TRANSFERASE ENZYMES
20230106588 · 2023-04-06
Inventors
- Anne OSBOURN (Norwich, Norfolk, GB)
- James REED (Norwich, Norfolk, GB)
- Anastasia ORME (Norwich, Norfolk, GB)
- Thomas LOUVEAU (Norwich, Norfolk, GB)
Cpc classification
C12P5/007
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
C12Y106/02004
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C12N15/8243
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention relates generally to genes and polypeptides which have utility in glycosylating quillaic acid in host cells, including enzymes capable of successive glycosylation at the C-3 position of quillaic acid. The invention further relates to systems, methods and products employing the same.
Claims
1. A method of converting a host from a phenotype whereby the host is unable to perform the biosynthesis of the 3-O branched trisaccharide quillaic acid (“QA”) derivative (“QA-3-O-TriS”), which QA-3-O-TriS is 3β-{[β-
2. A method as claimed in claim 1, wherein the heterologous nucleic acid encodes two or three of the following types of polypeptide (i), (ii) or (iii): (i) a QA 3-O glucuronosyl transferase (“QA-GlcAT”) capable of transferring D-glucuronic acid (“GlcpA”) at the 3-O position of quillaic acid to form 3β-{[β-
3. A method as claimed in claim 2, wherein the heterologous nucleic acid encodes all three types of polypeptide.
4. A method as claimed in claim 2, wherein the nucleotide sequences are from Q. saponaria.
5. A method as claimed in claim 3, wherein (i) the polypeptides are selected from the QA-GlcAT, QA-GalT, and QA-RhaT/XylT enzymes in Table 5 or 6, or from substantially homologous variants or fragments of any of said polypeptides in Tables 5 or 6, and/or (ii) the nucleotide sequences are selected from the QA-GlcAT, QA-GalT, and QA-RhaT/XylT nucleotide sequence in Table 5 or 6, or from substantially homologous variants or fragments of any of said nucleotides sequences in Tables 5 or 6.
6. A method as claimed in claim 4, wherein the respective polypeptides are selected from the lists consisting of: (i) the QA-GlcAT shown in SEQ ID: No 2 or 26; (ii) the QA-GalT shown in SEQ ID: No 4; (iii) a QA-RhaT/XylT shown in SEQ ID: No 6, 28, 30, or 32; or substantially homologous variants or fragments of any of said respective polypeptides.
7. A method as claimed in claim 1, wherein the heterologous nucleic acid further comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have QA biosynthesis activity (“QA polypeptide”), wherein the nucleic acid encodes all of the following QA polypeptides: (i) a β-amyrin synthase (bAS) for cyclisation of 2,3-oxidosqualene (OS) to a triterpene; (ii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid (“C-28 oxidase”); (iii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol (“C-16α oxidase”); and (iv) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde (“C-23 oxidase”), wherein each of the polypeptides is optionally from Q. saponaria.
8. A method as claimed in claim 7, wherein the C-28 oxidase, C-16α oxidase, and C-23 oxidase are all CYP450 enzymes.
9. A method as claimed in claim 8, wherein (i) the C-28 oxidase is a CYP716; (ii) the C-16α oxidase is a CYP716 or CYP87; (iii) the C-23 oxidase is a CYP714, CYP72, or CYP94.
10. A method as claimed in claim 7, wherein the QA polypeptides are selected from the list consisting of: the β-amyrin synthase (bAS) shown in SEQ ID: No 12; the C-28 oxidase shown in SEQ ID: No 14; the C-16α oxidase shown in SEQ ID: No 16; the C-23 oxidase shown in the SEQ ID: No 18; or substantially homologous variants or fragments of any of said polypeptides.
11. A method as claimed in claim 1, wherein the nucleic acid further comprises a plurality of nucleotide sequences encoding one or more of the following polypeptides: (i) an HMG-CoA reductase (HMGR); (ii) a squalene synthase (SQS) wherein the HMGR or SQS are optionally selected from the respective polypeptides in Table 7 or substantially homologous variants or fragments of any of said polypeptides, or are encoded by the respective polynucleotides in Table 7, or substantially homologous variants or fragments of any of said polynucleotides.
