Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB
2019
Paul, Eldho | Albert, Abhishek | Ponnusamy, Sasikumar | Venkatesan, Sangeetha | Govindan Sadasivam, Selvam
Lactobacillus plantarum WCFS1 (L. plantarum WCFS1) is commonly used as a potential cell factory because of its ‘generally recognized as safe’ status. The plasmid instability and the presence of antibiotic selection marker complicate the application of genetically modified L. plantarum in human clinical trials. In the present study, we aimed to integrate oxalate decarboxylase (oxdC) gene of Bacillus subtilis origin by targeted chromosomal mutation in L. plantarum using mobile genetic element Ll.LtrB as a therapeutic tool against calcium oxalate stone disease. oxdC expression cassette was constructed and integrated into a targeted gene, thymidylate synthase (thyA) in the L. plantarum genome. The dependence on external thymidine for growth and survival was established by live dead population assay and SEM (scanning electron microscopy) analysis. The western blotting assay showed the secretion of 44 kDa OxdC protein in the culture supernatant of L. plantarum ∆thyA:OxdC. The biologically contained recombinant strain significantly reduced the oxalate concentration by 53% and exhibited a loss of viability when introduced to environmental samples. Biologically contained L. plantarum secreting OxdC constructed using group II intron has the ability to degrade oxalate present in the extracellular environment and could be used as a therapeutic tool for the calcium oxalate stone disease.
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