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A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis.

Aerenchymatous phellem (AP) is important for internal aeration and adaptation to waterlogging in plants. Herein, the extensive accumulation of triterpenoids such as lupeol and betulinic acid was identified in AP. However, the biological and physiological roles of these triterpenoids in plants are largely unknown. Lupeol is converted from 2,3-oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Functional analysis of LUS genes in soybean revealed that GmLUS1 is crucial for triterpenoid biosynthesis in AP. Lupeol and betulinic acid were found to be the major components of epicuticular wax on the surface of AP cells, and they contributed to tissue hydrophobicity and oxygen transport to roots. Additionally, the lus1 mutant produced a shallow root system due to less oxygen transport via AP under waterlogged conditions. In conclusion, triterpenoid accumulation in AP aids internal aeration and root development for adaptation to waterlogging.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Uridine 5'-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1-4) showed UGD activity in vitro. GuUGD1-4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1-4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.

Orobanchaceae root parasitic weeds cause serious agricultural damage worldwide. Although numerous studies have been conducted to establish an effective control strategy for the growth and spread of root parasitic weeds, no practical method has been developed so far. Previously, metabolomic analyses were conducted on germinating seeds of a broomrape, Orobanche minor, to find novel targets for its selective control. Interestingly, planteose metabolism was identified as a possible target, and nojirimycin (NJ) selectively inhibited the germination of O. minor by intercepting planteose metabolism, although its precise mode of action was unclear. Here, transcriptome analysis by RNA-Seq was conducted to obtain molecular insight into the effects of NJ on germinating O. minor seeds. Differential gene expression analysis results suggest that NJ alters sugar metabolism and/or signaling, which is required to promote seed germination. This finding will contribute to understanding the effect of NJ and establishing a novel strategy for parasitic weed control.

Purpose: Artemisinin, a secondary metabolite in Artemisia annua is one of primary choice for the treatment of malaria, it is naturally produced in low concentration from this plant. This study was aimed to clone key enzymes of artemisinin production in order to enhance its production through the semi-synthetically production in Saccharomyces cerevisiae. Methods: Two key enzymes in artemisinin biosynthetic pathway which are farnesyl phosphate synthase (fps) and amorpha-4,11-diene synthase (ads) genes were transformed into S. cerevisiae using pBEVY vector. Successful transformation was checked by polymerase chain reaction (PCR) method and sequencing analysis Results: Recombinant plasmids which are pBEVY-GU_ads and pBEVY_GL_fps were successfully constructed. The optimized ads gene was amplified using PCR with a couple of primers that are designed in order to provide the homolog recombination between ads gene with the expression plasmid of pBEVY-GU respectively. While the A. annua optimized fps gene was cloned using classical method. Transformants were grown in selective media Synthetic Defined (SD) without leucine for transformants contain plasmid pBEVY-GL_fps and media without uracil for transformants contain plasmid pBEVY-GU_ads. Confirmation of colonies was done by PCR with primers to amplify fps and ads. DNA from yeast was isolated from positive colonies then transformed to E. coli. Plasmid from E. coli was isolated for restriction analysis and sequencing. Protein expression was induced by cultivating the yeast in the media with 2% galactose. Conclusion: Based on PCR, restriction and sequencing analysis, it could be concluded that fps and ads genes were successfully constructed, transformed and expressed in S. cerevisiae.

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane–localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins., Saponins such as glycyrrhizin, a natural sweetener found in licorice root, are a class of triterpenoids synthesized that are characterized by a glucoronic acid moiety at the C-3 position. Here the authors show that saponin glucuronosylation is catalyzed by cellulose-synthase like enzymes and reconstitute glycyrrhizin synthesisin yeast.

Potato (Solanum tuberosum) is one of the most important crops in the world. However, it is generally difficult to breed a new variety of potato crops because they are highly heterozygous tetraploid. Steroidal glycoalkaloids (SGAs) such as α-solanine and α-chaconine found in potato are antinutritional specialized metabolites. Because of their toxicity following intake, controlling the SGA levels in potato varieties is critical in breeding programs. Recently, genome-editing technologies using artificial site-specific nucleases such as TALEN and CRISPR-Cas9 have been developed and used in plant sciences. In the present study, we developed a highly active Platinum TALEN expression vector construction system, and applied to reduce the SGA contents in potato. Using Agrobacterium-mediated transformation, we obtained three independent transgenic potatoes harboring the TALEN expression cassette targeting SSR2 gene, which encodes a key enzyme for SGA biosynthesis. Sequencing analysis of the target sequence indicated that all the transformants could be SSR2-knockout mutants. Reduced SGA phenotype in the mutants was confirmed by metabolic analysis using LC-MS. In vitro grown SSR2-knockout mutants exhibited no differences in morphological phenotype or yields when compared with control plants, indicating that the genome editing of SGA biosynthetic genes such as SSR2 could be a suitable strategy for controlling the levels of toxic metabolites in potato. Our simple and powerful plant genome-editing system, developed in the present study, provides an important step for future study in plant science.

