Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9.
Jianhong AnWenli ZhangXiaoran JingYao NieYan XuPublished in: 3 Biotech (2020)
l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout sucAB and aceAK gene in the TCA cycle pathway of Escherichia coli (E. coli). For single-gene knockout, the whole process took approximately 7 days. However, the manipulation time was reduced by 2 days for each round of gene modification for multigene editing. Using the genome-edited recombinant strain E. coli BL21(DE3) ΔsucABΔaceAK/pET-28a(+)-ido (2Δ-ido), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to E. coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production.
Keyphrases
- crispr cas
- escherichia coli
- genome editing
- genome wide
- genome wide identification
- type diabetes
- copy number
- klebsiella pneumoniae
- computed tomography
- pet ct
- biofilm formation
- dna methylation
- genome wide analysis
- cardiovascular disease
- positron emission tomography
- emergency department
- transcription factor
- wastewater treatment
- gene expression
- staphylococcus aureus
- adipose tissue
- metabolic syndrome
- weight loss
- climate change
- cell free
- visible light
- candida albicans
- metal organic framework