4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O₂, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-P_brnFE was researched. The native P_brnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger P_brnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9–74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong P_tacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different P_brnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O₂ supply in ST04 by different combinations of P_brnFEN-odhI and P_brnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by P_brnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

中文翻译：

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通过改良的 l-异亮氨酸生物传感器在重组谷氨酸棒杆菌中动态控制 4-羟基异亮氨酸生物合成。

4-羟基异亮氨酸（4-HIL）是一种很有前景的治疗糖尿病的药物，它可以通过在谷氨酸棒杆菌中表达Ile双加氧酶基因ido由自产的l-异亮氨酸（Ile）合成。然而，Ile、α-酮戊二酸（α-KG）和O ₂三种底物的需求使得这种从头生物合成在静态工程条件下难以有效实现。在这项研究中，研究了 Ile 生物传感器 Lrp-P _brnFE对 4-HIL 生物合成的动态控制。即使在 Ile 诱导下，天然 Ile 生物传感器的天然 P _brnFE启动子仍然很弱。通过tetA通过双重遗传选择，从 Ile 生物传感器的合成文库中获得了几个经过修饰的更强 P _brnFE N 启动子。通过改良的 Ile 生物传感器对ido表达的动态调节将 4-HIL 滴度从 24.7 mM 增加到 28.9–74.4 mM。最好的菌株 ST04 产生的 4-HIL 甚至比通过强 P _tacM启动子 (69.7 mM)过表达ido的静态菌株SN02 多_一点。通过表达不同的 P _brnFE N 控制的odhI对 ST04 中 α-KG 供应的进一步动态调节降低了 4-HIL 的产生，但增加了l-谷氨酸或 Ile 的积累。然而，α-KG 供应和 O _{2 的}协同调节供应在ST04由P的不同组合_brnFE N- odhI和P _brnFE N- VGB提高了4-HIL生产显著，最高滴度（135.3毫摩尔）在ST17菌株获得用P调节所有三种基因_brnFE 7.本滴度高于所有构建的静态代谢工程谷氨酸棒杆菌菌株。因此，通过改良的 Ile 生物传感器进行动态调节是增强谷氨酸棒杆菌中4-HIL从头生物合成的主要策略。