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• W4367692392 abstract "Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites. Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites. The most abundant sterols in plants are the C24-alkyl phytosterol group such as campesterol (1) and β-sitosterol (2, Fig. 1). However, a significant number of important secondary metabolites such as steroidal saponins (1Challinor V.L. Smith D.M. De Voss J.J. Steroidal saponins isolated from an Australian Yam Dioscorea sp.Aust. J. Chem. 2011; 64: 545-549Google Scholar) and glycoalkaloids (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar) lack this C24-alkyl moiety, and this strongly implicates cholesterol (3) as their likely precursor. Biosynthesis of the C24-alkyl phytosterols begins with the 30-carbon precursor 2,3-oxidosqualene, which is cyclized by cycloartenol synthase (CAS) to yield cycloartenol (4, Fig. 1). Immediately following this, a C24-alkyl group is installed by a sterol methyl transferase (SMT) to generate 5. This means that cholesterol (3) biosynthesis must diverge before this occurs. Cholesterol (3) is usually present only in low amounts in most plants. However, some Dioscorea are known to contain an abundance of bioactive steroidal saponins, which comprise up to 2% of the plant’s dry weight in some species (1Challinor V.L. Smith D.M. De Voss J.J. Steroidal saponins isolated from an Australian Yam Dioscorea sp.Aust. J. Chem. 2011; 64: 545-549Google Scholar). Whilst the cholesterol (3) biosynthetic pathway in animals has been defined for decades (3Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Google Scholar), plant cholesterogenesis is still somewhat enigmatic. In animals, cholesterol (3) biosynthesis is initiated by the lanosterol synthase (LSS) catalyzed cyclization of 2,3-oxidosqualene to generate lanosterol (6) (4Thimmappa R. Wang S. Zheng M. Misra R.C. Huang A.C. Saalbach G. et al.Biosynthesis of saponin defensive compounds in sea cucumbers.Nat. Chem. Biol. 2022; 18: 774-781Google Scholar). Some plants, such as Arabidopsis thaliana and members of the Solanum genus, express LSS alongside CAS, which could permit cholesterogenesis via lanosterol (6) (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar). However, gene-knockout studies in these plants have revealed that LSS does not play a significant role in cholesterogenesis, and instead, an alternate pathway must yield cholesterol (3), presumably from cycloartenol (4) (5Cárdenas P.D. Sonawane P.D. Pollier J. Vanden Bossche R. Dewangan V. Weithorn E. et al.GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.Nat. Commun. 2016; 7: 1-16Google Scholar). One such pathway has been tentatively defined in Solanum lycopersicum. Members of the Solanum genus such as S. tuberosum (potato) and S. lycopersicum (tomato) generate steroidal glycoalkaloids, which, like steroidal saponins, are derived from cholesterol (3) (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar). In a gene-knockout study of S. tuberosum, it was found that this species may bifurcate its phytosterol metabolic pathway at cycloartenol (4), where the C24 of this precursor is either methylenated by SMT1 to initiate phytosterol biosynthesis or reduced by a unique and regioselective sterol side-chain reductase 2 (SSR2) to permit cholesterogenesis (Fig. 1) (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar). A later study putatively defined eight additional transformations that follow reduction by SSR2 to yield cholesterol (3) (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). From a combination of gene-knockout and biochemical studies, four of these subsequent reactions are reportedly performed by enzymes, which also participate in phytosterol metabolism concurrently, postulating that the enzymes exhibit substrate promiscuity for intermediates in both pathways (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). These enzymes are present as a single copy in the genome of S. lycopersicum, and silencing them decreases the level of all sterols in the plant. For example, a single CYP51 enzyme within this plant appears to be responsible for the key