4-羟基异亮氨酸(4-hydroxyisoleucine,4-HIL)在治疗Ⅱ型糖尿病方面极具潜力。异亮氨酸双加氧酶(isoleucine dioxygenase,IDO)能够将L-异亮氨酸(L-isoleucine,Ile)转变成4-HIL。为提高重组谷氨酸棒杆菌的4-HIL产量同时降低副产物L-赖氨酸(L-lysine,Lys)的合成,首先将Lys-OFF核糖体开关整合到Lys合成途径的关键基因dapA序列上游,得到D-RS菌株。该菌株能够根据胞内外Lys的浓度,动态弱化Lys合成,使Lys的含量降低了46.7%。其次,在该菌株中再利用Ile激活型传感器Lrp-PbrnFEN去控制密码子优化后的ido基因的表达,使4-HIL的产量提高至116.3 mmol/L。最后,为了进一步提高4-HIL产量,利用强启动子PbrnFE7动态控制odhI和vgb的表达,增强α-酮戊二酸和O2的供应。最终得到了1株摇瓶发酵4-HIL产量高达166.0 mmol/L,Lys含量降至6.5 mmol/L 的重组菌株D-RS-0I7O7V。研究所采用的动态调控策略为4-HIL的高效合成提供了一种新思路。
4-Hydroxyisoleucine (4-HIL) has great potential in the treatment of type II diabetes. Isoleucine dioxygenase (IDO) can convert L-isoleucine (Ile) into 4-HIL. To improve the product of 4-HIL and reduce the by-product L-lysine (Lys) synthesis in recombinant Corynebacterium glutamicum, the Lys-OFF riboswitch was integrated into the upstream of dapA gene, the key gene of Lys synthesis pathway, and the D-RS strain was obtained. The strain could dynamically minimize the synthesis to Lys by the concentration of intracellular and extracellular Lys, and decreased the content of Lys by 46.7%. Secondly, the Ile-activated biosensor Lrp-PbrnFEN was used to control the expression of codon-optimized ido in D-RS strain, and the titer of 4-HIL was increased to 116.3 mmol/L. In order to further improve the titer of 4-HIL, the strong promoter PbrnFE7 was used to dynamically control the expression of odhI and vgb to enhance the supply of α-ketoglutarate and oxygen. Finally, a recombinant strain D-RS-0I7O7V with the 4-HIL titer up to 166.0 mmol/L and Lys content down to 6.5 mmol/L after fermentation in shake flasks was obtained. The dynamic regulation strategy provides a new idea for the efficient biosynthesis of 4-HIL.
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