Lactic acid bacteria (LAB) are central to food fermentation, probiotic delivery, and emerging synthetic biology applications, yet their robust cell envelopes and restriction–modification systems complicate DNA uptake. This review synthesizes practical routes for introducing DNA into LAB—natural competence, electroporation, conjugation, phage-mediated transduction, and biolistics—and outlines vector systems for expression and chromosomal editing, including food-grade strategies. We highlight recent advances that broaden strain tractability while noting strain-to-strain variability and host-specific barriers that still require tailored solutions. These advances directly enable applications in food and probiotic biotechnology, including improving starter robustness, tailoring flavor and texture pathways, and installing food-grade traits without residual selection markers. We close with near-term priorities for standardizing protocols, widening replicon compatibility, and leveraging modern genome-editing platforms to accelerate safe, marker-free engineering of industrial and probiotic LAB.

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications.

Oxidized nicotinamide adenine dinucleotide (NAD⁺) is a biological molecule of systemic importance. Essential role of NAD⁺ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD⁺ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD⁺ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25–70 years old), our quantification of the blood concentration of NAD⁺ (18 μM, minimum 15, max 23) corresponds well to NAD⁺ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18–55 years old), the NAD⁺ levels significantly (p < 0.0001) decrease (to 14 μM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42–83 years old), the blood levels of NAD⁺ (13 μM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD⁺ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD⁺ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

Ribonucleoside hydrolases are enzymes that catalyze the cleavage of ribonucleosides to nitrogenous bases and ribose. These enzymes are found in many organisms: bacteria, archaea, protozoa, metazoans, yeasts, fungi and plants. Despite the simple reaction catalyzed by these enzymes, their physiological role in most organisms remains unclear. In this review, we compare the structure, kinetic parameters, physiological role, and potential applications of different types of ribonucleoside hydrolases discovered and isolated from different organisms.