demethylation of obtusifoliol (7) in phytosterol biosynthesis as well a potential intermediate in cholesterol biosynthesis. In contrast, the remaining four transformations are believed to be performed by unique enzymes that have presumably evolved from phytosterol biosynthetic enzymes to be specific for cholesterogenesis. Silencing these genes prevented cholesterol (3) formation, but phytosterols were still generated in similar quantities (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). As the phytosterol and cholesterol (3) pathways share a common precursor in Solanum, some mechanism must exist to control the metabolic flux through the cholesterol (3) biosynthetic pathway relative to phytosterol biosynthesis. One control mechanism previously postulated is through modulation of SMT1 expression levels, where a decrease in SMT1 expression favors cholesterogenesis (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). However, in S. tuberosum, SMT1 mRNA expression levels did not change appreciably; and instead increasing SSR2 expression favored cholesterogenesis by regioselective reduction of the Δ24,25 double bond of cycloartenol (4) (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar). The Australian native yam D. transversa (“Long yam” or “Kowar”), traditionally used mainly as a foodstuff (7Maiden J.H. The Useful Native Plants of Australia, (Including Tasmania). Turner and Henderson, Sydney1889: 23Google Scholar), provides an ideal system in which to study plant-derived cholesterol (3) as it possesses an unusually simple steroidal saponin profile. The tubers contain only two saponins, the furostanols 1β-hydroxyprotoneogracillin (8) and protoneogracillin (9), at high concentration (Fig. 1) (1Challinor V.L. Smith D.M. De Voss J.J. Steroidal saponins isolated from an Australian Yam Dioscorea sp.Aust. J. Chem. 2011; 64: 545-549Google Scholar). As the first step in exploring steroidal saponin biosynthesis, we set out in this work to define the cholesterogenesis pathway in D. transversa via a combination of transcriptomics alongside in vitro and in vivo biochemical characterization of two key enzymes in the pathway, a CYP51 and an SSR. A transcriptome of D. transversa was constructed to identify potential enzymes in the cholesterol (3) biosynthetic pathway. Many, but not all, secondary metabolites are stored in the same location in which they are originally synthesized (8Seigler D.S. Plant Secondary Metabolism.1st edn. Springer Science, New York1998: 427-455Google Scholar). Previous work with steroidal saponins from D. zingiberensis suggested that these secondary metabolites are both synthesized and stored in the tuber/rhizome of this plant rather than in the leaves (9Hua W. Kong W. Cao X.Y. Chen C. Liu Q. Li X. et al.Transcriptome analysis of Dioscorea zingiberensis identifies genes involved in diosgenin biosynthesis.Genes and Genomics. 2017; 39: 509-520Google Scholar). Hence, it was anticipated that D. transversa would behave in a similar fashion. As the steroidal saponins 1β-hydroxyprotoneogracillin (8) and protoneogracillin (9) were isolated from the rhizome of the plant (1Challinor V.L. Smith D.M. De Voss J.J. Steroidal saponins isolated from an Australian Yam Dioscorea sp.Aust. J. Chem. 2011; 64: 545-549Google Scholar), total RNA was extracted from the rhizome for the construction of the transcriptome and from the leaves for use as a transcriptome control. In addition, extraction of the steroidal saponins from the rhizome was performed to ensure that the tissue used to provide the total RNA contained steroidal saponins at the time of extraction. A portion of the same sample used to obtain the total RNA was extracted with 80% aqueous methanol and analyzed by HPLC and mass spectrometry (MS) confirming that this tissue was indeed producing both saponins (1Challinor V.L. Smith D.M. De Voss J.J. Steroidal saponins isolated from an Australian Yam Dioscorea sp.Aust. J. Chem. 2011; 64: 545-549Google Scholar). The total RNA from both the rhizome and the leaves was then used to generate a complementary DNA (cDNA) library that was sequenced on an Illumina HighSeq platform (Australian Genome Research Facility [AGRF]). The two libraries (rhizome and leaves) were sequenced on a single lane to produce 113,390,389 paired-end reads for the leaf sample and 116,072,936 paired-end reads for the rhizome sample. These sequenced libraries were used to construct a de novo assembly of the transcriptome using Trinity (https://github.com/trinityrnaseq/trinityrnaseq) (10Grabherr M.G. Haas B.J. Yassour M. Levin J.Z. Thompson D.A. Amit I. et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nat. Biotechnol. 