Metagenomic and culture-dependent analysis of Rhinopithecius bieti gut microbiota and characterization of a novel genus of Sphingobacteriaceae.

Culture-dependent and metagenomic binning techniques were employed to gain an insight into the diversification of gut bacteria in Rhinopithecius bieti, a highly endangered primate endemic to China. Our analyses revealed that Bacillota_A and Bacteroidota were the dominant phyla. These two phyla species are rich in carbohydrate active enzymes, which could provide nutrients and energy for their own or hosts' survival under different circumstances. Among the culturable bacteria, one novel bacterium, designated as WQ 2009T, formed a distinct branch that had a low similarity to the known species in the family Sphingobacteriaceae, based on the phylogenetic analysis of its 16S rRNA gene sequence or phylogenomic analysis. The ANI, dDDH and AAI values between WQ 2009T and its most closely related strains S. kitahiroshimense 10CT, S. pakistanense NCCP-246T and S. faecium DSM 11690T were significantly lower than the accepted cut-off values for microbial species delineation. All results demonstrated that WQ 2009T represent a novel genus, for which names Rhinopithecimicrobium gen. nov. and Rhinopithecimicrobium faecis sp. nov. (Type strain WQ 2009T = CCTCC AA 2021153T = KCTC 82941T) are proposed.

Paenibacillus baimaensis sp. nov., a bacterium isolated from mountain soil in the habitat of Rhinopithecus bieti.

A novel Gram-positive strain WQ 127069T that was isolated from the soil of Baima Snow Mountain, a habitat of highly endangered Yunnan snub-nosed monkeys (Rhinopithecus bieti), was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolate belongs to the genus Paenibacillus, showing 98.4 and 96.08 % sequence similarity to the type strains Paenibacillus periandrae PM10T and Paenibacillus foliorum LMG 31456T, respectively. The G+C content of the genomic DNA of strain WQ127069T was 45.6 mol%. The predominant isoprenoid quinone was MK-7, and meso-diaminopimelic acid was present in peptidoglycan. The major cellular fatty acids were antiiso-C15 : 0, iso-C15 : 0 and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmonomethylethanolamine. The whole genome average nucleotide identity and digital DNA-DNA hybridization values between strain WQ 127069T and strain PM10T were 93.2 and 52.5 %, respectively. Growth occurred at 5-40 °C (optimally at 20-35 °C), pH 6-8 (optimally at pH7.0) and with 0.5-2 % (w/v) NaCl (optimally at 0.5 %). On the basis of the taxonomic evidence, a novel species, Paenibacillus baimaensis sp. nov., is proposed. The type strain is WQ 127069T (=KCTC 43480T=CCTCC AB 2022381T).

Sphingobacterium bovistauri sp. nov., Isolated from the Faeces of Bos Taurus.

A novel bacterium designated WQ 366 T was isolated from the faeces of Bos taurus, foraging on the slopes of the Baima Snow Mountain in Yunnan, China. The isolate grew optimally at 30 â„ƒ and pH 7.0-8.0 without NaCl. The cells were Gram-stain-negative, aerobic, rod-shaped, non-gliding, catalase-positive, and produced yellow color colonies on Columbia Agar. A polyphasic study was applied to clarify its taxonomic position through 16S rRNA gene and genome sequence analysis, and other extensive biological typing. Phylogenetic analysis revealed that the isolate was affiliated to the genus Sphingobacterium and its 16S rRNA gene sequence was closely related to Sphingobacterium bovisgrunnientis YK2 T (97.3%), Sphingobacterium composti T5-12 T (96.4%), and Sphingobacterium cavernae 5.0403-2 T (96.4%). The calculated whole genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization values between strain WQ 366 T and the three related strains were 78.3, 78.6, 73.9 and 21.2, 21.2, 21.0%, respectively. The predominant fatty acids (>10%) were iso-C15:0, iso-C17:0 3-OH, Summed Feature 3 (C16:1 ω7c and/or C16:1 ω6c), and Summed feature 9 (iso-C17:1 ω9c and 10-methyl C16:0). The main polar lipids were PE, GPL, GL, and PL. MK-7 was the major menaquinone. The genome size and the G + C content of WQ 366 T was 4.1 Mb and 34.6%, respectively. All these results indicated that strain WQ 366 T represents a novel species of the Sphingobacterium genus. Therefore, the name Sphingobacterium bovistauri sp. nov. is proposed, and the type strain is WQ 366 T (= CCTCC AA 2020029 T = KCTC 82395 T).

