Luo Xiaoping, Su Buli, Lai Peixuan, Li Man, Deng Ming-Rong, Zhu Honghui
Key Laboratory of Agricultural Microbiomics and Precision Application (MARA), Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Key Laboratory of Agricultural Microbiome (MARA), State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, 510070, People's Republic of China.
Appl Biochem Biotechnol. 2025 Aug 4. doi: 10.1007/s12010-025-05351-9.
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.
烟酰胺腺嘌呤二核苷酸磷酸（NADPH）对于L-苏氨酸的生物合成至关重要，其供应不足会显著限制L-苏氨酸的生产。为应对NADPH可用性不足对L-苏氨酸合成产生不利影响这一挑战，我们开发了一种NADPH再生系统，旨在增加NADPH的供应，进而提高L-苏氨酸的产量。通过过表达参与磷酸戊糖途径（PPP）中NADPH生成的zwf和gnd基因，该菌株中的NADPH/NADP比率相比对照菌株提高了4.1倍，L-苏氨酸产量增加了2.0倍。随后，整合与NADPH消耗相关的asd和thrA1034基因，使L-苏氨酸产量提高了3.6倍。此外，启动子工程的应用使L-苏氨酸产量相比对照菌株提高了7.1倍。最后，我们使用CRISPR-Cas12f1系统删除pgi基因，以进一步研究其对L-苏氨酸生产的影响。结果表明，删除pgi基因后，NADPH/NADP比率增加，随后L-苏氨酸产量提高。因此，本研究开发的NADPH再生系统显示出有效提高L-苏氨酸产量的潜力，可能成为L-苏氨酸合成的一种新策略。
