Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs. Issue 2 (30th August 2016)

• Record Type: Journal Article
• Authors: Cho, Changhee; Lee, Sang Yup
• Is Part Of: Biotechnology and bioengineering. Volume 114:Issue 2(2017)
• Page Start: 374
• Page End: 383
• Publication Date: 2016-08-30
• Subjects: Clostridium acetobutylicum -- synthetic sRNAs -- gene expression control -- gene knockdown -- butanol -- Hfq; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26077
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)‐based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold‐based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold‐Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum . Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild‐type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta‐Hfq Eco ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed‐batch culture of PJC4BK (pPta‐Hfq Eco ) strain coupled with in situ gas stripping produced 105.5 g of total solvents (70.7 g butanol, 20.5 g acetone, and 14.3 g ethanol), demonstrating that the sRNA‐based engineered C. acetobutylicum strain can be cultured without instability. The synthetic sRNA system reported in this study will be useful for more efficient development of engineered C. acetobutylicum strains capable of producing valuable chemicals and fuels. Biotechnol. Bioeng. 2017;114: 374–383. © 2016 Wiley Periodicals, Inc. Abstract : Clostridium acetobutylicum is an industrially important bacterium, that has been known to be extremely difficult to genetically manipulate. Here the authors report development of synthetic sRNA‐based gene knockdown system that allows efficient knockdown of inherent and heterologous genes in C. acetobutylicum . By knocking down the pta gene in buk ‐negative mutant C. acetobutylicum strain, butanol production could be enhanced both in batch culture and fed‐batch culture coupled with in situ recovery.