Determination of fermentation conditions
Fermentation period
HPLC analysis was conducted to validate what time was suitable for the fermentation. The flasks were grouped by the different fermentation periods (every three days), then transferred to -20℃ refrigerator to terminate the reaction. Under HPLC conditions (same as the pilot experiment), we analyzed the extracts of the fungal β-asarone produced in different periods by external reference method with standard β-asarone. Final fungal fermentation products were analyzed by HPLC (Fig. 1 and Fig. 2 A). The results showed the peak positions and peak shapes were identical or very close to that of the chemical reference.
β-asarone producing position
Liquid fermentation supernatant, white mycelia and mycelia with black spores (as the fermentation proceeded, the black spores gradually formed at the surface of the mycelia) were centrifuged (Fig. 1 B) to test which part of them was the β-asarone producing position. The results showed that β-asarone only appeared in the mycelia with black spores instead of the other two parts. Furthermore, the total yield of β-asarone might increase as the fermentation period lasted.
NaCl/CaCl2 and pH stability
To determine the range of pHs in which the yield of cis-asarone is the most, we measured the content of cis-asarone under different pH conditions PDA plate and cultured for 3 weeks. The peak areas of cis-asarone were quite stable at pH 6 and 7(Fig.1 C), the minimum and maximum relative content came across at pH 5 and pH 9. While the A. niger had difficulty on growth at pH 4 and pH 9 with more time to grow and the product abundance was higher at pH 6 and pH 7 (data not shown) which meant the neutral culture was good to fermentation.
To determine whether the addition of Na+ and Ca2+ will increase the cis-asarone yield (Fig.1 D), different concentration of NaCl or CaCl2 was added into the PDA plate and cultured for 3 weeks. The peak areas appeared the most at 1% either NaCl or CaCl2, and generally decreased from 2% and both remained to 0 when the concentration was over 10%, and the plates had no A.niger when the concentration was over 25%.
Evaluation of the yield of β-asarone
The third column (Table1) presented that β-asarone was the major component in some species(Dan et al. 2010; Deng et al. 2004; Govinden-Soulange et al. 2004; Perumalsamy et al. 2010), while considering the essential oil yield was often the minor part showing in the second column. Hence, when estimating the yield of β-asarone (C) of the plant, the yield of essential oil (A) was multiplied by the yield of β-asarone (B). As not every study showed all these data at the same time, and based on the reference we could get, the total EO yield was no more than 5%, and the β-asarone yield from any essential oil was no more than 50%, when came across data deficiency, 5% and 50% were used to substitute the yield of EO and β-asarone, respectively. It was obvious that the theoretical yield of β-asarone obtained from this study was the highest, which provided a further evidence that this strain could be the potential β-asarone supplier.
Evaluation of the culture with extra additions
According to the KEGG pathway, some potential chemicals were inferred to be the precursor for asarone, and added into the PDB liquid cultures to evaluate whether the yield of β-asarone would be improved. From Fig.S1, isoeugenol and anethole inhibited the growth obviously, especially at the first 7 days. This strain would grow with almost all the chemicals at first, and No. 4 was the only one to have black spores first, while when the days lasted, the cultures changed hugely, and 3 of them had no black spores appeared in the end (No.8, 9 and 10).
Bio-informatic analysis
Annotation
The target strain (DFY1) was primarily classified to be A. niger. After we isolated the A. niger stain (DFY1) which could produce β-asarone, whole genome sequencing was conducted, and the results showed the A. niger strain (DFY1) genome was assembled approximately 2.668 Gb with 61 contigs and 47 scaffolds, containing 8,875 genes encoded proteins. The results and evaluation of genome assembly completeness showed in Table S2. Complete BUSCOs occupied nearly 99.7% and the purified GC content was around 50.04%, which was similar to the existing data(de Vries et al. 2017). Quality of sequence results showed in Table S2.
CAZy prediction (Fig.S2A) presented that glycoside hydrolases group was the majority that had 247 genes annotated in this class. Glycosyl transferases, auxillary activities and carbohydrate esterases groups had similar genes from around 100. Polysaccharide lyases and carbohydrate-binding modules were the minor parts that could be ignored. From this CAZy prediction, some maltases were found (Table S3) which might explain why this strain performed better in malt liquid culture than other cultures. All the functional annotation gene numbers showed in the Table S2. In total, 39,560 GO terms were assigned (Fig.S2B), from which 11,283 were GO terms related to Cellular component class, 12,641 – to Molecular function class, and 15,636 – to Biological process class. KEGG classification (Fig.S2C) revealed that genetic information (belonged to Brite hierarchies), signaling and cell (belonged to Brite hierarchies) and signal transduction (belonged to Environmental information processing) matched over 500 genes. EggNOG annotation (Fig.S2D) of protein coding genes was conducted by eggnog-mapper. Carbohydrate transport and metabolism (G)and secondary metabolites biosynthesis, transport and catabolism(Q) possessed the most major functions except for the unknown part (S).
Phylogenetic tree
As the fungi has difficulty on species identification, and there are too many Aspergillus genera presented black, single copy orthologues got from genomes were used to analyze the genetic relationship by using OrthoFinder. According to BIC score in IQ-Tree, the best-fit model was JTT+F+R6, the phylogenetic tree (Fig.3 A) showed that strain DFY1 was closer to A.niger rather than other Aspergillus genera or species with similar black morphological characteristics, which provided further identification that strain DFY1 was A.niger.
Isolation and morphological characteristics of culturable endophytic fungi in A. heterotropoides. The 6 endophytic fungal strains were isolated from the bark tissues of A. heterotropoides based on the morphological characteristics, and only one strain possessed the ability to produce β-asarone after 18 days fermentation during the pilot experiments (data not shown). The target fungus strain (named as DFY1) was stained by cotton-blue dyeing method (Fig. 3B and C). Under the light microscope, it was clear to see the hypha had black round conidium formed, which had extremely similar characteristic with A. niger (oil immersion,1000X, Fig.3.C). The mycelia were white at the beginning, and the black spores generally appeared in the liquid medium.