Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs
Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs
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DOI：
10.1002/bit.26077
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发表时间：
2017-02-01
影响因子：
3.8
通讯作者：
Lee, Sang Yup
中科院分区：
文献类型：
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作者：
Cho, Changhee;Lee, Sang Yup
梭菌被认为是生产有价值的工业化学品的有前途的微生物宿主。然而，梭菌是臭名昭著的遗传操作的困难，因此代谢工程。因此，已经进行了许多努力来开发用于梭菌属菌株的遗传和代谢工程的新工具。在这里，我们报告了一个合成的小调控RNA（sRNA）为基础的系统控制的基因表达丙酮丁醇梭菌，由一个目标识别位点，MicC sRNA支架，和RNA伴侣Hfq。研究C.首先用Evoglow荧光蛋白作为模型蛋白检查丙酮丁醇菌的表达控制。最初，C。将注释为Hfq的丙酮丁醇菌蛋白与基于大肠杆菌MicC支架的合成sRNA组合以敲低Evoglow表达。然而，C. acetobutylicum Hfq不与E. coli MicC中表达，而基于MicC支架的合成sRNA本身能够敲低Evoglow的表达。当E. colihfq基因的导入，通过荧光强度的测定来评估敲低效率，可以大大提高。然后，这个E。coliMicC scaffold-Hfq系统敲低adhE 1基因在C.丙酮丁醇使用合成的sRNA敲低adhE 1基因表达导致丁醇产量（2.5g/L）降低40%，与携带空载体的野生型菌株产生的丁醇产量（4.5g/L）相比。进一步扩展sRNA系统以敲低布克突变体C中pta基因的表达。用于提高丁醇生产的丙酮丁醇菌菌株PJC 4 BK。具有pta基因表达敲低的PJC 4 BK（pPta-Hfq（Eco））菌株能够产生16.9g/L的丁醇，这高于PJC 4 BK菌株产生的丁醇（14.9g/L），这主要是由于减少的乙酸产生。PJC 4 BK（pPta-Hfq（Eco））菌株的补料分批培养结合原位气提产生105.5g总溶剂（70.7g丁醇、20.5g丙酮和14.3g乙醇），表明基于sRNA的工程C.丙酮丁醇菌菌株可以无不稳定性地培养。本研究中报道的合成sRNA系统将有助于更有效地开发工程C。能够生产有价值的化学品和燃料的丙酮丁醇菌菌株。Biotechnol. Bioeng. 2017;114：374-383. (c)2016 Wiley Periodicals，Inc.
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 (2.5g/L), compared to that (4.5g/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 16.9g/L of butanol, which is higher than that (14.9g/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 105.5g of total solvents (70.7g butanol, 20.5g acetone, and 14.3g 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. (c) 2016 Wiley Periodicals, Inc.