Butyrate production in engineered Escherichia coli with synthetic scaffolds.

Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

Metabolic engineering of Enterobacter aerogenes for 2,3-butanediol production from sugarcane bagasse hydrolysate.

The pathway engineering of Enterobacter aerogenes was attempted to improve its production capability of 2,3-butanediol from lignocellulosic biomass. In the medium containing glucose and xylose mixture as carbon sources, the gene deletion of pflB improved 2,3-butanediol carbon yield by 40%, while the deletion of ptsG increased xylose consumption rate significantly, improving the productivity at 12 hr by 70%. The constructed strain, EMY-22-galP, overexpressing glucose transporter (galP) in the triple gene knockout E. aerogenes, ldhA, pflB, and ptsG, provided the highest 2,3-butanediol titer and yield at 12 hr flask cultivation. Sugarcane bagasse was pretreated with green liquor, a solution containing Na2CO3 and Na2SO3 and was hydrolyzed by enzymes. The resulting hydrolysate was used as a carbon source for 2,3-butanediol production. After 72 hr in fermentation, the yield of 0.395g/g sugar was achieved, suggesting an economic production of 2,3-butanediol was possible from lignocellulosic biomass with the metabolically engineered strain.

Reutilization of green liquor chemicals for pretreatment of whole rice waste biomass and its application to 2,3-butanediol production.

The performance of green liquor pretreatment using Na2CO3 and Na2SO3 and its optimization for whole rice waste biomass (RWB) was investigated. Incubation of Na2CO3-Na2SO3 at a 1:1 ratio (chemical charge 10%) for 12% RWB at 100°C for 6h resulted in maximum delignification (58.2%) with significant glucan yield (88%) and total sugar recovery (545mg/g of RWB) after enzymatic hydrolysis. Recovery and reusability of the resulting chemical spent wash were evaluated to treat RWB along with its compatibility for enzymatic digestibility. Significant hydrolysis and lignin removal were observed for up to three cycles; however, further reuse of Na2CO3 and Na2SO3 lowered their performance. Significant 2,3-butanediol (BDO) was produced by Klebsiella pneumoniae KMK-05 with the RWB enzymatic hydrolysate from each pretreatment cycle. BDO yield achieved using RWB-derived sugars was similar to those using laboratory-grade sugars. This pretreatment strategy constitutes an ecofriendly, cost-effective, and practical method for utilizing lignocellulosic biomass.