Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

Abstract

We have previously isolated a lactic acid bacterium (LAB), Pediococcus pentosaceus LP28, from the longan fruit Euphoria longana. Since the plant-derived LAB strain produces an extracellular polysaccharide (EPS), in this study, we analyzed the chemical structure and the biosynthesizing genes for the EPS. The EPS, which was purified from the LP28 culture broth, was classified into acidic and neutral EPSs with a molecular mass of about 50 kDa and 40 kDa, respectively. The acidic EPS consisted of glucose, galactose, mannose, and N-acetylglucosamine moieties. Interestingly, since pyruvate residue was detected in the hydrolyzed acidic EPS, one of the four sugars may be modified with pyruvate. On the other hand, the neutral EPS consisted of glucose, mannose, and N-acetylglucosamine; pyruvate was scarcely detected in the polysaccharide molecule. As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs) with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.

Keywords: Exopolysaccharide gene cluster; Pediococcus pentosaceus LP28; Pyruvic acid; Whole genome sequence.

Fig. 1

Fractionation of EPS produced by Pediococcus pentosaceus LP28 strain by ion-exchange chromatography. The elute fractions were analyzed for carbohydrate content by the phenol-sulfuric acid reaction.

Fig. 2

HPLC chromatogram of monosaccharide from hydrolyzed EPS. The hydrolyzed acidic and neutral EPSs were dissolved in purified water. After the filtration, each hydrolyzed EPS was analyzed by HPLC. Chromatographic peaks were identified by monosaccharide standard solutions (Thermo Scientific). Glc: Glucose, Gal: Galactose, GlcN: Glucosamine, Man: Mannose.

Fig. 3

HPLC analysis of pyruvic acid in the hydrolyzed EPSs from LP28 strain. Hydrolyzed neutral EPS (B), acidic EPS (C) and 1 mM of authentic pyruvic acid (A) were dissolved in 3.8 mM H₂SO₄ and analyzed by the HPLC.