Fig. 1
CRISPRi system design and construction for reducing byproduct formation in engineered E. coli producing n-butanol. a Schematic representation of the n-butanol-production pathway. The reconstituted n-butanol-production pathway consists of five enzymes involved in the six-step synthesis of n-butanol from acetyl-CoA. Glucose and glycerol mainly used for n-butanol production in E. coli are redirected into succinate, lactate, acetate, and ethanol production during glycolysis. b A plasmid encoding the five genes of the reconstructed n-butanol pathway. The expression of n-butanol-pathway genes was controlled by a constitutive lac promoter (lacP′). c The CRISPRi plasmid harboring an sgRNA array consisting of four sgRNAs targeting pta, frdA, ldhA, and adhE genes in E. coli. The CRISPRi system consisted of a dCas9 protein and sgRNAs governed by an l-rhamnose-inducible promoter (P rhaBAD ) and a J23119 constitutive promoter (PJ23119), respectively. P, F, L, and A are sgRNAs targeting endogenous pta, frdA, ldhA, and adhE gene, respectively. AdhE, aldehyde/alcohol dehydrogenase; AtoB, acetyl-CoA acetyltransferase; CamR, chloramphenicol-resistance gene; Crt, crotonase; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; FrdA, fumarate reductase; G3P, glyceraldehyde 3-phosphate; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; KanR, kanamycin resistance gene; LdhA, lactate dehydrogenase; PEP, phosphoenolpyruvate; Pta, phosphate acetyltransferase; Ter, trans-enoyl-CoA reductase