Boosting the Synthesis of Pharmaceutically Active Abietane Diterpenes in S. sclarea Hairy Roots by Engineering the GGPPS and CPPS Genes

Abstract

Abietane diterpenoids (ADs), synthesized in the roots of different Salvia species, such as aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol, have a variety of known biological activities. We have shown that aethiopinone has promising cytotoxic activity against several human tumor cell lines, including the breast adenocarcinoma MCF7, HeLa, epithelial carcinoma, prostate adenocarcinoma PC3, and human melanoma A375. The low content of these compounds in natural sources, and the limited possibility to synthesize them chemically at low cost, prompted us to optimize the production of abietane diterpenoids by targeting genes of the methylerythritol phosphate (MEP) pathway, from which they are derived. Here, we report our current and ongoing efforts to boost the metabolic flux towards this interesting class of compounds in Salvia sclarea hairy roots (HRs). Silencing the gene encoding the ent-copalyldiphosphate synthase gene (entCPPS), acting at the lateral geranylgeranyl pyrophosphate (GGPP) competitive gibberellin route, enhanced the content of aethiopinone and other ADs in S. sclarea HRs, indicating indirectly that the GGPP pool is a metabolic constraint to the accumulation of ADs. This was confirmed by overexpressing the GGPPS gene (geranyl-geranyl diphosphate synthase) which triggered also a significant 8-fold increase of abietane diterpene content above the basal constitutive level, with a major boosting effect on aethiopinone accumulation in S. sclarea HRs. A significant accumulation of aethiopinone and other AD compounds was also achieved by overexpressing the CPPS gene (copalyl diphosphate synthase) pointing to this biosynthetic step as another potential metabolic target for optimizing the biosynthesis of this class of compounds. However, by co-expressing of GGPPS and CPPS genes, albeit significant, the increase of abietane diterpenoids was less effective than that obtained by overexpressing the two genes individually. Taken together, the results presented here add novel and instrumental knowledge to a rational design of a hairy rootbased platform to yield reliable amounts of aethiopinone and other ADs for a deeper understanding of their molecular pharmacological targets and potential future commercialization.

Transcriptomic Characterization of Nitrate-Enhanced Stevioside Glycoside Synthesis in Stevia (Stevia rebaudiana) Bertoni

“…This was an important observation as these are the key catalytic enzymes responsible for ent-diterpenoid synthesis and are highly conserved in all terpenoid-synthesizing organisms [52]. Thus, enhanced diterpene synthesis and accumulation have been achieved by overexpressing the CPS or GGPPS gene in Salvia species [52,53] and tobacco [54]. Similarly, Jassbi et al [55] demonstrated that the silencing of GGPPS disrupted the synthesis of 17-hydroxygeranyllinalool diterpenoid glycosides, together with the increased susceptibility to tobacco hornworm.…”

“…Even more striking was the upregulation of genes encoding GGPPS and CPS, by NO 3 − feeding. This was an important observation as these are the key catalytic enzymes responsible for ent-diterpenoid synthesis and are highly conserved in all terpenoid-synthesizing organisms [52]. Thus, enhanced diterpene synthesis and accumulation have been achieved by overexpressing the CPS or GGPPS gene in Salvia species [52,53] and tobacco [54].…”

Transcriptome Analyses Revealed the Key Metabolic Genes and Transcription Factors Involved in Terpenoid Biosynthesis in Sacred Lotus

“…For example, FPPS overexpression increased the production of ganoderic acid [ 42 ]. A high number of monoterpenes was produced by overexpression of peppermint ( Mentha × piperita ) GPPS.SSU in transgenic tobacco plants [ 43 ] and the content of abietane diterpenes in Salvia sclarea L. hairy roots was increased by engineering the GGPPS and CPPS [ 44 ]. In this research, six DEGs encoding IPTS were identified, including one GPPS , two FPPS , and three GGPPS .…”

Transcriptome analysis of two Pogostemon cablin chemotypes reveals genes related to patchouli alcohol biosynthesis

“…of the terpene biosynthetic pathway have recently been successfully overexpressed in Salvia spp. to increase terpenoid yield ( Vaccaro et al, 2020 ; Shi et al, 2016 ; Kai et al, 2011 ). This is a feasible strategy for enhancing the production of natural products by manipulating a number of biosynthetic genes at regulatory points.…”