Xanthomonas campestris pv. campestris (Xcc), the pathogenic agent of black rot disease in cruciferous plants, produces large amount of extracellular polysaccharide (EPS), which has found wide applications in industry. For the great commercial value of the xanthan gum, many of the genes involved in EPS biosynthesis have been cloned and the mechanism of EPS biosynthesis also has been studied. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5 gusA5, and a number of EPS-defective mutants were isolated in our previous work. The Tn5 gusA5 inserted sites of these mutants were located by using thermal asymmetric interlaced PCR, and results showed that two EPS-defective mutants were insertion mutants of the gene wxcA which involved in lipopolysaccharide (LPS) biosynthesis. The gene wxcA involved in lipopolysaccharide biosynthesis but dose not extracellular polysaccharide in others' report. wxcA::Tn5 gusA5 mutant 021C12, the polar mutant, was complemented with recombinant plasmid pLATC8570 harboring an intact wxcA gene in this work, but the yield of EPS of the wxcA::Tn5 gusA5 mutant was not restored. In order to identify the function of wxcA gene of Xcc 8004 strain, the gene wxcA was deleted by gene replacement strategy, and the no-polar mutant of wxcA was obtained. DeltawxcA mutant strain, named Xcc 8570, was confirmed by using both PCR and southern analysis. Beside the LPS biosynthesis of deltawxcA mutant was affected, The EPS yield of deltawxcA mutant strain reduced by 50% as compared with the wild-type strain 8004. DeltawxcA mutant could be complemented in trans with the intact wxcA gene, and the EPS yield of the mutant was restored. The combined data showed that wxcA gene not only involved in LPS biosynthesis but also EPS yield in Xcc 8004 strain.
Cell Proliferation ; Genes, Bacterial ; physiology ; Lipopolysaccharides ; biosynthesis ; Mutation ; Polysaccharides, Bacterial ; biosynthesis ; Xanthomonas campestris ; genetics

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.
Biosynthetic Pathways ; Diosgenin/metabolism* ; Gene Expression Profiling ; Phytochemicals/biosynthesis* ; Polygonatum/metabolism* ; Polysaccharides/biosynthesis* ; Transcriptome

OBJECTIVETo investigate the submerged culture conditions and nutritional requirments for the production of mycelial biomass and exopolysaccharides (EPS) by medicinal mushroom Phellinus baumii.

METHODThe carbon sources, nitrogen sources, inoculum volume, initial pH and temperature were investigated based on shake flask cultures, respectively.

RESULTThe glucose was the most suitable carbon source for both mycelial biomass and EPS production, soy peptone was favorable nitrogen sources for both mycelial biomass and EPS production. The optimal inoculum volume, initial pH and temperature for both mycelial growth and EPS production were 6%, 6.0 and 28 degrees C, respectively.

CONCLUSIONThe study obtained basic datas for large-scale submerged culture of P. baumii.

Basidiomycota ; growth & development ; metabolism ; Biomass ; Hydrogen-Ion Concentration ; Mycelium ; growth & development ; metabolism ; Polysaccharides ; biosynthesis ; Temperature

Agitator is one of the essential factors to realize high efficient fermentation for high aerobic and viscous microorganisms, and the influence of different impeller combination on the fermentation process is very important. Welan gum is a microbial exopolysaccharide produced by Alcaligenes sp. under high aerobic and high viscos conditions. Computational fluid dynamics (CFD) numerical simulation was used for analyzing the distribution of velocity, shear rate and gas holdup in the welan fermentation reactor under six different impeller combinations. The best three combinations of impellers were applied to the fermentation of welan. By analyzing the fermentation performance, the MB-4-6 combination had better effect on dissolved oxygen and velocity. The content of welan was increased by 13%. Furthermore, the viscosity of production were also increased.
Alcaligenes ; metabolism ; Fermentation ; Hydrodynamics ; Industrial Microbiology ; methods ; Oxygen ; Polysaccharides, Bacterial ; biosynthesis ; Viscosity

