Clostridium is a promising industrial microorganism for valuable
chemical production. Metabolic engineering of Clostridium, however, has been retarded due to current inefficient
genetic manipulation techniques for
Clostridium. Here, we report a gene knockdown system for Clostridium acetobutylicum based on synthetic
small regulatory RNA (sRNA). In C.
acetobutylicum, heterologous Escherichia
coli MicC scaffold-based anti-Evoglow sRNA was found to knock down the
expression of Evoglow while the sRNA does not bind to the native C. acetobutylicum Hfq (CaHfq). Additional
introduction of E. coli Hfq (EcHfq) in
C. acetobutylicum, which forms
complex with sRNA, allowed stronger repression of Evoglow. Applying the results
for metabolic engineering in C.
acetobutylicum, heterologous expression of E. coli MicC scaffold-Hfq system to knock down adhE1 in C. acetobutylicum led
to 40% decrease in butanol production (2.5 g/L). In contrast, sRNA-based knockdown
of pta gene in a buk-mutant C. acetobutylicum
strain PJC4BK (PJC4BK (pPta-HfqEco)) allowed production of 16.9 g/L butanol,
which is higher than that produced by the original PJC4BK strain (14.9 g/L). In
fed-batch fermentation with in situ gas
stripping, the final strain PJC4BK (pPta-HfqEco) produced 105.5 g solvents
(70.7 g butanol, 20.5 g acetone, and 14.3 g ethanol, demonstrating the stable fermentation
of a strain harboring the sRNA system. [This work was supported by the
Intelligent Synthetic Biology Center through the Global Frontier Project
(2011-0031963) funded by the Ministry of Science, ICT and Future Planning
(MSIP) through the National Research Foundation (NRF) of Korea.]
