- Top of page
- Experimental procedures
Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to β-amyrin synthases (EC 5.4.99.–), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a β-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved β-amyrin production by 50%, achieving levels of 6 mg·L−1 culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher β-amyrin production. Options for further engineering efforts are explored.
Triterpenes belong to the isoprenoid family of compounds and are recognized by their C30 backbones. They are typically synthesized by the cyclization of the sterol precursor 2,3-oxidosqualene into a multi-ringed compound with a single alcohol group. Fungi and mammals convert 2,3-oxidosqualene to the triterpene compound lanosterol in the biosynthetic pathways to ergosterol and cholesterol, respectively. The equivalent step in plant primary metabolism is the cyclization of 2,3-oxidosqualene to cycloartenol for the production of membrane sterols. Cycloartenol is also the triterpene precursor of brassinosteroid phytohormones that regulate plant growth and development [1,2]. Based on chemical and genetic analyses performed to date, it appears that plants are more diverse than animals or fungi in the range of tritepene products synthesized . However, despite the fact that a large variety of triterpene compounds have been isolated from plant sources , the majority of triterpene synthase genes isolated to date have encoded either lupeol or β-amyrin synthases (EC 5.4.99.–) . β-amyrin in particular serves as the olefin precursor to a wide range of downstream products. The action of oxidative enzymes (typically cytochrome P450 monooxygenases) and glycosyltransferases convert β-amyrin to various triterpene saponins in different plant species [5–7]. These saponins may perform protective roles in the host plant, acting as antimicrobial  and insecticidal  agents, and many of these compounds are also of interest from a human health perspective. The effect of plant saponins on low-density lipoprotrein cholesterol absorption and arterial atherosclerosis has received much attention, leading to the development of several cholesterol-reducing dietary supplements . Saponins may also find applications in ruminant nutrition , as anticancer agents [12,13], and as vaccine adjuvants .
Although triterpene synthases have been expressed in microbial hosts such as Saccharomyces cerevisiae, there has been little effort made so far to engineer the metabolism of a microbial host for enhanced production of triterpenes. By contrast, there have been many considerable efforts to engineer microbes for higher production of mono-, sesqi- and diterpenes . These projects have mainly focused on the overexpression of enzymes involved in either of the two pathways (mevalonate or 1-deoxy-d-xylulose-5-phosphate) responsible for the biosynthesis of isoprenoids [16–18]. In S. cerevisiae, the mevalonate pathway is responsible for the biosynthesis of isoprenoids and sterols. A good deal is known about regulatory mechanisms within the pathway, although the majority of studies have focused on the upper part of the pathway, from acetyl-CoA to squalene. Our knowledge of how the lower half of the pathway, from squalene to ergosterol, is regulated remains somewhat limited. As the branch point for triterpene biosynthesis is located in this latter half of the pathway, the optimal steps to increase their production in yeast are not immediately apparent.
Artemisia annua, or sweet wormwood, has been used medicinally for centuries, predominantly in China . A sesquiterpene constituent, artemisinin, is one of the most important drugs used in the treatment of malaria. In an effort to isolate and characterize new terpene synthases from A. annua, we have designed degenerate primers for use in RT-PCR. Here, we describe the isolation of a β-amyrin synthase gene from A. annua and its expression in S. cerevisiae. Our findings on engineering overproduction of β-amyrin in S. cerevisiae should be relevant to the production of any triterpene.
- Top of page
- Experimental procedures
We have shown that it is possible to engineer increased production of tritepenes in S. cerevisiae without the need for feeding with exogenous sterols. The 50% increase in β-amyrin levels demonstrated in the present study should be considered in the light of the fact that triterpene production may not be as amenable to engineering efforts as the volatile sesquiterpenes and monoterpenes that readily diffuse out of the cell. However, it is apparent that further progress can be made and there are some clues as to what these next steps may comprise. We have achieved a 12-fold increase in squalene levels over the initial βamy1 strain, and a logical course of action would be to find a way to convert this squalene into β-amyrin.
M’baya et al.  demonstrated that ERG1 activity is reduced in the presence of excess sterols through a mechanism other than enzyme inhibition, most likely transcriptional repression. Not a great deal is known about how the latter half of the sterol pathway is regulated and exactly what role ERG1 plays in this process. However, the fact that we observe a further increase in squalene upon downregulation of ERG7 in strain βamy4 would indicate that 2,3-oxidosqualene can act as a repressor of ERG1. Tight regulation at ERG1 would make sense as it marks the beginning of the oxygen-dependent reactions in the pathway. If the feedback regulation is transcriptional in nature, then it should be possible to circumvent it by overexpressing ERG1 under the control of an independent promoter. Veen et al.  overexpressed ERG1 and tHMG1 together and observed a 50% increase in sterol concentrations.
We have subsquently tested this hypothesis by transforming the strain βamy4 with a high-copy expression vector harboring ERG1 under control of the GAL1 promoter (to create strain βamy5). A comparison between these two strains showed that β-amyrin production levels were essentially unchanged whereas squalene levels actually increased slightly in strain βamy5 (data not shown). This would indicate that flux from squalene to β-amyrin is not limited by ERG1 transcription levels, but the possibility remains that there is regulation of ERG1 at the protein level. It also is likely that there are other factors contributing to the lack of flux from squalene to β-amyrin. In particular, the availability of squalene for conversion by ERG1 may be limited by its biochemical state. In cases where tHMG1 has been overexpressed in yeast, squalene accumulates predominantly in an insoluble form that is not immediately available to the sterol pathway [25,27]. The sterol-acyl transferases ARE1 and ARE2 are responsible for esterification of excess squalene for storage in insoluble lipid particles . Thus, it appears that attenuation of this process would be the next logical step for further engineering triterpene production in S. cerevisiae. Additional studies into the possible regulation of ERG1 by 2,3-oxidosqualene at either post-translational or enzyme kinetic levels may also be warranted.