An Jianhong, Zhang Wenli, Jing Xiaoran, Nie Yao, Xu Yan
1School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122 China.
3International Joint Research Laboratory for Brewing Microbiology and Applied Enzymology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122 China.
3 Biotech. 2020 Apr;10(4):167. doi: 10.1007/s13205-020-2160-3. Epub 2020 Mar 13.
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 and gene in the TCA cycle pathway of (). 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 BL21(DE3) ΔΔ/pET-28a(+)- (2Δ-), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to BL21(DE3)/pET-28a(+)- [BL21(DE3)] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production.
L-异亮氨酸双加氧酶（IDO）是一种依赖于Fe（II）/α-酮戊二酸（α-KG）的双加氧酶，它能特异性地将L-异亮氨酸（L-Ile）转化为（2S，3R，4S）-4-羟基异亮氨酸（4-HIL）。4-HIL是治疗和预防1型和2型糖尿病的重要药物，但目前方法的产量较低。在本研究中，CRISPR-Cas9基因编辑系统被用于敲除（）三羧酸循环途径中的和基因。对于单基因敲除，整个过程大约需要7天。然而，对于多基因编辑，每轮基因修饰的操作时间减少了2天。使用基因组编辑的重组菌株BL21(DE3)ΔΔ/pET-28a(+)-（2Δ-），与BL21(DE3)/pET-28a(+)-[BL21(DE3)]菌株相比，L-Ile向4-HIL的生物转化率提高了约15%。CRISPR-Cas9编辑策略在更快速地修饰多个基因和优化工业生产菌株方面具有潜力。