Summary Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins., Significance Statement Glycyrrhizin, a licorice‐derived sweet triterpenoid diglycoside, is one of the most important phytoconstituents in the pharmaceutical and food industries. Here, we identified UGT73P12 as the enzyme catalyzing the sugar–sugar linkage to produce glycyrrhizin from its less glycosylated precursor in the presence of UDP‐glucuronic acid, and found that Arg32 of UGT73P12 is essential for the high specificity for UDP‐glucuronic acid, which was probably acquired during evolution to enable the functional specialization of UGT73P12 for glycyrrhizin biosynthesis.

Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography-mass spectrometry. Through transcriptome analyses, homology-based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α-amyrin synthase activity. B. forficata highly accumulated α-amyrin. We discovered an OSC with a preponderant α-amyrin-producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β-amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α-amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non-model legume plant.

α-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the Δ5,6 double bond; the dehydro-type). The aglycone of α-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3βHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3βHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3βHSD1 protein revealed that Sl3βHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3βHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3βHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3βHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3βHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.

Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.

Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of β-amyrin at position C-11 to produce 11-oxo-β-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply β-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-β-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3β-ol and oleana-11,13(18)-dien-3β-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-β-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized.

Orobanchaceae root parasitic weeds cause serious agricultural damage worldwide. Although numerous studies have been conducted to establish an effective control strategy for the growth and spread of root parasitic weeds, no practical method has been developed so far. Previously, metabolomic analyses were conducted on germinating seeds of a broomrape, Orobanche minor, to find novel targets for its selective control. Interestingly, planteose metabolism was identified as a possible target, and nojirimycin (NJ) selectively inhibited the germination of O. minor by intercepting planteose metabolism, although its precise mode of action was unclear. Here, transcriptome analysis by RNA-Seq was conducted to obtain molecular insight into the effects of NJ on germinating O. minor seeds. Differential gene expression analysis results suggest that NJ alters sugar metabolism and/or signaling, which is required to promote seed germination. This finding will contribute to understanding the effect of NJ and establishing a novel strategy for parasitic weed control.

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, SlS5αR1- or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.

Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.

Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.

Several enzymes of the CYP716A subfamily have been reported to be involved in triterpenoid biosynthesis. Members of this subfamily oxidize various positions along the triterpenoid backbone and the majority of them catalyze a three-step oxidation at the C-28 position. Interestingly, C-28 oxidation is a common feature in oleanolic acid, ursolic acid, and betulinic acid, which are widely distributed in plants and exhibit important biological activities. In this work, three additional CYP716A enzymes isolated from olive, sugar beet, and coffee, were characterized as multifunctional C-28 oxidases. Semi-quantitative comparisons of in vivo catalytic activity were made against the previously characterized enzymes CYP716A12, CYP716A15, and CYP716A52v2. When heterologously expressed in yeast, the isolated enzymes differed in both catalytic activity and substrate specificity. This study indicates that the screening of enzymes from different plants could be a useful means of identifying enzymes with enhanced catalytic activity and desired substrate specificity. Furthermore, we show that "naturally-evolved" enzymes can be useful in the heterologous production of pharmacologically and industrially important triterpenoids.

Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in the mevalonate pathway. In higher plants, mevalonate pathway involves in the production of precursor for isoprenoids biosynthesis, including essential components for cell functions. Previously, we confirmed that the Arabidopsis thaliana HMGR1S (AtHMGR1S) is phosphorylated at S577 by the combination of sucrose non-fermenting related kinase-1 (SnRK1) and geminivirus rep-interacting kinase-1 (GRIK1) in vitro. However, even in quantitative phosphoproteomics studies that were directed to find SnRK1 target substrates, AtHMGR1S phosphorylation at S577 has never been detected in planta. In this study, we expressed AtHMGR1S as a C-terminal FLAG-fusion protein in A. thaliana hmg1 mutant to confirm its phosphorylation in planta. Our results provide the first direct evidence that AtHMGR1S is phosphorylated at S577 in planta. Moreover, phosphatase inhibitors treatment to the A. thaliana seedlings induced AtHMGR1S phosphorylation at sites other than S577, suggesting the presence of a novel HMGR regulatory mechanism in planta.

Aluminum tolerance of aluminum-sensitive rice was enhanced under darkness by multiple changes in membrane sterols: decreased stigmasterol, increased precursor partitioning for sterols biosynthesis, and increased expression of HMG genes., Aluminum-sensitive rice (Oryza sativa L.) cultivars showed increased Al tolerance under dark conditions, because less Al accumulated in the root tips (1 cm) under dark than under light conditions. Under dark conditions, the root tip concentration of total sterols, which generally reduce plasma membrane permeabilization, was higher in the most Al-sensitive japonica cultivar, Koshihikari (Ko), than in the most Al-tolerant cultivar, Rikuu-132 (R132), but the phospholipid content did not differ between the two. The Al treatment increased the proportion of stigmasterol (which has no ability to reduce membrane permeabilization) out of total sterols similarly in both cultivars under light conditions, but it decreased more in Ko under dark conditions. The carotenoid content in the root tip of Al-treated Ko was significantly lower under dark than under light conditions, indicating that isopentenyl diphosphate transport from the cytosol to plastids was decreased under dark conditions. HMG2 and HMG3 (encoding the key sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase) transcript levels in the root tips were enhanced under dark conditions. We suggest that the following mechanisms contribute to the increase in Al tolerance under dark conditions: inhibition of stigmasterol formation to retain membrane integrity; greater partitioning of isopentenyl diphosphate for sterol biosynthesis; and enhanced expression of HMGs to increase sterol biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

HMG-CoA reductase (HMGR) is a key enzyme in the mevalonate pathway for sterols and cytosolic isoprenoid production. Although HMGR kinases from spinach, barley, and cauliflower tissues have been strongly suggested as members of SNF1-related protein kinases 1 (SnRK1), the phosphorylation and inactivation of HMGR by plant SnRK1s has not been demonstrated. In this study, we elucidated that AKIN10, an Arabidopsis SnRK1, acts as an HMGR kinase. The recombinant AKIN10 phosphorylates and inactivates AtHMGR1S using recombinant GRIK1 as the AKIN10 activator. In contrast, AKIN10-GRIK1 fails to inactivate AtHMGR1S-S577A, suggesting that this is achieved through Ser577 phosphorylation. Moreover, phosphorylation is detected not only in AtHMGR1S but also in AtHMGR1S-S577A, suggesting the presence of a novel regulatory mechanism of plant HMGR.

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.

The biosynthetic pathway of cytosolic isoprenoids bifurcates after farnesyl diphosphate into sesquiterpene and triterpene pathways. “Metabolic switching” has been used to increase sesquiterpene content in plants by suppressing the competitive triterpene pathway using transgenic technology. To develop “metabolic switching” without using transgenic technology, we developed a model system of “chemical metabolic switching” using inhibitors of the competitive pathway. Arabidopsis plants that overexpress the amorpha-4,11-diene synthase gene were treated with squalestatin, a squalene synthase inhibitor, or terbinafine, a squalene epoxidase inhibitor. We then analyzed total sterol content as major triterpenes and amorpha-4,11-diene in the plant. Plants treated with squalestatin showed decreased total sterol content and increased amorpha-4,11-diene content. In contrast, plants treated with terbinafine showed decreased total sterol content, but amorpha-4,11-diene accumulation was quite low. These results suggest that inhibition of the enzyme just below the branch point is more effective than inhibition of enzymes far from the branch point for “chemical metabolic switching”. In addition, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the cytosolic isoprenoid biosynthetic pathway, was upregulated in plants treated with squalestatin, suggesting that feedback regulation of 3-hydroxy-3-methylglutaryl-CoA reductase may contribute to amorpha-4,11-diene production. Here we demonstrated the effectiveness of “chemical metabolic switching” in plants.

CYP716A179, a cytochrome P450 monooxygenase expressed predominantly in tissue-cultured stolons of licorice ( Glycyrrhiza uralensis ), functions as a triterpene C-28 oxidase in the biosynthesis of oleanolic acid and betulinic acid. Cytochrome P450 monooxygenases (P450s) play key roles in the structural diversification of plant triterpenoids. Among these, the CYP716A subfamily, which functions mainly as a triterpene C-28 oxidase, is common in plants. Licorice (Glycyrrhiza uralensis) produces bioactive triterpenoids, such as glycyrrhizin and soyasaponins, and relevant P450s (CYP88D6, CYP72A154, and CYP93E3) have been identified; however, no CYP716A subfamily P450 has been isolated. Here, we identify CYP716A179, which functions as a triterpene C-28 oxidase, by RNA sequencing analysis of tissue-cultured stolons of G. uralensis. Heterologous expression of CYP716A179 in engineered yeast strains confirmed the production of oleanolic acid, ursolic acid, and betulinic acid from β-amyrin, α-amyrin, and lupeol, respectively. The transcript level of CYP716A179 was about 500 times higher in tissue-cultured stolons than in intact roots. Oleanolic acid and betulinic acid were consistently detected only in tissue-cultured stolons. The discovery of CYP716A179 helps increase our understanding of the mechanisms of tissue-type-dependent triterpenoid metabolism in licorice and provides an additional target gene for pathway engineering to increase the production of glycyrrhizin in licorice tissue cultures by disrupting competing pathways.