12. A method as claimed in claim 1, wherein the nucleotide sequences are present on two or more different nucleic acid molecules.
13. A method as claimed in claim 12, wherein the host is a plant and the nucleic acid molecules are introduced by co-infiltration with a plurality of Agrobacterium tumefaciens strains each carrying one or more of the nucleic acid molecules.
14. A method as claimed in claim 13, wherein the nucleic acid molecules are transient expression vectors, wherein each of the transient expression vectors comprises an expression cassette comprising: (i) a promoter, operably linked to (ii) an enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated; (iii) a nucleotide sequence encoding one of the polypeptides which in combination have said QA-3-O-TriS biosynthesis activity; (iv) a terminator sequence; and optionally (v) a 3′ UTR located upstream of said terminator sequence.
15. (canceled)
16. A method as claimed in claim 1, wherein the host is a plant which is converted such as to have a modified QA-3-O-TriS content.
17. A host cell containing or transformed with a heterologous nucleic acid which comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have QA-3-O-TriS biosynthesis activity, wherein expression of said nucleic acid imparts on the transformed host the ability to carry out QA-3-O-TriS biosynthesis.
18. A host cell as claimed in claim 17 containing or transformed with a heterologous nucleic acid which further comprises a plurality of nucleotide sequences each of which encodes a polypeptide which in combination have QA biosynthesis activity, wherein the nucleic acid encodes all of the following QA polypeptides: (i) a β-amyrin synthase (bAS) for cyclisation of 2,3-oxidosqualene (OS) to a triterpene; (ii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-28 position to a carboxylic acid (“C-28 oxidase”); (iii) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-16α position to an alcohol (“C-16α oxidase”); and (iv) an enzyme capable of oxidising β-amyrin or an oxidised derivative thereof at the C-23 position to an aldehyde (“C-23 oxidase”), wherein each of the polypeptides is optionally from Q. saponaria.
19. A process for producing the host cell of claim 17 by co-infiltrating a plurality of recombinant constructs comprising said nucleic acid into the cell for transient expression thereof.
20. A process for producing the host cell of claim 17 by transforming a cell with heterologous nucleic acid by introducing said nucleic acid into the cell via a vector and causing or allowing recombination between the vector and the cell genome to introduce the nucleic acid into the genome.
21. A method for producing a transgenic plant, which method comprises the steps of: (a) performing a process as claimed in claim 20 wherein the host cell is a plant cell, and (b) regenerating a plant from the transformed plant cell.
22. A transgenic plant which is obtained by the method of claim 21, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, wherein expression of said heterologous nucleic acid imparts an increased ability to carry out QA-3-O-TriS synthesis compared to a wild-type plant otherwise corresponding to said transgenic plant.
23. A transgenic plant comprising nucleic acid encoding the following types of polypeptide (i), (ii) or (iii): (i) a QA 3-O glucuronosyl transferase (“QA-GlcAT”) capable of transferring D-glucuronic acid (“GlcpA”) at the 3-O position of quillaic acid to form 3β-{[β-
24-34. (canceled)
35. A recombinant vector which comprises a nucleic acid, wherein said nucleic acid comprises a OA-3-O-TriS-biosynthetic nucleotide sequence which is selected from the group consisting of: (i) a nucleotide sequence which encodes all or part of polypeptide SEO ID NO: 2, 4, 6, 26, 28, 30 or 32; (ii) a nucleotide sequence which encodes a variant polypeptide which is a homologous variant of any of these SEO ID Nos which shares at least about 60% identity with said SEO ID NOs, which variant polypeptide in each case has the respective activity of said SEO ID NO shown in Table 5; and/or (iii) is selected from SEO ID NO: 1, 3, 5, 25, 27, 29, or 31 or the genomic equivalent thereof.
36. A vector as claimed in claim 35 wherein the nucleic acid is operably linked to a promoter for transcription in a host cell, wherein the promoter is optionally an inducible promoter.
37-38. (canceled)
39. A method which comprises the step of introducing one or more different vectors of claim 35 into a host cell, and optionally causing or allowing recombination between the vector and the host cell genome such as to transform the host cell.
