Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

Effects of mushroom-tobacco rotation on microbial community structure in continuous cropping tobacco soil.

AIMS: Rotation is an effective strategy for controlling crop diseases and improving plant health. However, the effect of a mushroom-tobacco rotation on the composition and structure of microbial communities in continuous cropping soil is unclear. METHODS AND RESULTS: This study analysed the structure and function of soil bacterial and fungal communities using Illumina MiSeq high-throughput 16S rRNA gene sequencing. The results showed that the physicochemical properties (organic matter, available nitrogen, available phosphorus, and available potassium) and enzymatic activity (phosphatase, catalase, urease and invertase activity) in the rotation treatments (Y1, M1, Y2 and M2) were significantly higher than those in the control (continuous cropping) treatment (CK) and reached peak values in the M2 treatment. PCA showed that the soil microbial community structure in each rotation treatment was different from that in the control. The dominant bacterial phyla of the different soil treatments were Proteobacteria and Actinobacteriota, and the dominant fungal phyla of the different soil treatments were Ascomycota and Basidiomycota. The M2 rotation significantly reduced the relative abundance of harmful fungi (Penicillium and Gibberella) compared to the other treatments. RDA showed that the most abundant bacterial taxa were negatively correlated with pH and positively correlated with physicochemical properties. However, the most abundant fungal taxa were positively correlated with pH and negatively correlated with physicochemical properties. CONCLUSIONS: The mushroom-tobacco rotation can effectively maintain the ecological balance of the substrate microbial environment, and provide a more effective way to prevent the continuous cropping of tobacco.

Two new C19-diterpenoid alkaloids from Aconitum straminiflorum.

Two new C19-diterpenoid alkaloids, straconitines A (1) and B (2), were isolated from the roots of Aconitum straminiflorum. Their structures were elucidated as 14-benzoylducloudine D (1) and 6-hydroxy-14-benzoylducloudine D (2) based on spectroscopic analysis, including IR, ESI-MS, HR-ESI-MS, 1D, and 2D NMR.

Three new sativene sesquiterpenoids from cultures of endophytic fungus Bipolaris eleusines.

Three new sativene sesquiterpenoids, bipolenins A-C (1-3), together with two known analogs (4 and 5), were obtained from cultures of endophytic fungus Bipolaris eleusines. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses. Compound 2 showed weak inhibitory activities against lung cancer A-549 and breast cancer MCF-7 with IC50 values of 33.2 and 19.1 µmol, respectively.

Arbuscular mycorrhiza and plant growth promoting endophytes facilitates accumulation of saponin under moderate drought stress.

