Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses.

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20. Then, the ultra-high-Mw (> 6.0 × 106 Da) γ-PGA produced by GXD-20 was characterized. Interestingly, GXD-20 could produce γ-PGA at 42°C, and exhibited a γ-PGA titer of up to 22.29 ± 0.59 g L-1 in a 5-L fermenter after optimization of the fermentation process. Comparative genomic analysis indicated that the specific protein sequence and subcellular localization of PgdS (a γ-PGA-degrading enzyme) were closely related to the ultra-high-Mw of γ-PGA. Transcriptomic analysis revealed that the high γ-PGA titer at 42°C was mainly related to the high expression of genes encoding enzymes for sucrose transportation and utilization, nitrogen transportation, endogenous glutamate synthesis, and γ-PGA synthesis. These results provide new insights into the production of ultra-high-Mw γ-PGA by Bacillus at high temperatures.

Enhanced Production of Poly-γ-glutamic Acid by Bacillus subtilis Using Stage-controlled Fermentation and Viscosity Reduction Strategy.

In this study, the production of poly-γ-glutamic acid (PGA) by Bacillus subtilis using stage-controlled fermentation and viscosity reduction strategy was investigated in detail. Based on the single-factor optimization experiment, temperature (42 °C and 37 °C), pH (7.0 and uncontrolled), aeration rate (1.2 vvm and 1.0 vvm), and agitation speed (700 rpm and 500 rpm) were selected for the two-stage controlled fermentation (TSCF). The time points for the TSCF of temperature, pH, aeration rate, and agitation speed were set at 18.52 h, 2.82 h, 5.92 h, and 3.62 h, respectively, based on the kinetic analysis. A PGA titer of 19.79 ~ 22.17 g/L was obtained from the TSCF, which did not increase significantly than that (21.25 ± 1.26 g/L) of non-stage controlled fermentation (NSCF). This may be due to the high viscosity and low dissolved oxygen of the PGA fermentation broth. Thus, the TSCF combined with a viscosity reduction strategy was developed to further improve the production of PGA. The PGA titer reached 25.00 ~ 30.67 g/L, which increased by 17.66 ~ 32.94% to that of NSCF. This study provided a valuable reference for the development of process control strategies for high-viscosity fermentation systems.

Green synthesis of silver nanoparticles using Phlebopus portentosus polysaccharide and their antioxidant, antidiabetic, anticancer, and antimicrobial activities.

Silver nanoparticles (AgNPs) by green synthesis from fungi polysaccharides are attracting increasing attention owing to their distinctive features and special applications in numerous fields. In this study, a cost-effective and environmentally friendly biosynthesizing AgNPs method with no toxic chemicals involved from the fruiting body polysaccharide of Phlebopus portentosus (PPP) was established and optimized by single factor experiment and response surface methodology. The optimum synthesis conditions of polysaccharide-AgNPs (PPP-AgNPs) were identified to be the reaction time of 140 min, reaction temperature of 94 °C, and the PPP: AgNO3 ratio of 1:11.5. Formation of PPP-AgNPs was indicated by visual detection of colour change from yellowish to yellowish brown. PPP-AgNPs were characterized by different methods and further evaluated for biological activities. That the Ultraviolet-visible (UV-Vis.) spectroscopy displayed a sharp absorption peak at 420 nm confirmed the formation of AgNPs. Fourier transform infrared (FTIR) analysis detected the presence of various functional groups. The lattice indices of (111), (200), (220), and (331), which indicated a faced-centered-cubic of the Ag crystal structure of PPP-AgNPs, was confirmed by X-ray diffraction (XRD) and the particles were found to be spherical through high resolution transmission electron microscopy (HRTEM). Energy dispersive X-ray spectroscopy (EDS) determined the presence of silver in PPP-AgNPs. The percentage relative composition of elements was determined as silver (Ag) 82.5 % and oxygen (O) 17.5 % for PPP-AgNPs, and did not exhibit any nitrogen peaks. The specific surface area of PPP-AgNPs was calculated to be 0.5750 m2/g with an average pore size of 24.33 nm by BET analysis. The zeta potential was -4.32 mV, which confirmed the stability and an average particle size of 64.5 nm was calculated through dynamic light scattering (DLS). PPP-AgNPs exhibited significant free radical scavenging activity against DPPH with an IC50 value of 0.1082 mg/mL. The MIC values of PPP-AgNPs for E. coli, S. aureus, C. albicans, C. glabrata, and C. parapsilosis are 0.05 mg/mL. The IC50 value of the inhibition of PPP-AgNPs against α-glucosidase was 11.1 µg/mL, while the IC50 values of PPP-AgNPs against HepG2 and MDA-MB-231 cell lines were calculated to be 14.36 ± 0.43 µg/mL and 40.05 ± 2.71 µg/mL, respectively. According to the evaluation, it can be concluded that these green-synthesized and eco-friendly PPP-AgNPs are helpful to improve therapeutics because of significant antioxidant, antimicrobial, antidiabetic, and anticancer properties to provide new possibilities for clinic applications.