40. (canceled)
41. A host cell containing or transformed with a vector according to claim 35.
42. A host cell as claimed in claim 41 which is microbial, optionally a yeast cell.
43. A host cell as claimed in claim 42 which further contains or is transformed with heterologous nucleic acid which comprises one or more nucleotide sequences each of which encodes a polypeptide which is a plant cytochrome P450 reductases (CPR), which is optionally the CPR shown in SEQ ID No: 23 or is a substantially homologous variant or fragment of said polypeptide.
44-45. (canceled)
46. A method for producing a transgenic plant, which method comprises the steps of: (a) performing a method as claimed in claim 39 wherein the host cell is a plant cell, and (b) regenerating a plant from the transformed plant cell.
47. A transgenic plant which is obtained by the method of claim 46, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, which in each case includes a heterologous nucleic acid of having a nucleotide sequence which is selected from the group consisting of: (i) a nucleotide sequence which encodes all or part of polypeptide SEO ID NO: 2, 4, 6, 26, 28, 30 or 32, (ii) a nucleotide sequence which encodes a variant polypeptide which is a homologous variant of any of these SEO ID Nos which shares at least about 60% identity with said SEO ID NOs, which variant polypeptide in each case has the respective activity of said SEO ID NO shown in Table 5, and/or (iii) a nucleotide sequence which is selected from SEO ID NO: 1, 3, 5, 25, 27, 29, or 31 or the genomic equivalent thereof.
48-55. (canceled)
56. A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or downstream product thereof, in a host, which is optionally a plant, which method comprises performing a method as claimed in claim 1, and optionally isolating the product from the host.
57. A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or downstream product thereof in a heterologous host, which method comprises culturing a host cell as claimed in claim 17, and purifying the product therefrom.
58. A method of producing a product which is a glycosylated QA, which is optionally QA-3-O-TriS, or a derivative thereof in a heterologous plant, which method comprises growing a plant as claimed in claim 23 and then harvesting the a glycosylated QA and purifying the product therefrom.
59-62. (canceled)
Description
FIGURES
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EXAMPLES
Example 1—Identification and Cloning of Glycosyltransferases from Q. saponaria
[0310] In order to augment the publicly available transcriptome, we generated genome sequence data (PacBio sequencing performed by the Earlham Institute, Norwich, Norfolk). The genome sequence was annotated using publicly available data (including the ‘1 KP’ leaf transcriptomic data from Q. saponaria [4]) and proteins from related plant species in Phytozome.
[0311] From this data, we shortlisted a series of sequences which were annotated as putative Family 1 UDP-dependent glycosyltransferases (UGTs)—an important class of enzymes which are known to participate in biosynthesis of many plant natural products, including triterpenes [5, 6]. We refined the initial list (containing ˜200 sequences) down to sequences which were also represented in the 1 KP database from which the original QA biosynthetic enzymes were found. The Q. saponaria contigs consist of a 4-letter code (OQHZ) followed by seven digits. Where possible, this seven digit code is included for all of the candidate genes below. To further refine this list, we performed phylogenetic analysis using a series of characterised GTs from other plant species (Table 3). This allowed us to prioritise the enzymes which fell into the same phylogenetic groups as currently characterised triterpene UGTs from other plant species (Groups A, D and L) and UGTs with relevant sugar-donor specificity (Group B).
[0312] Finally, in recent years it has been proposed that a number of chemically-diverse plant natural products are synthesised by enzymes encoded by physically co-localised genes. These so-called ‘biosynthetic gene clusters’ (BCGs) could facilitate identification of additional candidate genes. We therefore deployed the ‘PlantiSMASH’ genome mining tool [7] to predict possible BCGs within the Q. saponaria genome. This combination of approaches resulted in a final list of 30 candidate Q. saponaria UGTs (
[0313] As described above, the genes for quillaic acid biosynthesis appear to be expressed in leaf tissue and were previously amplified by PCR from leaf cDNA. The same approach was therefore utilised for amplification of the GT candidates. A series of oligonucleotide primers were designed which incorporated 5′ attB sites upstream of the target sequence to allow for Gateway® cloning. From this, genes were successfully amplified and cloned into pDONR 207. The clones were sequenced before transfer into the plant expression vector pEAQ-HT-DEST1 [14]. Finally, the expression constructs were transformed individually into Agrobacterium tumefaciens (LBA4404) for transient expression in N. benthamiana.
