Structure,Biological Activity Of Exopolysaccharides And Transcriptome-Differentially Expressed Gene Analysis From Two Strains Of Rhodotorula Mucilaginosa

Yeast exopolysaccharides?YEPS?,a class of major microbial polysaccharides with physiological function,can be used as organic immunomodulators and potential natural antioxidants in food and medicine with a broad market prospect.In this study,two single exopolysaccharide components,RmEPS-11 and REPS2-A,were isolated and purified from the liquid media of Rhodotorula mucilaginosa?R.mucilaginosa CM-1 and R.mucilaginosa CICC 33013?.Fourier transform infrared spectroscopy?FT-IR?,ion chromatography?ICS?,nuclear magnetic resonance spectrometer?NMR?,and methylation analysis were used to analyze the structures,anti-hepatoma pathway,and hepato-protective mechanism of the exopolysaccharides produced by these two strains.A new generation of high-throughput sequencing technology was used to perform global transcriptome analysis of the R.mucilaginosa strains,thereby successfully establishing a transcriptome information platform for R.Mucilaginosa,which may be beneficial for further research on the sugar production mechanisms of R.mucilaginosa and identification of sugar-producing genes.The main results are as follows:?1?First,the yield of the water-soluble exopolysaccharide RmEPS from the liquid mediumof R.mucilaginosa CM-1 was determined to be 7.35 g/L.Pure polysaccharide RmEPS-11 was obtained after isolation and purification of RmEPS,the structure of which was analyzed later.Results showed that RmEPS-11?with an average molecular weight of 2.3×10⁵ Da?was composed of arabinose,galactose,glucose,and mannose with a molar ratio of 1.3:4.4:7.8:86.4,respectively.According to FT-IR,NMR,and methylation analysis,the skeleton of RmEPS-11 consisted of 1,3-Man,while the branches were composed of 1,2-Glc,1,2,6-Gal,and 1,2,3,4-Ara.In addition,the isolation,purification,and structural analysis of exopolysaccharide metabolized by R.mucilaginosa CICC 33013 were performed.Results indicated that the yield of the water-soluble exopolysaccharide REPS from R.mucilaginosa CICC 33013was 6.2 g/L.REPS2-A,isolated and purified from REPS,consisted of galactose,arabinose,glucose,and mannose in a molar ratio of 63.1:0.2:18.3:18.3,respectively,with an average molecular weight of 7.125×10⁶ Da.Based on FT-IR,NMR,and methylation analysis,REPS2-A was shown to be a highly branched polysaccharide with a skeleton composed of 1,3-Gal,Man,Gal,and Ara residues,the branches of which consistedof 1,2-Glc,1,4-Man,1,3-Glc,1,4,6-Man,and 1,2,3,4-Ara.?2?The antioxidant activities of exopolysaccharides from different strains of R.mucilaginosa were investigated.RmEPS-11 showed high DPPH?66.15%?and ABTS free radical scavenging activities?82.8%?,reducing power?0.753?,and oxygen radical absorption capacity?560.38±6.45?MTE/g?at a concentration of 8 mg/mL.Comparative analysis showed significant free radical scavenging capacity of REPS2-A in DPPH,ABTS,and reducing power assays.REPS2-A?8 mg/mL?showed remarkable DPPH free radical scavenging activity?clearance rate was 49.1%?,ABTS free radical scavenging activity?51.2%?,and high reducing power?0.352?.However,the antioxidant activity of RmEPS-11was higher than that of REPS2-A under the same conditions.The reason is that the molecular weight of polysaccharide determines the strength of its antioxidant capacity,the smaller is molecular weight,the stronger is its antioxi-dant activity.?3?The hepato-protective mechanism of RmEPS-11 in isoniazid?INH?and rifampicin?RIF?-treated mice was investigated.Compared to mice treated with INH and RIF,the serum alanine aminotransferase?ALT?and aspartate aminotransferase?AST?levels and liver MDA content of RmEPS-11-treated mice were significantly reduced,with recovery of superoxide dismutase?SOD?activity and glutathione?GSH?content in the liver.Furthermore,the treatment reduced hepatic histopathological damage and the number of apoptotic hepatocytes.Studies on the protective mechanisms of hepatic function indicated that the generation of harmful free radicals and reactive oxygen species?ROS?was reduced,accompanied by significant induction of antioxidant capacity due to increase in CYP2E1 mRNA level.RmEPS-11 inhibited lipid peroxidation by suppressing CYP2E1 expression,which increased radical scavenging capacity and protected liver function.At the same time,it was verified that RmEPS-11has higher free radical scavenging capacity than REPS2-A.?4?The optimum concentration and time required to observe the inhibitory