Efficient Production of Melanin by Aureobasidium Melanogenum Using a Simplified Medium and pH-Controlled Fermentation Strategy with the Cell Morphology Analysis.

Natural melanin is a biopolymer with wide application prospects in medicine, food, cosmetics, environmental protection, agriculture, and so on. Microbial fermentation is an important and effective way to produce melanin. In this study, Aureobasidium melanogenum, known as black yeast with cellular pleomorphism, was used for the production of melanin. Based on the characteristic of A. melanogenum secreting melanin under oligotrophic stress, a simple medium containing only glucose, MgSO4·7H2O, and KCl was constructed for the production of melanin. The melanin titer of 6.64 ± 0.22 g/L was obtained after 20 days of fermentation without pH control. The cell morphological changes of A. melanogenum during the production of melanin were recorded, and the results showed that chlamydospore might be the most favorable cell morphology for melanin synthesis. Then, different fermentation strategies with cell morphology analysis were developed to further improve the production of melanin in a 5-L fermenter. Results showed that the maximum titer of melanin reached 18.50 g/L by using the fermentation strategy integrating pH control, ammonium salt addition, and H2O2 stimulation, which increased by 178.6% than that of the strategy without pH control. Furthermore, the melanin obtained from the fermentation broth was characterized as eumelanin containing an indole structure. This study provided a potentially feasible fermentation strategy for the industrial production of melanin.

Four New Species of Strobilomyces (Boletaceae, Boletales) from Hainan Island, Tropical China.

Strobilomyces, one of the most noticeable genera of Boletaceae (Boletales), is both ecologically and economically important. Although many studies have focused on Strobilomyces in China, the diversity still remains incompletely understood. In the present study, several collections of Strobilomyces from Hainan Island, tropical China were studied based on morphology and molecular phylogenetic analyses. Four species are described as new, viz. S. baozhengii, S. conicus, S. hainanensis, and S. pachycystidiatus. Detailed descriptions, color photos of fresh basidiomata, and line drawings of microstructures of the four species are presented.

Structural characterization, and in vitro hypoglycemic activity of a polysaccharide from the mushroom Cantharellus yunnanensis.

A polysaccharide CY-2 from C. yunnanensis was obtained through a process of consecutive water extraction, alcohol precipitation, and DEAE-52 fast-flow chromatography. CY-2, with an average molecular weight of 2.69 × 104 Da mainly consisted of glucose and mannose with a molar ratio of 33.5: 56.9. Infrared spectrum (IR), methylation analysis, and nuclear magnetic resonance (NMR) results revealed that CY-2 may have a backbone consisting of →6)-α-D-Manp-(1 â†’ 3)-ß-D-Glcp-(1→, and branch chain ß-D-Glcp-(1→. Meanwhile, CY-2 had a higher inhibition rate on α-glucosidase activity compared with other fractions (CY-0, CY-1, and CY-4) and was a mixed competitive inhibitor. In addition, CY-2 at the concentration of 10 µg/mL presented a superior power to improve glucose consumption and metabolism in HepG2 cells compared with metformin. Overall, these findings highlight the potential value of CY-2 as a hypoglycemic agent.

Extraction, Characterization, and In Vitro Hypoglycemic Activity of a Neutral Polysaccharide from the New Medicinal Mushroom Cantharellus yunnanensis (Agaricomycetes).

