Identification and characterization of six glycosyltransferases involved in the biosynthesis of a new bacterial exopolysaccharide in Paenibacillus elgii

• Published in: Applied microbiology and biotechnology Vol. 102; no. 3; pp. 1357 - 1366
• Main Authors: Ou, Li, Ang, Li, Chujun, Zhang, Jingyu, Huang, Yongli, Meng, Shenjing, Yuan, Junhua, Huang, Xu, Gao, Yulong, Yao, Rui, Yin, Jinpan, Hu, Bin, Ding, Xiufang, Hu
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.02.2018; Springer; Springer Nature B.V
• Subjects: Applied Genetics and Molecular Biotechnology; Bacillus; Bacteria; Biomedical and Life Sciences; Biosynthesis; Biosynthetic Pathways; Biotechnology; Chemical properties; E coli; Escherichia coli - genetics; Flocculation; Gene clusters; Gene Deletion; Gene Knockout Techniques; Genes; Genes, Bacterial; Glycosides - chemistry; Glycosyltransferases; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Identification and classification; Life Sciences; Linkages; Mass Spectrometry; Mass spectroscopy; Microbial Genetics and Genomics; Microbial polysaccharides; Microbiological synthesis; Microbiology; Multigene Family; Observations; Paenibacillus; Paenibacillus - enzymology; Paenibacillus - genetics; Phenotype; Physiological aspects; Pollutants; Polysaccharides, Bacterial - biosynthesis; Priming; Waste Water - microbiology; Wastewater; Xylose; Xylose - chemistry; Glycosyltransferase; Bacterial exopolysaccharide; Gene knockout; Enzymatic reactions; Paenibacillus elgii
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-017-8673-y

Paenibacillus elgii B69 produces a new xylose-containing exopolysaccharide (EPS) that effectively removes the pollutants from wastewater through flocculation. However, information about the biosynthesis of this EPS is limited. In this study, sequence analysis showed six putative glycosyltransferases (GTs) genes in polysaccharide gene clusters involved in glycosidic linkages of repeating units. Each gene was deleted and phenotypes were examined to understand the functions of these genes. Two of the genes were deleted successfully to encode a priming glucose GT and a side-chain xylose GT, but other genes were unsuccessfully deleted because of the accumulation of toxic intermediate products. The six genes were cloned and expressed in Escherichia coli , and the corresponding enzymes were purified. The activity of GTs was analyzed through mass spectrometry by using the purified membrane fraction as a lipid carrier receptor after a hexasaccharide repeated unit was reconstructed in vitro. The specificities of six different GTs and the building order of the hexasaccharide were characterized. This study provided a basis for future research on the biosynthetic pathway of EPS in Paenibacillus or other genera.