Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum

•De novo biosynthesis of an antidiabete, 4-HIL, was improved in C. glutamicum.•Optimization of RBS sequences for ido expression enhanced 4-HIL biosynthesis.•Fine-tuning cosubstrates pool by odhI-vgb expression promoted 4-HIL synthesis.•avtA or ldhA-pyk2 deletion decreased byproducts content and increased 4-HIL yield. 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.