## 2 Results
### Structural Annotation and Compound Classification of Compound-1 by Using CSI:FIngerID and CANOPUS  
SIRIUS has recently been integrated with CSI: FingerID web service to identify the chemical structure of the query subjects [55]. In this step, the annotated mass spectrum (Figure 5B) was compared against several compounds in the chosen molecular structure databases (e.g., PubChem, MeSH, and COCONUTS). Of more than 100 possible structures retrieved from all databases and locally predicted by the tool, ferulenol (2) as the target product was ranked as the tenth candidate structure with a percentage similarity of 43.62% (Figure 6A). Since ferulenol (2) is a natural product exclusively produced by _F. commu-nis_, we narrowed the scope of the structural elucidation by using Natural Products and COCONUTS databases and found the rank of the candidate was substantially improved from tenth to fifth (Figure 6A). By searching against the structural compounds from the NORMAN database, our prenylated 4 HC analog (compound-1) was perfectly matched to that of ferulenol (2) as the top ranked candidate (Figure 6A). Besides, CSI:FingerID can provide the crucial information of so-called \"molecular fingerprints\" to verify various substructures that can be found in the query subjects (Figure 6B). In this instance, several molecular fingerprints belonging to ferulenol (2) were predicted to be present in this prenylated product (_m/z_ 365.21 [M \(-\) H]\({}^{-}\)). For example, substructures encoded by a SMARTS string \"[#6]c1c([#8])ccc1 (Cc1c(O)ccc1)\" with a score of 98% (F1 = 0.823) correspond to the benzene ring attached to the pyrone ring of 4 HC (1). The basis pyrone ring of 4 HC encoded by [#8]=;[#6]-,[#6]-,[#6]-,[#8](O=C-C-C-O) possessed 85% similarity (F1 = 0.815) and was verified to be present in same candidate structure. Biosynthetically, they were all obtained from the 4 HC (1) fed in the culture medium of clones bearing pCDFDuet-AtaPT-CsTDXS. Equally important, there were several substructures belonging to the farnesyl moiety which originated from the DXP pathway, predicted to be present in the same candidate structure. Several substructures representing the basic benzene and pyrone rings along with the isoprene building block belonging to the query ferulenol (2) were predicted to be present in the trained structures of SIRIUS tool (Figure 6C). SIRIUS has also been developed to connect with CANOPUS (class assignment and ontology prediction using mass spectrometry) for logical classification of unknown metabolites based on the high-resolution MS/MS data [56]. According to the molecular properties annotated by CSI:FingerID web service, our query prenylated product _m/z_ 365.21 [M \(-\) H]\({}^{-}\) was systematically classified as coumarins and derivatives (class), where phenylpropanoids/polyketides and organic compounds served as the superclass and kingdom, respectively (Figure 6D). CANOPUS also provides alternative classes of the query subjects. In this case, the putative ferulenol (2) was mainly classified as a coumarin derivative, but aromatic monoterponoids, benzenoids, and lactones were recognized as the alternative classes of this product. Based on the  
Figure 5: Molecular formula annotation of the prenylated 4 HC analog produced by clones carrying pCDFDuet-AtaPT-CstDXS using SIRIUS (version 4.8.2). (**A**) High-resolution isotope pattern analysis reveals the molecular formula of query subject is best annotated as C\({}_{24}\)H\({}_{30}\)O\({}_{3}\). (**B**) The mass spectra is annotated to match with the local databases of SIRIUS (indicated by the green spectrum with corresponding molecular formula), while the spectra with no annotations are considered to be noise peaks (indicated by the black spectrum).