Stimulating Novel and Bioactive Metabolite Production by Cocultivation of Two Fungi─Aspergillus oryzae and Epicoccum dendrobii.

Fungi produce various bioactive secondary metabolites (SMs) as protective and weaponized tools to enhance survival in shared ecological niches. By mimicking a competitive ecosystem, cocultivation has been proven to be particularly successful in stimulating SM discovery. Here, we reported the identification of four novel metabolites, epiclactones A and B, epioxochromane and aoergostane, from the coculture of two biotechnologically important strains, Aspergillus oryzae and Epicoccum dendrobii. Transcriptome and metabolome analyses revealed widespread silent gene activation during fungal-fungal interaction. The majority of differentially expressed gene clusters were summarized for both strains. Based on these highly activated biosynthetic pathways, we suggested that a bidirectional chemical defense occurred under cocultivation. E. dendrobii enhanced the production of the spore inhibitor, fumigermin. Moreover, A. oryzae highly accumulated the antifungal agent kojic acid with a yield of up to 1.10 g/L. This study provides an excellent example for the discovery of hidden natural products by cocultivation.

Zbtb14 Promotes Non-Alcoholic Fatty Liver Disease-Associated Fibrosis in Gerbils via the ß-Catenin Pathway.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a popular chronic liver disorder with high morbidity and with no approved therapeutic drugs. Fibrosis is a crucial drug efficacy indicator for NAFLD. Thus, investigating the mechanisms of NAFLD-associated fibrosis and exploring effective therapeutic targets is imperative. METHODS: Gerbil NAFLD-associated fibrosis model was constructed by feeding a high-fat and high-cholesterol diet. The hematoxylin and eosin staining and the alanine transaminase (ALT) and aspartate transaminase (AST) assays were used to determine liver tissue injury. Masson staining and hydroxyproline (Hyp) level determination were used to assess liver fibrosis. High-throughput mRNA sequencing was used to screen differentially expressed genes in the NAFLD-associated fibrosis model. Cell Counting Kit-8 was utilized to test cell viability. RESULTS: Liver injury and fibrosis were observed in the gerbil NAFLD-associated fibrosis model with increased ALT, AST, and Hyp levels. The screened differentially expressed genes were mainly enriched in "negative regulation of hemopoiesis", "response to interleukin-1", and "granulocyte migration". Zinc Finger and BTB Domain Containing 14 (Zbtb14) was upregulated in liver tissues of the gerbil NAFLD-associated fibrosis model, patients with liver fibrosis, and hepatic stellate cells (HSCs). Additionally, Zbtb14 regulated primary HSCs activation via the ß-catenin pathway. CONCLUSIONS: Zbtb14 regulated NAFLD-associated fibrosis via the ß-catenin pathway, for the first time, and it serves as the probable target for NAFLD therapy.

Advances in nanocarriers to improve the stability of dsRNA in the environment.

RNAi technology, known as a revolutionary technology in the history of pesticides, has been identified as a very promising novel approach for crop protection, which is of great significance for achieving the sustainable agricultural development of the United Nations Food and Agriculture Organization. Although many studies have shown that RNA biopesticides have strong application prospects, its stability seriously restricts the commercial use. As the core component of RNAi, double-stranded RNA (dsRNA) is unstable in its natural form. Therefore, how to ensure the stability of dsRNA is one of the most significant challenges in realizing the commercial use of RNA biopesticides. Nanomaterials such as cationic polymers and lipofectamine can improve the stability of dsRNA in the environment, which has been proved. This paper reviews the recent research progress of nanomaterials that can be used to improve the environmental stability of dsRNA, and discusses the advantages and limitations of different nanomaterials combined with dsRNA, which provides reference for the selection of dsRNA nanoformulations.

Bacillus amyloliquefaciens 35 M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium.