[0314] Screening of the 31 candidate GTs was performed using transient expression in N. benthamiana. All infiltrations included the four A. tumefaciens strains carrying the constructs for QA biosynthesis (QsbAS and C-28/C-23/C-16α oxidases) along with a strain carrying tHMGR, a key yield-enhancing enzyme for triterpene production.
Example 2—Identification of Quillaic Acid 3-O Glucuronosyl Transferase
[0315] Following LC-MS analysis of the samples, it was discovered that, unexpectedly, one candidate, a predicted ‘cellulose synthase-like’ (CSL) enzyme (named herein QsCSL1) was active upon quillaic acid. Co-expression of this enzyme with the five A. tumefaciens strains for QA-production resulted in significant depletion of the QA peak at 19.2 minutes, accompanied by the appearance of a new peak at 13.9 minutes (
Example 3—Identification of QA-GlcpA Galactosyl Transferase
[0316] Following the identification of a putative glucuronosyl transferase, the next proposed step was the addition of the β-
[0317] A triterpene 3-O-glucuronoside-β-1,2-galactosyltransferase, GmUGT73P2 has been previously identified in soybean (Glycine max) (Shibuya et al, 2010). This enzyme catalyses the addition of
[0318] Interestingly, the phylogenetic analysis of the Q. saponaria UGT enzymes showed that one candidate, Qs_2073886_D6, is closely related to GmUGT73P2 (
[0319] Qs_2073886_D6 was coexpressed with the six genes required for production of the putative QA-GlcpA (tHMGR/QsbAS/CYP716-C-28/CYP716-C-16α/CYP714-C23/QsCSL1). HPLC-MS analysis revealed that Qs_2073886_D6 appeared to convert the putative QA-GlcpA product to a new, more polar product at 12.6 minutes (
[0320] To establish further evidence for the identity of the new product, we utilised the soybean (Glycine max) triterpene 3-O-glucuronoside-β-1,2-galactosyltransferase enzyme, GmUGT73P2. It was reasoned that this enzyme may show similar galactosyltransferase activity towards the putative QA-GlcpA product. An infiltration was thus also performed with coexpression of the six enzymes necessary for synthesis of the QA-GlcpA and GmUGT73P2. LC-MS analysis of the infiltrated leaf extracts revealed that a peak could indeed be observed in the GmSGT2-expressing samples which had a matching retention time and mass spectrum to the product seen at 12.6 minutes in the Q. saponaria galactosyltransferase-expressing samples (
[0321] We additionally performed a large scale infiltration of tHMGR/QsbAS/CYP716-C-28/CYP716-C-16α/CYP714-C23/QsCSL1/Qs-3-O-GalT in N. benthamiana as previously described [19] to purify this compound (32.1 g) to assign its structure by NMR. This confirmed it to be 3β-{[β-
Example 4—Identification of QA-GlcpA-Galp Dual Rhamnosyl/Xylosyl Transferase
[0322] We next repeated the process of screening the remaining GT candidates against the QA-GlcpA-Galp product. As before, GT candidates were screened by co-expression with the seven genes required to make QA-GlcpA-Galp (tHMGR/QsbAS/CYP716-C-28/CYP716-C-16α/CYP714-C23/QsCSL1/Qs-3-O-GalT). With this strategy we identified a UGT enzyme which resulted in depletion of the QA-GlcpA-Galp product. However, rather than a single new product, we observed the appearance of two new products with very close retention times to the former QA-GlcpA-Galp (
[0323] Q. saponaria is known to produce in excess of 100 different saponins [16]. Within these saponins, the 3-O-GlcpA-β-1,2-
[0324] Previously, chemical profiling of Q. saponaria trees has demonstrated the existence of distinct ‘chemotypes’ which vary in their ability to produce saponins containing either Rhap or Xylp attached to GlcpA-3-O (see WO 2018/057031). One explanation for these observations is the presence of two distinct alleles of the terminal sugar transferase with differing sugar specificity as previously demonstrated for soybean [18]. Notwithstanding this, the present disclosure provides an enzyme which is capable of catalysing addition of two distinct sugars at the same position.
