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Application of CRISPR/Cas9 mediated genome editing for improvement of bacteriocin production in selected lactic acid bacteria
Dissertation Abstract:
Lactic acid bacteria (LAB) are powerful cell factories capable to produce high-value biochemical compounds. They have generally recognized as safe (GRAS) status and are known to produce an efficient biopreservative compound called bacteriocin (nisin). Bacteriocin is not toxic and effective in inhibiting food spoilage and pathogenic bacteria, however, high production cost and low-level production by the producing strain limit its utilization at the industrial scale. This study aims to isolate and characterize LAB from fermented foods and to enhance bacteriocin yield in the LAB model strain Lactococcus lactis ATCC 11454 and selected LAB strains. 55 LAB strains have been isolated from 20 samples of fermented foods and beverages in Malaysia. The isolated strains exhibited tolerance to a wide range of temperature, pH, and salt concentrations. 16S rRNA gene analysis showed that nine different species were identified, i.e., L. rhamnosus (34.5%), L. plantarum (20%), L. fermentum (20%), L. paracasei (12.7%), L. casei (3.6%), Lactobacillus sp. (1.8%), E. faecalis (3.6%), E. faecium (1.8%), and E. durans (1.8%). To screen LAB with desirable characters for genome edit study, the isolated LAB were subjected to phenotypic and genotypic characterization. LABs subtyping was determined through Random Amplified Polymorphic DNA (RAPD), antibiotyping, and plasmid profiling. Dendrogram of M13 primer produced clear strains differentiation and antibiogram revealed distinct differences in antibiotic susceptibility profiles. Plasmid studies showed fifteen plasmidless strains, while others harboured approximately 1 to 5 plasmids. LAB strains with antimicrobial activity were screened, and all cell-free supernatants (CFS) inhibited the growth of at least one out of eight pathogens with inhibition zones from 7 to 22.3 mm. In the present study, Clustered regularly interspaced short palindromic repeats (CRlSPR)/CRlSPR-associated enzyme (Cas) based genome editing was applied to enhance the yield of bacteriocin as it targets DNA at a precise location and can act as a programmable restriction enzyme. A two-plasmid CRISPR/Cas9 system has been developed for lactate dehydrogenase (ldh) gene knockout. The lactic acid deficient strains (ldhΔ) show no reduction in pH as compared to the wildtype (WT). Antibacterial activity of the ldh-strains of L. lactis ATCC 11454, L. paracasei, L. plantarum, L. rhamnosus, and E. faecalis was increased by 6.15 to 100% with an average of 62.07, 27.64, 20.4, 18.4, and 100%, respectively. The highest inhibition against all studied pathogens was shown by L. lactis ATCC 11454-Δldh. To increase nisin production in L. lactis ATCC 11454, the nisin producer strain, nisin regulatory genes (nisRK) was overexpressed. Overexpression of nisRK was also performed in L. lactis ATCC 11454-Δldh to investigate the synergistic effect of double genetic modifications on nisin synthesis. nisRK overexpressed L. lactis ATCC 11454 (nisRKox exhibited increased nisin production in correlation with the increase of antibacterial activity by at least 33.3% in comparison to the WT. Overexpression of nisRK in the ldhΔ-strain (ldhΔnisRKox) increased antibacterial activity by 80.4%. This finding offers a novel approach for the improvement of nisin production. Overall, the present study provides a framework model for LAB and other beneficial bacteria to synthesize and increase the yield of bacteriocin and other valuable compounds.