Engineering solventogenic clostridia

mda kimal Engineering solventogenic clostridia. mmm . ISSN nn

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Abstract

Solventogenic clostridia are strictly anaerobic, endospore forming bacteria that produce a large array of primary metabolites, like butanol, by anaerobically degrading simple and complex carbohydrates, including cellulose and hemicellulose. Two genomes have been sequenced and some genetic tools have been developed, but more are now urgently needed. Genomic tools for designing, and assessing the impact of, genetic modifications are well developed. Early efforts to metabolically engineer these organisms suggest that they are promising organisms for biorefinery applications. Pathway engineering efforts have resulted in interesting strains, but global engineering of their transcriptional machinery has produced better outcomes. Future efforts are expected to undertake the development of complex multigenic phenotypes, such as aerotolerance, solvent tolerance, high-cell density fermentations, abolished sporulation without impacting product formation, and genetic stability for continuous bioprocessing. Section snippets Introduction: solventogenic clostridia: from outcasts to workhorses? The development of renewable chemicals and biofuel technologies has been on the scientific and technological agenda in the US and worldwide for over 35 years now, but never quite with the urgency and high priority of the past two years, when finally this technology has been elevated to a high priority status from being underappreciated (as many working in this field have experienced) by the broader scientific community, industry, and the funding agencies. Significantly, combustion of The tools: to transform, overexpress, knockout (KO), knockdown (KD), to report, and to analyze in vivo fluxes, and the transcriptome Although not as easy to employ, as reliable, and as fast as in well established model organisms (like Escherichia coli and Bacillus subtilis), solventogenic clostridia have now reasonably developed tools that have been recently reviewed, for example [15]. Briefly, transformation by electroporation and means to overcome the restriction system in C. acetobutylicum and plasmids to overexpress genes was published over 15 years ago [16], and this approach has been adapted to other clostridia. How to fix the major generic issues: aerointolerance, low cell densities, and limited sustainable viability The major advantage of butyric acid and related clostridia is their powerful central primary metabolism (Figure 1). Specific carbon fluxes are very good (e.g. [13, 14]), but cell densities are relatively low (around a max of 10–11 of absorbance at 600 nm (A600)), and the ability to sustain them viable over prolonged time periods is limited. Significantly, while anaerobiosis is essential for their powerful primary metabolism, their typically low aerotolerance complicates bioprocessing. The issue How to make them grow on complex cellulosic substrates: the cellulosome and how to fix it Cellulolytic clostridial degrade cellulose via the cellulosome [38]. This enzymatic complex is generally bound to the cell surface, contains motifs that bind to insoluble cellulose, and is made up of various cellulases that cleave oligosaccharides from insoluble cellulose. There are several sequenced organisms that contain complete and functional cellulosomes (coded by 11 to ca. 26 genes), and of notable interest are cellulolytic clostridia (incl. C. phytofermentans, C. thermocellum, and C. To undo the sporulation, and thus increase productivity and simplify bioprocessing: differentiation engineering? The metabolism of C. acetobutylicum and of other solventogenic clostridia is biphasic in batch culture: first producing acetate and butyrate and later butanol, acetone, and ethanol. During growth, the production of acids lowers the pH of the culture, which combined with butyrate accumulation shifts the metabolism toward solvent production. Solvent formation is associated with re-uptake of the acids that are then converted into solvents (Figure 1). Solvent formation coincides with initiation of

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