Advenella mandrilli sp. nov., a bacterium isolated from the faeces of Mandrillus sphinx.

A novel Gram-negative strain WQ 585T, isolated from the faeces of mandrills (Mandrillus sphinx) collected at Yunnan Wild Animal Park, Yunnan province, China, was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belongs to the genus Advenella, sharing 98.5% and 98.2% sequence similarity with the type strain Advenella alkanexedens LAM0050T and Advenella faeciporci M-07T, respectively. The predominant ubiquinone was Q-8. The major cellular fatty acids (> 10%) were C16:0, C17:0 cyclo and Summed Feature 2. The G + C content of the genomic DNA of strain WQ 585T was 49.0%. The whole genome average nucleotide identity (gANI) values of strain WQ 585T with strain A. alkanexedens LAM0050T and A. faeciporci M-07T were 86.7% and 86.7%, and the digital DNA-DNA hybridization values of strain WQ 585T with strain A. alkanexedens LAM0050T and A. faeciporci M-07T were 64.5% and 62.5%, respectively. Growth occurred at 10-45 °C (optimally at 20-30 °C), pH 6.0-9.0 (optimally at pH 7.0), and 0-5% (w/v) NaCl (optimally at 0.5-2.0%). On the basis of the taxonomic evidence, a novel species, Advenella mandrilli sp. nov., is proposed. The type strain is WQ 585T (= KCTC 82396 T = CCTCC AA 2020028 T).

Functional Characterization of a New Bifunctional Terpene Synthase LpNES1 from a Medicinal Plant Laggera pter odonta.

Laggera pterodonta, known in China as 'Choulingdan' for its stimulous odor, has long been used as traditional herbal medicine. The essential oil of L. pterodonta, which exhibits various pharmacological activities, is a rich resource of monoterpenes and sesquiterpenes. To date, however, the terpene synthases responsible for their production remain unknown. In present study, a new terpene synthase gene (LpNES1) was identified from L. pterodonta, transcript level of which was significantly upregulated in response to methyl jasmonate treatment. Recombinant LpNES1 could synthesize (E)-nerolidol and minor ß-farnesene from farnesyl diphosphate and linalool from geranyl diphosphate in vitro. Whereas, only sesquiterpenes including (E)-nerolidol and minor ß-farnesene were released when LpNES1 was reconstituted in yeast, even coexpressed with a geranyl diphosphate synthase (ERG20WW). Combined with subcellular localization experiment, the result indicated that the cytosol-targeted LpNES1 was responsible for (E)-nerolidol biosynthesis exclusively in L. pterodonta. Additionally, the expression level of LpNES1 gene was more prominent in floral buds than that in other tissues. LpNES1 characterized in present study not only lays the molecular foundation for sesquiterpene biosynthesis of L. pterodonta, but provides a key element for further biosynthesis of bioactive compound in microbes.

Faecalibacter rhinopitheci sp. nov., a bacterium isolated from the faeces of Rhinopithecus bieti.

