Functional diversification of kaurene synthase-like genes

The Lamiaceae family contains numerous diterpenoids, offering a valuable model system for the study of diterpenoid chemical diversity. In this study we report that the ent -kaurene diterpenoids in 38 Isodon rubescens have the largest diTPS gene family identified in Lamiaceae. Three genes are 39 predicted to be involved in ent -kaurene diterpenoid synthesis while another two KSL genes are 40 functional enzymes utilizing ent -CPP as a substrate. We also showed the presence of a normal-CPP 41 mediated biosynthesis pathway in I. rubescens and that three KSL genes possessing different 42 domain architectures are involved in this pathway. This study provides new information regarding 43 the understanding of diterpenoid chemical diversity and this compounds’ evolution with in the 44 Lamiaceae family. ABSTRACT 48 Ent -kaurene diterpenoids are the largest group of known Isodon diterpenoids. Among them, 49 oridonin is accumulated in the leaves, and is the most frequently studied compound because of its 50 anti-tumor and anti-bacterial activities. We have identified five copalyl diphosphate synthase (CPS) 51 and six kaurene synthase-like (KSL) genes by transcriptome profiling of I. rubescens leaves. An i n 52 vitro assay assigns ten of them to five different diterpene biosynthesis pathways, except IrCPS3 53 that has a mutation in the catalytic motif. The Lamiaceae-specific clade genes (IrCPS1 and IrCPS2) 54 synthesize the intermediate copalyl diphosphate (normal-CPP), while IrCPS4 and IrCPS5 55 synthesize the intermediate ent -copalyl diphosphate ( ent -CPP). IrKSL2, IrKSL4 and IrKSL5 react 56 with ent -CPP to produce an ent -isopimaradiene-like compound, ent -atiserene and ent -kaurene, 57 respectively. Correspondingly, the Lamiaceae-specific clade genes IrKSL1 or IrKSL3 combined 58 with normal-CPP led to the formation of miltiradiene. The compound then underwent 59 aromatization and oxidization with a cytochrome P450 (CYP76AH30) forming two related 60 compounds, abietatriene and ferruginol, which were detected in the root bark . IrKSL6 reacts with 61 normal-CPP to produce isopimaradiene. IrKSL3 and IrKSL6 have the γβα tri-domain structure, as 62 these proteins tend to possess the bi-domain structure of IrKSL1, highlighting the evolutionary 63 history of KSL gene domain loss and further elucidating chemical diversity evolution from a 64 macroevolutionary stance in Lamiaceae. The diterpenoid biosynthesis has been explored in nine species in Lamiaceae, including Salvia 85 miltiorrhiza (Gao et al., 2009; Cui et al., 2015; Su et al., 2016), S. sclarea (Caniard et al., 2012; 86 Schalk et al., 2012), Isodon eriocalyx (Li et al., 2012), Coleus forskohlii (Pateraki et al., 2014), 87 Rosmarinus officinalis (Bruckner et al., 2014), S. fruticosa ( Božić et al., 2015 ), S. pomifera ( Trikka et 88 al. , 2015), and S. divinorum (Pelot et al., 2017) from the biggest subfamily Nepetoideae, and Marrubium vulgare (Zerbe et al., 2014) from the subfamily Lamioideae. These enzymes have the ability to produce abietane, labdane, clerodane, and ent -kaurane diterpenes via an enzymatic 91 hydrolysis catalyzed reaction (Gao et al., 2009; Cui et al., 2015; Caniard et al., 2012; Schalk et al., 2012; Li et al., 2012; Pateraki et al., 2014; Zerbe et al., 2014; Bruckner et al., 2014; Božić et al., 93 2015; Trikka et al., 2015; Pelot et al., 2017). Based on the stereochemistry of the intermediate that is initially cyclized by CPS, these enzymes can be divided into two groups. One group produces an sequencing project. We identified IrCPS4 that is predicted to be involved in oridonin biosynthesis, 126 three KSL genes involved in ent -CPP interactions, and a normal-CPP mediated miltiradiene and 127 isopimaradiene biosynthesis pathway. The three KSL genes bearing different domain structures are 128 involved in the normal-CPP mediated biosynthesis, revealing information regarding the 129 evolutionary origin of the γ domain loss and further shedding light on the macroevolution of 130 diterpenoid chemical diversity in Lamiaceae . 95 °C. Primer specificity was assessed by agarose gel and 428 melting curve analysis. The results were normalized with housekeeping gene actin. Relative 429 expression levels were calculated as the mean of three technical replicates of 3 biological 430 replicates.