Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens

Abstract

Ent-kaurene diterpenoids are the largest group of known Isodon diterpenoids. Among them, oridonin is accumulated in the leaves, and is the most frequently studied compound because of its antitumor and antibacterial activities. We have identified five copalyl diphosphate synthase (CPS) and six kaurene synthase-like (KSL) genes by transcriptome profiling of Isodon rubescens leaves. An in vitro assay assigns ten of them to five different diterpene biosynthesis pathways, except IrCPS3 that has a mutation in the catalytic motif. The Lamiaceae-specific clade genes (IrCPS1 and IrCPS2) synthesize the intermediate copalyl diphosphate (normal-CPP), while IrCPS4 and IrCPS5 synthesize the intermediate ent-copalyl diphosphate (ent-CPP). IrKSL2, IrKSL4, and IrKSL5 react with ent-CPP to produce an ent-isopimaradiene-like compound, ent-atiserene and ent-kaurene, respectively. Correspondingly, the Lamiaceae-specific clade genes IrKSL1 or IrKSL3 combined with normal-CPP led to the formation of miltiradiene. The compound then underwent aromatization and oxidization with a cytochrome P450 forming two related compounds, abietatriene and ferruginol, which were detected in the root bark. IrKSL6 reacts with normal-CPP to produce isopimaradiene. IrKSL3 and IrKSL6 have the γβα tridomain structure, as these proteins tend to possess the bidomain structure of IrKSL1, highlighting the evolutionary history of KSL gene domain loss and further elucidating chemical diversity evolution from a macroevolutionary stance in Lamiaceae.

Figure 1.

The major medicinally active diterpenoids in I. rubescens. A, The plant of I. rubescens. B, The typical ent-kaurene diterpenoid of oridonin. C, The typical abietane diterpenoid of rubesanolide D.

Figure 2.

Alignment of the conserved DXDD motif and the positive selection site (Ser-362/Trp-364) of CPS from I. rubescens and other characterized CPSs. The DXDD motif is labeled above the alignment with a solid line, and the conserved Ser or Trp position is marked with an asterisk. The second Asp of the DXDD motif is substituted by Asn in IrCPS3. The enzymes are separated by the horizontal line according to normal-CPP/LDPP or ent-CPP stereochemistry. The CPS in the upper section (including IrCPS1 and IrCPS2 from this study) were characterized as being involved in normal-CPP/LDPP formation while those in the lower section (including IrCPS4 and IrCPS5 from this study) were characterized to be involved in ent-CPP production. CfTPS1 and CfTPS2 are from C. forskohlii, SmCPS1, SmCPS2, SmCPS5 from S. miltiorrhiza, MvCPS3 from M. vulgare, SsLPS from S. sclarea, and AtCPS from Arabidopsis.

Figure 3.

Phylogeny of I. rubescens diterpene synthases. The maximum likelihood tree illustrates the phylogenetic relationship of I. rubescens diterpene synthases with 96 representative characterized diTPS (Supplemental Table S5). Numbers on branches indicate the bootstrap percentage values calculated from 1000 bootstrap replicates. Physcomitrella patens CPS/kaurene was used as an outgroup. Blue lines show diTPS genes from Lamiaceae involved in normal-CPP/LDPP-mediated diterpenoid metabolism and red lines show the loss of the N-terminal γ domain found in eudicot and monocot KSLs. Yellow lines show genes from Lamiaceae involved in the specialized ent-CPP/LDPP-related diterpenoid metabolism. Red-marked enzymes show diTPS from I. rubescens in this study.

Figure 4.

GC-MS analysis of in vitro assays with I. rubescens diTPS on a Cyclodex-β GC column. A, Extracted ion chromatograms of m/z 257 of in vitro assays with IrCPS coupled with different IrKSL. The characterized SmCPS5 (ent-CPP synthase from S. miltiorrhiza), SmCPS1 (normal-CPP synthase from S. miltiorrhiza), SmKSL1 (miltiradiene synthase from S. miltiorrhiza), AtKS (ent-kaurene synthase from Arabidopsis), and PcmISO1 (pimaradiene from P. banksiana) were used as positive controls, along with the corresponding authentic standard ent-atiserene. (1) Miltiradiene, (2) isopimaradiene, (3) ent-kaurene, (4) ent-atiserene, and (5) ent-isopimaradiene-like compound. Asterisk indicates the unexpected enzyme products identified in this study. B, Corresponding mass spectra of recombinant enzyme assay products and authentic standard. EIC, Extracted ion chromatograms.

Figure 5.

The mRNA expression levels of the 11 candidate diTPS in root, stem, leaf, and flower tissues from I. rubescens. The expression level was normalized to that of actin. The error bars show the SDs from mean value (n = 3 experiments).

Figure 6.

GC-MS detection of abietane diterpenoids in the periderm of the root and identification of CYP76AH30 in I. rubescens on a TR-5ms capillary column. A, Total ion chromatograms of abietane diterpenoids in the hexane extract of the root periderm and the ferruginol production from strain YJ26 transformed with the plasmid pESC-Leu::CYP76AH30. Strain YJ26 with the plasmid pESC-Leu was used as a control, along with the authentic standard of ferruginol. B, Mass spectra of (1) abietatriene, (2) miltiradiene, and (3) ferruginol from the root periderm (upper halves) and yeast (lower halves). TIC, Total ion chromatograms.

Figure 7.

The proposed diterpenoid biosynthesis in I. rubescens. Two sequential diterpene synthases CPS and KSL, and corresponding reactions are indicated, along with the possible downstream natural products. Dashed arrows indicate multiple enzymatic reactions.

Figure 8.

Proposed process for the evolution of bidomain terpene synthases in Lamiaceae. The purple arrows show the proposed evolutionary steps from the ancient tridomain kaurene synthase to the bidomain KSLs in Lamiaceae.