No abstract

Plant-derived diterpene synthases (PdiTPSs) play a critical role in the formation of structurally and functionally diverse diterpenoids. However, the relationship between PdiTPSs and the specificity or promiscuity of their products remains unclear. To explore this correlation, the sequences of 199 functionally characterized PdiTPSs and their corresponding 3D structures were collected and manually corrected. Using this compiled annotated database, the correlations among PdiTPSs sequences, domains, structures and their corresponding products were comprehensively analyzed. However, utilizing sequence similarity network (SSN), phylogenetic trees, and structural topology features alone was insufficient for effective functional classification of PdiTPSs as these methods could not establish a clear mapping between the enzymes and products. Surprisingly, residues verified to play a function through mutagenesis experiments were located within 8Å of the substrate. Aromatic residues surrounding the substrate exhibited selectivity towards its chemical structure. Specifically, tryptophan (W) was preferentially located around the linear substrate geranylgeranyl pyrophosphate (GGPP), while phenylalanine (F) and tyrosine (Y) were preferentially located around the initial cyclized diterpene intermediate. This analysis revealed the functional space of residues surrounding the substrate of PdiTPSs, most of which have not been experimentally explored. These findings provide guidance for screening specific residues for mutation studies to change the catalytic products of PdiTPSs, allowing us to better understand the correlation between PdiTPSs and their products.

Biosynthesis of β-ionone by microbial cell factories has become a promising way to obtain natural β-ionone. The catalytic activity of carotenoid cleavage dioxygenase 1 (CCD1) in cleavage of β-carotene to β-ionone severely limits its biosynthesis. In this study, NtCCD1-3 from Nicotiana tabacum with high ability to cleave β-carotene was screened. Multiple strategies for improving the β-ionone yield in Saccharomyces cerevisiae were performed. The results showed that NtCCD1-3 could cleave a variety of caroteniods at the 9,10 (9′,10′) double bonds and lycopene at the 5,6 (5′,6′) positions. The insertion site delta for NtCCD1-3 gene was more suitable for enhancing the yield of β-ionone, showing 19.1-fold increase compared with the rox1 site. More importantly, mutant K38A of NtCCD1-3 in membrane-bonding domains could greatly promote β-ionone production by more than 3-fold. We also found that overexpression of the NADH kinase Pos5 could improve β-ionone yield up to 1.5 times. These results may provide valuable references for biosynthesis of β-ionone.