The ß-1,3-glucan synthase gene GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis in Grifola frondosa.

ß-1,3-Glucans are well-known biological and health-promoting compounds in edible fungi. Our previous results characterized a glucan synthase gene (GFGLS) of Grifola frondosa for the first time to understand its role in mycelial growth and glucan biosynthesis. In the present study, we identified and functionally reannotated another glucan synthase gene, GFGLS2, based on our previous results. GFGLS2 had a full sequence of 5944 bp including 11 introns and 12 exons and a coding information for 1713 amino acids of a lower molecular weight (195.2 kDa) protein with different conserved domain sites than GFGLS (5927 bp with also 11 introns and a coding information for 1781 aa). Three dual-promoter RNA-silencing vectors, pAN7-iGFGLS-dual, pAN7-iGFGLS2-dual, and pAN7-CiGFGLS-dual, were constructed to downregulate GFGLS, GFGLS2, and GFGLS/GFGLS2 expression by targeting their unique exon sequence or conserved functional sequences. Silencing GFGLS2 resulted in higher downregulation efficiency than silencing GFGLS. Cosilencing GFGLS and GFGLS2 had a synergistic downregulation effect, with slower mycelial growth and glucan production by G. frondosa. These findings indicated that GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis and provides a reference to understand the biosynthesis pathway of mushroom polysaccharides. KEY POINTS: • The 5944-bp glucan synthase gene GFGLS2 of G. frondosa was cloned and reannotated • GFGLS2 showed identity and significant differences with the previously identified GFGLS • GFGLS2 played a major role in fermentation and glucan biosynthesis.

ARTP mutation and adaptive laboratory evolution improve probiotic performance of Bacillus coagulans.

Bacillus coagulans is a thermophilic, facultative anaerobic, spore-forming Gram-positive bacterium, which is used as a probiotic in animal feed and human dietary supplements. In the present study, a bile-resistant thermophilic B. coagulans WT-03 strain was isolated and genetically identified. Atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the probiotic performance of B. coagulans WT-03. After 15 s of ARTP mutation and 40 days of ALE culture, a mutant artp-aleBC15 was obtained and showed the improved tolerance to pH 2.5 and 0.3% bile salt with a survival rate of 22.4%. Further studies showed that the artp-aleBC15 mutant exhibited a relatively stable morphology, lower permeability, and higher hydrophobicity of cell membrane compared with the parent strain of B. coagulans. Additionally, artp-aleBC15 could maintain homeostasis with an intracellular pH of over 4.5 and had the altered contents of saturated fatty acids/unsaturated fatty acids in the cell membrane at pH 2.5. Our study proved that ARTP mutation combined with ALE is an efficient mutagenesis strategy to improve the probiotic performance of B. coagulans for potential industrial use.Key Points• A B. coagulans strain that can grow at 80 °C and 0.3% bile salt was screened.• ARTP combined with ALE effectively mutated B. coagulans WT-03.• B. coagulans artp-aleBC15 mutant showed an improved probiotic performance.• The mutant exhibited the lower permeability and altered fatty acid contents in the cell membrane.

Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc.

BACKGROUND: Mushroom showed pellet, clump and/or filamentous mycelial morphologies during submerged fermentation. Addition of microparticles including Talc (magnesium silicate), aluminum oxide and titanium oxide could control mycelial morphologies to improve mycelia growth and secondary metabolites production. Here, effect of microparticle Talc (45 µm) addition on the mycelial morphology, fermentation performance, monosaccharide compositions of polysaccharides and enzymes activities associated with polysaccharide synthesis in G. frondosa was well investigated to find a clue of the relationship between polysaccharide biosynthesis and morphological changes. RESULTS: Addition of Talc decreased the diameter of the pellets and increased the percentage of S-fraction mycelia. Talc gave the maximum mycelial biomass of 19.25 g/L and exo-polysaccharide of 3.12 g/L at 6.0 g/L of Talc, and mycelial polysaccharide of 0.24 g/g at 3.0 g/L of Talc. Talc altered the monosaccharide compositions/percentages in G. frondosa mycelial polysaccharide with highest mannose percentage of 62.76 % and lowest glucose percentage of 15.22 % followed with the corresponding changes of polysaccharide-synthesis associated enzymes including lowest UDP-glucose pyrophosphorylase (UGP) activity of 91.18 mU/mg and highest UDP-glucose dehydrogenase (UGDG) and GDP-mannose pyrophosphorylase (GMPPB) activities of 81.45 mU/mg and 93.15 mU/mg. CONCLUSION: Our findings revealed that the presence of Talc significantly changed the polysaccharide production and sugar compositions/percentages in mycelial and exo-polysaccharides by affecting mycelial morphology and polysaccharide-biosynthesis related enzymes activities of G. frondosa.

A macromolecular α-glucan from fruiting bodies of Volvariella volvacea activating RAW264. 7 macrophages through MAPKs pathway.

