Calcium (Ca2+)-regulated exopolysaccharide biosynthesis in probiotic Lactobacillus plantarum K25 as analyzed by an omics approach

Abstract

Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca²⁺ on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl₂ at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca²⁺ also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca²⁺, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca²⁺ upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca²⁺ activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca²⁺, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.

Keywords: Lactobacillus plantarum; biosynthesis gene; calcium; exopolysaccharides.