Regulation Of Extracellular Polysaccharide In Paenibacillus Mucilaginosus And Transcriptome Study On Its Related Genes

Paenibacillus mucilaginosus, featured with multifunction and high resistance, is the top choice for microbial fertilizers. However, the strain of P. mucilaginosus produced thick exopolysaccharides during its growth, which consumes much nutrition, develops zoogloea, depresses continuous growth, reduces fermentation density, and restricts its application. Based on previous studies, P. mucilaginosus 3016 was used to explore the main controlling factors influencing exopolysaccharides production. In order to increase fermentation density and efficiency, reduce exopolysaccharides production, delay the production of exopolysaccharides, Respones Surface Methodology was used to optimize components of seed medium. Transcriptomics of the two cultures induced by different media, revealed the mechanism of exopolysaccharides formation and the results could be used to improve the quality of the microbial fertilizer. Three main conclusions were shown as follows.1. The components of seed medium for P. mucilaginous were optimized to reduce the amount of extracellular polysaccharide and increase fermentation density. Based on the results of single factor experiments, the optimum carbon and nitrogen sources were maltose and tryptone. By the Plackett-Burman(PB) designing method, nine factors affecting the exopolysaccharides production were evaluated and the concentration of maltose and MgSO4·7H2O are demonstrated to be the most important factors. RSM analysis was used to choose the optimal condition, which can reduce exopolysaccharides production and increase fermentation density. The optimal seed medium contained maltose 2.5g/L, tryptone 1.0g/L, MgSO4·7H2O 0.73g/L, K2HPO4·3H2O 0.4g/L, NaCl 0.06 g/L, FeCl3 0.6mg/L, salicylic acid 10mg/Land CaCO3 1.0g/L. Under the optimized medium, the production of exopolysaccharides was reduced to 0.0302 mg/mL, and the concentration of fermentation density could be up to 4.12×108 cfu/mL, increased by nearly 10 times.2. The transcriptional expression were followed.. The strain of P. mucilaginous was induced by two different media, one lead P. mucilaginosus produce more extracellular polysaccharide(9hN-) while the other less(9hN+). The RNA-Seq results showed that, totally 13,632,780 and 10,007,128 reads were sequenced for 9hN- and 9hN+, respectively. RNA-Seq datas showed that, there are 1580 genes differentially expressed under 9hN- and 9hN+, which were mainly involved in biological process(i.e. regulation, localization, metabolic process, regulation of biological progress and single-organism process), cell part(i.e. cell, cell part and macromolecular complex in cellular component cluster) and molecular function(i.e.bing and catalytic activity). Among the different expressed genes, 1084 genes were up-regulated, while the other were down-regulated.3. The transcriptional genes involved in the formation of exopolysaccharides were analyzed. Under the induction of 9hN- for glucolysis, the first and second key enzymes were up-regulated, while the last key enzyme(pyruvate kinase) was down-regulated. However, it was not the case for gluconeogenesis, because the first and second key enzymes were up-regulated, but the other two enzymes did not express significantly. The results suggested that, there was a commom intermediate product fructose phosphate in gluconeogenesis and glycolysis, and fructose phosphate was substrate for the synthesis of nucleotide sugar. We also found the up-regulated genes pgm and galE coding activated nucleotide sugar in amino sugar and sugar nucleotides pathway, which suggested that nucleotide was vital in expolysaccharide synthesis. In this study, results showed all the glycosyl transferase genes were up-regulated significantly. This suggested that, glycosyl transferase may play an important role in exopolysaccharide synthesis. In addition, under the induction of 9hN-, lip, galE and cps genes related with transporting, adjustment and CPS forming, were all up-regulated, respectively. GlnA, coding glutamine syntheses involved in nitrogen metabolism, was up-regulated significantly, which imply the gene may be correlated with the synthesis of exopolysaccharide in P. mucilaginosus.