Objective: Paris polyphylla var. yunnanensis, one of the important medicinal plant resources in Yunnan, China, usually takes 6-8 years to be harvested. Therefore, it is urgent to find a method that can not only shorten its growth years, but also improve its quality. In this study, we examined the effects of a combination treatment of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting endophytes (PGPE) and drought stress on the accumulation of saponins in it. Methods: P. polyphylla var. yunnanensis was infected with a mixture of AMF and PGPE under drought stress. The content of saponins, as well as morphological, physiological, and biochemical indicators, were all measured. The UGTs gene related to saponin synthesis was obtained from transcriptome data by homologous comparison, which were used for RT-PCR and phylogenetic analysis. Results: Regardless of water, AMF treatment could infect the roots of P. polyphylla var. yunnanensis, however double inoculation with AMF and PGPE (AMF + PGPE) would reduce the infection rate of AMF. Plant height, aboveground and underground fresh weight did not differ significantly between the single inoculation AMF and the double inoculation treatment under different water conditions, but the inoculation treatment significantly increased the plant height of P. polyphylla var. yunnanensis compared to the non-inoculation treatment. Single inoculation with AMF considerably increased the net photosynthetic rate, stomatal conductance, and transpiration rate of P. polyphylla var. yunnanensis leaves under various water conditions, but double inoculation with AMF + PGPE greatly increased the intercellular CO2 concentration and chlorophyll fluorescence parameter (Fv/Fm). Under diverse water treatments, single inoculation AMF had the highest proline content, whereas double inoculation AMF + PGPE may greatly improve the amount of abscisic acid (ABA) and indoleacetic acid (IAA) compared to normal water under moderate drought. Double inoculation AMF + PGPE treatment improved the proportion of N, P, and K in the rhizome of P. polyphylla var. yunnanensis under various water conditions. Under moderate drought stress, AMF + PGPE significantly enhanced the contents of P. polyphylla var. yunnanensis saponins I, II, VII, and total saponins as compared to normal water circumstances. Farnesyl diphosphate synthase (FPPS), Geranyl pyrophosphate synthase (GPPS), Cycloartenol synthase (CAS), and Squalene epoxidase (SE1) were the genes that were significantly up-regulated at the same time. The amount of saponins was favorably linked with the expression of CAS, GPPS, and SE1. Saponin VI content and glycosyl transferase (UGT) 010922 gene expression were found to be substantially associated, as was saponin II content and UGT010935 gene expression. Conclusion: Under moderate drought, AMF + PGPE was more conducive to the increase of hormone content, nutrient absorption, and total saponin content in P. polyphylla var. yunnanensis, and AMF + PGPE could up regulate the expression of key genes and UGTs genes in one or more steroidal saponin synthesis pathways to varying degrees, thereby stimulating the synthesis and accumulation of steroidal saponins in the rhizome of P. polyphylla var. yunnanensis. The combination of AMF and PGPE inoculation, as well as adequate soil drought, reduced the buildup of saponins in P. polyphylla var. yunnanensis and increased its quality.

Mycorrhizal fungi reduce the photosystem damage caused by drought stress on Paris polyphylla var. yunnanensis.

Drought stress (DS) is one of the important abiotic stresses facing cash crops today. Drought can reduce plant growth and development, inhibit photosynthesis, and thus reduce plant yield. In this experiment, we investigated the protective mechanism of AMF on plant photosynthetic system by inoculating Paris polyphylla var. yunnanensis(P.py) with a clumping mycorrhizal fungus (AMF) under drought conditions. The drought environment was maintained by weighing AMF plants and non-AMF plants. The relative water content (RWC) of plant leaves was measured to determine its drought effect. DS decreased the RWC of plants, but AMF was able to increase the RWC of plants. chlorophyll a fluorescence curve measurements revealed that DS increased the OKJIP curve of plants, but AMF was able to reduce this trend, indicating that AMF increased the light absorption capacity of plants. DS also caused a decrease in plant Y(I) and Y(II). ETRI and ETRII, and increased Y(NO) and Y(NA) in plants, indicating that DS caused photosystem damage in plants. For the same host, different AMFs did not help to the same extent, but all AMFs were able to help plants reduce this damage and contribute to the increase of plant photosynthesis under normal water conditions.

Mechanisms associated with the synergistic induction of resistance to tobacco black shank in tobacco by arbuscular mycorrhizal fungi and ß-aminobutyric acid.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, is one of the most harmful diseases of tobacco. There are many studies have examined the mechanism underlying the induction of disease resistance by arbuscular mycorrhizal fungi (AMF) and ß-aminobutyric acid (BABA) alone, but the synergistic effects of AMF and BABA on disease resistance have not yet been studied. This study examined the synergistic effects of BABA application and AMF inoculation on the immune response to TBS in tobacco. The results showed that spraying BABA on leaves could increase the colonization rate of AMF, the disease index of tobacco infected by P.nicotianae treated with AMF and BABA was lower than that of P.nicotianae alone. The control effect of AMF and BABA on tobacco infected by P.nicotianae was higher than that of AMF or BABA and P.nicotianae alone. Joint application of AMF and BABA significantly increased the content of N, P, and K in the leaves and roots, in the joint AMF and BABA treatment than in the sole P. nicotianae treatment. The dry weight of plants treated with AMF and BABA was 22.3% higher than that treated with P.nicotianae alone. In comparison to P. nicotianae alone, the combination treatment with AMF and BABA had increased Pn, Gs, Tr, and root activity, while P. nicotianae alone had reduced Ci, H2O2 content, and MDA levels. SOD, POD, CAT, APX, and Ph activity and expression levels were increased under the combined treatment of AMF and BABA than in P.nicotianae alone. In comparison to the treatment of P.nicotianae alone, the combined use of AMF and BABA increased the accumulation of GSH, proline, total phenols, and flavonoids. Therefore, the joint application of AMF and BABA can enhance the TBS resistance of tobacco plants to a greater degree than the application of either AMF or BABA alone. In summary, the application of defense-related amino acids, combined with inoculation with AMF, significantly promoted immune responses in tobacco. Our findings provide new insights that will aid the development and use of green disease control agents.

Two new C19-diterpenoid alkaloids from Aconitum transsectum.

Two new C(19)-diterpenoid alkaloids, named aconitramines D (1) and E (2), were isolated from the roots of Aconitum transsectum. Based on comprehensive spectroscopic analyses including IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR, their structures were elucidated as 18-demethoxyltransconitine A (1) and 8-O-anisoyl-14-hydroxylacoforesticine (2).

Two new compounds from an endophytic fungus Alternaria solani.

Two new secondary metabolites, named 7-dehydroxyl-zinniol (1) and 20-hydroxyl-ergosta-4,6,8(14),22-tetraen-3-one (2), were isolated from the culture of Alternaria solani, an endophytic fungal strain residing in the roots of Aconitum transsectum. Their structures were elucidated on the basis of comprehensive spectroscopic analyses including IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR. Biological activity tests indicated that compound 1 showed moderate anti-HBV activity.

Probing the transcriptome of Aconitum carmichaelii reveals the candidate genes associated with the biosynthesis of the toxic aconitine-type C19-diterpenoid alkaloids.

Aconitum carmichaelii has long been used as a traditional Chinese medicine, and its processed lateral roots are known commonly as fuzi. Aconitine-type C19-diterpenoid alkaloids accumulating in the lateral roots are some of the main toxicants of this species, yet their biosynthesis remains largely unresolved. As a first step towards understanding the biosynthesis of aconitine-type C19-diterpenoid alkaloids, we performed de novo transcriptome assembly and analysis of rootstocks and leaf tissues of Aconitum carmichaelii by next-generation sequencing. A total of 525 unigene candidates were identified as involved in the formation of C19-diterpenoid alkaloids, including those encoding enzymes in the early steps of diterpenoid alkaloids scaffold biosynthetic pathway, such as ent-copalyl diphosphate synthases, ent-kaurene synthases, kaurene oxidases, cyclases, and key aminotransferases. Furthermore, candidates responsible for decorating of diterpenoid alkaloid skeletons were discovered from transcriptome sequencing of fuzi, such as monooxygenases, methyltransferase, and BAHD acyltransferases. In addition, 645 differentially expressed genes encoding transcription factors potentially related to diterpenoid alkaloids accumulation underground were documented. Subsequent modular domain structure phylogenetics and differential expression analysis led to the identification of BAHD acyltransferases possibly involved in the formation of acetyl and benzoyl esters of diterpenoid alkaloids, associated with the acute toxicity of fuzi. The transcriptome data provide the foundation for future research into the molecular basis for aconitine-type C19-diterpenoid alkaloids biosynthesis in A. carmichaelii.

Four new C18-diterpenoid alkaloids with analgesic activity from Aconitum weixiense.

Four new C18-diterpenoid alkaloids, weisaconitines A-D (1-4), were isolated from Aconitum weixiense. Based on extensive UV, IR, MS, 1D and 2D NMR analyses, their structures were elucidated as 8-O-ethyldolaconine (1), 4-demethylgenicunine B (2), 14-oxoaconosine (3), and 8-O-ethylaconosine (4). The analgesic activity of compound 4 was studied with CH3COOH-induced writhing model in mice. Compound 4 showed writhing inhibitions of 24% (50 mg/kg), 26% (100 mg/kg) and 34% (200 mg/kg), respectively, as compared to the reference drug aspirin (63%) at a dose of 200 mg/kg.