ArticleMicrobial cell factories2019
A rapid and versatile tool for genomic engineering in Lactococcus lactis.
Article in Microbial cell factories, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 44 papers.
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Who cites it
44 citing papers in PubMed, 87 citations in OpenAlex.
- Menaquinone biosynthesis inEngineering microbiology · 2026Review
- Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.Archives of microbiology · 2026Review
- An optimized CRISPR/Cas9-based genome editing platform enhances bacteriocin production in Lactiplantibacillus plantarum.Journal of biological engineering · 2026Article
- Prospects and challenges in using engineered lactic acid bacteria in aquaculture applications.Engineering microbiology · 2026Review
- Genetically Modified Lactic Acid Bacteria in the EU Food Chain: Applications, Benefits, and Risk Assessment.International journal of molecular sciences · 2026Review
- Application and Research Prospects of CRISPR/Cas Gene Editing Technology in Lactic Acid Bacteria.Microorganisms · 2026Review
- Review
- Enhanced editing ofApplied and environmental microbiology · 2026Article
- Occurrence and applications of CRISPR-Cas systems in bifidobacteria.Applied and environmental microbiology · 2026Review
- CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.Pharmaceutics · 2026Review
- Advances in gene editing tools for four typical Gram-positive bacteria.Frontiers in microbiology · 2026Review
- Probiotic-Based Materials as Living Therapeutics.Advanced materials (Deerfield Beach, Fla.) · 2026Review
- Clinical and Genomic Characterization of Recalcitrant Enterococcal Bacteremia: A Multicenter Prospective Cohort Study (VENOUS).The Journal of infectious diseases · 2025Article
- Being a better version of yourself: genetically engineered probiotic bacteria as host defense enhancers in the control of intestinal pathogens.Gut microbes · 2025Review
- Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.World journal of microbiology & biotechnology · 2025Article
- Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions.International journal of molecular sciences · 2025Review
- Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.Microbial biotechnology · 2024Article
- Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.Applied microbiology and biotechnology · 2024Article
- Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000.Biotechnology letters · 2023Article
- Genetic Engineering of Resident Bacteria in the Gut Microbiome.Journal of bacteriology · 2023Review
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
backgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.
resultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h.
conclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.
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