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.

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

“…CRISPR and CRISPRi have been individually harnessed to engineer genome or rewire metabolic networks in different bacteria such as cyanobacteria (Huang et al, ) and E. coli (Y. Li et al, ) to promote the production of bio‐derived chemicals. However, virtually all previous studies exploiting CRISPR or CRISPRi for E. coli engineering have used SpCas9 (Ding, Weng, Du, Chen, & Kang, ; Heo et al, ; Y. Jiang et al, ; Kiani et al, ; Liang et al, ; Liu et al; Pyne, Moo‐Young, Chung, & Chou, ; Reisch & Prather, ; Ronda, Pedersen, Sommer, & Nielsen, ; Umenhoffer et al, ; Zhao et al, ) or SpdCas9 (Cress et al, , ; Kim et al, ; S. Li, Jendresen, et al, ; Lv et al, ; Rath, Amlinger, Hoekzema, Devulapally, & Lundgren, ; Wu et al, ). Furthermore, in previous studies the CRISPRi modules were maintained in plasmids and have yet to be integrated into the chromosome using the CRISPR system, which is at least partly because the expressed SpCas9 may complex with the sgRNA designed for SpdCas9‐mediated suppression and lead to off‐target mutagenesis.…”

“…Li et al, 2015) to promote the production of bio-derived chemicals. However, virtually all previous studies exploiting CRISPR or CRISPRi for E. coli engineering have used SpCas9 (Ding, Weng, Du, Chen, & Kang, 2017;Heo et al, 2017;Y. Jiang et al, 2015;Kiani et al, 2015;Liang et al, 2017;Liu et al;Pyne, Moo-Young, Chung, & Chou, 2015;Reisch & Prather, 2015;Ronda, Pedersen, Sommer, & Nielsen, 2016;Umenhoffer et al, 2017;Zhao et al, 2016) or SpdCas9 (Cress et al, 2015(Cress et al, , 2016Kim et al, 2016;S.…”

“…In E. coli , the CRISPR‐induced DSB was first coupled with recombineering to promote genome editing (W. Jiang, Bikard, Cox, Zhang, & Marraffini, ), and subsequently exploited for metabolic engineering of E. coli to produce bio‐derived chemicals such as β‐carotene (Y. Li et al, ), isopropanol (Liang et al, ), n‐butanol (Heo, Jung, Um, Lee, & Oh, ), malate (Z. J. Li, Hong, Da, Li, & Stephanopoulos, ), and 3‐hydroxypropionic acid (Liu et al, ). Furthermore, CRISPR interference (CRISPRi) was developed to specifically block target gene transcription, by using catalytically inactive SpCas9 (SpdCas9) and cognate sgRNA (Qi et al, ).…”

“…In E. coli, the CRISPR-induced DSB was first coupled with recombineering to promote genome editing (W. Jiang, Bikard, Cox, Zhang, & Marraffini, 2013), and subsequently exploited for metabolic engineering of E. coli to produce bio-derived chemicals such as βcarotene (Y. Li et al, 2015), isopropanol (Liang et al, 2017), n-butanol (Heo, Jung, Um, Lee, & Oh, 2017), malate (Z. J. Li, Hong, Da, Li, & Stephanopoulos, 2018), and 3-hydroxypropionic acid .…”

Advances in bio‐based production of dicarboxylic acids longer than C4

“…Therefore, fine-tuning expression with a combination of suitable promoters and ribosome binding sites, and dynamic regulation of key enzymes are useful for enhancing the production from synthetic pathways [57][58][59][60][61][62]. The primary consideration is to tune the expression levels of multiple enzymes of the synthetic pathway to achieve a good balance.…”