Example 5—Purification and NMR Validation of the Trisaccharide Produced in N. benthamiana
[0325] To verify the structures of the compounds 1 and 2 (
Example 6—Use of QA-3-O-TriS Genes, Optionally in Combination with QA Genes, for Production of Stably Transformed Plants
[0326] Triterpenes have previously been produced using engineered transgenic plant lines (e.g. Arabidopsis, Wheat). A series of Golden Gate [23] vectors which allows for construction of multigene vectors and allows integration of an entire pathway into a single locus have been reported. These can be applied analogously to the present invention, in the light of the disclosure herein.
[0327] The QA-3-O-TriS genes described herein, optionally in conjunction with QA genes of prior-filed unpublished PCT/EP2018/086430 (subsequently published as WO 2019/122259), may thus be used to produce stable transgenic plants in the light of the present disclosure in combination with known transgenic technologies.
Example 7—Identification of Quillaic Acid 3-O Glucuronosyl Transferase CSLG2 (QsCSLG2)
[0328] As described in the preceding Examples, the ‘1 KP’ Q. saponaria leaf transcriptome was used to identify genes involved in the biosynthesis of quillaic acid (QsbAS, QsCYP716-C-16α, QsCYP714-C-23 and QsCYP716-C-28) and the trisaccharide at the C-3 position of QS-21 (QsCSL1, Qs-3-O-GalT and Qs-3-O-RhaT/XylT).
[0329] Genes involved in triterpene glycoside biosynthesis are typically co-expressed [25]. In order to investigate the expression pattern of the characterised QS-21 biosynthetic genes across multiple tissues, RNA-seq data were generated for six Q. saponaria tissues (primordia, expanding leaf, mature leaf, old leaf, green stem and root). The gene expression profiles for QsbAS, QsCYP716-C-16α, QsCYP714-C-23, QsCYP716-C-28 and Qs-3-O-GalT showed a pattern of low expression in old leaf and high expression in primordia, with some variability in expression levels in root, expanding leaf, green stem and mature leaf (
[0330] As the expression profile for QsCSL1 did not follow the general pattern seen for the other characterised QS-21 genes, it was investigated whether there might be genes related to QsCSL1 that did have the QS-21 gene expression pattern and which therefore might be involved in QS-21 biosynthesis. QsCSL1 was used in a BLASTp search to identify cellulose synthase-like genes in the Q. saponaria annotated genome. This identified 39 additional cellulose synthase superfamily genes, of which five (named CslG2 to CslG6) were in the same subfamily as QsCSL1 (
[0331] Analysis of the expression profile of these genes show that CslG3-CslG6 are expressed most highly in old leaf or in the root (
Example 8—Identification of QA-GlcpA-Galp Xylosyltransferase and Rhamnosyltransferases
[0332] As explained in Example 4, the DNA sequence for the dual glycosyltransferase Qs-3-O-RhaT/XylT was not identified in the Quillaja saponaria genomic dataset. Instead, this gene appeared to be a chimera between two adjacent genes, Qs_0283860 (a pseudogene) and Qs_0283870 (
[0333] It is theoretically possible that there are alleles of these genes that are not represented in the genomic Q. saponaria dataset or that this region was incorrectly resolved. As an alternative database, a de novo transcriptome assembly was generated from the Q. saponaria primordia RNA-seq reads [26]. A BLASTn search using the three genomic genes and Qs-3-O-RhaT/XylT as queries identified two full-length transcripts: DN20529_c0_g2_i6, which was identical to the sequence of Qs_0283870, corroborating the sequence of this gene; and DN20529_c0_g2_i8, which had 99% DNA sequence identity to the Qs_0283860 pseudogene and 98% DNA sequence identity to Qs_0283850 (Table 9).