effect of REPS2-A on Hep-G2 hepatoma cells were determined.The inhibition rate of Hep-G2 was higher than that of IC₅₀ when the concentration of REPS2-A was 1 mg/mL.Meanwhile,the maximum inhibitory effect was achieved,in contrast to that at other concentrations.The spontaneous apoptosis rate of Hep-G2 was 0.40%.The apoptosis rate of Hep-G2 was 77.70%,88.18%,and97.08%for treatment with 1 mg/mL REPS2-A for 24,48,and 72 h,respectively.The time taken for observing the effect of REPS2-A on Hep-G2 showed that REPS2-A blocked cells in the G1/S phase.REPS2-A can effectively inhibit the proliferation of tumor cells,and there is a dose effect.Compared to RmEPS-11,REPS2-A has a significant in vitro anti-tumor activity due to REPS2-A consists of?1?3?-linked active galactose?Gal?as the backbone composition.?5?Analysis of differential gene expression?DGE?led to the identification of 1,016 differentially expressed genes?DEGs?,among which 527 DEGs were up-regulated and 489 DEGs were down-regulated.GO enrichment analysis of DEGs showed that genes enriched in transmembrane transport and cellular component were most abundant.The results of KEGG analysis on DEGs demonstrated that 14 cells were significantly enriched in the yeast cell cycle pathway,and there was no significant enrichment of down-regulated genes.The key enzymes involved in the glycolytic pathway included glyceraldehyde-3-phosphate dehydrogenase?GAPDH?,acetyl-CoA synthetase?ACSS?,acetaldehyde dehydrogenase?ALDH?,and fructo-diphosphate aldolase?FBAI?.GAPDH,ACSS,ALDH,and FBAI showed different degrees of up-regulation in the KEGG pathway,whereas no down-regulated genes were observed during entire glycolysis.The findings of analysis of gene differential expression is that the transcription level of fructose diphosphate aldolase encoding gene c14925_gl and glyceraldehyde-3-phosphate dehydrogenase encoding gene c14905_gl is significantly higher than that of R.mucilaginosa CICC 33013 in R.mucilaginosa CM-1?RN?,increasing 5.2279 times and6.4615times respectively.ATP synthesized by glycolysis can provide energy for the polymerization of EPS and promote the synthesis and transport of EPS.The comparison of the differential expressed genes between two strains of yeast transcriptome find that there were significant differences in the expression of glycosyl transferase genes between R.mucilaginosa CM-1?RN?and R.mucilaginosa CICC 33013.Firstly,the transcriptional level of?-1,2-mannose transferase gene c13430_gl is 1.3967 times higher than that of R.mucilaginosa CICC 33013 in R.mucilaginosa CM-1,which is consistent with the result of the previous reserch that difference exists in the primary structure of the two polysaccharides in the early stage.The results of transcriptome sequencing show that there are different types of glycosyltransferases in R.mucilaginosa CM-1 and R.mucilaginosa CICC 33013.Different transcriptional levels of glycosyltransferase result in the difference of the primary structure of polysaccharides synthesized by two strains.Transcriptome sequencing also indicate that the transcriptional level of glucoside hydrolase encoding gene c19749_gl is 6.5689 times higher than that of R.mucilaginosa CICC 33013 in R.mucilaginosa CM-1 cells,and the difference in transcription level is obvious.It is speculated that the molecular weight of the EPS of R.mucilaginosa CM-1 is lighter because its cells may degrade its polysaccharides by a large number of synthetic glucosidases,resulting in a decrease in the molecular weight of the polysaccharides.In addition,genes encoding citrate synthase?GLTA?and ATP citrate lyase?ACLY?,two key enzymes in the tricarboxylic acid cycle?TCA?,were differentially expressed,with up-regulation of GLTA and down-regulation of ACLY,indicating that GLTA and ACLY are involved in the differential expression of R.mucilaginosa EPS.The results of pathway analysis show that the key genes in the MAPK signaling pathway and p53 signaling pathway,which are closely related to the regulation of physiological activities such as antioxidation and anti-tumor activity of R.mucilaginosa,also show differences in expression.The expression of STE12,MSN2O4 and TEAD was up-regulated in the MAPK signaling pathway,and the expression of CDC42 and GNG was down-regulated.Meanwhile,gene ATR and CHK1 are up-regulated in p53signaling pathway.The above genes play an important role in regulating the free radical scavenging activity of EPS of R.mucilaginosa and inhibiting the differential expression of liver cancer cells and thus help protecting liver.