Some microbial strains are ideal producers of extracellular enzymes that can be used in various industries. However, in many fields, especially in the pharmaceutical field, these enzymes need to be recovered and purified through multistep processes and tedious procedures before they can be used. The recovery process is difficult and increases the cost of enzyme production. Therefore, reducing purification steps will greatly benefit the utilization of microbial enzymes. The 35 M strain of Bacillus amyloliquefaciens, which has high extracellular protease production, was isolated from a phosphate mine. When cultured in a medium with soybean meal as the main component, the maximum activity of extracellular protease reached 16,992 U/mL. SDS-PAGE showed that there were two main proteins in the fermentation supernatant, with a paucity of other defined protein bands. Mass spectrometry and zymogram analysis showed that the two main bands were two proteases, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively. Gene cloning, sequencing, and further comparisons were used to confirm AprM and NprM correspond to these proteases from B. amyloliquefaciens. Notably, SDS-PAGE and zymogram analysis showed that NprM had obviously higher catalytic efficiency toward casein than did AprM. Strain 35 M is a promising protease producer with great potential for applications in industrial protease production. Additionally, this study demonstrates strain 35 M may be particularly well suited to use in degrading anti-nutritional factors in soybean meal, so as to improve the nutritional value of soybean meal.

Transcriptional Differences Guided Discovery and Genetic Identification of Coprogen and Dimerumic Acid Siderophores in Metarhizium robertsii.

Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, biosynthesis gene clusters of coprogens and dimerumic acids were identified by transcriptional level differences of genes related to iron deficiency conditions in Metarhizium robertsii. This leads to the characterization of new coprogen metachelin C (1) and five known siderophores metachelin A (2), metachelin A-CE (3), metachelin B (4), dimerumic acid 11-mannoside (5), and dimerumic acid (6). The structure of metachelin C (1) was elucidated by NMR spectroscopy and HR-ESI-MS analysis. Genetic deletions of mrsidA, and mrsidD abolished the production of compounds 1-6 that implied their involvement in the biosynthesis of coprogen and dimerumic acid. Interestingly, NRPS gene mrsidD is responsible for biosynthesis of both coprogen and dimerumic acid, thus we proposed plausible biosynthetic pathways for the synthesis of coprogen and dimerumic acid siderophores. Therefore, our study provides the genetic basis for understanding the biosynthetic pathway of coprogen and dimerumic acid in Metarhizium robertsii.

Rapid and Accurate Screening of Lysine-Producing Edible Mushrooms via the Homocitrate Synthase Gene as a Universal Molecular Marker.

Edible mushrooms are important nutraceutical sources of foods and drugs, which can produce various nutritional ingredients including all essential amino acids. The method of rapid screening for the strains producing specific functional components is very indispensable. Homocitrate synthase is one of the key enzymes in the α-aminoadipate pathway for lysine biosynthesis and has preferable sequence conservation in Agaricales. Based on the blast of homocitrate synthase homologous genes of strains of Agaricales, we achieved combinations of degenerate primers as molecular markers to rapidly screen the lysine-producing edible mushrooms. The experimental results revealed that the consistency between PCR amplification and HPLC analysis attained 82 and 75% in strains of Agaricales and Polyporales, respectively. The finding showed that the molecular marker has higher universality for screening edible mushroom resources of Agaricales. This PCR-based approach shows excellent potential in evaluating and discriminating edible wild-grown mushrooms with high lysine content in Agaricales.

Discovery and genetic identification of amphiphilic coprogen siderophores from Trichoderm hypoxylon.

Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, we discovered and identified five amphiphilic coprogen siderophores including three new natural products according to LC-MS-guided separation strategy from Trichoderm hypoxylon. The structures of three new coprogens were elucidated by NMR spectroscopy, and high-resolution (HR)-ESI-MS analysis. Genetic deletions of dfcA and dfcB abolished the production of compounds 1-5 that implied their involvement in the biosynthesis of coprogens. Interestingly, cultivations of ΔdfcA and ΔdfcB mutants with pathogenic fungi Fusarium oxysporum and Mucor corcinelloides showed the weaker inhibitions in comparison to wild type that demonstrated coprogen's role in combating the pathogenic fungi. Our study not only enriched the diversities of siderophores but also provided an approach for finding the rare amphiphilic coprogen siderophores in fungi. Furthermore, this work provided a basis for investigation on the biosynthesis of fungal amphiphilic siderophores and their ecological roles in nature. KEY POINTS: • A series of amphiphilic coprogens were found. • The gene cluster of amphiphilic coprogens and ecological roles were elucidated.

Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana.

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

Molecular Evolution of Lysine Biosynthesis in Agaricomycetes.

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.

Modulatory Effect of Fermented Papaya Extracts on Mammary Gland Hyperplasia Induced by Estrogen and Progestin in Female Rats.

Fermented papaya extracts (FPEs) are obtained by fermentation of papaya by Aspergillus oryzae and yeasts. In this study, we investigated the protective effects of FPEs on mammary gland hyperplasia induced by estrogen and progestogen. Rats were randomly divided into 6 groups, including a control group, an FPE-alone group, a model group, and three FPE treatment groups (each receiving 30, 15, or 5 ml/kg FPEs). Severe mammary gland hyperplasia was induced upon estradiol benzoate and progestin administration. FPEs could improve the pathological features of the animal model and reduce estrogen levels in the serum. Analysis of oxidant indices revealed that FPEs could increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, decrease malondialdehyde (MDA) level in the mammary glands and serum of the animal models, and decrease the proportion of cells positive for the oxidative DNA damage marker 8-oxo-dG in the mammary glands. Additionally, estradiol benzoate and progestin altered the levels of serum biochemical compounds such as aspartate transaminase (AST), total bilirubin (TBIL), and alanine transaminase (ALT), as well as hepatic oxidant indices such as SOD, GSH-Px, MDA, and 8-oxo-2'-deoxyguanosine (8-oxo-dG). These indices reverted to normal levels upon oral administration of a high dose of FPEs. Taken together, our results indicate that FPEs can protect the mammary glands and other visceral organs from oxidative damage.

Comamonas phosphati sp. nov., isolated from a phosphate mine.

A Gram-stain-negative, facultatively anaerobic, non-pigmented, non-sporulating, rod-shaped bacterial strain (WYH 22-41T) was isolated from a phosphate mine in Yunnan Province, China. The cells were motile with a single polar flagellum. The 16S rRNA gene of strain WYH 22-41T was phylogenetically related to the corresponding gene of Comamonas terrae DSM 27221T (98.4 % 16S rRNA gene sequence similarity), Comamonas odontotermitis LMG 23579T (97.6 %) and Comamonas aquatica LMG 2370T (97.4 %). DNA-DNA hybridizations of strain WYH 22-41T with these three strains showed relatedness values of 33.2 %, 20.5 % and 27.7 %, respectively. The DNA G+C content of strain WYH 22-41T was 62.4 mol%. The predominant respiratory quinone was ubiquinone-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major fatty acids of strain WYH 22-41T were C16 : 0, C17 : 0 cyclo, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). On the basis of phenotypic properties, phylogenetic characteristics, DNA-DNA hybridization, as well as whole-cell fatty acid composition, strain WYH 22-41T represents a novel species of the genus Comamonas, for which the name Comamonas phosphati sp. nov. is proposed. The type strain is WYH 22-41T ( = CGMCC 1.12294T = DSM 26017T).

Methylopila henanense sp. nov., a novel methylotrophic bacterium isolated from tribenuron methyl-contaminated wheat soil.

A bacterial strain, designated LYBFD3-16A2(T), was isolated from tribenuron methyl contaminated wheat soil. Cells were observed to be Gram-negative short rods with a single flagellum. The strain was found to utilize methanol, glucose, maltose and mannitol as carbon and energy sources, and utilized glutamate, leucine, phenylalanine as organic nitrogen sources. Strain LYBFD3-16A2(T) was found to be aerobic, to form urease, produce hydrogen sulfide and reduce nitrate to nitrite. The indole test in tryptone broth was observed to be positive. The major cellular fatty acids were identified as C18:1ω7c (81.3 %), 11-methylC18:1ω7c (7.9 %), C18:0 (3.0 %) and C16:0 (3.0 %). The major phospholipids were identified as phosphatidylcholine, phosphatidylethanolamine, phosphatidyglycerol and diphosphatidylglycerol. The main ubiquinone was identified as Q-10. The DNA G+C content was determined to be between 70.2 and 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated the affiliation of strain LYBFD3-16A2 to members of the genus Methylopila. The DNA-DNA hybridization values of the novel strain with the type strains of the most closely related species Methylopila musalis MUSA(T) and Methylopila jiangsuensis JZL-4(T) were 35.4 % and 31.4 % respectively. The genotypic and phenotypic characterization, along with chemotaxonomic properties of strain LYBFD3-16A2(T), showed that the strain represents a novel species of the genus Methylopila for which the name Methylopila henanense sp. nov. is proposed. The type strain is LYBFD3-16A2(T) (=CGMCC1.10703(T) = LMG 25959(T)).