[0334] To investigate the presence and function of these genes, we attempted to amplify the sequences from Q. saponaria leaf cDNA. Qs_0283850 and Qs_0283870 were successfully amplified. Primers designed to amplify the pseudogene Qs_0283860 amplified a full-length sequence with 100% sequence identity in the coding region of the gene predicted by the de novo transcriptome, DN20529_c0_g2_i8. This amplified sequence is subsequently referred to as DN20529_c0_g2_i8. These three amplified genes (Qs_0283850, Qs_0283870 and DN20529_c0 g2_i8) were cloned into the plant expression vector pEAQ-HT-DEST1 and transformed into A. tumefaciens for transient expression in Nicotiana benthamiana.
[0335] As described above, co-expression of Qs-3-O-RhaT/XylT with the seven genes able to make QA-GlcpA-Galp (tHMGR/QsbAS/CYP716-C-28/CYP716-C-16α/CYP714-C23/QsCSL1/Qs-3-O-GalT) resulted in the appearance of trisaccharides QA-GlcpA-[Galp]-Rhap (retention time=12.5 min, MW=970) and QA-GlcpA-[Galp]-Xylp (retention time=12.75 min, MW=956), which have very close retention times to the former QA-GlcpA-Gal (retention time=12.6 min, MW=824) (
[0336] Similarly, co-expression of either Qs_0283850, Qs_0283870 or DN20529_c0 g2_i8 with the genes required to make QA-GlcpA-Galp revealed that all three enzymes were able to convert QA-GlcpA-Galp, but resulted in the production of one new product each (
[0337] Co-expression of Qs_0283870 with the genes required to make QA-GlcpA-Galp also reduced the QA-GlcpA-Galp peak, however it accumulated a less polar compound with the same retention time (12.75 min) and molecular weight (MW=956) as QA-GlcpA-[Galp]-Xylp (
[0338] This suggests that Qs_0283870 is primarily a xylosyltransferase and can produce QA-GlcpA-[Galp]-Xylp without producing significant amounts of QA-GlcpA-[Galp]-Rhap.
Materials and Methods
Phylogenetic Analysis of UGT Candidates
[0339] Amino acid sequences were deduced from the predicted full-length coding sequences of the Q. saponaria UGTs. Representative amino acid sequences of characterised glycosyltransferase family 1 UGTs from other plant species (Table 3) were obtained from the NCBI database and incorporated into the phylogenetic analysis. Protein sequences were aligned using MAFFT (https://mafft.cbrc.jp/alignment/software/). The unrooted trees were constructed in MEGA7 by the Neighbor-Joining method with 1000 bootstrap replicates [20, 21].
Primers and Cloning
[0340] The genes encoding the enzymes described herein (QsCSL1, Qs-3-O-GalT, Qs-3-O-RhaT/XylT, QsCslG2, Qs_0283850, DN20529_c0_g2_i8 and Qs_0283870) were amplified by PCR from cDNA derived from leaf tissue of Q. saponaria. PCR was performed using the primers detailed in Tables 2 and 10, using iProof polymerase with thermal cycling according to the manufacturer's recommendations. The resultant PCR products were purified (Qiagen PCR cleanup kit) and each cloned into the pDONR207 vector using BP clonase according to the manufacturer's instructions. The BP reaction was transformed into E. coli and the resulting transformants were cultured and the plasmids isolated by miniprep (Qiagen). The isolated plasmids were sequenced (Eurofins) to verify the presence of the correct genes. Next each of the three genes were further subcloned into the pEAQ-HT-DEST1 expression vector using LR clonase. The resulting vectors were used to transform A. tumefaciens LBA4404 by flash freezing in liquid N.sub.2.
Agroinfiltration of N. benthamiana Leaves
[0341] Agroinfiltration was performed using a needleless syringe as previously described [19]. All genes were expressed from pEAQ-HT-DEST1 binary expression vectors [14] in A. tumefaciens LBA4404 as described above. Cultivation of bacteria and plants is as described in [19].
