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

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0802-x) contains supplementary material, which is available to authorized users.

Regulation of Microbial Metabolic Rates Using CRISPR Interference With Expanded PAM Sequences

“…While nonsense point mutation can affect cells permanently by deleting the function of the target gene, dCas9mediated gene expression control allows the selection of desired time points (Quebatte and Dehio, 2017). Endogenous competing pathway genes can be repressed by CRISPRi in E. coli for metabolic engineering (Kim et al, 2017). Even if the genes are essential for growth, metabolic engineering with CRISPRi provides the necessary metabolites without disturbing cell growth (Cress et al, 2017).…”

Escherichia coli as a host for metabolic engineering

“…CRISPRi is often used as a tool to efficiently modulate expression of metabolic pathways (Kim et al, 2017;Cress et al, 2017). Strain optimization can also be performed concurrently with use of CRISPR for gene editing .…”

“…Strain optimization can also be performed concurrently with use of CRISPR for gene editing . In one study, CRISPRi was used to enhance butanol productivities and yields by downregulating pathways that compete for carbon usage and NADH (Kim et al, 2017). CRISPR and CRISPRi were used simultaneously to improve production of 1,4butanediol (Wu et al, 2017).…”

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

“…Furthermore, CRISPR interference (CRISPRi) was developed to specifically block target gene transcription, by using catalytically inactive SpCas9 (SpdCas9) and cognate sgRNA (Qi et al, ). CRISPRi has been harnessed to fine‐tune metabolic pathways in E. coli for the production of biopolymers (Lv, Ren, Chen, Wu, & Chen, ), flavonoid (J. Wu, Du, Chen, & Zhou, ), violacein (Cress et al, ), anthocyanin (Cress et al, ), malate (C. Gao et al, ), lycopene (Kim et al, ), pinosylvin (J. Wu, Zhang, et al, ), and n ‐butanol (Kim, Seong, Han, Lee, & Lee, ). All the aforementioned studies used SpCas9 for CRISPR‐ and SpdCas9 for CRISPRi‐mediated metabolic engineering.…”

“…Wu, Du, Chen, & Zhou, 2015), violacein (Cress et al, 2016), anthocyanin (Cress et al, 2017), malate (C. Gao et al, 2018), lycopene , pinosylvin (J. Wu, Zhang, et al, 2017), and n-butanol (Kim, Seong, Han, Lee, & Lee, 2017). All the aforementioned studies used SpCas9 for CRISPR-and SpdCas9 for CRISPRi-mediated metabolic engineering.…”