Novosphingobium kunmingense sp. nov., isolated from a phosphate mine.

A yellow-pigmented, Gram-stain-negative, strictly aerobic, rod-shaped, round-ended bacterium, designated strain 18-11HK(T), was isolated from a phosphate mine situated in the suburb of Kunming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, strain 18-11HK(T) was shown to belong to the genus Novosphingobium, showing the highest levels of sequence similarity with respect to 'Novosphingobium ginsenosidimutans' FW-6 (97.2%), Novosphingobium subterraneum DSM 12447(T) (96.7%), Novosphingobium aromaticivorans DSM 12444(T) (96.7%) and Novosphingobium tardaugens DSM 16702(T) (96.3%). Strain 18-11HK(T) had a genomic DNA G+C content of 65.3 mol% and Q-10 as the predominant respiratory quinone. DNA-DNA hybridizations of strain 18-11HK(T) with N. subterraneum DSM 12447(T), N. aromaticivorans DSM 12444(T) and N. tardaugens DSM 16702(T) showed low relatedness values of 29.6, 33.5 and 32.3%, respectively. The predominant fatty acids of strain 18-11HK(T) were summed feature 8 (C18 : 1ω7c and/or C18: 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0, and the major 2-hydroxy fatty acid was C14 : 0 2-OH. The polar lipid profile revealed the presence of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and some unidentified lipids. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain 18-11HK(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium kunmingense sp. nov. is proposed. The type strain is 18-11HK(T) ( = CGMCC 1.12274(T) = DSM 25975(T)).

Pseudomonas kunmingensis sp. nov., an exopolysaccharide-producing bacterium isolated from a phosphate mine.

A Gram-stain-negative, rod-shaped, exopolysaccharide-producing, strictly aerobic bacterium with a single polar flagellum, designated strain HL22-2(T), was isolated from a phosphate mine situated in a suburb of Kunmming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HL22-2(T) was related to members of the genus Pseudomonas. 16S rRNA gene sequence similarities between strain HL22-2(T) and Pseudomonas xanthomarina KMM 1447(T), Pseudomonas alcaliphila AL15-21(T) and Pseudomonas stutzeri ATCC 17588(T) were 98.9, 98.10% and 98.06%, respectively. The major cellular fatty acids were C(18 : 1)ω7c, C(16 : 0) and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 60.3 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, strain HL22-2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas kunmingensis sp. nov. is proposed. The type strain is HL22-2(T) ( = CGMCC 1.12273(T) = DSM 25974(T)).

Pseudomonas linyingensis sp. nov.: a novel bacterium isolated from wheat soil subjected to long-term herbicides application.

A strain of genus Pseudomonas, LYBRD3-7(T) was isolated from long-term sulfonylurea herbicides applied wheat-field soil in Linying located in Henan province of China. This strain is a strictly aerobic and Gram-negative short rod-shaped bacterium with single flagellum. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate as a member of Pseudomonas, and most closely to Pseudomonas tuomuerensis CGMCC 1.1365(T) (97.1 %) and P. alcaligenes IAM12411(T) (97.1 %). Morphological characters and chemotaxonomic data confirmed the affiliation of strain LYBRD3-7(T) to the genus Pseudomonas. The results of phylogenetic analysis, physiological and biochemical studies, and DNA-DNA hybridization allowed the differentiation of genotype and phenotype between strain LYBRD3-7(T) and the phylogenetic closest species with valid names. The name proposed for the new species is Pseudomonas linyingensis sp. nov. The type strain is LYBRD3-7(T) (=CGMCC 1.10701(T ) =LMG 25967(T)).

