Location: Renewable Product Technology ResearchTitle: Purification and characterization of phosphonoglycans from Glycomyces sp. NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338) Author
Submitted to: Journal of Bacteriology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/1/2014
Publication Date: 4/1/2014
Citation: Yu, X., Price, N.P., Evans, B.S., Metcalf, W.W. 2014. Purification and characterization of phosphonoglycans from Glycomyces sp. NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338. Journal of Bacteriology. 196(9):1768-1779. Interpretive Summary: Polysaccharides are produced from a variety of agriculture sources, and are used as thickening and emulsifying agents in food (for example, xanthan gum, pectin), in fracking fluids (guar gum), and as adhesives for wood products. We have discovered an entirely new type of polysaccharide, called phosphonoglycans, that are produced by two soil bacteria which are housed at the ARS Microbial Collection. The phosphonoglycans were recovered from the bacterial cultures (Glycomyces and Stackebrandtia), and have been purified by a variety of chemical means. Using sugar analysis methods it was shown that both of these polysaccharides have unusual phosphonate chemical groups attached to them, making them distinctly acidic. They are large (~ 50 KDa) and form viscous solutions when dissolved in water. Both phosphoglycans contain 2-hydroxyethylphosphonate (2-HEP) groups, and unusual partially methylated sugars (methylgalactoses), as well as galactose and xylose. We have also identified several of the bacterial genes involved in phosphonoglycan biosynthesis. Expression of the pepM gene clusters resulted into production of free 2-HEP. These are a new group of acid sugar polymers, that might find uses as thickeners, acidifiers, or ion exchange materials.
Technical Abstract: Phosphonate biosynthetic gene clusters from two actinomycete strains, Glycomyces sp. NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified by screening for the PEP mutase gene, which is required for the biosynthesis of most phosphonates. Subsequent examination of the two strains by 31P NMR led to the detection of phosphonate-containing exopolysaccharides (EPS) (also known as phosphonoglycans). The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP). The EPS from the Glycomyces strain has a mass of 40-50 kDa and is composed of galactose, xylose, and five distinct partially O-methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal; 2,4-linked 3-MeGal; 2,3-linked Gal; 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O-methylated galactose residues. Isotopic labeling indicated that the O-methyl groups in the Stackebrandtia phosphonoglycan arise from S-adenosylmethionine. Partial acid hydrolysis of the purified EPS from Glycomyces followed by purification and size-selection yielded a 2-HEP linked hexose; LC-MS analysis demonstrated that 2-HEP is ether-linked to the hexose through either O-5 or O-6. Besides, 2-HEP mono (2,3-dihydroxypropyl) ester was detected. A combination of 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and LC-MS analysis applied to Stackebrandtia EPS after partial acid hydrolysis also revealed the presence of 2-HEP mono (2,3-dihydroxypropyl) ester. Heterologous expression of the pepM-containing gene clusters from the two strains led to production of free 2-HEP in Streptomyces lividans. This result, coupled with bioinformatics analysis, suggests that the similar pepM gene clusters of the two organisms are required for phosphonoglycan synthesis.