Xylitol, a five-carbon sugar alcohol, has many interesting applications in the food, pharmaceutical, and odontological industries, owing to its high sweetening power, its anticariogenic properties, and its insulin-independent metabolism. The bioconversion of detoxified hemicellulosic hydrolysate to xylitol by microorganisms could be a cheaper alternative to the current chemical process, since it is a simple process, with great specificity and low energy requirements. However, the success of fermentations for xylitol production depends on the productivity of the strain and its tolerance to different toxic or inhibitory compounds existing in the hydrolysates. In addition, a number of culture process parameters proved to have significant effects on xylitol production in hemicellulosic hydrolysate media. One of the most important control variables in this bioconversion is the aeration level, which affects the biochemical pathways in the xylose metabolism. The production of biomass is favored by aerobic conditions, while under anaerobic conditions xylose cannot be assimilated by yeast, whereas xylitol is formed in oxygen-limited incubation conditions. An adapted Candida sp. with enhanced resistance to the inhibitors in the hydrolysate can directly ferment the simply detoxified corn cob hemicellulosic hydrolysate to xylitol. In the present study, the combined effects of shaking speed, C/ N ratio, initial pH, and inoculum level on the fermentation of corn cob hemicellulosic hydrolysate to xylitol by an adapted Candida sp. were investigated using an orthogonal experimental design in flask. As a result, the optimum fermentation conditions were as follows: 180 r/min, a C/N ratio of 50, initial pH 5.5, and an inoculum level of 5% (volume ratio). Moreover, the optimum concentration factor of hydrolysate varied between 3.0 and 3.72 was obtained. Based on these results, in order to evaluate the effect of aeration rate on the fermentation of corn cob hemicellulosic hydrolysate to xylitol in fermentor, batch fermentations were carried out in a 3.7 L stirred fermentor using four different aeration strategies, including three kind of two-stage aeration strategies, which provided relatively high aeration rate in the early stage but reduced it in the later stage, and including a one-stage aeration strategy provided a constant aeration rate. With respect to xylitol yield, the results indicated that two-stage aeration strategy was significantly superior to one-stage aeration strategy. The highest xylitol yield (0.75 g/g) was obtained with oxygen supply strategy C (3.75 L/min for first 24 h, then lowered it to 1.25 L/min, 2.5 L fermentation medium was employed). In this process, without extensive detoxification of hydrolysate, an adapted Candida sp. can efficiently ferment the simply treated corn cob hemicellulosic hydrolysate to xylitol under the optimized fermentation conditions. This work should help the development of an efficient process for producing xylitol from corn cob hemicellulosic hydrolysate on a larger scale by bioconversion.
Aerobiosis ; Candida tropicalis ; metabolism ; Fermentation ; Hydrolysis ; Polysaccharides ; metabolism ; Xylitol ; biosynthesis ; Zea mays ; metabolism

The effect of inorganic phosphate on cell growth, accumulation of polysaccharides together with nutrient utilization was investigated in suspension cultures of protocorm-like bodies of Dendrobium huoshanense. Thirty-day-old cells were transferred into liquid medium with the inoculum density of 100 g/L cells. The results indicate that the optimal concentration of phosphate in medium for cell growth of protocorm-like bodies of Dendrobium huoshanense was 2.5 mmol/L and biomass was 496.5 g/L (fresh weight). Phosphate was a limited factor for cell growth and there was relationship between the levels of intracellular phosphate and cell growth. 2.5 mmol/L of medium phosphate was beneficial to the absorption of carbohydrate and nitrate source. 0.312 mmol/L of medium phosphate was better for accumulation of polysaccharides and production of polysaccharides was 2.22 g/L at 36 d.
Cell Proliferation ; drug effects ; Dendrobium ; growth & development ; metabolism ; Phosphates ; metabolism ; pharmacology ; Polysaccharides ; biosynthesis ; Suspensions

The synthetic characteristic of extracellular polysaccharide (EPS) of Ganoderma lucidum performed in batch fermentation was studied. The result showed that the production EPS was partially growth-associated. The cell dry weight (CDW) and EPS reached 15.56 g x L(-1), 3.02 g x L(-1) respectively. The yield of EPS to cell dry weight ( Y(p/x)) was 0.19. Based on the test result of batch fermentation, a kinetic model was proposed by using the Logistic equation for cell growth, the Luedeking Piret equation for EPS production and the Luedeking-piret-like equation for consumption of glucose as substrate. The calculated results of models were compared satisfactorily with experimental data under various glucose concentrations, the average relative error was no more than 5%. The kinetic model had practically guiding producing PES in fermentation of Ganoderma lucidum.
Culture Techniques ; methods ; Extracellular Space ; metabolism ; Fermentation ; Kinetics ; Models, Biological ; Polysaccharides ; biosynthesis ; Reishi ; chemistry ; growth & development

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.
Codonopsis ; enzymology ; genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases ; genetics ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis ; Transcriptome

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
Base Sequence ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis

Our previous work has indicated that mycelium growth and exopolysaccharide accumulation in submerged fermentation by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 are strongly affected by many internal and external factors, including medium constituents and fermentation conditions. In this study, we use an effective two-phase statistical approach to enhance exopolysaccharide production. In the first phase, Plackett-Burman design was undertaken to evaluate the effects of the twenty factors, i.e., glucose, fructose, maltose, yeast extract, tryptone, K2HPO4, KH2PO4, (NH4)2SO4, NaNO3, FeSO4, MgSO4, MnCl2, ZnCl2, FeCl3, CuSO4.5H2O, vitamin B1, initial pH, the temperature, the medium volume and the duration, to the fermentation. By regression analysis, yeast extract, tryptone, fructose, MgSO4, MnCl2, initial pH and temperature were found to be important for exopolysaccharide production, while glucose, maltose, NaNO3, ZnCl2, vitamin B1, the duration and the volume are important to the mycelium biomass. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimal concentration levels and the relationships between these factors. Based on the results of the first phase, a five-level six-factor (yeast extract, fructose, MgSO4, maltose, ZnCl2 and initial pH) central composite rotatable design (CCRD) was employed. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentrations for obtaining 876.32 microg exopolysaccharide per milliliter of fermentation liquor were calculated as: 6.0g/L yeast extract, 11.5g/L fructose, 0.5g/L MgSO4, 9.6g/L maltose, 38.6mg/L ZnCl2 and with the initial pH 5.3. The experimental data under various conditions have validated the theoretical values.
Culture Media ; Fermentation ; Fructose ; metabolism ; Hydrogen-Ion Concentration ; Maltose ; metabolism ; Pholiota ; growth & development ; metabolism ; Polysaccharides ; analysis ; biosynthesis ; Temperature