Search

Long-term energy stability and environmental concerns have driven development toward the production of transportation fuels from renewable feedstocks such as sugars or lignocellulosic biomass. n-Butanol and isobutanol have emerged as prominent advanced transportation biofuels because of their favorable fuel properties, compatibility with current infrastructure, and ability to serve as chemical feedstocks. Microbial productions of such compounds from sugars have been demonstrated with reasonably encouraging yields and productivities. Ultimately, production of butanol from cellulosic materials will be the goal. To this end, cellulolytic Clostridium species are among the most promising organisms to serve a host because they provide an opportunity for consolidated bioprocessing. These microbes are natively equipped with robust cellulose-degrading machinery, but they require introduction and optimization of pathways for producing n-butanol or isobutanol. This chapter will describe the benefits of cellulolytic Clostridia for economical biomass degradation, outline pathways for microbial n-butanol and isobutanol production, and discuss progress and strategies for improving butanol production as well as the current status and future directions of clostridial butanol.

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50 °C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3 g/l of isobutanol was produced from glucose and 0.6 g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48 h at 50 °C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.

On page 1, Introduction, Column 2, line 8 from bottom “An exception is G. thermoglucosidasius, for which transformation procedures enabling metabolic manipulation have been established (Cripps et al., 2009)” Should read “An exception is G. thermoglucosidasius, for which transformation procedures enabling metabolic manipulation have been established (Taylor et al., 2008; Cripps et al., 2009; Bartosiak-Jentys et al., 2012, 2013).” On page 7, Discussion, Column 2, the first sentence “Although this organism has an established transformation protocol and an integration vector system for gene replacements (Cripps et al., 2009), no expression system has been established. Here, we identified a robust promoter to drive gene expression from a plasmid, and we characterized enzymes for isobutanol biosynthesis at elevated temperature.” Should read “Previously, transformation procedures, plasmid-based expression systems, and gene replacement protocols have been established for this organism (Taylor et al., 2008; Cripps et al., 2009; Bartosiak-Jentys et al., 2012, 2013). In this work, we determined that the ldh promoter from G. thermodenitrificans is suitable for driving the expression of the isobutanol pathway genes, and we characterized enzymes for isobutanol biosynthesis at 50 1C.”