## 3 Discussion
SIRIUS is one of the in silico toolsdeveloped to unravel various chemical features hidden in the MS/MS data of the query subjects [52]. This tool has typically been used in the field of metabolomics and has been shown to be helpful in the field of metabolic engineering, in which it was used to identify 2,4,6-trihydroxybenzophenone produced by the engineered _E. coli_[58]. The results from SIRIUS showed that our query product (compound-1) possessed the neutral molecular formula C\({}_{24}\)H\({}_{30}\)O\({}_{3}\), which corresponded to that of ferulenol (2) deposited in the PubChem database ([[https://pubchem.ncbi.nlm.nih.gov/compound/Ferulenol](https://pubchem.ncbi.nlm.nih.gov/compound/Ferulenol)]([https://pubchem.ncbi.nlm.nih.gov/compound/Ferulenol](https://pubchem.ncbi.nlm.nih.gov/compound/Ferulenol)) (accessed on 26 September 2021)). SIRIUS also locally computed the relevant MS/MS spectra and fragmentation pathway which might be involved in the fragmentation process of compound-1 in which eight mass peaks were explained, meanwhile, the rest of the three peaks were considered as the noises. Based on the eight mass peaks explained by SIRIUS, CSI:FingerID analysis suggested that the compound-1 was annotated as ferulenol (2) as the tenth-ranked candidate after searching against all databases provided by the SIRIUS tool. Although the expected ferulenol (2) was not perfectly categorized as the first candidate for the compound-1, it might be speculated that CSI:FingerID integrated in SIRUS exhibited extremely good performance in the case of the independent MS/MS being trained and deposited in the training data, and the rate of correct identification was substantially decreased when the trained data were removed from this tool [54]. This can be confirmed by the fact that the MS/MS data for the authentic ferulenol (2) (encoded by InChI=1S/C24H30O3/c1-17(2)9-7-10-18(3)11-8-12-19(4)15-16-21-23(25)20-13-5-6-14-22(20)27-24(21)26/h5-6,9,11,13-15,25H,7-8,10,12,16H2,1-4H3/b18-11+,19-15+) has not yet been trained in the negative mode mass data of the SIRIUS tool ([[https://www.csi-fingerid.uni-jena.de/v1.6.0/api/fingerid/trainingstructures?predictor=2](https://www.csi-fingerid.uni-jena.de/v1.6.0/api/fingerid/trainingstructures?predictor=2)]([https://www.csi-fingerid.uni-jena.de/v1.6.0/api/fingerid/trainingstructures?predictor=2](https://www.csi-fingerid.uni-jena.de/v1.6.0/api/fingerid/trainingstructures?predictor=2)) (accessed on 23 June 2021)). Based on the LC-MS search (for the molecular ion _m_/_z_ 365.21 [M \(-\) H]\({}^{-}\)), it might also be possible to exclude the rest of candidates (ranked first to ninth retrieved from all included databases, and ranked first to fifth from the Natural Products and COCONUTS databases) as incorrect structures since they are not natural occurring in _E. coli_, and there was only 3b-allotetrahydrocortisol showing the proximal _m_/_z_ 365.23 [M \(-\) H]\({}^{-}\) found in the _E. coli_ metabolomics database (ECMDB) ([[https://ecmdb.ca/spectra/ms/search](https://ecmdb.ca/spectra/ms/search)]([https://ecmdb.ca/spectra/ms/search](https://ecmdb.ca/spectra/ms/search)) (accessed on 23 June 2021)). This indicated that only ferulenol (2) as the product of pathway engineering could be accepted as the correct structure. In the case of the query metabolites acquired from the biological samples, SIRIUS also provides the biological databases as the choices to narrow the scope of natural molecules. When MS/MS spectra of compound-1 were annotated against biological databases, e.g., Natural Products, COCONUTS, and NORMAN, the rank annotated as \"ferulenol (2)\" was greatly improved as the fifth and first candidate, respectively. Based on the substructures predicted by CSI:Finger ID and CANOPUS, it subsequently provided the vital information that the compound-1 was classified as coumarins and derivatives. According to [PERSON] and colleagues [47], the 4 HC (1) can exist in three different tautomeric structures: 4-hydroxy-2-chromenone (a), 2,4-chromadione (b), and 2-hydroxy-4-chromenone (c) (Figure 3E).