State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
ACS Synth Biol. 2020 Sep 18;9(9):2378-2389. doi: 10.1021/acssynbio.0c00127. Epub 2020 Sep 2.
4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene in . However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O, makes such biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-P was researched. The native P promoter of natural Ile biosensor was still weak even under Ile induction. Through dual genetic selection, several modified stronger PN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing by the strong P promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PN-controlled decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O supply in ST04 by different combinations of PN- and PN- improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by P7. This titer was higher than those of all the static metabolic engineered strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL biosynthesis in .
4-羟基异亮氨酸（4-HIL）是一种有前途的治疗糖尿病药物，可通过在 中表达异亮氨酸双加氧酶基因，从自身产生的 L-异亮氨酸（Ile）合成。然而，该生物合成需要 3 种底物，即 Ile、α-酮戊二酸（α-KG）和 O，使得在静态工程条件下难以有效地满足这种生物合成。在本研究中，通过 Ile 生物传感器 Lrp-P 研究了 4-HIL 生物合成的动态控制。即使在 Ile 诱导下，天然 Ile 生物传感器的天然 P 启动子仍然较弱。通过双基因选择，从 Ile 生物传感器的合成文库中获得了几个经过修饰的更强的 PN 启动子。通过修饰后的 Ile 生物传感器的动态表达调控，将 4-HIL 产量从 24.7mM 提高到 28.9-74.4mM。最佳菌株 ST04 甚至比过表达强 P 启动子的静态菌株 SN02 产生更多的 4-HIL（69.7mM）。进一步通过表达不同的 PN 控制的 来动态调节 ST04 中的α-KG 供应，降低了 4-HIL 的产量，但增加了 l-谷氨酸或 Ile 的积累。然而，通过不同的 PN-和 PN-组合对 ST04 中的α-KG 供应和 O 供应进行协同动态调节，显著提高了 4-HIL 的产量，在 ST17 菌株中通过 P7 调控所有 3 个基因获得了最高产量（135.3mM）。该产量高于以往构建的所有静态代谢工程 菌株。因此，通过修饰后的 Ile 生物传感器进行动态调节是增强 中的 4-HIL 生物合成的主要策略。
