Two reductases complete steroidal glycoalkaloids biosynthesis in potato

Abstract

Introduction

Steroidal glycoalkaloids (SGAs), a class of specialized metabolites derived from cholesterol and containing nitrogen, are typically found in the Solanaceae plant family. This includes staple food crops such as potato (Solanum tuberosum), tomato (Solanum lycopersicum), and eggplant (Solanum melongena) (Friedman, 2002, 2006, 2015). SGAs serve as a chemical protectant against a wide range of plant pathogens and herbivores (Milner et al., 2011). Some SGAs are considered antinutritional factors in foods, causing gastrointestinal and neurological disorders in humans, in addition to exhibiting unpleasant tastes described as bitter, burning, scratchy, or acrid.

SGAs are composed of two structural elements: the aglycone unit, an anitrogen-containing steroid derived from cholesterol, and a glycoside residue attached to the hydroxy group at C-3 (Milner et al., 2011; Sawai et al., 2014). The skeletal structure of the aglycone allows SGAs to be classified into two general classes: spirosolane and solanidane (Friedman et al., 1997; Milner et al., 2011). Tomatoes exclusively contain spirosolane-type SGAs, with α-tomatine and dehydrotomatine being the most abundant in green tissues (Friedman, 2002). Conversely, potatoes produce solanidane-type SGAs, specifically α-solanine and α-chaconine, which constitute over 90% of the total SGAs in cultivated potatoes (Shakya & Navarre, 2008).

Recent research on tomato and potato plants has uncovered numerous biosynthetic genes involved in SGA biosynthesis (Fig. 1). Three cytochrome P450 monooxygenases (PGA2/GAME6, PGA1/GAME8, and PGA3/GAME4), a 2-oxoglutarate-dependent dioxygenase (16DOX/GAME11), and an aminotransferase (PGA4/GAME12) have been identified as key players in the early stages of SGA biosynthesis from cholesterol (Itkin et al., 2013; Umemoto et al., 2016; Nakayasu et al., 2017, 2021). Additionally, GAME15, a member of the cellulose synthase-like family, has been identified as an SGA biosynthetic gene (Jozwiak et al., 2024). GAME15 catalyzes the transglucuronidation of the C3-hydroxy group of cholesterol, which represents the first step in the SGA biosynthetic pathway from cholesterol (Jozwiak et al., 2024). Furthermore, GAME15 functions as a scaffold protein, facilitating physical interactions among the SGA biosynthetic enzymes and enabling efficient substrate channeling (Boccia et al., 2024; Jozwiak et al., 2024). The final products, such as α-solanine and α-tomatine, do not retain the glucuronic acid moiety, suggesting that the glucuronic acid is hydrolyzed at some point in the biosynthetic pathway, although the exact timing of this removal remains unknown. These enzymes, common to both potato and tomato, participate in the biosynthesis of the spirosolane-type aglycone, tomatidenol (Fig. 1). In tomatoes, the C-5,6 double bond from tomatidenol is removed by Sl3βHSD1 and SlS5αR2, resulting in tomatidine (Akiyama et al., 2019; Lee et al., 2019). Tomatidenol and tomatidine then undergo further modifications catalyzed by four UDP-glycosyltransferases (UGTs) to produce dehydrotomatine and α-tomatine (Itkin et al., 2011, 2013). In potatoes, dioxygenase for solanidane synthesis (DPS) was recently identified as a crucial enzyme for solanidane skeleton formation (Akiyama et al., 2021b). DPS-silenced potato plants show a decrease in α-solanine and α-chaconine levels, accompanied by a significant accumulation of α-solamarine and β-solamarine, glycosides of tomatidenol. DPS exhibits ring rearrangement activity against α-solamarine/β-solamarine, forming zwittersolanine/zwitterchaconine that contain the solanidane scaffold. Following this transformation, the biosynthesis of α-solanine/α-chaconine is anticipated to be completed through reduction and dehydration from zwittersolanin/zwitterchaconine.

Fig. 1

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Steroidal glycoalkaloids biosynthetic pathway in potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Common steps between potato and tomato are highlighted in green. Steps unique to tomato or potato are indicated by red or yellow shading, respectively. The reaction step characterized in this work by reductase for potato glycoalkaloids biosynthesis enzymes, is shown with a red arrow. 16DOX, 22,26-hydroxycholesterol 16α-hydroxylase; 3βHSD1, 3β-hydroxysteroid dehydrogenases 1; UGT, uridine diphosphate-dependent glycosyltransferases; Xyl, xylose; Gal, galactose; GAME, glycoalkaloids metabolism; Glc, glucose; GlcA, glucuronic acid; PGA, potato glycoalkaloids biosynthesis; Rha, rhamnose; S5αR2, steroid 5α-reductase 2.

In this study, we identified two genes, reductase for potato glycoalkaloid biosynthesis 1 (RPG1) and RPG2, involved in the final steps of α-solanine/α-chaconine biosynthesis in potato (Fig. 1). RPG1 and RPG2 were co-expressed with previously identified SGA biosynthetic genes in potato. Potato hairy roots with disrupted RPG1 and RPG2 did not produce α-solanine or α-chaconine, but instead accumulated zwittersolanine and zwitterchaconine. Our in vitro assay showed that the sequential reactions catalyzed by RPG1 and RPG2 complete the conversion from zwittersolanine/zwitterchaconine to α-solanine/α-chaconine. Here, we have successfully filled the critical missing link in the biosynthetic pathway of a notorious toxin in potato.