State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Biotechnol Lett. 2023 Sep;45(9):1169-1181. doi: 10.1007/s10529-023-03407-6. Epub 2023 Jul 3.
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-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L) were similar with control strain S-I (15.73 ± 2.66 g 4-HIL L). 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-I and KIRSA-3-I, and Ile concentration was maintained less than 35 mmol L under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-I produced 22.46 ± 0.96 g 4-HIL L.
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.
探索一种 L-异亮氨酸（Ile）诱导的生物传感器，用于下调 Corynebacterium glutamicum SN01 中的 Ile 合成途径并提高 4-羟基异亮氨酸（4-HIL）的产量。
从 TPP 核糖开关的突变文库中筛选出 4 种不同强度的 Ile 诱导型核糖开关（IleRSN）。首先，将 IleRSN 整合到 SN01 菌株的染色体上，位于 ilvA 基因的上游。携带 P 驱动的 IleRS1 或 IleRS3 的菌株（14.09±1.07、15.20±0.93 g 4-HIL L）的 4-HIL 产量与对照菌株 S-I（15.73±2.66 g 4-HIL L）相似。然后，在 SN01 衍生的菌株 D-RS 中，将另一个 IleRS3-ilvA 拷贝整合到染色体 cg0963 基因的下游，以降低 L-赖氨酸（Lys）的生物合成。在 ilvA 双拷贝菌株 KIRSA-3-I 和 KIRSA-3-I 中，Ile 供应和 4-HIL 产量增加，并且在 IleRS3 的控制下，发酵过程中 Ile 浓度保持在 35 mmol L 以下。所得菌株 KIRSA-3-I 产生 22.46±0.96 g 4-HIL L。
筛选出的 IleRS 可有效下调 C. glutamicum 中的 Ile 合成途径，并且具有不同强度的 IleRSN 可应用于各种条件。