A novel Gram-stain-negative strain, WQ 117T, isolated from the faeces of Rhinopithecus bieti collected at Yunnan Snub-nosed Monkey National Park, Yunnan province, PR China, was subjected to a polyphasic taxonomic study. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate represented a member of the genus Faecalibacter, sharing 97.64 % sequence similarity with the type strain Faecalibacter macacae YIM 102668T. The G+C content of the genomic DNA of WQ117T was 30.5 mol%. The predominant isoprenoid quinone was MK-6. The major cellular fatty acids was iso-C15 : 0. The whole genome average nucleotide identity (gANI) values and the digital DNA-DNA hybridization values between WQ 117T and YIM 102668T were 79.66 % and 22.20 %, respectively. Growth occurred at 0-50 °C (optimally at 28-35 °C), pH 7.0-9.0 (optimally at pH 8.0) and with 0-2 % (w/v) NaCl (optimally without NaCl). On the basis of the taxonomic evidence, a novel species, Faecalibacter rhinopitheci sp. nov., is proposed. The type strain is WQ 117T (=KCTC 82394T=CCTCC AA 2020027T).

Sphingobacterium rhinopitheci sp. Nov., isolated from the faeces of Rhinopithecus bieti in China.

A novel bacterium, WQ 047T, was isolated from the faeces of Rhinopithecus bieti, a highly endangered primate endemic to China. The cells were aerobic, oval/rod-shaped, Gram-stain-negative, non-motile, catalase positive, and produced yellow pigmented colonies on Columbia Agar. The taxonomic position of WQ 047T was clarified by applying a polyphasic study based on 16S rRNA gene sequence phylogenetic analysis, extensive biological typing, and whole genome sequencing. Phylogenetic analysis indicated that stain WQ 047T belonged to the genus Sphingobacterium and its 16S rRNA gene sequence exhibited 96.47% pairwise similarity with that of the closest relatives Sphingobacterium nematocida M-SX103T. The calculated whole genome average nucleotide identity (ANI) value between strain WQ 047T and strain M-SX103 was 72.3%. The digital DNA-DNA hybridization value of strain WQ 047T and M-SX103T was 15.73%, which was obtained by calculating the genome-to-genome distance. The major fatty acids were C15:0 iso, C17:0 iso 3-OH, Summed Feature 3 (C16:1 ω7c/C16:1 ω6c) and Summed feature 9 (iso-C17:1ω9c and/or 10-methyl C16:0). The predominant polar lipids were PE, PL and APL. MK-7 was the predominant menaquinone. The G + C content of WQ 047T was 34.89 mol% according to genome analysis. All these characteristics were consistent with those of the genus of Sphingobacterium. Therefore, based on these results, we propose a novel species for which the name Sphingobacterium rhinopitheci sp. Nov. is proposed, with the type strain WQ 047T (= CCTCC AA 2020026T = KCTC82393T).

[Recent advances in biosynthesis of forskolin].

Forskolin is a complex labdane plant diterpenoid, which has been used in the treatment of a variety of diseases based on its activity as an activator of adenosine monophosphate(cAMP) cyclase. Natural forskolin exists only in the cork layer of the root of Coleus forskohlii. Due to the complexity of the extraction and chemical synthesis processes, the yield and purity of forskolin cannot meet commercial requirements. In recent years, with the rapid development of synthetic biology and the analysis and interpretation of many diterpene biosynthetic pathways, a new approach has been provided for the green production of forskolin. In this paper, the structure, activity, biosynthetic pathway and the heterologous biosynthesis of forskolin were reviewed. The problems and solutions in the heterologous biosynthesis of forskolin were also discussed and summarized, which will provide references for the construction of high-yielding forskolin engineering strains.

Cloning and Characterization of a Ginsenoside-Hydrolyzing α-L-Arabinofuranosidase, CaAraf51, From Cellulosimicrobium aquatile Lyp51.

