Targeted Proteomics Enabled Metabolic Engineering of Clostridium Cellulolyticum for n-Butanol Production

The sustainable production of biofuels and chemicals require the use of environmentally friendly carbon neutral feedstock’s, which most importantly do not compete with food production. An abundant and widely available source of such feedstock’s is lignocellulosic material, like agricultural residues, woody material like energy crops or municipal solid waste (MSW). Unfortunately, these materials are highly resistant towards microbial digestion as they are composed of complex polymers of various compositions, e.g. cellulose and hemicellulose. Common pretreatment processes to break down these polymers require harsh conditions leading to the formation of inhibitory substances like HMF as well as the addition of costly degrading enzymes. Clostridium cellulolyticum naturally excretes a cellulosome, a multi subunit enzyme complex able to degrade lignocellulosic components. Thus the cost and condition of the pretreatment process could be tremendously reduced by using C. cellulolyticum as a production organism for various compounds.

As a proof of concept, we report on metabolic engineering C. cellulolyticum to produce n-butanol, an advanced biofuel and industrial important bulk chemical directly from crystalline cellulose. Also, great progress has been made towards developing better and novel genetic tools for Clostridia, a still troublesome bottleneck is the functional expression and regulation of heterologous genes. Moreover, fine tuning of engineered pathways is difficult, because little is known about genetic regulatory elements especially on the posttransciptional level. Only a few promoters are currently used in engineering heterologous pathways, which are mostly constitutive. Here, we report on developing a method involving targeted proteomics combined with metabolite profiling to facilitate metabolic engineering of heterologous pathways and fine tuning thereof. Combining the information of the protein level (targeted proteomics) and the flux through the pathway (fluxomics), changes to regulatory elements in the engineered pathway towards transcription, translation or post translation can easily be accessed with our newly developed method and used to fine tune the entire biosynthetic pathway. The information about the regulation of expression can furthermore be used in the metabolic engineering of additional heterologous pathways leading to the production of various desirable chemicals.