## 5 Conclusions  
In this study, we demonstrated that the _E coli_ BL21(DE3)pLysS strain could be engineered as the prenylation system of 4 HC (1) via co-expression of the genes encoding for AtaPT and CstDXS. Based on the high-resolution LC-ESI(-)QTOF-MS/MS, feeding 4 HC (1) into the engineered strain resulted in the formation of the preynylated 4 HC analog (namely compound-1) showing \(m\)/\(z\) 365.21 [M \(-\) H]\({}^{-}\), which was detected from the culture medium. The mass fragmentation pattern of this product was highly identical to the authentic feruzelenol (2) established by the previous work [(66)]. MetFrag analysis clearly showed that the compound was best explained as \"ferulenol (2)\" among 2267 candidated compounds from the PubChem database. Further annotation using SIRIUS integrated with CSI:FingerID and CANOPUS led to unraveling the chemical features hidden in compound-1, e.g., molecular formula (defined as C\({}_{24}\)H\({}_{30}\)O\({}_{3}\)), ferulenol (2) as a potential structure, and classes (defined as coumarin derivatives), indicating the preynlate-4 HC derivative was indeed ferulenol (2). Meanwhile, compound-2 (\(m\)/\(z\) 365.21 [M \(-\) H]\({}^{-}\) and 367.22 [M \(+\) H]\({}^{+}\)) was elucidated as the farnesylated of 2-hydroxy-4-chromenone (c), which was proposed to be formed through the tautomerization of ferulenol (2). It is worthwhile to emphasize that both products could be formed without the external supply of prenyl donors (e.g., DMAPP, GPP, and FPP), which indicated the capacity of _E. coli_ to supply the prenyl donors, particularly FPP, and verified the functional expression of AtaPT and CstDXS in this bacterial system. Equally important, they were secreted in the culture medium, providing a great benefit from an economic point of view since breaking cell pellets is not needed. Further experiments, e.g., overexpression of rate-limiting step enzymes in the DXP pathway along with the optimized culture conditions, are required to improve the yields of two products. Based on the substrate promiscuity of AtaPT, the in vivo prenylation by clones carrying pCDFDuet-AtaPT-CstDXS could be applied towards other types of natural products such as quercetin and mangiferin to generate diverse classes of prenylated derivatives. The following are available online, Figure S1: The insertion of AtaPT in MCS-1 of pCDFDuet-1 vector, Figure S2: The insertion of CstDXS in MCS-2 of pCDFDuet-1 vector, Figure S3: The verified insertion of CstDXS (\(\sim\)1992 bp) in pCDFDuet-AtaPT (\(\sim\)5020 bp) via _Kpn1_ and _Xlnl_ digestion, Figure S4: The raw mass (MS2) data for putative ferulenol (2) (compound-1) obtained from negative-ESI mode, Figure S5: The proposed reaction mechanisms illustrating the various chemical losses present in the MS/MS spectrum of compound-1, Figure S6: The proposed reaction mechanisms illustrating the various chemical losses present in the MS/MS spectrum of compound-2, Figure S7: The postulated mechanisms underlying the formation of ion _m/z_ 189.05 compound-1, Figure S8: The postulated mechanisms underlying the formation of ion _m/z_ 189.05 of compound-2. Conceptualization, N.N. and A.K. ; methodology, A.K., J.N., P.P. and N.N. ; validation, A.K., J.N., P.P. and N.N. ; formal analysis, A.K., N.N., P.P. and J.N. ; investigation, A.K. and J.N. ; resources, N.N. ; writing--original draft preparation, A.K. and N.N. ; writing--review and editing, A.K., J.N., P.P. and N.N. ; supervision, P.P. and N.N. ; funding acquisition, N.N. All authors have read and agreed to the published version of the manuscript. This work was supported by the National Research Council of Thailand-Khon Kaen University, 2018 (NRCT-KKU 2018): 610034; Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand. Not applicable. A.K. wishes to thank the Graduate School, Khon Kaen University for the overseas research scholarship. The authors would like to thank Assistant Professor [PERSON], Suandusit University, Bangkok, Thailand for the valuable comments and advice on the mass spectrometry elucidation.