Moutai Jiuqu is a famous aromatic raw material of Maotai flavor liquor in China. It is brewed at high temperature and contains many kinds of bacteria, molds, and yeasts. There are many useful glycoside hydrolases in these microfloras, from which efficient glycoside hydrolases can be screened for biotransformation of natural saponins. In this study, an α-L-arabinofuranosidase gene (CaAraf51, 1524 bp, 507 amino acid, 55.07 kDa, and pI = 4.8) was cloned from Cellulosimicrobium aquatile Lyp51, which was isolated from the Maotai Jiuqu. The CaAraf51 was heterogeneously expressed in E. coli BL21 (DE3) and purified by N-terminal His-tag with the Ni2+-affinity column chromatography. The results show that purified CaAraf51 has a 6.8-fold purification factor and specific activity of 15 U/mg. Under optimal conditions (pH 5.0, temperature 40 °C), kinetic parameters Km of CaAraf51 for pNPαAraf and Rc were 1.1 and 0.57 mM, the Vmax were 25 and 6.25 µmol/min/mg, respectively. 90% of 0.87 mg Rc substrate can be transformed by 9.6 U purified CaAraf51 in 1 mL reaction system under suitable conditions (30 °C, pH 7.5 phosphate buffer, 1 h). In addition, we also tested the effects of metal ions and chemical agents on the activity of CaAraf51. According to systematically studied its function and enzymatic properties, CaAraf51 has excellent value and potential of biotransformation Rc into Rd.

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

A new cyclohexenone from the tin mine tailings-derived fungus Aspergillus flavus YIM DT 10012.

A new cyclohexenone, named phomaligol D (1), together with two known compounds, kojic acid (2) and phomaligol A (3) were isolated from the tin mine tailings-derived fungus Aspergillus flavus YIM DT 10012. Their structures were elucidated by detailed analysis of spectroscopic data.

Two new diketopiperazines from the tin mine tailings-derived fungus Schizophyllum commune YIM DT 10058.

Two new diketopiperazines, named diphenylalazine C (1) and epicoccin U (2), together with six known compounds (3-8) were isolated from the EtOAc extract of the fermentation broth of the tin mine tailings-derived fungus Schizophyllum commune YIM DT 10058. Their structures were elucidated by detailed analysis of spectroscopic data and comparison with related known compounds. Compounds 1 and 2 exhibited weak antibacterial and cytotoxic activities.

[Thermodynamic analysis of water adsorption and desorption process of Chinese herbal decoction pieces].

Based on the basic theory of thermodynamics, the thermodynamic parameters and related equations in the process of water adsorption and desorption of Chinese herbal decoction pieces were established, and their water absorption and desorption characteristics were analyzed. The physical significance of the thermodynamic parameters, such as differential adsorption enthalpy, differential adsorption entropy, integral adsorption enthalpy, integral adsorption entropy and the free energy of adsorption, were discussed in this paper to provide theoretical basis for the research on the water adsorption and desorption mechanism, optimum drying process parameters, storage conditions and packaging methods of Chinese herbal decoction pieces.

[Growth inhibition effect of immobilized pectinase on Microcystis aeruginosa].

To confirm the growth inhibition effect of immobilized pectinase on algae, co-cultivation method was used to investigate the effect of immobilized pectinase on the growth of Microcystis aeruginosa. After co-cultivation, the damage status of the algae was observed through electron microscope, and the effect of immobilized pectase on the physiological and biochemical characteristics of the algae was also measured. The results showed that the algae and immobilized pectase co-cultivated solution etiolated distinctly on the third day and there was a significantly positive correlation between the extent of etiolation and the dosage as well as the treating time of the immobilized pectinase. Under electron microscope, plasmolysis was found in the slightly damaged cells, and the cell surface of these cells was rough, uneven and irregular; the severely damaged cells were collapsed or disintegrated completely. The algal yield and the chlorophyll a content decreased significantly with the increase of the treating time. The measurement of the malondiadehyde (MDA) value showed that the antioxidation system of the treated algal cells was destroyed, and their membrane lipid was severely peroxidated. The study indicated that the immobilized pectinase could efficiently inhibit the growth of M. aeruginosa, and the inhibitory rate reached up to 96%.