Preparation of N. benthamiana Leaf Extracts for LC-MS Analysis
[0342] Leaves were harvested 5 days after agroinfiltration and freeze-dried. Freeze-dried leaf material (10 mg per sample) was ground at 1000 rpm for 1 min (Geno/Grinder 2010, Spex SamplePrep). Extractions were carried out in 550 μL 80% methanol with 20 pg/mL of digitoxin (internal standard; Sigma) for 20 min at 40° C., with shaking at 1400 rpm (Thermomixer Comfort, Eppendorf). The sample was partitioned twice with 400PL hexane. The aqueous phase was dried under vacuum at 40° C. (EZ-2 Series Evaporator, Genevac). Dried material was resuspended in 75 μL of 100% methanol and filtered at 12, 500 g for 30 sec (0.2 μm, Spin-X, Costar). Filtered samples were transferred to glass vials and analysed as detailed below.
LC-MS Analysis of N. benthamiana Leaf Extracts
[0343] Analysis was carried out using a Prominence HPLC system with single quadrupole mass spectrometer LCMS-2020 (Shimadzu) and Corona Veo RS Charged Aerosol Detector (CAD) (Dionex). Detection: MS (dual ESI/APCI ionization, DL temp 250° C., neb gas flow 15 L.Math.min−1, heat block temp 400° C., spray voltage Pos 4.5 kV, Neg −3.5 kV) CAD: data collection rate 10 Hz, filter constant 3.6 s, 925 evaporator temp. 35° C., ion trap voltage 20.5 V. Method: Solvent A: [H.sub.2O+0.1% formic acid] Solvent B: [acetonitrile (CH.sub.3CN)+0.1% formic acid. Injection volume: 10 μL. Gradient: 15% [B] from 0 to 1.5 min, 15% to 60% [B] from 1.5 to 26 min, 60% to 100% [B] from 26 to 26.5 min, 100% [B] from 26.5 to 28.5 min, 100% to 15% [B] from 28.5 to 29 min, 35% [B] from 29 to 30 min. Method was performed using a flow rate of 0.3 mL.Math.min-1 and a Kinetex column 2.6 μm XB-C18 100 Å, 50×2.1 mm (Phenomenex). Analysis was performed using LabSolutions software (Shimadzu).
Large Scale Vacuum Infiltration of N. benthamiana
[0344] A total of 198 plants were infiltrated by vacuum as previously described [19, 22] with the A. tumefaciens strains carrying the pEAQ-HT-DEST1 constructs for tHMGR, QsbAS, CYP716-C-28, CYP716-C-16α, CYP714-C-23, QsCSL1, Qs-3-O-GalT and Qs-3-O-RhaT/XylT. Plants were harvested after 4 days and freeze dried, resulting in a total of 175.25 g dry leaf material.
Purification of Compounds from Large Scale Infiltrations of N. benthamiana
General Procedures
[0345] Organic solvents used for extraction and flash chromatography were reagent grade and used directly without further distillation. HPLC mobile phases were prepared using HPLC grade solvents. LC-MS spectral data were recorded on SHIMADZU-2020, single quad, using Kinetex-XB-C.sub.18 (50×10 mm i.d.; 2.6 μm; USA), (JIC, UK). 1D and 2D NMR spectra were recorded on Bruker Avance 600 MHz spectrometer equipped with a BBFO Plus Smart probe and a triple resonance TCI cryoprobe, respectively (JIC, UK). The chemical shifts are relative to the residual signal solvent (MeOH-d.sub.4: δ.sub.H 3.31; δ.sub.C 49.15). Preparative HPLC experiments were performed on Ultimate 3000 using Luna C18 column (250×10 mm i.d.; 5 μm; USA). Flash column chromatography (FCC) was performed using an Isolera One (Biotage), using SNAP Ultra 50 g columns. Analytical TLC experiments were performed on silica gel precoated aluminium plates (F254, 20×20 cm, Merck KGaA, Germany). TLC plates were visualized under UV light (254 nm) followed by staining with p-anisaldehyde (2% v/v p-anisaldehyde, 2% v/v, Conc. H.sub.2SO.sub.4).