2013; 29: 644-652Google Scholar). A total of 108,310 transcript isoforms (66,562 unigenes) were assembled, with 48,886 of these transcripts having ORFs. The assembled transcriptome had an N50 of 1915 bp and an average transcript length of 1147 bp, indicating many transcripts were nonfragmented and likely to be of full length. Other Dioscorea transcriptomes have reported similar transcript statistics: D. zingiberensis, 56,993 unigenes with an average length of 1142 bp (9Hua W. Kong W. Cao X.Y. Chen C. Liu Q. Li X. et al.Transcriptome analysis of Dioscorea zingiberensis identifies genes involved in diosgenin biosynthesis.Genes and Genomics. 2017; 39: 509-520Google Scholar); D. alata, 60,020 unigenes with an average length of 592 bp (11Wu Z.G. Jiang W. Mantri N. Bao X.Q. Chen S.L. Tao Z.M. Transciptome analysis reveals flavonoid biosynthesis regulation and simple sequence repeats in yam (Dioscorea alata L.) tubers.BMC Genomics. 2015; 16: 1-12Google Scholar); and D. composita, 62,341 unigenes with an average length of 1368 bp (12Wang X. Chen D. Wang Y. Xie J. De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of dioscorea composita.PLoS One. 2015; 10: 1-18Google Scholar). Upon analysis of the transcriptome, no transcript corresponding to an LSS was observed indicating that D. transversa must not generate cholesterol (3) from its usual precursor lanosterol (6). In addition, a single CAS (TR2_c1_g1) was present and expressed in both the leaf (L) and rhizome (R) tissue (38:104 L:R transcripts per million [TPMs], Table S1). This suggested that cycloartenol (4) was the initial precursor of both the phytosterol and cholesterol (3) biosynthetic pathway(s) (Fig. 1). Overlap between these biosynthetic pathways has previously been proposed following transcriptome analysis of D. zingiberensis (9Hua W. Kong W. Cao X.Y. Chen C. Liu Q. Li X. et al.Transcriptome analysis of Dioscorea zingiberensis identifies genes involved in diosgenin biosynthesis.Genes and Genomics. 2017; 39: 509-520Google Scholar) and experimentally observed in Solanaceae (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). Thus, candidates for the enzymes involved in the biosynthesis of phytosterols and cholesterol (3) from cycloartenol (4) were sought. Homologs with high identity (ID) and similarity (SIM) to 11 of the 12 enzymes proposed for the transformation of cycloartenol (4) into cholesterol (3) (10 steps, Fig. 1) were identified (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar), with the single missing homolog being a second SSR. The additional SMTs required for the generation of the C24-alkyl sterols via 5 were also observed in the transcriptome (Table S1). In some cases (SSR, 3βHSD, CPI, CYP51, cytochrome P450 reductase [CPR], and 7-DR), only a single transcript was found for the steps between cycloartenol (4) and the later steroids (1–3, Fig. 1), implying enzyme functionality in both the cholesterol (3) and phytosterol biosynthetic pathways. In other cases (C14-R, 8,7-SI, and C5SD1), duplicates were detected, and this suggests that these transcripts encode enzymes that may be specific for either the biosynthesis of cholesterol (3) or the C24-alkyl phytosterols. This type of gene duplication/apparent substrate promiscuity has previously been observed in the tomato S. lycopersicum (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). One significant difference between the transcriptomes reported for members of the Solanum genus and that of D. transversa is that only a single SSR homolog (SSRDt) is expressed in the latter (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar, 6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). In S. lycopersicum and S. tuberosum, cholesterogenesis is thought to be initiated by the chemospecific reduction of cycloartenol (4) to cycloartanol (10) by a unique SSR (SSR2) that is specific to the cholesterol (3) pathway (2Sawai S. Ohyama K. Yasumoto S. Seki H. Sakuma T. Yamamoto T. et al.Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.Plant Cell. 2014; 26: 3763-3774Google Scholar, 6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar). However, only one complete SSR homolog was expressed in D. transversa that shared significant homology to SSR1 from A. thaliana (80% amino acid ID and 090% SIM). A second transcript also shared homology with SSR1 of A. thaliana (74% ID and 87% SIM), but the complete gene was not obtained (326 residues out of 560), and the transcript has low expression levels, especially in the rhizome where we postulated cholesterogenesis occurs (0.5:0 L:R TPM). Little functional information could be obtained from a phylogenetic analysis of SSRDt; a phylogram showing the gene is simply clustered with the related monocotyledonous Oryza sativa (Fig. 2). This is distant from both the aforementioned Δ24,25 reductases (S. lycopersicum SSR2 and S. tuberosum SSR2) and Δ24,28 reductases (S. lycopersicum SSR1, S. tuberosum SSR1, and A. thaliana DWF1). If D. transversa generates 3 in a similar manner to that which is postulated for S. lycopersicum, it would need to be initiated by a multifunctional SSR, which can catalyze the reduction of the Δ24,25 double bond of cycloartenol (4) as well as reduction of the Δ24,28 double bond of 24-methylenecholesterol (11) and isofucosterol (12) in phytosterol formation. This dual function implies the need for a mechanism to control the metabolic flux between these pathways distinct from the regiospecific SSR2 proposed for Solanum spp. Our SSRDt transcript was preferentially expressed in the rhizome (39:359 L:R TPM), and it is possible that the significant increase in relative mRNA expression level permits rapid reduction of the Δ24,25 olefin of cycloartenol (4). Lower-level expression in the leaves would allow methylation by SMT1 to be competitive with SSR reduction and allow phytosterol production to predominate. We observed that the SMT1 candidate (TR37901_c0_g1) exhibited similar expression levels in both tissues, suggesting that the metabolic flux may be controlled by differential expression of SSRDt. If so, SSRDt must be a promiscuous and multifunctional catalyst capable of reducing both cycloartenol (4) and advanced precursors of the phytosterols 1 and 2, such as 11 and 12. To investigate the reduction activities of SSRDt, the confirmed genetic sequence was codon optimized and overexpressed in several strains of Saccharomyces cerevisiae (yeast), which featured mutations in the well-characterized biosynthetic pathway leading to ergosterol (13), the major sterol in this organism. Within this pathway, erg4 encodes a Δ24, 28 SSR, which generates ergosterol (13) from Δ24,28 substrate ergosta-5,7,22,24(28)-tetraenol (14) and accordingly erg4Δ yeast accumulates 14 (13Johnston E.J. Moses T. Rosser S.J. The wide-ranging phenotypes of ergosterol biosynthesis mutants, and implications for microbial cell factories.Yeast. 2020; 37: 27-44Google Scholar). The activity of ERG4 is therefore analogous to one of the predicted functions of SSRDt: reduction of Δ24,28 in 24-methylenecholesterol (11) and isofucosterol (12) in D. transversa. Thus, observing complementation of ERG4 by SSRDt by analyzing the sterol profile of a complemented ERG4 mutant would allow us to confirm the role of this enzyme in phytosterol metabolism. ERG6 in yeast is an SMT, which methylates C24 of zymosterol. Yeasts that are deficient in this gene accumulate variants of the ergosterol (13) pathway intermediates, which retain the Δ24,25 double bond, such as zymosterol (13Johnston E.J. Moses T. Rosser S.J. The wide-ranging phenotypes of ergosterol biosynthesis mutants, and implications for microbial cell factories.Yeast. 