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.

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