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

• Published in: Microbial cell factories Vol. 16; no. 1; p. 188
• Main Authors: Kim, Seong Keun, Seong, Wonjae, Han, Gui Hwan, Lee, Dae-Hee, Lee, Seung-Goo
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 03.11.2017; BioMed Central; BMC
• Subjects: Acetic acid; Bacteria; Biodegradable materials; Biosynthesis; Butanol; Byproducts; Case studies; CRISPR; CRISPR interference; Deoxyribonucleic acid; DNA; E coli; Endogenous gene; Escherichia coli; Ethanol; Gene expression; Gene silencing; Genes; Genetic aspects; Genetic engineering; Genomes; Industrial engineering; Interference; Lactic acid; Manufacturing engineering; Metabolic engineering; Metabolic flux; Metabolism; Metabolites; Methods; Microbial genetic engineering; Microbiological synthesis; Microorganisms; Multiple gene knockdown; Multiplexing; Mutation; n-Butanol; NADH; Nicotinamide adenine dinucleotide; Organic chemistry; Physiological aspects; Productivity; Regulators; Technical Notes; Transcription; South Korea; Endogenous gene; n-Butanol; CRISPR interference; Escherichia coli; Multiple gene knockdown
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-017-0802-x

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. 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. 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.