Introduction

The vast majority of naturally occurring sterols contain an alkyl group at carbon-24 in the side-chain. 24-Alkyl sterols are collectively knownas phytosterols and possess chemical structures similar to that of cholesterol (cholest-5-en-3β-ol). Sitosterol is the major C₂₉-Δ⁵-phytosterol and ergosterol is the major C₂₈-Δ⁵-phytosterol of plants and fungi, respectively. Cholesterol is the major C₂₇-Δ⁵-zoosterol of animals and insects (Fig. 9.1). Pure sitosterol resembles cholesterol, white in color and waxy in nature. The physical resemblance of the two sterols is mirrored in the similarities of the biosynthetic pathways and functions of these compounds. A striking feature of phytosterol synthesis is that the pathway is directed to form membrane components, in a similar fashion as for cholesterol production and processing in animal systems. However, the subtle differences in the structures of the sterol side chains, 24-methyl (or ethyl) group compared to a 24-hydrogen atom, make for plant-specific functions of sterols.

FIGURE 9.1 Major sterols found in living systems.

Phytosterolsmake up greater than 80%of the total sterol content of the vegetative parts of plants and accumulate to 500–3,000 fg/cell or 1 mg/blade, mostly as free sterol (Nes, 1990; Nes and McKean, 1977). High phytosterol levels are found esterified to fatty acids in seed oils and in leaves of aging plants (Nes, 1990; Wojciechowski, 1991). In humans, cholesterol is present in cells about 500– 3,000 fg/cell, similar to the sterol content of plant cells, and about 10 mg/liter in the serum (Nes, 1990; Nes et al., 2000). Alternatively, under normal physiological conditions phytosterols are found at 800–1,000 times lower concentration than that of endogenous cholesterol in the serum. When the concentration of phytosterol is maintained at a high level, ca. 300 mg up to 5 g/day, by a diet rich in vegetables and fruits or through vegetable oil spreads, such as Benecol or Take Control, there are important benefits. For example, phytosterols have been shown experimentally to inhibit colon, breast, and prostate cancers; induce anti-inflammatory effects; and reduce cholesterol levels (Awad and Fink, 2000; Moreau et al., 2002; Tapiero et al., 2003). The exact mechanism by which sitosterol offers protection from cancer is not known and several theories have been advanced as reviewed (Ling and Jones, 1995). On the other hand, it is generally assumed that cholesterol reduction in the blood stream results directly frominhibition of food-based cholesterol absorption through displacement of cholesterol from micelles (Akihisa et al., 2000; Moreau et al., 2002). For these reasons, phytosterols are considered nutraceuticals and the engineering of plants to increase the phytosterol content as a nonpharmacological approach to prevent certain diseases is underway. It is worth pointing out that there is a portion of the literature that continues to use beta (β)-sitosterol; the beta in this case does not refer to the stereochemistry of the molecule but is used to distinguish the compound from α- and γ-sitosterol. The notation is dropped in common usage (Nes, 2000).

In contrast to other plant lipids, such as fatty acids defined on the basis of their physical properties, phytosterols are defined by their common chemical structure and biosynthetic reasoning related to the cyclization of squalene oxide (Nes and McKean, 1977). Phytosterols, which are insoluble in water and can be extracted from cells by nonpolar organic solvents (such as hexane or chloroform), are characterized by a cyclopentanoperhydrophenanthrene structured nucleus, a flexible side-chain of 9 or 10 carbon atoms and equatorial attachments of a polar head (3β-hydroxyl group) and nonpolar tail (17β-side-chain). The three-dimensional shape is established by the alternating all trans–anti stereochemistry of the ring system and the 20R-configuration that directs the side-chain to a ‘‘right-handed’’ conformation (Nes et al., 1984; Parker and Nes, 1992; Fig. 9.2). When the side-chain of, for example, sitosterol is oriented to the ‘‘right,’’ the sterol has the appropriate length—ca. 19Å to fit the monolayer—that is, one-half of the lipid leaflet structure. The combination of asymmetry and electronics in the sitosterol structure gives rise to an amphipathic molecule, basically flat with a length suitable for the sterol to insert the membrane. The presence of 24-ethyl sterols in plant membranes correlates with their efficiency in developing interactions with plant phospholipids to affect membrane fluidity (Mckersie and Thompson, 1979; Schuler et al., 1991).

FIGURE 9.2 Sitosterol distribution in the intact plant: (A) whole plant; (B) leaf; (C) cell structure; (D) membrane lipid leaflet; (E) conformational perspective of sterol; (F) structure and stereochemistry in sitosterol.

The C-24 alkylated family of phytosterols contains a greater number of individual compounds in plants than fungi or protozoa. The variant structures and health benefits of generating a modified phytosterol composition provide the basis for investigations of phytosterol profiling and metabolic engineering. Studies on testing inhibitors of sterol methyltransferase (SMT) action in cultured cells and genetic manipulation of the phytosterol pathway in several plants have revealed a physiological requirement for a 24-alkyl substituted steroid in plant growth, the central position of C-methylation in the plant sterol pathway and the connection of the SMT to manufacturing value-added traits (Chappell et al., 1995; Harker et al., 2003; Nes et al., 1991c; Rahier et al., 1980). Since the biosynthesis and functions of sterols in plants are covered in recent review articles (Benveniste, 2004; Clouse, 2002; Lindsey et al., 2003; Nes, 2003; Schaller, 2004), we have included only background material pertinent to the phytosterol pathway as influenced by the SMT. Initially, the pathways of sterol biosynthesis will be described. After a brief coverage of phytosterolomics, with emphasis on structure determination, we review the enzymology and evolution of the SMT and conclude with the current state of metabolic engineering sterol pathways in plants. We have not attempted to be comprehensive in our presentation of the literature; rather the focus is on illustrative examples that may serve to indicate future opportunities to engineer plants with unusual sterol profiles or traits.