Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified <scp>l</scp>-Isoleucine Biosensor in Recombinant <i>Corynebacterium glutamicum</i>

Abstract

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 <i>ido</i> in <i>Corynebacterium glutamicum</i>. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O₂, makes such <i>de novo</i> 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_<i>brnFE</i> was researched. The native P_<i>brnFE</i> promoter of natural Ile biosensor was still weak even under Ile induction. Through <i>tetA</i> dual genetic selection, several modified stronger P_<i>brnFE</i>N promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of <i>ido</i> 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 <i>ido</i> by the strong P_<i>tacM</i> promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different P_<i>brnFE</i>N-controlled <i>odhI</i> decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O₂ supply in ST04 by different combinations of P_<i>brnFE</i>N-<i>odhI</i> and P_<i>brnFE</i>N-<i>vgb</i> improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by P_<i>brnFE</i>7. This titer was higher than those of all the static metabolic engineered <i>C. glutamicum</i> strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL <i>de novo</i> biosynthesis in <i>C. glutamicum</i>.