Enhancement of substrate supply and ido expression to improve 4-hydroxyisoleucine production in recombinant...

4-Hydroxyisoleucine (4-HIL) has potential value in treating diabetes. L-isoleucine dioxygenase (IDO) catalyzes the hydroxylation of L-isoleucine (Ile) to form 4-HIL with the concomitant oxidation of α-ketoglutarate (α-KG) and oxygen consumption. In our previous study, by expressing the ido gene in the Ile producer Corynebacterium glutamicum ssp. lactofermentum SN01, 4-HIL was de novo-synthesized from glucose without adding either Ile or α-KG. In this study, synergistically improving the substrates supply and IDO activity was applied to enhance the de novo biosynthesis of 4-HIL. Deletion of aceA and blocking of the glyoxylate pathway effectively enhanced α-KG supply and Ile synthesis and thus improved 4-HIL production to 69.47 ± 2.18 mM, 18.9% higher than in the original strain. Coexpression of mqo with ido further improved Ile synthesis but decreased 4-HIL production, partially due to the inadequate activity of IDO. Coexpression of another gene, ido3, with mqo and ido efficiently promoted IDO activity, thus improving 4-HIL production to 91.54 ± 8.29 mM. Further expression of vgb and promotion of the oxygen uptake rate did not change the 4-HIL titer significantly but increased the 4-HIL production rate in the first 72 h of fermentation. After fermentation in the optimized medium, 4-HIL production by the final strains increased to 112-117 mM, indicating these strains are promising candidates for producing 4-HIL. These results demonstrate that synergistically promoting substrate supply and improving IDO activity are efficient approaches to enhance 4-HIL production in C. glutamicum.