Enhanced Expression of PEPC Promotes the Synthesis of 4-Hydroxyisoleucine by Corynebacterium glutamicum

Abstract:

[Objective] This study aims to achieve the efficient biosynthesis of 4-hydroxyisoleucine (4-HIL) in Corynebacterium glutamicum (C. glutamicum). [Method] The isoleucine (Ile) inducible promoters (P_brnFEN) of varying strengths were used to dynamically upregulate the expression of the isoleucine dioxygenase (IDO) gene ido and the Vitreoscilla hemoglobin (VHb) gene vgb. On this basis, constitutive promoters of different strengths were employed to enhance the expression of the phosphoenolpyruvate carboxylase (PEPC) gene ppc and the pyruvate carboxylase gene pyc. [Result] Among the recombinant strains harboring different P_brnFEN promoters, strain C. glutamicum SN-I₇V₇ (SN-I₇V₇) was selected. After 144 h of fermentation with this strain, the concentration of 4-HIL reached (109.14±1.03) mmol/L, representing a 46.69% increase compared with the control strain C. glutamicum SN02 (SN02). Subsequently, overexpression of ppc with the strong promoter P_tacM led to the development of strain C. glutamicum RS-MP (RS-MP), an elite 4-HIL producer. This strain achieved a 4-HIL concentration of (145.76±6.04) mmol/L, a 33.55% improvement over the control strain SN-I₇V₇. [Conclusion] The screened P_brnFEN combinations and the ppc overexpression strategy provide a theoretical basis for the efficient biosynthesis of 4-HIL in C. glutamicum.