Polysaccharides serve as promising ingredients for health-beneficial functional foods, while there were no investigations into the structural characterizations and bioactivities of an edible mushroom Cantharellus yunnanensis. In the study, crude polysaccharides from this mushroom were extracted by hot water and isolated by ethanol precipitation. Then, a neutral polysaccharide (named CY-1) was purified from the crude polysaccharide by deproteinization with Sevag reagent, decolorization with a kind of macroporous adsorption resin SP-825, DEAE-52 cellulose column chromatography and dialysis. The physicochemical properties of CY-1 were characterized by UV, IR, SEM, NMR, and HPLC analyses. Structural characterizations revealed that CY-1 is a homogeneous heteropolysaccharide with an average molecular weight of 3.06 × 104 Da. CY-1 exhibited a honeycomb structure with an irregular branching shape, and it was composed of mannose, glucose, fucose, xylose, arabinose, galactose, rhamnose, and glucuronic acid, with molar fractions of 54.83%, 25.11%, 10.28% 4.53%, 2.12%, 1.64%, 0.83%, and 0.64%, respectively. In vitro hypoglycemic tests showed that CY-1 had an inhibitory effect on α-glucosidase. In addition, CY-1 of 160 µg/mL promoted glucose consumption in normal HepG2 cells. These results suggested that CY-1 may be a potential hypoglycemic agent.

Updated taxonomy of Chinese Cantharellus subgenera Afrocantharellus and Magni (Hydnaceae, Cantharellales): Three new taxa and amended descriptions of one previous species.

Cantharellus, one of the main genera of Hydnaceae (Cantharellales), is both ecologically and economically important. Although many studies have focused on this genus in China, the taxonomy should be further updated. In the present study, Cantharellus subgenera Afrocantharellus and Magni were investigated based on morphology and molecular phylogenetic analyses with new collections from China. Five phylogenetic species were recognized among the studied collections, three of which were described as new: C. bellus, C. cineraceus, and C. laevigatus; one was previously described taxon: C. hygrophoroides; and the remaining species was not defined due to the paucity of the materials. Among the four described species, both C. bellus and C. laevigatus are members of subgen. Magni, whereas C. cineraceus and C. hygrophoroides belong to subgen. Afrocantharellus.

Notes on four species of Russula subgenus Heterophyllidiae (Russulaceae, Russulales) from southern China.

Heterophyllidiae, one of the main subgenus of Russula (Russulaceae, Russulales), is both ecologically and economically important. Although many studies have focused on subgenus Heterophyllidiae in China, the diversity, taxonomy, and molecular phylogeny still remained incompletely understood. In the present study, two new species, R. discoidea and R. niveopicta, and two known taxa, R. xanthovirens and R. subatropurpurea, were described based on morphology and molecular phylogenetic analyses of ITS and 28S DNA sequences with new collections of subgenus Heterophyllidiae from southern China. Both morphological and phylogenetic analyses consistently confirmed that R. niveopicta and R. xanthovirens belong to the subsect. Virescentinae, R. discoidea and R. subatropurpurea come under subsect. Heterophyllae, and R. prasina is synonymized with R. xanthovirens.

Purification and Structure Characterization of the Crude Polysaccharide from the Fruiting Bodies of Butyriboletus pseudospeciosus and Its Modulation Effects on Gut Microbiota.

Polysaccharides from the species of Boletaceae (Boletales, Agaricomycetes, Basidiomycota) are economically significant to both functional foods and medicinal industries. The crude polysaccharide from Butyriboletus pseudospeciosus (BPP) was prepared, and its physicochemical properties were characterized through the use of consecutive experimental apparatus, and its impact on the gut microbiota of Kunming mice was evaluated. Analyses of the structure characteristics revealed that BPP was mainly composed of Man, Glc, and Gal, possessing the pyranose ring and ß/α-glycosidic linkages. TG analysis exhibited that BPP had great heat stability. The SEM observation performed demonstrated that BPP appeared with a rough, dense, and porous shape. Through the BPP intervention, the serum and fecal biochemical index in mice can be improved obviously (p < 0.05). The abundance of beneficial microbiota in the BPP-treated group was significantly increased, while the abundance of harmful microbiota was significantly decreased (p < 0.05). Based on the Tax4Fun, we also revealed the relationship between the species of gut microbiota and showed that the high dose of BPP has significantly changed the functional diversities compared with those in other groups (p < 0.05). The results suggest that B. pseudospeciosus could serve as potential functional food or medicine.

Preparation, structural characterisation, and antioxidant activities of polysaccharides from eight boletes (Boletales) in tropical China.