A novel macromolecular polysaccharide VGPⅠ-a was purified from Volvariella volvacea fruiting bodies with ultrasound-assisted extraction, ion exchange and gel chromatography. VGPⅠ-a was proved as a α- glucan with Mw of 1435.6 kDa and contained a 1,4-linked d-Glcp backbone with the substitution at C-6 with 1-linked d-Glcp residue. Congo-red test, AFM and SEM analysis showed VGPⅠ-a had a triple-helical conformation and the interacted chains to form a small screw-rod and dispersed appearance. VGPⅠ-a had no cytotoxic effect on macrophage RAW264.7 cells in vitro and significantly enhanced the production and mRNA expression of NO, TNF-α, IL-6 and IL-1ß in a dose-dependent manner. Further analyses demonstrated that VGPⅠ-a activated the MAPK signaling pathway by improving the phosphorylated levels of p38, JNK and ERK in RAW264.7 cells to promote the expression and secretion of above cytokines. These findings would provide a better understanding of V. volvacea glucan and its potential immunomodulating mechanisms.

The role of Rho1 gene in the cell wall integrity and polysaccharides biosynthesis of the edible mushroom Grifola frondosa.

Grifola frondosa polysaccharides, especially ß-glucans, showed the significant antitumor, hypoglycemic, and immune-stimulating activities. In the present study, a predominant regulatory subunit gfRho1p of ß-1,3-glucan synthase in G. frondosa was identified with a molecular weight of 20.79 kDa and coded by a putative 648-bp small GTPase gene gfRho1. By constructing mutants of RNA interference and over-expression gfRho1, the roles of gfRho1 in the growth, cell wall integrity and polysaccharide biosynthesis were well investigated. The results revealed that defects of gfRho1 slowed mycelial growth rate by 22% to 33%, reduced mycelial polysaccharide and exo-polysaccharide yields by 4% to 7%, increased sensitivity to cell wall stress, and down-regulated gene transcriptions related to PKC-MAPK signaling pathway in cell wall integrity. Over-expression of gfRho1 improved mycelial growth rate and polysaccharide production of G. frondosa. Our study supports that gfRho1 is an essential regulator for polysaccharide biosynthesis, cell growth, cell wall integrity and stress response in G. frondosa.

Enhanced Acid Tolerance in Lactobacillus acidophilus by Atmospheric and Room Temperature Plasma (ARTP) Coupled with Adaptive Laboratory Evolution (ALE).

The aim of this study was to improve the acid tolerance of Lactobacillus acidophilus by combining atmospheric and room temperature plasma (ARTP) mutation with adaptive laboratory evolution (ALE). To achieve a high mutation efficiency, 60 s was determined as the ideal exposure time for ARTP mutation of L. acidophilus with a survival rate of 5.91%. The ARTP-ALE mutant strain LAartp-ale2 displayed increased lactic acid stress tolerance with survival rates of 75.67% and 25.78% when cultured in pH 3.0 and 2.5, respectively, for 3 h. Physiological analysis revealed that the ARTP-ALE mutant exhibited a lower inner membrane permeability than that of the parental strain during acid stress. Furthermore, the mutant LAartp-ale2 produced more biofilm in response to lactic acid-induced acid stress and showed an increased hydrophobicity (87.2%) when compared to the parent strain (76.2%) at pH 2.5. LAartp-ale2 exhibited a higher unsaturated fatty acid (UFA) to saturated fatty acid (SFA) ratio that affected the physical state of the cell membrane for increased survival in pH 3.0 and 2.5. The mutation with ARTP coupled with ALE in the present study proved to be effective in enhancing the acid tolerance of L. acidophilus for potential industrial use.

UDP-glucose pyrophosphorylase gene affects mycelia growth and polysaccharide synthesis of Grifola frondosa.

To elucidate potential roles of UDP-glucose pyrophosphorylase (UGP) in mycelial growth and polysaccharide synthesis of Grifola frondosa, a putative 2036-bp UDP-glucose pyrophosphorylase gene gfugp encoding a 53.17-kDa protein was cloned and re-annotated. Two dual promoter RNA silencing vectors of pAN7-iUGP-P-dual and pAN7-iUGP-C-dual were constructed to down-regulate gfugp expression by targeting its promoter or conserved functional sequences, respectively. Results showed that silence of gfugp promoter sequence had a higher down-regulating efficiency with slower mycelial growth and polysaccharide production than those of conserved sequence. The monosaccharide compositions/percentages of mycelial and exo-polysaccharides significantly changed with the increase of galactose and arabinose contents possibly due to block of UDP-glucose supply by gfugp silence and alteration of sugar metabolism via up-regulation of UDP-glucose-4-epimerase (gfuge) and UDP-xylose-4-epimerase (gfuxe) transcription. Our findings would provide a reference to know the biosynthesis pathway of mushroom polysaccharides and improve their production by metabolic regulation.

Functions of a Glucan Synthase Gene GFGLS in Mycelial Growth and Polysaccharide Production of Grifola frondosa.

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.