farnesyl-pyrophosphate and cycloartenol

Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to cycloartenol or β-amyrin, which contained Arabidopsis (Arabidopsis thaliana)-derived squalene synthase (AtSQS) and squalene epoxidase (AtSQE) genes in addition to the Arabidopsis cycloartenol synthase (AtCAS1) gene, or the β-amyrin synthase (EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase (HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene (zwf or gdh) as well as bacterial redox partner protein genes (camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase (OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants.

Topics: Arabidopsis; Escherichia coli; Farnesyl-Diphosphate Farnesyltransferase; Intramolecular Transferases; Metabolic Engineering; Metabolic Networks and Pathways; Oleanolic Acid; Phytosterols; Polyisoprenyl Phosphates; Sesquiterpenes; Squalene Monooxygenase; Triterpenes

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Carbon Radioisotopes; Cell Line; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hydroxymethylglutaryl CoA Reductases; Methyltransferases; Naphthalenes; Nicotiana; Oxygenases; Phosphoric Monoester Hydrolases; Phytosterols; Polyisoprenyl Phosphates; Sesquiterpenes; Squalene; Squalene Monooxygenase; Terbinafine; Tricarboxylic Acids; Triglycerides; Triterpenes; Up-Regulation