Construction and performance analysis of NADPH-auxotrophic Corynebacterium glutamicum recombinant

Abstract:

[Objective] The biosynthetic pathway of NADPH in Corynebacterium glutamicum was modified to block NADPH production, thus constructing an NADPH-auxotrophic C. glutamicum recombinant.[Methods] To block NADPH production in cell, we firstly inactivated the glucose-6-phosphate dehydrogenase (Zwf) and malic enzyme (MalE) in an L-lysine high-producing strain C. glutamicum Lys-χ, and replaced the native NADP⁺-dependent isocitrate dehydrogenase (NADP⁺-Icd_Cg) with NAD⁺-Icd_Sm from Streptococcus mutans. Then, we introduced the proton-pumping nicotinamide nucleotide transhydrogenase (PntAB) from Escherichia coli with different expression level controlled by different strength promoters into NADPH-auxotrophic C. glutamicum recombinant. Lastly, we analyzed the changes of intracellular redox level and production intensity of L-lysine in the recombinant strains with different PntAB expression levels. [Results] There was no detectable NADPH in the recombinant strain C. glutamicum Lys-χ ΔZMI_Cg::I_Sm (i.e., Lys-χ1), indicating that strain Lys-χ1 was an NADPH-auxotrophic C. glutamicum. Strain Lys-χ1 grew well and accumulated L-lysine in the basic medium with gluconolactone as carbone source, whereas it could not grow with glucose, pyruvate, α-ketoglutaric acid and oxalacetic acid as carbon source. In addition, overexpression of PntAB in strain Lys-χ1 replenished the intracellular NADPH level, but the NADPH level in different recombinant strains was different because there was the different expression level of PntAB under the different intensity of promoters. And these affected the cell growth and L-lysine production.[Conclusion] The strain Lys-χ1 could be used as a chassis cell for the capacity of strategies to regenerate NADPH in vivo, thus obtaining recombinant strains with different intracellular NADPH levels. Therefoere, this study provided a basis for investigating the regulatory mechanism of NADPH on physiology and metabolism of microbial cells.