Polysaccharides in boletes (Boletales) are economically significant to both function food and medicinal industries. The polysaccharides were extracted from the fruit bodies of eight boletes, namely, Aureoboletus longicollis, Butyriboletus hainanensis, Crocinoboletus rufoaureus, Hemioporus japonicus, Neoboletus infuscatus, Neoboletus obscureumbrinus, Tylopilus otsuensis, Xanthoconium fusciceps, which were collected from tropical China; their physicochemical properties and antioxidant activities were characterised and evaluated, respectively. The results revealed that the polysaccharides among the eight boletes were mainly composed of glucose, mannose, and galactose, with a broad molecular weight range, and contained a pyranose ring revealed by FT-IR and NMR spectral analyses. Many factors such as different species of boletes, geographic conditions, molecular weight, configuration, and monosaccharide content may affect the antioxidant power of polysaccharides, simultaneously, instead of one single factor. The antioxidant activities of the polysaccharides were measured according to in vitro assays of DPPH scavenging, superoxide anion scavenging, and ferrous ion reducing tests. The polysaccharide of C. rufoaureus has greatly superior antioxidant activity and it could serve as potential functional food or medicine.

A Contribution to Knowledge of Craterellus (Hydnaceae, Cantharellales) in China: Three New Taxa and Amended Descriptions of Two Previous Species.

Species of Craterellus (Hydnaceae, Cantharellales) in China are investigated on the basis of morphological and molecular phylogenetic analyses of DNA sequences from nuc 28S rDNA D1-D2 domains (28S) and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 region. Five species are recognized in China, of which three of them are described as new, viz. C. fulviceps, C. minor, and C. parvopullus, while two of them are previously described taxa, viz. C. aureus, and C. lutescens. A key to the known Chinese taxa of the genus is also provided.

Morphological and Phylogenetic Evidences Reveal Four New Species of Cantharellus Subgenus Cantharellus (Hydnaceae, Cantharellales) From China.

Species of Cantharellus subgenus Cantharellus are interesting and important for their mycorrhizal properties, medicinal values, and edibility. In China, there are many undescribed species of the subgenus. In this study, four new species of subg. Cantharellus, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus pinetorus, and Cantharellus ravus from Hainan and Hunan Provinces, respectively, were described based on morphological and phylogenetic evidence as a contribution to the knowledge of the species diversity in China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of these four new species are presented as well as comparisons with related species.

Rubroboletus flammeus (Boletaceae, Boletales), a novel species unveiled from subtropical China based on morphological and phylogenetic evidences.

Rubroboletus flammeus is described as a new species from subtropical China based on morphological and molecular phylogenetic analyses. It is morphologically characterized by a red to fiery-red basidioma with a dry pileus, a context in pileus white but that in stipe bright yellow, a stipe densely covered with spots, a blue color change of tissues, and a trichoderm-type pileipellis. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microscopic features of the new species are presented. A key to the known Chinese taxa of Rubroboletus is provided.

Purification and Characterization of a Fibrinolytic Enzyme from Marine Bacillus velezensis Z01 and Assessment of Its Therapeutic Efficacy In Vivo.

Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In the present study, we purified and characterized an extracellular fibrinolytic serine metalloprotease (named Velefibrinase) that is produced by marine Bacillus velezensis Z01 and assessed its thrombolysis in vivo. SDS-PAGE and MALDI-TOF-MS analyses showed that the molecular mass of Velefibrinase was 32.3 KDa and belonged to the peptidase S8 family. The optimal fibrinolytic activity conditions of Velefibrinase were 40 °C and pH 7.0. Moreover, Velefibrinase exhibited high substrate specificity to fibrin, and a higher ratio of fibrinolytic/caseinolytic (1.48) values, which indicated that Velefibrinase had excellent fibrinolytic properties. Based on the degradation pattern of fibrin and fibrinogen, Velefibrinase could be classified as α/ß-fibrinogenase. In vitro, Velefibrinase demonstrated efficient thrombolytic ability, anti-platelet aggregation, and amelioration of blood coagulation (APTT, PT, TT, and FIB), which were superior to those of commercial anticoagulant urokinase. Velefibrinase showed no hemolysis for erythrocyte in vitro and no hemorrhagic activity in vivo. Finally, Velefibrinase effectively prevented mouse tail thrombosis in a dose-dependent (0.22-0.88 mg/kg) manner. These findings suggested that Velefibrinase has the potential to becoming a new thrombolytic agent.

Neoboletus infuscatus, a new tropical bolete from Hainan, southern China.

