Study on secondary metabolites of 

Antarctic fungus Aspergillus sp. NJ49

Abstract: The polar region is a special ecosystem harboring a variety of fauna and microorganisms including bacteria, actinomycetes and fungi. To survive in conditions of low temperature, strong wind, low nutrient and high UV radiation, polar organisms require a wide range of biochemical and physiological adaptations that are essential for survival. Organisms from the polar regions are rich sources of various chemical scaffolds and novel natural products with good biological activity. In recent years, a large number of novel natural biological compounds with various activities such as antibacterial, antitumor and antiviral have been isolated from microorganisms, mollusks and other polar organisms. Aspergillus fungi have been reported as one of the most productive and potential resources for the production of structurally diverse secondary metabolites, mainly including alkaloids, polypeptides, terpenoids and polyketides. Researchers have discovered many different kinds of secondary metabolites from Antarctic fungi with novel structures and biological activities. Antarctic krill is an important species in the Antarctic ecosystem and is one of the most abundant single biological resources on the earth. At present, studies on the intestinal microbes of Antarctic marine organisms are relatively limited, especially for Antarctic krill, mainly focusing on their distribution, abundance, growth status and krill oil. The research on the intestinal microbes of Antarctic krill is still in the exploratory stage, and few studies involve the secondary metabolites of Aspergillus fungi in the intestinal microbes of Antarctic krill. Isolating microorganisms from Antarctic krill and obtaining natural products is an important way to enrich natural product research. To continue exploring the application potential of valuable Antarctic fungal resources, molecular identification, isolation of secondary metabolite and structural identification of an Antarctic krill symbiotic fungal strain were carried out in this study. The fungal DNA was extracted using Takara kit, amplified by PCR, and the amplified product was sequenced. The ITS rDNA sequences obtained from the sequencing were subjected to BLAST analysis. The phylogenetic tree was constructed through comparative analysis. ITS rDNA sequence analysis showed that the ITS rDNA sequence of the strain NJ49 and A. niger MK457457 had high similarity. According to the constructed phylogenetic tree, strains NJ49 and A. niger were on the same branch with the highest similarity. Combined with morphological methods, the strain was identified as A. niger NJ49. For strain NJ49, we used the original medium, Rose Bengal Agar Medium, for activation. The seed solution was first cultured and mass fermentation was carried out using Martin’s liquid medium as fermentation medium. A total of 54 L fermentation liquid was obtained by shaking culture. The fermentation liquid was filtered to obtain mycelium and fungal fermentation filtrate, and the mycelium was extracted with ethanol ultrasonic to obtain the extraction liquid, and the fermentation broth and the fungal fermentation filtrate were combined to obtain the ethyl acetate and aqueous phases. After the extraction liquid and mycorrhiza were combined, the ethyl acetate phase and water phase were extracted. The ethyl acetate fraction was separated by Sephadex LH20 gel column and high performance liquid chromatography (HPLC). And the mobile phase prepared by HPLC was acetonitrile water mixed system, gradient elution, flow rate of 2.5ml·min-1, UV detection wavelength of 214 nm, 234 nm, and 254 nm. Compounds 2, 5, 6 were obtained from ethyl acetate. The water part was roughly divided into four parts by macroporous adsorption resin: NJ49MeI, NJ49MeII, NJ49MeIII and NJ49MeIV. NJ49MeI was separated by G25 gel column and HPLC to obtain compounds 3, 4, 8, 10, 12. NJ49MeII was separated by G25 gel column and HPLC to obtain compounds 1, 2, 7, 9, 11. And the structures were identified by modern spectroscopic analysis methods, such as high performance liquid chromatography time of flight mass spectrometry (HPLCTOFMS), nuclear magnetic resonance spectrum (NMR) (1HNMR and 13CNMR) and other analytical methods. A total of 12 compounds were isolated and identified from A. niger NJ49, including TrpValVal(1), PheValVal(2), cyclo(His, Leu)(3), cyclo(Pro, Arg)(4), (3S,12aS)3(Isopropyl)2,3,6,7,12,12ahexahydropyrazino［1′,2′∶1,6］pyrido［3,4b］indole1,4dione(5), cyclo(Pro, Phe) (6), cyclo (Val, Phe) (7), cyclo(Pro, Tyr) (8), cyclo(Pro, Val) (9), PheVal (10), sclerin (11)and phydroxyphenylethylamine (12). 10 peptide compounds were subdivided into chainlike tripeptides (1, 2), dipeptide (10) and cyclic dipeptide (39). According to the chemical structure types of the secondary metabolites of NJ49, the secondary metabolites of the fungi of Aspergillus Antarctica were mainly polyketones, alkaloids and peptides. Compounds 3, 6, 9, 11 had a variety of antibacterial activity. The results showed that strain NJ49 might have a good application prospect in antibacterial activity, and the production of these active compounds could be increased by expanding the fermentation yield.  Compounds 3, 9, and 11 were firstly isolated from Antarctic fungi Aspergillus. The results of the study provide a basis for deepening the understanding of Antarctic marine microbial resources and enriching the secondary metabolite pool of Antarctic fungi Aspergillus. The findings are useful for further understanding the structural types of secondary metabolites and the discovery of active natural products of Aspergillus Antarctica fungi reference significance. Comparatively speaking, marine natural products are mainly studied in temperate and tropical organisms. The research on the active natural products of Aspergillus Antarctica is still in its infancy at present, which may be limited by the extreme environment and scientific research conditions in Antarctica. The resources of Aspergillus Antarctica can continue to be excavated in the future and the medicinal potential of Aspergillus antarctica fungi can be fully utilized. In the systematic isolation study of this strain, it was found that the metabolites of this symbiotic fungal strain were limited under the conditions in this paper, and no terpenoids and alkaloids reported in the literature were isolated. The structural novelty of the secondary metabolites isolated from the NJ49 ferment is still lacking. This may be due to different culture conditions. In the subsequent experiments, we will try to use rice medium to optimize the existing culture conditions, and combine with LCMS analytical guidance, and screen suitable culture conditions before carrying out LCMS guided separations to isolate structurally novel active compounds and improve the efficiency of isolation and identification of secondary metabolites with complex or novel structures.

Keywords: Antarctic fungi; Aspergillus; secondary metabolites; structure identification