Light calcium carbonate improves pullulan biosynthesis by Aureobasidium pullulans under high concentration of sugar.

The effects of light calcium carbonate (CaCO3) on pullulan biosynthesis by Aureobasidium pullulans NCPS2016 were investigated. Light CaCO3 enhanced pullulan production by 12.4 % when added to the low concentration of fructose broth compared with K2HPO4. Pullulan production was further improved when increasing both the concentrations of light CaCO3 and fructose. Compared to K2HPO4, light CaCO3 improved the activities of UDP-glucose pyrophosphorylase, α-phosphoglucose mutase, UDP-glucosyltransferase, and glucosyltransferase relevant to pullulan biosynthesis, and the gene transcriptional levels of UDP-glucose pyrophosphorylase, α-phosphoglucose mutase, UDP-glucosyltransferase, and glucose kinase were enhanced. During 30-liter fermentation, 144.3 g/L of purified pullulan was produced from 200 g/L of fructose and 15 g/L of light CaCO3 within 168 h, with the yield and productivity of 0.72 g/g and 0.86 g/L/h respectively. This is the first report that light CaCO3 improves pullulan production significantly.

High-level production of pullulan from high concentration of glucose by mutagenesis and adaptive laboratory evolution of Aureobasidium pullulans.

The cost of carbon sources and the low efficiency of the fermentation titer limit the industrial application of pullulan. In this study, a hypertonic-tolerant strain with efficient utilization of glucose was obtained using a double strategy. Initially, a strain for efficient synthesis of pullulan from glucose was generated by mutagenesis. Subsequently, the mutant was directionally evolved on the plate containing a high glucose concentration to enhance high osmotic resistance. The enzyme activities and the transcriptional levels involved in pullulan biosynthesis and high osmotic tolerance in mutants were increased. Nitrogen source and inorganic salts also significantly affected the production of pullulan by M233-20 from high concentration of glucose. The pullulan titer of 162.1 g/L was obtained using the response surface methodology in the flask. The strain M233-20 produced 162.3 g/L pullulan in a 30-L bioreactor with a yield of 0.82 g/g glucose. Hence, this work provides a potential industrial pullulan producer M233-20 and a strategy to develop excellent strain.

Evaluation of the Efficacy of Enterococcus faecium L3 as a Feed Probiotic Additive in Chicken.

The study was devoted to the comparison of the probiotic effect of enterococcal Enterococcus faecium L3 to the antibiotic enramycin as a chicken feed additive. Two hundred and sixteen chickens were divided into three groups and tested by different parameters including weight gain, food consumption, blood biochemistry, immunology, and caecal microbiome at two checkpoints, 21 and 39 days after birth. By the end of the experiment, a group of chickens getting probiotic demonstrated weight gain of more than 100 g at the average relative to the control group with no additive in animal feed (P < 0.05). Blood serum biochemistry showed a significant increase in HDL level (P < 0.05) relative to the control group. The 16S RNA sequencing demonstrated the growth abundance of Lachnospiraceae and the decrease of Proteobacteria in probiotic fed group. On the contrary, the antibiotic fed group showed a noticeable increase in the abundance of Proteobacteria which included the genus Salmonella. Thus, probiotic E. faecium L3 being added to chicken food as a single additive may be considered as a possible replacement of antibiotic enramycin.

Identification of Lipopeptide Iturin A Produced by Bacillus amyloliquefaciens NCPSJ7 and Its Antifungal Activities against Fusarium oxysporum f. sp. niveum.

Bacillus amyloliquefaciens NCPSJ7 showed potential fungicidal activities for the effective control of fungal infection. From the PCR test, the key genes (srfAA, sfp, fenD, bmyB, ituD, and ituC) were detected in B. amyloliquefaciens NCPSJ7. These genes were closely related to the lipopeptides (LPs) synthesis. Next, three LPs families were identified with liquid chromatography-mass spectrometry (LC/MS), including iturin A, fengycin A, and surfactin. After purification with C18, the main active antifungal compound was proven to be C14-iturin A by ESI-HRMS, which has significant activities against fungi. These results proved that C14-iturin A played an important role in inhibiting the growth of fungi for B. amyloliquefaciens NCPSJ7. Furthermore, the isolated LP could inhibit mycelial growth and conidia germination at 30 µg/mL. SEM allowed us to observe that mycelial morphology and conidia germination were also affected. The mycelial ultrastructure TEM observations showed that the external electron-dense outer layer cell wall, which mainly consisted of glycoproteins, was affected. Furthermore, swollen mitochondria, enriched glycogen, and increased vacuoles were also found. LP also affected the intact wall and membranes, leading to their increased permeability, which was proved by propidium iodide (PI) staining and conductivity measurements. Meanwhile, the ergosterol, which has an affinity for iturin A, also increased. These results indicated that LP caused fungal dysfunction and membrane permeability increase, leading to fungal inhibition. Identifying and studying LPs is important in exploring the fungicidal activities of B. amyloliquefaciens, which promotes the use of B. amyloliquefaciens NCPSJ7 as a potential candidate for biocontrol.

