Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Seed oils are characterized by diverse fatty acid (FA) structures (1–3). Many of these deviate from typical C16 (e.g., palmitic acid, 16:0) and C18 (e.g., oleic acid, 18:1Δ9cis) FA that occur widely in seed oils, including major commercial vegetable oils. Structurally variant FAs, often referred to as “unusual” FAs, can have chain lengths other than 16 or 18 carbon atoms, double bonds in nontypical positions or cis-trans configurations, or carbon-chain modifications, such as hydroxyl or epoxy groups (4). The study of unusual FA biochemistry has increased understanding of enzyme structural determinants of substrate binding and reaction outcomes (4). These studies have also revealed how variations in biosynthetic and metabolic pathways allow for high levels of unusual FA biosynthesis and accumulation and have also facilitated research on the evolution of biochemical diversity in plants. Unusual FAs can occur in selected species or arise throughout a genus or family, and their accumulation may be associated with compensatory mutations in enzymes beyond the initial biosynthetic enzyme (5). Moreover, genes for unusual FA biosynthesis and metabolism can be useful for biotechnological research in plants and microbes to develop vegetable oils with enhanced nutritional or industrial value (6).In a variety of taxa, metabolic variations result in “extreme” unusual FA accumulation to ≥90% of seed oil, which can account for ≤50% of seed weight (2). Examples of extreme unusual FA production include medium chain-length FAs (C8–C14) in seeds of Cuphea species and ricinoleic acid, a hydroxylated C18 monounsaturated FA in castor seeds (Ricinus communis) (7, 8). Studies of FA metabolism in Cuphea have uncovered structurally variant FatB acyl-acyl carrier protein (ACP) thioesterases and acyltransferases associated with the biosynthesis and accumulation of medium chain-length FAs. These variant thioesterases have provided primary sequences for uncovering information about amino acid residues that control their substrate recognition properties (9–11). Studies of FA metabolism in castor seeds led to the discovery that structural variations in FAD2 genes for Δ12-oleic acid desaturases can lead to variant FA modifications, including insertion of hydroxyl and epoxy groups, triple bonds, and conjugated double bonds (12–14). FA biosynthetic and metabolic genes associated with these pathways have also been used to develop new oil functionalities in existing oilseed crops.Studies of unusual monounsaturated FAs have provided a wealth of biochemical information and biotechnological utility (15). Oleic acid, the most widely occurring monounsaturated FA in plants, is synthesized by the soluble Δ9-stearoyl-ACP desaturase (16). Structural variants of this enzyme result in a number of unusual FAs, including palmitoleic acid (16:1Δ9), sapienic acid (16:1Δ6), and petroselinic acid (18:1Δ6) (15). Studies of variant acyl-ACP desaturases in combination with three-dimensional (3D) structural data (17, 18) have revealed amino acid residues that control FA chain length- and regio-specificities of this enzyme class (19). This information has facilitated biotechnological development of oilseeds with high levels of unusual monounsaturated FAs using rationally designed acyl-ACP desaturases (20). In the case of petroselinic acid, a biosynthetic pathway has been deduced that involves not only a variant Δ4-16:0-ACP desaturase but also a coevolved variant β-ketoacyl-ACP synthase I and acyl-ACP thioesterase that together generate petroselinic acid at ≤85% of the total FAs of Apiaceae and Araliaceae seeds (21–24). This highlights that unusual FA biosynthesis can result from the initial evolution of a specialized biosynthetic enzyme, followed by evolution of additional enzymes that facilitate unusual FA production and accumulation.As part of our larger efforts to characterize the evolution of unusual FA biosynthesis in plants, we examined the FA composition of seeds in diverse Thunbergia species. It has been previously shown that Thunbergia alata seeds accumulate sapienic acid (16:1Δ6cis) to ∼85% of total FAs via the activity of a Δ6-16:0-ACP desaturase (25, 26). As reported here, we discovered that seeds of several Thunbergia species accumulate petroselinic acid instead of sapienic acid. In these seeds, petroselinic acid accumulates >90% of total FAs, which is among the highest naturally occurring levels of monounsaturated FA in plant seed oils. We provide biochemical, structural, and genetic evidence to explain the divergent evolution of increased sapienic and petroselinic acid production in Thunbergia. Furthermore, we show that the petroselinic acid biosynthetic pathway in Thunbergia is distinct from that in Apiaceae and Araliaceae (21) and can guide biotechnological production of unusual monounsaturated FAs in engineered crops and microbes.