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Title 

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

 

유전자가위 간섭 기반의 대장균 유전자 발현 저해를 통한 대사 흐름의 조절

Authors 

Seong Keun KimWonjae SeongGui Hwan HanDae-Hee LeeSeung Goo Lee

Publisher 

BioMed Central

Issue Date 

2017

Citation 

Microbial Cell Factories

Keywords 

CRISPR interferenceEndogenous geneEscherichia coliMultiple gene knockdownn-Butanol

Abstract 

Background: Multiplex 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. Results: In 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. Conclusions: We 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

ISSN 

1475-2859

Link 

http://dx.doi.org/10.1186/s12934-017-0802-x

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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