Dynamic control of 4-hydroxyisoleucine biosynthesis by multi-biosensor in Corynebacterium glutamicum.

4-hydroxyisoleucine (4-HIL) has a potential value in treating diabetes. The α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) can catalyze the hydroxylation of L-isoleucine (Ile) to form 4-HIL by consuming O2. In our previous study, the ido gene was overexpressed in an Ile-producing Corynebacterium glutamicum strain to synthesize 4-HIL from glucose. Here, a triple-functional dynamic control system was designed to regulate the activity of IDO, the supply of α-KG, O2, and Ile and the synthesis of by-product L-lysine (Lys) for promoting 4-HIL synthesis. Firstly, the codon-optimized ido was positively regulated by seven Ile biosensors Lrp-PbrnFEN with different intensities, and the resulting seven D-NI strains produced 38.7-111.1 mM 4-HIL. Then on the basis of D-NI, odhI and vgb were simultaneously regulated by three PbrnFEN with different intensities to synergistically control α-KG and O2 supply. The 4-HIL titer of twelve D-NINONV strains was more than 90 mM, with D-0I7O7V generating the highest titer of 141.1 ± 15.5 mM. Thirdly, ilvA was negatively regulated by an Ile attenuator PilvBNC on the basis of D-NI strains and some D-NINONV strains to balance the synthesis and conversion of Ile. The resulting D-NIPA strains produced 73.6-123.2 mM 4-HIL, while D-7I7O1VPA accumulated 127.1 ± 20.2 mM 4-HIL. Finally, dapA was negatively regulated by a Lys-OFF riboswitch and Lys content decreased by approximately 70% in most D-RS-NIPA strains. A strain D-RS-5IPA with the highest 4-HIL titer (177.3 ± 8.9 mM) and the lowest Lys concentration (6.1 ± 0.6 mM) was successfully obtained. Therefore, dynamic regulation of main and branch pathway by three functional biosensors can effectively promote 4-HIL biosynthesis in C. glutamicum. KEY POINTS: • Three biosensors were coordinated for dynamic 4-HIL biosynthesis in C. glutamicum • Bidirectional regulation of Ile synthesis and conversion promoted 4-HIL synthesis • Negative regulation of Lys synthesis further increased 4-HIL production.