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Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB

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Abstract

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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Acknowledgements

This study was supported by Department of Biotechnology (DBT), New Delhi, India (BT/PR4203/MED/30/681/2011). The first author thanks, CSIR, New Delhi for Senior Research Fellowship. The authors thank UGC-CEGS, -CAS, -NRCBS, DST-FIST, -PURSE, -SERB and DBT-IPLS program for the central instrumentation facility at SBS, MKU. The authors are grateful to Professor Michiel Kleerebezem, Wageningen, Centre for Food Sciences, Netherlands, for providing the strain Lactobacillus plantarum WCFS1, Professor Alan M. Lambowitz, School of Biological Sciences, University of Texas at Austin, USA for providing the plasmid pACD4C, Dr. Geir Mathiesen, Norwegian University of Life Sciences, Norway for providing plasmid pLP_0373AmyA, Dr. Stephen Bornemann for providing the plasmid pLB36 consisting oxdC gene.

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Authors and Affiliations

  1. Department of Biochemistry, Centre for Excellence in Genomics Science, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India

    Eldho Paul, Abhishek Albert, Sangeetha Venkatesan & Selvam Govindan Sadasivam

  2. Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY, USA

    Sasikumar Ponnusamy

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  1. Eldho Paul
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  2. Abhishek Albert
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  4. Sangeetha Venkatesan
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  5. Selvam Govindan Sadasivam
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Correspondence to Selvam Govindan Sadasivam.

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Communicated by Yusuf Akhter.

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Paul, E., Albert, A., Ponnusamy, S. et al. Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB. Arch Microbiol 201, 467–476 (2019). https://doi.org/10.1007/s00203-018-1585-0

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