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

Abstract

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-P_brnFEN 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 P_brnFEN with different intensities to synergistically control α-KG and O₂ supply. The 4-HIL titer of twelve D-_NI_NO_NV 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 P_ilvBNC on the basis of D-_NI strains and some D-_NI_NO_NV 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.

Keywords: 4-Hydroxyisoleucine; Corynebacterium glutamicum; Dynamic control; Lrp-PbrnFEN; Lys-OFF riboswitch; PilvBNC.