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

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• 4-hydroxyisoleucine (4-HIL) is a valuable compound with potential applications in diabetes treatment.
• The biosynthesis of 4-HIL involves the α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) enzyme, which hydroxylates L-isoleucine (Ile) using O2.
• Previous efforts focused on overexpressing the ido gene in an L-isoleucine-producing Corynebacterium glutamicum strain.

Purpose of the Study:

• To design and implement a triple-functional dynamic control system for optimizing 4-hydroxyisoleucine (4-HIL) biosynthesis in Corynebacterium glutamicum.
• To enhance the production of 4-HIL by dynamically regulating key metabolic pathways, including IDO activity, α-KG and O2 supply, L-isoleucine synthesis and conversion, and L-lysine byproduct formation.

Main Methods:

• Engineered Corynebacterium glutamicum strains using a codon-optimized ido gene regulated by Lrp-PbrnFE N biosensors for positive control of 4-HIL synthesis.
• Simultaneous dynamic regulation of odhI and vgb genes, controlled by PbrnFE N, to manage α-KG and O2 supply.
• Negative regulation of the ilvA gene using an Ile attenuator (P_ilvBNC) to balance L-isoleucine metabolism.
• Downregulation of the dapA gene via a Lys-OFF riboswitch to minimize L-lysine byproduct accumulation.

Main Results:

• Positive regulation of ido resulted in 4-HIL titers ranging from 38.7 to 111.1 mM.
• Combined regulation of IDO, α-KG, and O2 supply (D-NI N OV strains) yielded over 90 mM 4-HIL, with a peak of 141.1 mM.
• Negative regulation of ilvA (D-NIPA strains) produced 73.6–123.2 mM 4-HIL, and D-7I7O1VPA reached 127.1 mM.
• Downregulation of dapA significantly reduced L-lysine levels by ~70%, leading to the highest 4-HIL titer of 177.3 mM (D-RS-5IPA strain) with minimal L-lysine (6.1 mM).

Conclusions:

• A triple-functional dynamic control system effectively enhances 4-HIL biosynthesis in Corynebacterium glutamicum.
• Coordinated regulation of main and branch metabolic pathways using biosensors significantly boosts 4-HIL production.
• Bidirectional control of L-isoleucine synthesis and conversion, coupled with reduced L-lysine byproduct formation, is crucial for high-yield 4-HIL fermentation.