Structural Characterization And Identification Of Biosynthetic Pathway Of The Exopolysaccharide From Paenibacillus Elgii B69

Polysaccharides are polymeric carbohydrates consisting of linear repeats of mono-or oligosaccharide subunits joined by glycosidic bonds. Polysaccharides are involved in various metabolic functions in biology and are one of the four essential components along with nucleic acids, proteins, and lipids that regulate normal physiological activities. There are several types of polysaccharides, which are classified on the basis of their source of availability into plant polysaccharides, animal polysaccharides, algal polysaccharides, and microbial polysaccharides (bacteria and fungi polysaccharide). Bacterial exopolysaccharides are synthesized and secreted into the extracellular environment when cultured in the medium with a high carbon to nitrogen ratio. In the past decade, the production of the polymeric materials from petrochemical resources has drastically decreased owing to high cost and increased demand and limited availability of oil. In contrast, bacterial exopolysaccharides have drawn considerable attention owing to their short production cycles, relatively low toxicity, unique structure, excellent physical properties, and biodegradable and promising biological activities. Bacterial exopolysaccharides have a wide range of applications for their antioxidant, immunostimulatory, anti-ulcer, hepatoprotective, antitumor effects as well as wastewater bioflocculating activity in food, pharmaceutical, chemical, petrochemical, environmental pollutant treatment and other industries.Recently, the bacterial strain, Paenibacillus elgii B69was shown to be efficient in producing high yields of exopolysaccharide. A series of experiments showed that the exopolysaccharide from P. elgii B69possesses high flocculant activities toward various wastewaters. Monosaccharide composition analysis indicated that the polysaccharide from P. elgii B69contained xylose, which is unusual for bacterial exopolysaccharide. To further explore the role and nature of xylose involved in the synthesis of the exopolysaccharide, the present study aimed to characterize the structure and identify the biosynthetic pathway of the xylose-containing exopolysaccharide from P. elgii B69. The major findings of the study include the following:1. The P. elgii B69exopolysaccharide was purified by deproteinization by combining the enzyme and the Sevag reagent. The main fraction was obtained after fractionating through Q sepharose fast flow and Sepharose CL-6B column. Monosaccharide composition analysis revealed that the exopolysaccharide comprised glucose, xylose, glucuronic acid, and mannose at a ratio of1.95:2.07:1:0.93. The exopolysaccharide was studied by periodate oxidation, Smith degradation, partial hydrolysis, methylation, Gas chromatography-mass spectrometry (GC-MS) analysis, and enzymolysis together with collision-induced dissociation-MS/MS analysis. The resulting structure of the exopolysaccharide is shown below:2. Sequence analysis was performed to search for the genes encoding the UDP-glucuronic acid (UDP-GlcA) decarboxylase required for synthesis of UDP-xylose, the nucleotide sugar precursor of xylose-containing exopolysaccharide and two candidate sequences, designated as Peuxsl and Peuxs2, were found. The UDP-GlcA decarboxylase activities were measured by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of Peuxsl and Peuxs2on the synthesis of the exopolysaccharide were further investigated by a thymidylate synthase gene-based gene knockout system in a newly isolated thymine autotrophy strain. This system was used to substitute the conventional antibiotic resistance gene system in the P. elgii B69strain. The results showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1or Peuxs1knockout strains; the knockout of either Peuxs1or Peuxs2decreased the production and changed the monosaccharide ratio. Exopolysaccharide synthesis was completely abrogated when both the genes were knocked out.3. Sequence analysis indicated six different genes in the EPS gene cluster of P. elgii B69encoding glycosyltransferase that were involved in linkage of the glycosidic bonds of the oligosaccharide subunits. To understand the function of these genes, we aimed to delete each gene and study the deletion phenotypes. Two of the genes proved to encode a priming glucose glycosyltransferase and a side-chain-xylose glycosyltransferase, respectively. Attempts to delete the other genes were unsuccessful due to the accumulation of the toxic intermediate products. All the six genes were cloned and expressed in E. coli, and the enzymes were purified. The activity of the glycosyltransferase was analyzed by MS using membrane fraction as the lipid carrier receptor. Eventually, we were able to characterize the specificities of six different glycosyltransferases.4. Even though several polysaccharides were reported to be produced by Paenibacillus, the biosynthetic pathway has not been completely characterized. The exopolysaccharide biosynthetic pathway is a multistep process that includes the following stages:1. synthesis of sugar-activated precursors;2. assembly of repeat subunits; and3. polymerization and export. In this study, we found that the exopolysaccharide biosynthesis gene clusters from P. elgii B69consist of chromosomal DNA regions of20.2kb encoding18ORFs with identical transcription direction controlled by two promoters. Besides the six glycosyltransferase genes identified in this study, another four genes involved in the biosynthesis of the sugar nucleotide direct precursors, including UDP-glucose, UDP-xylose, UDP-glucuronic acid, and GDP-mannose, are found in the biosynthetic gene clusters; whereas, the genes involved in the synthesis of the indirect precursor of the intermediate substrate are not co-localized and are located outside. Additional six genes encode the proteins required for polymerization and export of the polymer. The abovementioned16genes are co-transcribed by the same promoter, while another two ORFs encoding putative lyases are expressed under the control of a different promoter. The results of this study provide the foundation for future research to further understanding the EPS biosynthetic pathway in Paenibacillus and its related mechanism.