Paul Eldho, Albert Abhishek, Ponnusamy Sasikumar, Venkatesan Sangeetha, Govindan Sadasivam Selvam
Department of Biochemistry, Centre for Excellence in Genomics Science, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India.
Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY, USA.
Arch Microbiol. 2019 May;201(4):467-476. doi: 10.1007/s00203-018-1585-0. Epub 2018 Oct 22.
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.
植物乳杆菌WCFS1（L. plantarum WCFS1）因其“一般认为安全”的地位而常被用作潜在的细胞工厂。质粒不稳定性和抗生素选择标记的存在使转基因植物乳杆菌在人体临床试验中的应用变得复杂。在本研究中，我们旨在利用移动遗传元件Ll.LtrB作为治疗草酸钙结石病的工具，通过在植物乳杆菌中进行靶向染色体突变来整合枯草芽孢杆菌来源的草酸脱羧酶（oxdC）基因。构建了oxdC表达盒并将其整合到植物乳杆菌基因组中的靶向基因胸苷酸合成酶（thyA）中。通过死活菌数量测定和扫描电子显微镜（SEM）分析确定了对外部胸苷生长和存活的依赖性。蛋白质印迹分析表明，在植物乳杆菌∆thyA:OxdC的培养上清液中分泌了44 kDa的OxdC蛋白。生物封闭的重组菌株使草酸盐浓度显著降低了53%，并且在引入环境样品时表现出活力丧失。使用II组内含子构建的分泌OxdC的生物封闭植物乳杆菌具有降解细胞外环境中存在的草酸盐的能力，可作为草酸钙结石病的治疗工具。
