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BACKGROUND: Nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide, plays an important in anti‐aging and disease. Lactococcus lactis, an important probiotic lactic acid bacteria (LAB), has shown great potential for the biosynthesis of NMN, which will significantly affect the probiotic effects of the dairy products. RESULTS: We used the CRISPR/nCas9 technique to knockout nadR gene of L. lactis NZ9000 to enhance the accumulation of NMN by 61%. The nadE* gene from Francisella tularensis with codon optimization was heterologous in L. lactis NZ9000ΔnadR and has a positive effect on NMN production. Combined with optimization of the concentration of substrate nicotinamide, a final intracellular NMN titer was 2289 μmol L–1 mg–1 with 10 g L–1 nicotinamide supplement, which was 5.7‐fold higher than that of the control. The transcription levels of key genes (pncA, nadD and prs1) involved in NMN biosynthesis were up‐regulated by more than two‐fold, indicating that the increase of NMN titer was attributed to FtnadE* heterologous expression. CONCLUSION: Our study provides a better understanding of the NMN biosynthesis pathway in L. lactis, and can facilitate NMN production in LAB via synthetic biology approaches. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Streptococcus thermophilus, one of the most important lactic acid bacteria, is widely used in food fermentation, which is beneficial to improve food quality. However, the current genetic transformation systems are inefficient for S. thermophilus S‐3, which hinders its further study. RESULTS: We developed three electroporation transformation methods for S. thermophilus S‐3, and optimized various parameters to enhance the transformation efficiency up to 1.3 × 106 CFU/μg DNA, which was 32‐fold higher than that of unoptimized. Additionally, transcriptional analysis showed that a series of competence genes in S. thermophilus S‐3 were remarkedly up‐regulated after optimization, indicating that improvement of transformation efficiency was attributed to the expression level of competence genes. Furthermore, to prove their potential, expression of competence genes (comEA, cbpD and comX) were employed to increase transformation efficiency. The maximum transformation efficiency was obtained by overexpression of comEA, which was 14‐fold higher than that of control. CONCLUSION: This is the first report of competence gene expression for enhancing transformability in S. thermophilus, which exerts a positive effect on the development of desirable characteristics strains. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]