Pseudomonas nitritireducens sp. nov., a nitrite reduction bacterium isolated from wheat soil.

A Gram-negative, non-mobile, polar single flagellum, rod-shaped bacterium WZBFD3-5A2(T) was isolated from a wheat soil subjected to herbicides for several years. Cells of strain WZBFD3-5A2(T) grow optimally on Luria-Bertani agar medium at 30 °C in the presence of 0-4.0 % (w/v) NaCl and pH 8.0. 16S rRNA gene sequence analysis revealed that strain WZBFD3-5A2(T) belongs to the genus Pseudomonas. Physiological and biochemical tests supported the phylogenetic affiliation. Strain WZBFD3-5A2(T) is closely related to Pseudomonas nitroreducens IAM1439(T), sharing 99.7 % sequence similarity. DNA-DNA hybridization experiments between the two strains showed only moderate reassociation similarity (33.92 ± 1.0 %). The DNA G+C content is 62.0 mol%. The predominant respiratory quinine is Q-9. The major cellular fatty acids present are C(16:0) (28.55 %), C(16:1ω6c) or C(16:1ω7c) (20.94 %), C(18:1ω7c) (17.21 %) and C(18:0) (13.73 %). The isolate is distinguishable from other related members of the genus Pseudomonas on the basis of phenotypic and biochemical characteristics. From the genotypic, chemotaxonomic and phenotypic data, it is evident that strain WZBFD3-5A2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitritereducens sp. nov. is proposed. The type strain is WZBFD3-5A2(T) (=CGMCC 1.10702(T) = LMG 25966(T)).

Gene cloning and recombinant expression of a novel fungal immunomodulatory protein from Trametes versicolor.

Homology-based cloning was used to obtain a new gene (named FIP-tvc) from the genomic DNA of the Chinese traditional medicinal mushroom Trametes versicolor. FIP-tvc is a member of the fungal immunomodulatory protein family, is composed of 336bp encoding 111 amino acids, and is highly homologous with other fungal immunomodulatory proteins. In addition, an expression system for FIP-tvc was constructed. The results indicated that recombinant protein FIP-tvc could be expressed in Escherichia coli and that about 20% of the expressed protein was in soluble form. Recombinant FIP-tvc was capable of agglutinating mouse and rat red blood cells. Furthermore, recombinant protein FIP-tvc could selectively enhance the expression of interleukin (IL)-1α, IL-2, IL-5, IL-6, tumor necrosis factor-alpha (TNF-α), and lympotoxin (LT) in mouse spleen cells.

[Correlation of bacterial diversity in rot Chinese cabbage with the habitat].

OBJECTIVE: To investigate the diversity of bacteria in soft rot Chinese cabbage and analyze their correlation with rhizosphere bacteria, we analyzed the bacterial population structures of soft rot Chinese cabbage and the rhizosphere in different habitat. METHODS: Based on the initial medium and artificial Chinese cabbage medium, we isolated the bacteria from soft rot tissues and rhizospheric soils from two typical habitats. According to the analysis of 16S rRNA gene sequence homology, we identified the isolated strains and analyzed the strains population structure. RESULTS: The total bacteria in soft rot tissues were 4.0 x 10(8) cell g(-1) and 1.2 x 10(11) cell g(-1), the number of pure strains were 56 and 85, the dominant strains were Curtobacterium flaccumfaciens pv. flaccumfaciens and Pseudomonas spp. (P. hibiscicola, P. taiwanensis, P. tuomuerensis, P. mosselii). The total bacteria in rhizospheric soils were 2.7 x 10(5) cell g(-1) and 6.2 x 10(7) cell g(-1), the number of pure strains were 36 and 70, the dominant strains were Bacillus megatherium and Pseudomonas spp. (P. plecoglossicida, P. hibiscicola, P. parafulva, P. monteilii, P. geniculata). CONCLUSION: The methods used in this study were effective in analyzing bacterial diversity in soft rot Chinese cabbage and the results correlated well with the soil bacteria analysis, suggesting that soft rot Chinese cabbage may be induced by various environmental bacteria. Our results infer that soft rot of Chinese cabbage might be pathogen-complex, and provide the clues for the mechanism study and protection.