CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli

Microbial Cell Factories

Technical Notes

Seong Keun Kim¹  Gui Hwan Han¹  Seung-Goo Lee²  Wonjae Seong²  Dae-Hee Lee² 

[1] Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 34141, Daejeon, Republic of Korea;Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 34141, Daejeon, Republic of Korea;Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), 34113, Daejeon, Republic of Korea;

关键词: CRISPR interference;    Escherichia coli;    n;    Multiple gene knockdown;    Endogenous gene;   

DOI  :  10.1186/s12934-017-0802-x

 received in 2017-07-10, accepted in 2017-10-30,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundMultiplex control of metabolic pathway genes is essential for maximizing product titers and conversion yields of fuels, chemicals, and pharmaceuticals in metabolic engineering. To achieve this goal, artificial transcriptional regulators, such as clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi), have been developed to specifically repress genes of interest.ResultsIn this study, we deployed a tunable CRISPRi system for multiplex repression of competing pathway genes and, thus, directed carbon flux toward production of molecules of interest in Escherichia coli. The tunable CRISPRi system with an array of sgRNAs successfully repressed four endogenous genes (pta, frdA, ldhA, and adhE) individually and in double, triple, or quadruple combination that are involved in the formation of byproducts (acetate, succinate, lactate, and ethanol) and the consumption of NADH in E. coli. Single-target CRISPRi effectively reduced the amount of each byproduct and, interestingly, pta repression also decreased ethanol production (41%), whereas ldhA repression increased ethanol production (197%). CRISPRi-mediated multiplex repression of competing pathway genes also resulted in simultaneous reductions of acetate, succinate, lactate, and ethanol production in E. coli. Among 15 conditions repressing byproduct-formation genes, we chose the quadruple-target CRISPRi condition to produce n-butanol in E. coli as a case study. When heterologous n-butanol-pathway enzymes were introduced into E. coli simultaneously repressing the expression of the pta, frdA, ldhA, and adhE genes via CRISPRi, n-butanol yield and productivity increased up to 5.4- and 3.2-fold, respectively.ConclusionsWe demonstrated the tunable CRISPRi system to be a robust platform for multiplex modulation of endogenous gene expression that can be used to enhance biosynthetic pathway productivity, with n-butanol as the test case. CRISPRi applications potentially enable the development of microbial “smart cell” factories capable of producing other industrially valuable products.

【 授权许可】

CC BY   
© The Author(s) 2017

【 图 表 】

12951_2016_246_Article_IEq12.gif

Fig. 1

Fig. 2

Scheme 1

12951_2016_246_Article_IEq8.gif