Neoboletus infuscatus (Boletaceae, Boletales) is described as a new species from Yinggeling of Hainan Tropical Rainforest National Park, southern China. It is morphologically characterized by a large basidioma with a nearly glabrous, brownish yellow, yellowish brown to pale brown pileus, pores orangish red when young, yellowish brown to brown when old, context and hymenophore staining blue when injured, a yellow stipe with red punctuations, surfaces of the pileus and the stipe usually covered with a thin layer of white pruina when young. Phylogenetic analyses of DNA sequences from part of the 28S gene, the nuclear rDNA internal transcribed spacer (ITS) region, and part of the translation elongation factor 1-α gene (TEF1) also confirm that N. infuscatus forms an independent lineage within Neoboletus. Detailed morphological description, color photos of fresh basidiomata and line-drawings of microstructures are provided.

Effects of cell physiological structure on the fermentation broth viscosity during poly-γ-glutamic acid production by Bacillus subtilis GXA-28.

The high viscosity of fermentation broth limited the further improvement of PGA titer. Our previous studies indicated that adding KCl to the medium could decrease the fermentation broth viscosity and improve the PGA titer. In order to clarify the reason, effects of cell physiological structure on the fermentation broth viscosity were investigated. Results from cell morphology observation showed that the reduction of cell aggregation caused by the weakened cross-linking between PGA and cells might be an important reason for the decrease in the fermentation broth viscosity. Besides, when 201.2 mM KCl was added to the medium, the zeta potential of cell surface decreased from - 70.48 ± 3.35 mV to - 81 ± 2.46 mV. The cell membrane integrity was reduced and permeability was enhanced. Furthermore, the percentage of lauric acid C12:0 in cell membrane increased by 12.36%, but palmitic acid C16:0 and stearic acid C18:0 decreased by 6.83% and 5.64%, respectively, which improved the fluidity of cell membrane. The above changes in cell membrane further affect the cross-linking between PGA and cells, thereby playing an important role in reducing the fermentation broth viscosity. This study provided some novel information for understanding the decrease of PGA fermentation broth viscosity by KCl.

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

BACKGROUND: As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). RESULTS: After codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25-4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly. CONCLUSIONS: The combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.

Poly(malic acid) production from liquefied corn starch by simultaneous saccharification and fermentation with a novel isolated Aureobasidium pullulans GXL-1 strain and its techno-economic analysis.

In this study, a novel Aureobasidium pullulans GXL-1 strain without melanin secretion was isolated for efficient polymalic acid (PMA) production. The PMA produced by GXL-1 was characterized, and its molecular mass was determined to be 1.621 kDa by gel permeation chromatography. Liquefied corn starch was shown to replace glucose for PMA production by GXL-1 through simultaneous saccharification and fermentation. The PMA titer obtained from batch fermentation was up to 49.0 ± 1.6 g/L in a 10 L fermentor, and the PMA yield and productivity obtained from repeated-batch fermentation were up to 0.50 g/g and 0.34 g/L·h, respectively. Furthermore, process design and techno-economic analysis were performed at an annual output level of 5000 metric tons by SuperPro Designer. Results showed that the production cost of $2.046/kg and payback period of 6.9 years were achieved by repeated-batch fermentation; this provides an economically feasible strategy for industrial-scale production of PMA.

Investigations in ultrasound-assisted anticoagulant production by marine Bacillus subtilis ZHX.

Anticoagulants are the main drugs for the prevention and treatment of thromboembolism. However, most of the present anticoagulants have shortcomings and novel anticoagulants are in great demand. Marine microorganisms are an important source of new drugs. Therefore, in this study, ultrasound was applied to enhance anticoagulant accumulation by marine Bacillus subtilis ZHX. Ultrasound parameters were optimized by single-factor experiments exploring the effects of ultrasound power, duration, duty cycle and the cell growth phases. The optimum conditions were exponential prophase (5 h) with 25 kHz frequency, 140 W power, and a 40% duty cycle for 5 min. The maximum anticoagulant activity (55.36 U/mL) was 1.73 times that of the control group, and the fermentation time was shortened by 3 h. Under optimal conditions, ultrasound increased the carbon utilization by Bacillus subtilis ZHX without significant changes in morphology, favoring cell growth and anticoagulant production. However, excessive ultrasound caused intracellular damage, which inhibited biomass accumulation, decreasing anticoagulant activity and even leading to cell rupture. This is the first report on the use of ultrasound to enhance anticoagulant production by Bacillus, and it provides useful information for scaling-up the process.