Genome Mining Leads to Diverse Sesquiterpenes with Anti-inflammatory Activity from an Arctic-Derived Fungus.

With the advancement of bioinformatics, the integration of genome mining with efficient separation technology enables the discovery of a greater number of novel bioactive compounds. The deletion of the key gene responsible for triterpene cyclase biosynthesis in the polar strain Eutypella sp. D-1 instigated metabolic shunting, resulting in the activation of dormant genes and the subsequent production of detectable, new compounds. Fifteen sesquiterpenes were isolated from the mutant strain, with eight being new compounds. The structural elucidation of these compounds was obtained through a combination of HRESIMS, NMR spectroscopy, and ECD calculations, revealing six distinct skeleton types. Compound 7 possessed a unique skeleton of 5/10 macrocyclic ether structure. Based on the gene functions and newly acquired secondary metabolites, the metabolic shunting pathway in the mutant strain was inferred. Compounds 6, 8, 11, 14, and 15 exhibited anti-inflammatory effects without cytotoxicity through the release of nitric oxide from lipopolysaccharide-stimulated RAW264.7 cells. Notably, acorane-type sesquiterpene 8 inhibited nitric oxide production and modulated the MAPK and NLRP3/caspase-1 signaling pathways. Compound 8 also alleviated the CuSO4-induced systemic neurological inflammation symptoms in a transgenic fluorescent zebrafish model.

Pimarane-Type Diterpenes with Anti-Inflammatory Activity from Arctic-Derived Fungus Eutypella sp. D-1.

The Arctic-derived fungus Eutypella sp. D-1 can produce numerous secondary metabolites, and some compounds exhibit excellent biological activity. Seven pimarane-type diterpenes, including three new compounds eutypellenone F (1), libertellenone Y (2), and libertellenone Z (3), and four known compounds (4-7), were isolated from fermentation broth of Eutypella sp. D-1 by the OSMAC strategy of adding ethanol as a promoter in the culture medium. Compound 2 has a rare tetrahydrofuran-fused pimarane diterpene skeleton. The anti-inflammatory activity of all compounds was evaluated. Compounds 3-6 showed a significant inhibitory effect on cell NO release at 10 µmol/L by in vitro experiments, of which 3-5 had inhibitory rates over 60% on nitric oxide (NO) release. Subsequently, the anti-inflammatory activity of 3-5 was evaluated based on a zebrafish model, and the results showed that 3 had a significant inhibitory effect on inflammatory cells migration at 40 µmol/L, while 4 and 5 had a significant inhibitory effect at 20 µmol/L. Moreover, compounds 3-5 have the same conjugated double bond structure, which may be an important group for these compounds to exert anti-inflammatory activity.

Saturated and Polyunsaturated Fatty Acids Production by Aurantiochytrium limacinum PKU#Mn4 on Enteromorpha Hydrolysate.

Thraustochytrids are unicellular marine heterotrophic protists, which have recently shown a promising ability to produce omega-3 fatty acids from lignocellulosic hydrolysates and wastewaters. Here we studied the biorefinery potential of the dilute acid-pretreated marine macroalgae (Enteromorpha) in comparison with glucose via fermentation using a previously isolated thraustochytrid strain (Aurantiochytrium limacinum PKU#Mn4). The total reducing sugars in the Enteromorpha hydrolysate accounted for 43.93% of the dry cell weight (DCW). The strain was capable of producing the highest DCW (4.32 ± 0.09 g/L) and total fatty acids (TFA) content (0.65 ± 0.03 g/L) in the medium containing 100 g/L of hydrolysate. The maximum TFA yields of 0.164 ± 0.160 g/g DCW and 0.196 ± 0.010 g/g DCW were achieved at 80 g/L of hydrolysate and 40 g/L of glucose in the fermentation medium, respectively. Compositional analysis of TFA revealed the production of equivalent fractions (% TFA) of saturated and polyunsaturated fatty acids in hydrolysate or glucose medium. Furthermore, the strain yielded a much higher fraction (2.61-3.22%) of eicosapentaenoic acid (C20:5n-3) in the hydrolysate medium than that (0.25-0.49%) in the glucose medium. Overall, our findings suggest that Enteromorpha hydrolysate can be a potential natural substrate in the fermentative production of high-value fatty acids by thraustochytrids.

Application of Gene Knockout and Heterologous Expression Strategy in Fungal Secondary Metabolites Biosynthesis.

The in-depth study of fungal secondary metabolites (SMs) over the past few years has led to the discovery of a vast number of novel fungal SMs, some of which possess good biological activity. However, because of the limitations of the traditional natural product mining methods, the discovery of new SMs has become increasingly difficult. In recent years, with the rapid development of gene sequencing technology and bioinformatics, new breakthroughs have been made in the study of fungal SMs, and more fungal biosynthetic gene clusters of SMs have been discovered, which shows that the fungi still have a considerable potential to produce SMs. How to study these gene clusters to obtain a large number of unknown SMs has been a research hotspot. With the continuous breakthrough of molecular biology technology, gene manipulation has reached a mature stage. Methods such as gene knockout and heterologous expression techniques have been widely used in the study of fungal SM biosynthesis and have achieved good effects. In this review, the representative studies on the biosynthesis of fungal SMs by gene knockout and heterologous expression under the fungal genome mining in the last three years were summarized. The techniques and methods used in these studies were also briefly discussed. In addition, the prospect of synthetic biology in the future under this research background was proposed.

Optimization of Protoplast Preparation and Establishment of Genetic Transformation System of an Arctic-Derived Fungus Eutypella sp.

Arctic-derived fungus Eutypella sp. D-1 has attracted wide attention due to its huge ability to synthesize secondary metabolites. However, current studies only focus on stimulating its production of new secondary metabolites by OSMAC strategies, and the relationship between secondary metabolites and biosynthetic gene clusters (BGCs) has not been explored. In this study, the preparation and regeneration conditions of Eutypella sp. D-1 protoplasts were explored to lay a foundation for the study of genetic transformation of this fungus. Orthogonal experiment showed that the optimal preparation conditions were 0.75 M NaCl, 20 g/L of lysing enzyme, and 20 g/L of driselase, 28°C for 6 h. The maximum yield of Eutypella sp. D-1 protoplasts could reach 6.15 × 106 cells·ml-1, and the concentration of osmotic stabilizer NaCl was the most important factor for Eutypella sp. D-1 protoplasts. The results of FDA staining showed that the prepared protoplasts had good activity. Besides, the best protoplasts regeneration medium was YEPS, whose maximum regeneration rate is 36%. The mediums with nitrogen sources, such as SR and RM, also had good effects on the Eutypella sp. D-1 protoplast regeneration, indicating that nitrogen sources played an important role on the Eutypella sp. D-1 protoplast regeneration. Subsequent transformation experiments showed that hygromycin resistance genes (hrg) could be successfully transferred into the genome of Eutypella sp. D-1, indicating that the prepared protoplasts could meet the needs of subsequent gene manipulation and research. This study lays a foundation for the genetic transformation of Eutypella sp. D-1.