2020; 37: 27-44Google Scholar). Consequently, it was envisaged that overexpressing SSRDt in erg6Δ yeast would reveal whether this enzyme is multifunctional as predicted and able to reduce sterol Δ24,25 double bonds, such as that found in cycloartenol (4) in the predicted cholesterol (3) biogenesis (Fig. 1). Both the erg4Δ and erg6Δ yeast mutants were separately generated by subcloning a HISMX gene fragment to disrupt each gene. Following this, a pRS426GPD plasmid was manipulated to carry SSRDt, and the construct was subsequently cloned into each yeast mutant. The cells were lysed, and after organic extraction, metabolites were analyzed by GC–MS. The major sterols (Fig. 3) in each case were identified by comparison of the mass spectra with known metabolites of the WT, erg4Δ, and erg6Δ yeast or to authentic standards where possible. As expected, the erg4Δ yeast did not generate any ergosterol (13), and instead, the major sterol accumulated by the yeast was identified by GC–MS analysis as 14 (Fig. 3A). When SSRDt was expressed in this mutant, ergosterol (13) was present as the major sterol in the extract, indicating that the SSR complemented the missing ERG4 activity. From this, it is clear that SSRDt is capable of reducing the Δ24,28 alkene required to generate campesterol (1) and β-sitosterol (2) in phytosterol biosynthesis (Fig. 1). Disruption of erg6 resulted in the generation of a complex mix of sterols (Fig. 3B). Through careful analysis of the fragmentation pattern and comparison with known metabolites of erg6Δ yeast (13Johnston E.J. Moses T. Rosser S.J. The wide-ranging phenotypes of ergosterol biosynthesis mutants, and implications for microbial cell factories.Yeast. 2020; 37: 27-44Google Scholar, 14Kaneshiro E.S. Johnston L.Q. Nkinin S.W. Romero B.I. Giner J.-L. Sterols of Saccharomyces cerevisiae erg6 knockout mutant expressing the Pneumocystis carinii S-adenosylmethionine:sterol C-24 methyltransferase.J. Eukaryot. Microbiol. 2015; 62: 298-306Google Scholar), we were able to identify two major components of the profile (15, 16), which possessed a Δ24,25 unsaturation. Upon expression of SSRDt in this mutant, a complete change in the steroid profile occurred (Fig. 3B). None of the steroids previously identified in the erg6Δ yeast were detected. Analysis of the fragmentation pattern of two peaks led to the tentative identification of two new metabolites (17, 18). The fragmentation patterns of these compounds correlate with loss of the Δ24,25 unsaturation observed for the erg6Δ yeast metabolites earlier, indicating that SSRDt is reducing the Δ24,25 double bond on the sterol side chain. From this, it can be concluded that SSRDt is a promiscuous and multifunctional enzyme capable of reducing both sterol Δ24,28 and Δ24,25 double bonds (Fig. 4). Having shown that variation in SSR level of expression was a potential mechanism by which D. transversa controlled cholesterol (3) biosynthesis relative to that of C24-alkyl phytosterols, overlap between these two pathways was investigated. One of the key steps in the biosynthesis of all steroids is the 14α-demethylation reaction catalyzed by a cytochrome P450 sterol, 14α-demethylase (CYP51). This ancient enzyme family is found in all kingdoms of life, and its function is almost exclusively this key demethylation step in sterol biosynthesis with only a single known exception (15Geisler K. Hughes R.K. Sainsbury F. Lomonossoff G.P. Rejzek M. Fairhurst S. et al.Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants.Proc. Natl. Acad. Sci. 2013; 110: E3360-E3367Google Scholar). Usually, this activity is limited to lanosterol (6) or its analogs as precursors in cholesterol (3) biosynthesis and obtusifoliol (7) and its analogs as precursors for phytosterol production. However, in gene-knockout studies, Δcyp51 mutants of S. lycopersicum (6Sonawane P.D. Pollier J. Panda S. Szymanski J. Massalha H. Yona M. et al.Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.Nat. Plants. 