2-Furoic acid associated with the infection of nematodes by Dactylellina haptotyla and its biocontrol potential on plant root-knot nematodes.

Dactylellina haptotyla is a typical nematode-trapping fungus that has garnered the attention of many scholars for its highly effective lethal potential for nematodes. Secondary metabolites play an important role in D. haptotyla-nematode interactions, but which metabolites perform which function remains unclear. We report the metabolic functions based on high-quality, chromosome-level genome assembly of wild D. haptotyla YMF1.03409. The results indicate that a large variety of secondary metabolites and their biosynthetic genes were significantly upregulated during the nematode-trapping stage. In parallel, we identified that 2-furoic acid was specifically produced during nematode trapping by D. haptotyla YMF1.03409 and isolated it from fermentation production. 2-Furoic acid demonstrated strong nematicidal activity with an LD50 value of 55.05 µg/mL against Meloidogyne incognita at 48 h. Furthermore, the pot experiment showed that the number of galls of tomato root was significantly reduced in the experimental group treated with 2-furoic acid. The considerable increase in the 2-furoic acid content during the infection process and its virulent nematicidal activity revealed an essential synergistic effect during the process of nematode-trapping fungal infection. IMPORTANCE Dactylellina haptotyla have significant application potential in nematode biocontrol. In this study, we determined the chromosome-level genome sequence of D. haptotyla YMF1.03409 by long-read sequencing technology. Comparative genomic analysis identified a series of pathogenesis-related genes and revealed significant gene family contraction events during the evolution of D. haptotyla YMF1.03409. Combining transcriptomic and metabolomic data as well as in vitro activity test results, a compound with important application potential in nematode biocontrol, 2-furoic acid, was identified. Our result expanded the genetic resource of D. haptotyla and identified a previously unreported nematicidal small molecule, which provides new options for the development of plant biocontrol agents.

Identification and Mechanism of Action of the Global Secondary Metabolism Regulator SaraC in Stereum hirsutum.

DNA methylation is an important factor in the regulation of gene expression. In analyzing genomic data of Stereum hirsutum FP-91666, we found a hypothetical bifunctional transcription regulator/O6Meguanine-DNA methyltransferase (named SaraC), which is widely present in both bacteria and fungi, and confirmed that its function in bacteria is mainly for DNA reparation. In this paper, we confirmed that SaraC has the function of DNA binding and demethylation through surface plasma resonance and reaction experiments in vitro. Then, we achieved the overexpression of SaraC (OES) in S. hirsutum, sequenced the methylation and transcription levels of the whole-genome, and further conducted untargeted metabolomics analyses of the OES transformants and the wild type (WT). The results confirmed that the overall-methylation levels of the transformants were significantly downregulated, and various genes related to secondary metabolism were upregulated. Through comparative untargeted metabolomic analyses, it showed that OES SA6 transformant produced a greater number of hybrid polyketides, and we identified 2 novel hybrid polyketides from the fermentation products of SA6. Our results show that overexpression SaraC can effectively stimulate the expression of secondary-metabolism-related genes, which could be a broad-spectrum tool for discovery of metabolites due to its cross-species conservation. IMPORTANCE Fungi are one of the important sources of active compounds. However, in fungi, most of the secondary metabolic biosynthetic gene clusters are weakly expressed or silenced under conventional culture conditions. How to efficiently excavate potential new compounds contained in fungi is becoming a research hot spot in the world. In this study, we found a DNA demethylation protein (SaraC) and confirmed that it is a global secondary metabolism regulator in Stereum hirsutum FP-91666. In the past, SaraC-like proteins were mainly regarded as DNA repair proteins, but our findings proved that it will be a powerful tool for mining secondary metabolites for overexpression of SaraC, which can effectively stimulate the expression of genes related to secondary metabolism.

Recent Advances in the Synthetic Biology of Natural Drugs.

Natural drugs have been transformed and optimized during the long process of evolution. These compounds play a very important role in the protection of human health and treatment of human diseases. Sustainable approaches to the generation of raw materials for pharmaceutical products have been extensively investigated in drug research and development because chemical synthesis is costly and generates pollution. The present review provides an overview of the recent advances in the synthetic biology of natural drugs. Particular attention is paid to the investigations of drugs that may be mass-produced by the pharmaceutical industry after optimization of the corresponding synthetic systems. The present review describes the reconstruction and optimization of biosynthetic pathways for nine drugs, including seven drugs from plant sources and two drugs from microbial sources, suggesting a new strategy for the large-scale preparation of some rare natural plant metabolites and highly bioactive microbial compounds. Some of the suggested synthetic methods remain in a preliminary exploration stage; however, a number of these methods demonstrated considerable application potential. The authors also discuss the advantages and disadvantages of the application of synthetic biology and various expression systems for heterologous expression of natural drugs. Thus, the present review provides a useful perspective for researchers attempting to use synthetic biology to produce natural drugs.

Metabolites from the endophytic fungus colletotrichum sp. F168.

An endophytic fungus, Colletotrichum sp. F168, was isolated from plant Huperzia serrata (Thunb. ex Murray) Trev. and was subjected to phytochemical investigation. Nine compounds including two new structures were obtained from SDA solid fermentation products of strain F168, which were elucidated by extensive spectroscopic analyses, including 1 D- and 2 D-NMR, and HR-MS experiments. The acetylcholinesterase inhibitory activity of two new compounds 1-2 was tested in vitro. Two new compounds didn't show evident acetylcholinesterase inhibitory activity.

Griseaketides A-D, New Aromatic Polyketides from the Pathogenic Fungus Magnaporthe grisea.

Magnaporthe grisea is the causal agent of rice blast disease, which is the most serious disease of cultivated rice. Aromatic polyketides are its typical metabolites and are involved in the infection process. In the search for novel lead compounds, chemical investigation of the fungus M. grisea M639 has led to the isolation of four new aromatic polyketides (salicylaldehyde skeleton bearing an unsaturated side chain), griseaketides A-D (1-4), as well as 15 known compounds (5-19). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses, including HR-MS, 2D NMR. Compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h. This is the first report describing the nematicidal activity of this type aromatic polyketide.

[Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2].

To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.