title: Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli.
abstract: Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate that redistributing carbon flux using genome editing is an efficient engineering tool for metabolite overproduction.
authors.affiliation: Department of Chemical & Biological Engineering, Korea University , 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, South Korea.
authors.forename: Min-Ji
authors.initials: MJ
authors.lastname: Heo
authors.affiliation: Department of Chemical & Biological Engineering, Korea University , 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, South Korea.
authors.forename: Hwi-Min
authors.initials: HM
authors.lastname: Jung
authors.affiliation: Department of Chemical & Biological Engineering, Korea University , 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, South Korea.
authors.forename: Jaeyong
authors.initials: J
authors.lastname: Um
authors.affiliation: Department of Chemical & Biological Engineering, Korea University , 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, South Korea.
authors.forename: Sang-Woo
authors.initials: SW
authors.lastname: Lee
authors.affiliation: Department of Chemical & Biological Engineering, Korea University , 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, South Korea.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Oh
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D020121:5' Untranslated Regions / Q000235:genetics; D000214:Acyl Coenzyme A / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002950:Citrate (si)-Synthase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods; D005798:Genes, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods
chemical_list: D020121:5' Untranslated Regions; D000214:Acyl Coenzyme A; C010667:acetoacetyl CoA; D020001:1-Butanol; D002950:Citrate (si)-Synthase
keywords: 5′-untranslated region; CRISPR/Cas9; Escherichia coli; genome editing; n-butanol