title: Genetically engineered Lactobacillus plantarum WCFS1 constitutively secreting heterologous oxalate decarboxylase and degrading oxalate under in vitro.
abstract: Hyperoxaluria is a major risk factor for urinary stone disease, where calcium oxalate (CaOx) is the most prevalent type of kidney stones. Systemic treatments of CaOx kidney stone patients are limited and comprise drawbacks including recurrence of stone formation and kidney damages. In the present work Lactobacillus plantarum (L. plantarum) was engineered to constitutively secrete oxalate decarboxylase (OxdC) for the degradation of intestinal oxalate. The homologous promoter PldhL and signal peptide Lp_0373 of L. plantarum were used for constructing recombinant vector pLdhl0373OxdC. Results showed that homologous promoter PldhL and signal peptide Lp_0373 facilitated the production, secretion, and functional expression of OxdC protein in L. plantarum. SDS-PAGE analysis revealed that 44 kDa protein OxdC was seen exceptionally in the culture supernatant of recombinant L. plantarum (WCFS1OxdC) harboring the plasmid pLdhl0373OxdC.The culture supernatant of L. plantarum WCFS1OxdC showed OxdC activity of 0.06 U/mg of protein, whereas no enzyme activity was observed in the supernatant of the wild type WCFS1 and the recombinant NC8OxdC strains. The purified recombinant OxdC from the WCFS1OxdC strain showed an activity of 19.1 U/mg protein. The recombinant L. plantarum strain secreted 25 % of OxdC protein in the supernatant. The recombinant strain degraded more than 70 % of soluble oxalate in the culture supernatant. Plasmid segregation analysis revealed that the recombinant strain lost almost 70-89 % of plasmid in 42nd and 84th generation, respectively. In conclusion, recombinant L. plantarum strain containing plasmid pLdhl0373OxdC showed constitutive secretion of bioactive OxdC and also capable of degrading externally available oxalate under in vitro conditions.
authors.affiliation: Department of Biochemistry, School of Biological Sciences, Centre for Advanced Studies in Organismal and Functional Genomics, Madurai Kamaraj University, Madurai, 625 021, India.
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Sasikumar
authors.forename: Sivasamy
authors.initials: S
authors.lastname: Gomathi
authors.forename: Kolandaswamy
authors.initials: K
authors.lastname: Anbazhagan
authors.forename: A Ebenezer
authors.initials: AE
authors.lastname: Baby
authors.forename: J
authors.initials: J
authors.lastname: Sangeetha
authors.forename: Govindan Sadasivam
authors.initials: GS
authors.lastname: Selvam
mesh_terms: D001711:Biotransformation; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D042822:Genomic Instability; D048191:Lactiplantibacillus plantarum / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering; D008970:Molecular Weight; D010070:Oxalates / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010070:Oxalates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase