Exploring L-isoleucine riboswitches for enhancing 4-hydroxyisoleucine production in Corynebacterium glutamicum

To explore an L-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01.

Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying P_tacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L^-1) were similar with control strain S-_D5I (15.73 ± 2.66 g 4-HIL L^-1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated L-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-_D5I and KIRSA-3-₉I, and Ile concentration was maintained less than 35 mmol L^-1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-₉I produced 22.46 ± 0.96 g 4-HIL L^-1.

The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.