Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii.

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1-4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1-4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.

Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis.

Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

Squalene epoxidase (SQLE) promotes the growth and migration of the hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with a poor response to chemotherapy. It is very important to identify novel therapeutic targets. Squalene epoxidase (SQLE), one of the rate-limiting enzymes in the cholesterol biosynthesis, recently has been found to be involved in the tumorigenesis. However, its expression profile and function in the progression of HCC remain largely unknown. Here, we found that the expression of SQLE was upregulated in the HCC tissues. Moreover, overexpression of SQLE in HCC cells promoted cell proliferation and migration, while downregulation of SQLE inhibited the tumorigenicity of HCC cells in vitro and in vivo. Mechanistically, SQLE positively regulated the ERK signaling. Taken together, our study suggests that SQLE is a promising therapeutic target in HCC.

[Development of the devices for synthetic biology of triterpene saponins at an early stage: cloning and expression profiling of squalene epoxidase genes in panax notoginseng].

Synthetic biology of traditional Chinese medicine (TCM) is a new and developing subject based on the research of secondary metabolite biosynthesis for nature products. The early development of synthetic biology focused on the screening and modification of parts or devices, and establishment of standardized device libraries. Panax notoginseng (Burk.) F.H.Chen is one of the most famous medicinal plants in Panax species. Triterpene saponins have important pharmacological activities in P. notoginseng. Squalene epoxidase (SE) has been considered as a key rate-limiting enzyme in biosynthetic pathways of triterpene saponins and phytosterols. SE acts as one of necessary devices for biosynthesis of triterpene saponins and phytosterols in vitro via synthetic biology approach. Here we cloned two genes encoding squalene epoxidase (PnSE1 and PnSE2) and analyzed the predict amino acid sequences by bioinformatic analysis. Further, we detected the gene expression profiling in different organs and the expression level of SEs in leaves elicited by methyl jasmonate (MeJA) treatment in 4-year-old P notoginseng using real-time quantitative PCR (real-time PCR). The study will provide a foundation for discovery and modification of devices in previous research by TCM synthetic biology. PnSE1 and PnSE2 encoded predicted proteins of 537 and 545 amino acids, respectively. Two amino acid sequences predicted from PnSEs shared strong similarity (79%), but were highly divergent in N-terminal regions (the first 70 amino acids). The genes expression profiling detected by real-time PCR, PnSE1 mRNA abundantly accumulated in all organs, especially in flower. PnSE2 was only weakly expressed and preferentially in flower. MeJA treatment enhanced the accumulation of PnSEI mRNA expression level in leaves, while there is no obvious enhancement of PnSE2 in same condition. Results indicated that the gene expressions of PnSE1 and PnSE2 were differently transcribed in four organs, and two PnSEs differently responded to MeJA stimuli. It was strongly suggested that PnSEs play different roles in secondary metabolite biosynthesis in P. notoginseng. PnSE1 might be involved in triterpenoid biosynthesis and PnSE2 might be involved in phytosterol biosynthesis.

Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus.

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.