Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9

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) Δ sucAB Δ aceAK /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.