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Morolic acid is a plant‐derived triterpenoid that possesses pharmacological properties such as cytotoxicity, as well as anti‐HIV, anti‐HSV, anti‐inflammatory, and antidiabetic effects. The significant therapeutic properties of morolic acid are desirable in the context of pharmacological and drug development research, but the low accessibility of morolic acid from natural resources limits its applications. In the present study, we developed a microbial system for the production of morolic acid. Using a combinatorial biosynthesis approach, a novel production pathway was constructed in Saccharomyces cerevisiae by co‐expressing BfOSC2 (germanicol synthase) from Bauhinia forficata and CYP716A49 (triterpene C‐28 oxidase) from Beta vulgaris. Moreover, we reconstructed the cellular galactose regulatory network by introducing a chimeric transcriptional activator (fusion of Gal4dbd.ER.VP16, GEV) to overdrive the genes under the control of the galactose promoter. We also overexpressed truncated HMG1, encoding feedback‐inhibition‐resistant form of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase 1 and sterol‐regulating transcription factor upc2‐1, to increase the isoprenoid precursors in the mevalonate pathway. Using this yeast system, we achieved morolic acid production up to 20.7 ± 1.8 mg/L in batch culture. To our knowledge, this is the highest morolic acid titer reported from a heterologous host, indicating a promising approach for obtaining rare natural triterpenoids. This article is protected by copyright. All rights reserved.

Plants produce various prenylated phenolic metabolites, including flavonoids, phloroglucinols, and coumarins, many of which have multiple prenyl moieties and display various biological activities. Prenylated phenylpropanes, such as artepillin C (3, 5-diprenyl-p-coumaric acid), exhibit a broad range of pharmaceutical effects. To date, however, no prenyltransferases (PTs) involved in the biosynthesis of phenylpropanes and no plant enzymes that introduce multiple prenyl residues to native substrates with different regio-specificities have been identified. This study describes the isolation from Artemisia capillaris of a phenylpropane-specific PT gene, AcPT1, belonging to UbiA superfamily. This gene encodes a membrane-bound enzyme, which accepts p-coumaric acid as its specific substrate and transfers two prenyl residues stepwise to yield artepillin C. These findings provide novel insights into the molecular evolution of this gene family, contributing to the chemical diversification of plant specialized metabolites. These results also enabled the design of a yeast platform for the synthetic biology of artepillin C., アルテピリンC合成酵素の発見とその生産 --雑草の遺伝子から生理活性物質の生産へ--. 京都大学プレスリリース. 2019-10-25.

Pentacyclic triterpenes may be active agents and provide a rich natural resource of promising compounds for drug development. The inhibitory activities of 29 natural oleanane and ursane pentacyclic triterpenes were evaluated against four major enzymes involved in the inflammatory process: 5-LOX, 15-LOX-2, COX-1, and COX-2. It was found that 3-O-acetyl-β-boswellic acid potently inhibited human 15-LOX-2 (IC50 = 12.2 ± 0.47 μM). Analysis of the structure-activity relationships revealed that the presence of a hydroxy group at position 24 was beneficial in terms of both 5-LOX and COX-1 inhibition. Notably, the introduction of a carboxylic acid group at position 30 was important for dual 5-LOX/COX inhibitory activity; furthermore, its combination with a carbonyl group at C-11 considerably increased 5-LOX inhibition. Also, the presence of an α-hydroxy group at C-2 or a carboxylic acid group at C-23 markedly suppressed the 5-LOX activity. The present findings reveal that the types and configurations of polar moieties at positions C-2, -3, -11, -24, and -30 are important structural aspects of pentacyclic triterpenes for their potential as anti-inflammatory lead compounds.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae., One goal of potato breeding is to reduce the accumulation of toxic solanidane glycoalkaloids. Here the authors show that potato DPS, a 2-oxoglutarate dependent dioxygenase, catalyzes ring rearrangement of a biosynthetic precursor to differentiate solanidanes from spirosolanes that are found in other solanaceous plants.