Application of antagonist Bacillus amyloliquefaciens NCPSJ7 against Botrytis cinerea in postharvest Red Globe grapes.

We investigated the effects and possible mechanisms of Bacillus amyloliquefaciens NCPSJ7 against the gray mold caused by Botrytis cinerea in the postharvest Red Globe grapes. The disease incidence, lesion diameter, decay index, and some resistance-related enzymes were evaluated. The antioxidant capacity of grape treated with 1 × 104 CFU/ml B. cinerea alone and combined with 1 × 107 CFU/ml NCPSJ7 was also determined. The results showed that NCPSJ7 + B. cinerea reduced the disease incidence, lesion diameter, and decay index of postharvest grapes and enhanced the activities of polyphenol oxidase, peroxidase, chitinase, and ß-1,3-glucanase during different storage periods. Furthermore, the oxidative resistance, demonstrated by an escalating trend in the total phenolic content, DPPH free radical clearance rate, reducing power, and superoxide anion clearance rate after lesion presence, was improved. However, NCPSJ7 showed an inhibitory effect on gray mold, but resulted in the reduced antioxidant capacity in the grapes.

Melanin biosynthesis in the desert-derived Aureobasidium melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1.

The melanin produced by Aureobasidium melanogenum XJ5-1 obtained from the Taklimakan Desert can play an important role in adaptation of the yeast strain to various stress treatments. It is very important to know how the desert-derived yeast sense, respond and adapt to the harsh environments. However, it is still unclear how melanin is genetically controlled by signaling pathways and transcriptional factors. In this study, it was found that the mitogen-activated protein kinase (MAPK) Slt2 in the cell wall integrity (CWI) signal pathway could regulate activity of the transcriptional activator Swi4; in turn, the Swi4 could control the expression of the CMR1 gene. The melanin-specific transcriptional activator Cmr1 encoded by the CMR1 gene was specifically bound to the promoter with the sequence TTCTCTCCA of the PKS1 gene and strongly stimulated expression of the PKS1 gene and any other genes responsible for melanin biosynthesis, so that a large amount of melanin could be produced by A. melanogenum XJ5-1. Therefore, melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 was controlled mainly by the CWI signal pathway among the cell wall-related signal pathways via a transcriptional activator Cmr and regulation of the melanin biosynthesis in A. melanogenum XJ5-1 was completely different from that of the melanin biosynthesis in any other fungi. This is the first time to show that melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1. This would provide the fundamentals for further research on the desert-derived yeast to sense, respond and adapt to the harsh environments.

Statistical Optimization of Medium for Pullulan Production by Aureobasidium pullulans NCPS2016 Using Fructose and Soybean Meal Hydrolysates.

Pullulan, with its excellent characteristics of film-forming, water solubility, and biodegradability, is attracting more and more attention in agricultural products preservation. However, high pullulan production cost largely restricts its widely application due to its low production. In order to improve pullulan production by Aureobasidium pullulans NCPS2016, the medium was optimized using single factor experiment and response surface methodology. Based on the single factor experiments, the contents of soybean meal hydrolysates (SMHs), (NH4)2SO4, and K2HPO4·3H2O were considered to be main factors influencing the extracellular polysaccharide (EPS) production, and were further optimized by Box⁻Behnken design. The optimal content of SMHs of 7.71 g/L, (NH4)2SO4 of 0.35 g/L, and K2HPO4·3H2O of 8.83 g/L were defined. Finally, EPS production of 59.8 g/L was obtained, 39% higher in comparison with the production in the basal medium. The purified EPS produced by NCPS2016 was confirmed to be pullulan. This is the first time fructose is reported to be the optimal carbon source for pullulan production by Aureobasidium pullulans, which is of great significance for the further study of the mechanism of the synthesis of pullulan by NCPS2016. Also, the results here have laid a foundation for reducing the industrial production cost of pullulan.

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

Extracellular overexpression of chitosanase from Bacillus sp. TS in Escherichia coli.

The chitosanase gene from a Bacillus sp. strain isolated from soil in East China was cloned and expressed in Escherichia coli. The gene had 1224 nucleotides and encoded a mature protein of 407 amino acid residues. The optimum pH and temperature of the purified recombinant chitosanase were 5.0 and 60 °C, respectively, and the enzyme was stable below 40 °C. The K m, V max, and specific activity of the enzyme were 1.19 mg mL(-1), 674.71 µmol min(-1) at 50 °C, and 555.3 U mg(-1), respectively. Mn(2+) was an activator of the recombinant chitosanase, while Co(2+) was an inhibitor. Hg(2+) and Cu(2+) inhibited the enzyme at 1 mM. The highest level of enzyme activity (186 U mL(-1)) was achieved in culture medium using high cell-density cultivation in a 7-L fermenter. The main products of chitosan hydrolyzed by recombinant chitosanase were (GlcN)3-6. The chitosanases was successfully secreted to the culture media through the widely used SecB-dependent type II pathway in E. coli. The high yield of the extracellular overexpression, relevant thermostability, and effective hydrolysis of commercial grade chitosan showed that this recombinant enzyme had a great potential for industrial applications.