Analysis of ethnobotanical literature led scientists to discover the Aztec sweet herb (Lippia dulcis), a highly aromatic plant containing the potent sesquiterpene ketone sweetener, hernandulcin. Despite the intense sweetness of L. dulcis, the main volatile constituent is the bitter-tasting and toxic monoterpenoid, camphor. To date, the biosynthesis of hernandulcin and camphor have yet to be fully elucidated in L. dulcis. Identification of key genes involved in both pathways could be used to enhance the safety and quality of hernandulcin-based alternative sweetener product.
Two terpene synthases (TPSs) were identified from L. dulcis transcriptomics, one of which exhibited bornyl diphosphate synthase (LdBPPS) activity, catalyzing the first step of camphor biosynthesis in L. dulcis. Interestingly, the N-terminal region of LdBPPS contains a duplicated RRX8W motif that was found to be inhibitory in vitro, suggesting that it may be cleaved with the chloroplast transit peptide. Phylogenetic analysis demonstrated that LdBPPS evolved independently from the homologous enzyme in sage, despite both performing the same biochemical reaction.
A cytochrome P450 monooxygenase (P450) was hypothesized to convert (+)-epi-α-bisabolol to hernandulcin. The induction of hernandulcin biosynthesis by methyl jasmonate was investigated to generate an elicitor-based transcriptome library but was found to be ineffective. Therefore, a homology-based cloning was used to identify ten putative P450s and their expression and activity were tested in yeast. A total of seven P450s were expressed in yeast, determined by immunoblot analyses, in either the native form or after N-terminal replacement. Six P450s showed no unique product formation after GC-MS analysis while one P450 (LdO10) showed minor affinity to (+)-epi-α-bisabolol substrate and produced bisabolol oxide B. Future efforts should be used to generate better bioinformatic resources to facilitate the mining for
genes involved in hernandulcin biosynthesis.