# Biosynthesis of Camphane Volatile terpenes in _Annomum_ _villosum_ Lour: Involved Genes and Enzymes
## 3 Discussion
These candidates' functions may be verified using in vitro enzymatic assays and heterologous expressions in yeast or _Escherichia coli_. In this study, although the transcriptome of _A. villusum_ fruit offered a highly effective technique to find the genes linked to recognized enzymes involved in the production of secondary metabolites, as well as candidate genes that might be linked to as yet unidentified steps in the pathway (Table S3), no DEGs were found to be involved in the 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythnitol kinase (CMK), acetyl-CoA acetyltransferasearase (AACT), PMK and isopentenyl-diphosphate Delta-isomerase (IPI), and unigenes associated with BPPH, and also were not found in the sequence annotation. It is difficult to screen the enzymes actually involved in the downstream pathway of volatile terpene biosynthesis. BPPS belongs to the family of terpenoid cyclases (TPSs), and is responsible for the cyclization of geranyl diphosphate to generate (+)-bornyl diphosphate, which is vital for the cyclizing process in monoterenoid biosynthesis [50]. It was found in _Salvia officinalis_ that (+)-bornyl diphosphate synthase might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase, which were previously assumed to be distinct enzymes [51]. BDH, an enzyme that catalyzes borneol to camphor, was named as a short-chain dehydrogenase in _Zingiber zerumbet_, which belongs to the short-chain dehydrogenase/reductase superfamily (SDR), displaying a bisubstrate feature corresponding with its flexible substrate-binding pocket [37; 38]. Moreover, it was reported that (+)-(3S)-neomenthol reductase could catalyze different terpene products (such as (+)-(3S)-neomenthol, (-)-(3R)-menthol, (+)-(3S)-isomenthol, and (+)-(3R)-neoisomenthol), and the diversity of substrates and biosynthesis results in more than one product catalyzed by terpene synthase [52]. TPSs are responsible for the initial cyclization cascade in the multistep synthesis of more than 60,000 known natural compounds, and due to the extremely small pool of substrates based on linear isoprenoids, these are often promiscuous and should face the challenge of a tremendous catalytic reaction [53]. Some TPSs with diverse domains might sometimes take the place of the catalytic function of known enzymes. Moreover, using de novo assembly without a reference genome, short transcripts or low expression genes may not be correctly spliced. The expression level is low. If the expression level of these enzyme genes is lower than the technical detection value (such as FPKMIRPKMITPM < 0.1), they may be filtered out or marked as \"not expressed\". The counts/TPM values of these genes in the original expression matrix will be checked, or their expression will be verified by qPCR. These might illustrate why no DEGs were found in some genes and BPPH was not obtained from the database. Incomplete annotations can be supplemented by homologous gene alignment (BLAST, [[https://blast.ncbi.nlm.nih.gov/Blast.cgi](https://blast.ncbi.nlm.nih.gov/Blast.cgi)]([https://blast.ncbi.nlm.nih.gov/Blast.cgi](https://blast.ncbi.nlm.nih.gov/Blast.cgi)), 7 June 2024) or de novo prediction. It is acknowledged that the expression of genes in the plant secondary metabolism pathway is regulated by various transcription factors [54; 55]. Moreover, several types of transcription factors belonging to the families bHLH, MYB, AP2/ERF, and WRKY have been identified in the regulation of secondary metabolism pathway in plants [56; 57; 58; 59]. In this analysis, a total of 143 unigenes were found in the DEGs. The top three were the families of bHLH (24.5%), MYB (12.6%), and bZIP (9.1%), which is almost in agreement with the results of one report [44]. The bHLH and MYB have been reported as master regulators in the terpenoid biosynthesis pathway [60]. It is possible that some TFs that we classified were involved in the regulation of volatile terpene biosynthesis in _A. villosum_. Further research should be carried out to verify these inferences.