Development of an NADPH Regeneration System for L-threonine Production in Escherichia coli

NADPH is essential for the biosynthesis of L-threonine, and a deficiency in its supply significantly constrains L-threonine production. To address the challenge of inadequate NADPH availability that adversely affects L-threonine synthesis, we developed an NADPH regeneration system aimed at enhancing the NADPH supply and subsequently improving L-threonine production. Through overexpression of the zwf and gnd genes, which are involved in NADPH generation within the pentose phosphate pathway (PPP), the NADPH/NADP⁺ ratio in the strain was elevated 4.1-fold compared with the control strain, resulting in a 2.0-fold increase in L-threonine production. Subsequently, integration of the asd and thrA1034 genes, which are linked to NADPH consumption, enhanced L-threonine production by 3.6-fold. Moreover, the application of promoter engineering facilitated a 7.1-fold increase in L-threonine production compared with the control strain. Finally, we employed the CRISPR-Cas12f1 system to delete the pgi gene to further examine its impact on L-threonine production. The results indicated an increase in the NADPH/NADP⁺ ratio and a subsequent enhancement in L-threonine production following deletion of the pgi gene. Consequently, the NADPH regeneration system developed in this study demonstrates potential to effectively improve L-threonine production and may serve as a novel strategy for L-threonine synthesis.