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<p>The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of <i>ldh1</i> and <i>ldh2</i> in <i>Lacticaseibacillus paracasei</i> NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δ<i>ldh1</i>, Δ<i>ldh2</i>, and Δ<i>ldh1</i>Δ<i>ldh2</i>). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δ<i>ldh1</i>, Δ<i>ldh2</i>, and Δ<i>ldh1</i>Δ<i>ldh2</i>, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21&#xa0;g/L L-lactic acid and 10.74 ± 0.19&#xa0;g/L D-lactic acid, the Δ<i>ldh1</i> mutant produced 76.31 ± 2.22&#xa0;g/L L-lactic acid and 7.72 ± 0.36&#xa0;g/L D-lactic acid, while the Δ<i>ldh2</i> mutant yielded 81.73 ± 0.46&#xa0;g/L L-lactic acid and ND (not detected) D-lactic acid. The Δ<i>ldh1</i>Δ<i>ldh2</i> double mutant generated 75.57 ± 2.96&#xa0;g/L L-lactic acid and ND (not detected) D-lactic acid. The Δ<i>ldh1</i>Δ<i>ldh2</i> double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of <i>ldh2</i> in D-lactate biosynthesis. These results indicate that targeted deletion of <i>ldh</i> genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of <i>ldh2</i> was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of <i>ldh1</i> alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of <i>ldh1</i> and <i>ldh2</i> in controlling lactic acid stereospecificity in <i>L. paracasei</i> NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.</p> Graphical Abstract <p></p>

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CRISPR/Cas9-Mediated Knockout of the NAD-Dependent Lactate Dehydrogenases for Altered Stereospecific Lactic Acid Production in Lacticaseibacillus paracasei NC4

  • 期刊论文

  • 发表日期：

    2026年4月7日

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  • Nhat Huy Chu,
  • Hoang Duc Le,
  • Ngoc Tung Quach,
  • Thi Khanh Linh Pham,
  • Ngoc Anh Ho,
  • Thi Hong Ha Nguyen,
  • Xuan Khoi Tran,
  • Bich Ngoc Pham,
  • Hoang Ha Chu

摘要

The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of ldh1 and ldh2 in Lacticaseibacillus paracasei NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δldh1, Δldh2, and Δldh1Δldh2). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δldh1, Δldh2, and Δldh1Δldh2, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21 g/L L-lactic acid and 10.74 ± 0.19 g/L D-lactic acid, the Δldh1 mutant produced 76.31 ± 2.22 g/L L-lactic acid and 7.72 ± 0.36 g/L D-lactic acid, while the Δldh2 mutant yielded 81.73 ± 0.46 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant generated 75.57 ± 2.96 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of ldh2 in D-lactate biosynthesis. These results indicate that targeted deletion of ldh genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of ldh2 was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of ldh1 alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of ldh1 and ldh2 in controlling lactic acid stereospecificity in L. paracasei NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.

Graphical Abstract