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Heterologous Production of n-Butanol at High Yield Using a Complete and Fully Functionnal Clostridial Pathway

Tian, L., LISBP, Toulouse University

N-butanol, an important industrial intermediate chemical and a potential biofuel, is naturally produced in slow-growing native hosts that belong to genus Clostridium, as a mixture with acetone and ethanol in biphasic fermentation. Even if the acetone-butanol-ethanol (ABE) fermentation was one the first large scale industrial fermentation and despite the long research history on the fermentation process, obstacles still remain today. Butanol titer, yield, and productivity are still low mainly due to the co-production of acetone, ethanol and acetic and butyric acids. Advances in genetic manipulation of clostridia have led to large increases in yield and productivity. However, the regulation of solvent production is highly complex and cannot yet be achieved in a single-stage fermentation at high yields. To overcome some of the problems associated with clostridial n-butanol production processes, the clostridial CoA dependent n-butanol pathway was expressed in several heterologous organisms such as E. coli B or K12 or Saccharomyces cerevisiae to convert acetyl-CoA to butanol. Reported results showed that the seven C. acetobutylicum known genes of the pathway are functionally expressed whatever the heterologous organism is, but the final butanol titer and yield are rather low. Neither species could produce more than 1g/l of butanol. A hypothesis could be that some key enzymes involved in butyryl-CoA synthesis in C. acetobutylicum, unknown up to now, were missing in the heterologous host.

To solve this problem and further improve final titer and yield of heterologous n-butanol production, our objectives were to i) identify all the enzymes involved in butyryl-CoA synthesis in C. acetobutylicum ii) demonstrate the neediness of these enzymes for in vivo butanol synthesis in C. acetobutylicum through knockout mutants construction and characterization iii) express the gene(s) encoding the novel enzyme (s) with genes encoding the complete already known metabolic butanol pathway from the C. acetobutylicum in a E. coli MG1655 strain. Results showed that the novel recombinant E. coli strain was able to produce n-butanol from glucose at high yield (73 % of the theoretical yield) never obtained before with any microorganisms (natural or non-natural n-butanol producers). Moreover, when the strain was grown in continuous culture, a stable production (for more than four months) of butanol with a low amount of other by-products was obtained.


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