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

4-Hydroxyisoleucine (4-HIL) has potential value for treating diabetes. alpha-Ketoglutarate (alpha-KG)-dependent alpha-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 alpha-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 alpha-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 alpha-KG was redirected into alpha-glutamate synthesis. Next, the O-2 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 alpha-KG and O-2 is critical for the high-yield production of 4-HIL. Finally, the avtA gene and the IdhA-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.