4-Hydroxyisoleucine (4-HIL) has potential value for treating diabetes. α-Ketoglutarate (α-KG)-dependent l-isoleucine dioxygenase (IDO) can convert l-isoleucine (Ile) into 4-HIL. In our previous study, 4-HIL was de novo synthesized from glucose by expressing the ido gene in Corynebacterium glutamicum strain SN01, an Ile producer, and neither Ile nor α-KG was added. In this study, ribosomal binding site (RBS) engineering was applied for gene expression and 4-HIL biosynthesis in C. glutamicum. The 18 tested RBS sequences showed greatly differing strengths for expressing ido, and 8.10-104.22 mM 4-HIL was produced. To supply the cosubstrate α-KG at different levels, the odhI gene was then expressed using the RBS sequences of high, medium, and low strength in the above mentioned optimal strain SF01 carrying R8-ido. However, 4-HIL production decreased to varying amounts, and in some strains, the α-KG was redirected into l-glutamate synthesis. Next, the O2 supply was further enhanced in three ido-odhI coexpressing strains by overexpressing the vgb gene, and 4-HIL production changed dramatically. 4-HIL (up to 119.27 ± 5.03 mM) was produced in the best strain, SF08, suggesting that the synchronic supply of cosubstrates α-KG and O2 is critical for the high-yield production of 4-HIL. Finally, the avtA gene and the ldhA-pyk2 cluster were deleted separately in SF08 to reduce pyruvate-derived byproducts, and 4-HIL production increased to 122.16 ± 5.18 and 139.82 ± 1.56 mM, respectively, indicating that both strains were promising candidates for producing 4-HIL. Therefore, fine-tuning ido expression and the cosubstrates supply through RBS engineering is a useful strategy for improving 4-HIL biosynthesis in C. glutamicum.

中文翻译：

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重组谷氨酸棒杆菌基因表达和4-羟基异亮氨酸生物合成的核糖体结合位点序列优化

4-羟基异亮氨酸 (4-HIL) 具有治疗糖尿病的潜在价值。α-酮戊二酸 (α-KG) 依赖性 l-异亮氨酸双加氧酶 (IDO) 可以将 l-异亮氨酸 (Ile) 转化为 4-HIL。在我们之前的研究中，4-HIL 是通过在谷氨酸棒杆菌菌株 SN01（一种 Ile 生产者）中表达 ido 基因而从葡萄糖从头合成的，并且既没有添加 Ile 也没有添加 α-KG。在这项研究中，核糖体结合位点 (RBS) 工程应用于谷氨酸棒杆菌中的基因表达和 4-HIL 生物合成。18 个测试的 RBS 序列显示出非常不同的表达 ido 的强度，并且产生了 8.10-104.22 mM 4-HIL。为了提供不同水平的共底物α-KG，然后在上述携带R8-ido的最佳菌株SF01中使用高、中、低强度的RBS序列表达odhI基因。然而，4-HIL 产量下降到不同程度，在一些菌株中，α-KG 被重新定向到 l-谷氨酸合成。接下来，通过过表达 vgb 基因，在三个 ido-odhI 共表达菌株中进一步增强了 O2 供应，并且 4-HIL 的产生发生了巨大变化。4-HIL（高达 119.27 ± 5.03 mM）在最佳菌株 SF08 中产生，这表明共底物 α-KG 和 O2 的同步供应对于 4-HIL 的高产生产至关重要。最后，在 SF08 中分别删除了 avtA 基因和 ldhA-pyk2 簇以减少丙酮酸衍生的副产物，4-HIL 产量分别增加到 122.16 ± 5.18 和 139.82 ± 1.56 mM，表明这两种菌株都是有希望的候选菌株4-HIL。所以，