2016; 3: 1-13Google Scholar) accumulated obtusifoliol (7), the classical plant CYP51 substrate, as well as 4-desmethyl-24,25-dihydrolanosterol (19), a possible intermediate for cholesterol (3) production (Fig. 1). As such, it is believed that the CYP51 expressed by these plants may be multifunctional, but this had not been confirmed by biochemical characterization previously. The transcriptome of D. transversa contained a single cyp51 candidate transcript (TR348_c1_g1) that was highly expressed in both the leaf (L) and rhizome (R) of the plant (224:236 L:R TPM, Table S1). When translated, this sequence shares 79% ID (90% SIM) and 80% ID (93% SIM) to the A. thaliana and S. lycopersicum orthologous proteins, respectively (Fig. S5). As such, it was predicted that a single CYP51 (CYP51Dt) expressed by D. transversa may exhibit substrate promiscuity and function in both the cholesterol (3) and phytosterol pathways. In order for P450-mediated catalysis to occur, an NADPH cytochrome P450 reductase (CPR) is required. Generally, for eukaryotic systems, two electrons are funneled from NADPH to the P450 via a CPR, permitting activation of molecular oxygen and allowing the oxidation to proceed (16Hannemann F. Bichet A. Ewen K.M. Bernhardt R. Cytochrome P450 systems--biological variations of electron transport chains.Biochim. Biophys. Acta - Gen. Subj. 2007; 1770: 330-344Google Scholar). Having the conspecific CPR usually results in better catalytic activity for a given P450 (17Gillam E.M. Engineering cytochrome P450 enzymes.Chem. Res. Toxicol. 2008; 21: 220-231Google Scholar). Thus, analysis of the transcriptome data was undertaken and revealed a single full-length CPR transcript that could be the necessary redox partner for D. transversa P450s. This putative reductase (CPRDt) has high homology to both CPR1 (67% ID and 80% SIM) and CPR2 (70% ID and 82% SIM) from A. thaliana and contained both the expected FAD- and FMN-binding domains (Fig. S6). In addition, the transcript levels of the putative CPR were high (100:46 L:R TPM, Table S1) in both the rhizome and the leaf, unsurprising given that this protein (CPRDt) appears to be the sole CPR responsible for the delivery of electrons to D. transversa P450s. The genetic sequences of candidate sterol 14α-demethylase (CYP51Dt) and its redox partner (CPRDt) were verified by Sanger sequencing. The coding sequences were then codon optimized for expression in Escherichia coli. Native eukaryotic P450s often have a hydrophobic N-terminal region, and heterologous protein expression in E. coli is frequently enhanced by replacing this with a shorter and less hydrophobic sequence while retaining the proline-rich hinge region (Pro Pro Ile in CYP51Dt) that is important for directing protein folding (17Gillam E.M. Engineering cytochrome P450 enzymes.Chem. Res. Toxicol. 2008; 21: 220-231Google Scholar). For cyp51Dt, the first 111 base pairs were replaced with a gene sequence encoding MAKKTSSKGKL (18Ohnishi T. Watanabe B. Sakata K. Mizutani M. CYP724B2 and CYP90B3 function in the early c-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.Biosci. Biotechnol. Biochem. 2006; 70: 2071-2080Google Scholar). A C-terminal hexa-histidine tag was introduced to the cyp51Dt sequence to facilitate protein purification. The truncated cyp51Dt sequence and the full-length cprDt sequence were cloned into pCW, a vector that is commonly utilized for P450 expression (19Shimada T. Wunsch R.M. Hanna I.H. Sutter T.R. Guengerich F.P. Gillam E.M.J. Recombinant human cytochrome P450 1B1 expression in Escherichia coli.Arch. Biochem. Biophys. 1998; 357: 111-120Google Scholar). CYP51Dt was expressed in E. coli from pCW at 25 °C accompanied by the chaperone proteins GroES and GroEL (20Notley L.M. De Wolf C.J.F. Wunsch R.M. Lancaster R.G. Gillam E.M.J. Bioactivation of tamoxifen by recombinant human cytochrome p450 enzymes.Chem. Res. Toxicol. 2002; 15: 614-622Google Scholar). Following successful expression of CYP51Dt, purification of the protein was carried out via immobilized nickel affinity chromatography and" @default.
• W4367692392 created "2023-05-03" @default.
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• W4367692392 creator A5026387564 @default.
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• W4367692392 date "2023-06-01" @default.
• W4367692392 modified "2023-09-28" @default.
• W4367692392 title "Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa" @default.
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