as well as not environmentally friendly. There is an urgent need to develop a novel cost-effective and environmentally friendly method to produce MEK other than through 2-butanol. The cellulosic biomass is the most abundant natural renewable resource and has great potential for the production of valuable biocommodities for both short- and long-term sustainability. However, the process for converting non-food lignocellulosic material into MEK is not yet economically feasible due to the high cost of the cellulase involved in cellulose hydrolysis and the use of fastidious culture media. Using synthetic pathway and metabolic engineering, this project will convert noncellulose-utilizing B. subtilis into an efficient cellulose utilizer to produce MEK with high yield and titer, suitable for industrial fermentation.
The broader impact/commercial potential of this project, if successful, will be a low-cost platform for producing MEK from nonfood biomass in a process called consolidated bioprocessing. MEK may then be used as a solvent for paint, and serve as an intermediate in the production of other chemicals. Therefore, MEK could easily find a market in the paint industry and in plastics manufacturing. More importantly, MEK could be converted by subsequent hydrogenation into octane isomers that can be used to produce high-grade aviation fuel. Currently, MEK is synthesized from petroleum-derived chemicals via a method involving greenhouse gas emissions. So far, few efforts have been made to produce bio-based MEK due to low process economics. The proposed recombinant cellulolytic B. subtilis would have advantages over developing other microorganisms. In addition, the novel green technology will satisfy operational cost considerations, environmental concerns, and health and safety regulations. Compared to traditional mechanism, this novel route will be more cost-effective and environmentally friendly. If successfully commercialized after the completion of the Phase II project, this bio-based MEK production technology will have a significant competitive advantage over traditional methods because it is more commercially attractive and supports sustainable societal development.
The goal of this STTR Phase I project is to demonstrate the feasibility of a novel synthetic route to ferment cellulosic biomass to MEK as the major product using a single strain of cellulolytic B. subtilis, without the addition of costly cellulases. We planned two major tasks: 1) to engineer the critical enzymes, i.e. diol dehydratase (DDH) and acetoin reductase (AR), and manipulate the metabolic pathways to achieve production of MEK as the major fermentation product in recombinant B. subtilis, and 2) to validate the one-step conversion of cellulosic biomass to MEK by recombinant cellulolytic B. subtilis. The critical enzyme for MEK production, diol dehydratase (DDH) has been successfully amplified from Klebsiella and Lactobacillus strains using their genomic DNAs and the primers designed based on reported and predicted sequences. These wild type DDHs were cloned into expression vectors, and soluble expression with a desired ratio of subunits was successfully achieved. Two purification approaches were tested and optimized for successful production of active DDHs. Further, the enzymatic activity assays for DDH was established for accurate estimation of the turnover number (kcat) of the purified enzymes. Our results suggested that the purified wt DDHs had expected activities towards 1,2-propanediol with less but detectable activity for 2,3-butanediol. In addition, these purified DDHs were not stable in oxidized conditions and prone to lose their activity during storage. For improved activity toward 2,3-butanediol, site-directed mutagenesis were identified according to DDH’s crystal structure. Mutation clones, including F374A, V300G, T222S etc. were successfully constructed. Due to the instability of the mutated enzymes, the purification of the active mutant enzyme failed. This work is the key part for our project and later the direct cloning of the mutant enzyme without the purification and characterization steps was also tried with great efforts but still failed. For the metabolic engineering aspect, by inactivating the lactate dehydrogenase Ldh in B. subtilis, the major fermentation product was successfully changed from lactate to R,R-2,3-BDO. The native acetoin reductase (AR) in B. subtilis was replaced with heterologous AR from B. licheniformis, the fermentation product was successfully switched from R,R-2,3-BDO to meso-2,3-BDO, which is the desired precursor for MEK synthesis. The engineered B. subtilis strain was further subjected to adaptive evolution and the titer of BDO was successfully increased by 6-fold to ~15 g/L. The attempt to construct the recombinant vector for expression of either wild type or mutant DDH complexes in B. subtilis failed. Without the success in this task, we were unable to test activities of the mutant DDHs we generated and failed to engineer the B. subtilis strain for producing the target product MEK. In summary, great progresses had been made on the cloning, purification and characterization of wildtype DDH enzymes and the metabolic engineering of B. subtilis strain for the production of MEK precursor meso-2,3-BDO with high titer and yield. However, we encountered tremendous difficulties on the purification and heterologous expression of DDH enzymes in B. subtilis, which unfortunately impeded the whole progress of the project.
