Enhanced Butanol Production in Clostridium acetobutylicum Using Small Regulatory RNAs for Metabolic Engineering

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The Ohio State University

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This project uses an RNA-based gene expression control system for metabolic engineering of Clostridium acetobutylicum. The primary focus is enhancing production of n-butanol, a green alternative to fossil fuels. Butanol is an attractive alternative fuel with higher energy density than other biofuels, and can directly replace gasoline. To improve the production of butanol using Clostridium, a small RNA (sRNA) platform is utilized. RNA is useful as a genetic regulatory tool because it provides flexible expression tuning compared to the on/off DNA knockout method. sRNAs are used by bacteria for regulating gene expression, as they bind to protein-coding mRNA sequences. Through binding, sRNA can enhance or reduce mRNA translation and thus protein expression. Two genes in the metabolic pathway, buk and hydA, will be down regulated using sRNA, potentially increasing butanol titer and yield without compromising cell growth. Down-regulation of these genes presents a novel opportunity to modify Clostridium, as hydA is essential to cellular function and cannot be completely turned off. Furthermore, using sRNA allows for simultaneous targeting of both genes. This is done using genetic engineering techniques to transform wild type cells with the desired genes. The recombinant plasmid for sRNA production is derived from E. coli and then ported over. Mutants are then screened and tested to determine performance as compared to the original strain. One mutant and a control plasmid have been successfully transformed. Preliminary results show that the mutant targeting hydA successfully down regulates the gene and reduces production of butyric acid, while also increasing butanol production. This project could have significant contribution to improving economic viability of biologically derived n-butanol. Furthermore, the sRNA platform has potential for broad applications in metabolically engineering various bacterial species.



Metabolic Engineering, sRNA, Butanol, Clostridium