Extraction and Isolation
[0346] Dried N. benthamiana powder was mixed with quartz sand (0.3-0.9 mm). This mixture was layered on top of a bottom layer of quartz sand (0.3-0.9 mm) 3 cm in depth within a 120 mL extraction cell. Extraction was performed using a Speed Extractor E-914 (Büchi) with three cycles at 100° C. and a pressure of 130 bar. Cycle one had zero hold time, and cycles two and three had 5 min hold times. The run finished with a 1 min solvent flush and 12 min N.sub.2 flush. The dried leaves were initially extracted by hexane for defatting, followed by subsequent exhaustive extraction using methanol. Organic layers were combined together and evaporated under reduced pressure. The crude methanolic extract was dissolved in the least amount of methanol and diluted with equivalent volume of water, then it was successfully partitioned using separation funnel against hexane, dichloromethane, ethyl acetate and n-butanol. The butanol layer was recollected and dried over anhydrous NaSO.sub.4, evaporated under reduced pressure and subjected to a normal phase silica-gel flash chromatography (35-70 μm), using a long gradient of DCM/MeOH [100/0-0/100] along 30 min. The column was further washed with ethyl acetate/acetone/water/formic acid (5/3/0.5/0.5). All fractions were monitored by TLC using different eluent systems and combined together according to their polarities. Based on the LC-MS profiling and .sup.1H NMR as well, promising fractions were introduced for further reparative chromatographic purifications by reversed phase (preparative/semipreparative C.sub.18-HPLC) using the eluent system water/acetonitrile containing 0.1% formic acid, to finally afford pure saponins. The detailed isolation scheme of the isolated compounds for the purification of compounds 1 and 2 (see Examples 4 and 5) and their quantities is given (
NMR Analysis
[0347] NMR spectra were recorded in Fourier transform mode at a nominal frequency of 600 MHz for .sup.1H NMR and 150 MHz for .sup.13C NMR in deuterated methanol unless otherwise indicated. Chemical investigation of the n-butanol fraction of N. benthamiana leaves (Examples 4 and 5) afforded the isolation of two previously reported triterpene saponins, namely 3β-{[α-
Alignment of RNA Sequences and Heatmaps
[0348] RNA-seq data (Illumina-sequenced reads) were aligned to the Q. saponaria genome using the STAR package (version 2.5) [27] and quantified using the featureCounts program (http://subread.sourceforge.net/, version 1.6.0). The heatmaps were drawn in R using heatmap.2, https://CRAN.R-proiect.org/packaae=gplots).
REFERENCES
[0349] 1. Del Giudice, G., R. Rappuoli, and A. M. Didierlaurent, Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Seminars in Immunology, 2018. 39: p. 14-21. [0350] 2. Marciani, D. J., Elucidating the Mechanisms of Action of Saponin-Derived Adjuvants. Trends in Pharmacological Sciences, 2018. 39(6): p. 573-585. [0351] 3. FDA. Available from: https://www.fda.qov/vaccines-blood-biologics/vaccines/shinqrix. [0352] 4. Johnson, M. T. J., et al., Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes. PLOS ONE, 2012. 7(11): p. e50226. [0353] 5. Bowles, D., et al., Glycosyltransferases of lipophilic small molecules. Annu Rev Plant Biol, 2006. 57: p. 567-97. [0354] 6. Louveau, T. and A. Osbourn, The Sweet Side of Plant-Specialized Metabolism. Cold Spring Harb Perspect Biol (In Press), 2019. [0355] 7. Kautsar, S. A., et al., plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters. Nucleic Acids Res, 2017. [0356] 8. Luang, S., et al., Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates. J Biol Chem, 2013. 288(14): p. 10111-23. [0357] 9. Matsuba, Y., et al., A novel glucosylation reaction on anthocyanins catalyzed by acyl—glucose-dependent glucosyltransferase in the petals of carnation and delphinium. Plant Cell, 2010. 22(10): p. 3374-89. [0358] 10. Miyahara, T., et al., Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus. J Plant Physiol, 2012. 169(13): p. 1321-6. [0359] 11. Miyahara, T., et al., Isolation of anthocyanin 7-O-glucosyltransferase from Canterbury bells (Campanula medium). Plant Biotechnology, 2014. advpub. [0360] 12. Nishizaki, Y., et al., p-Hydroxybenzoyl-glucose is a zwitter donor for the biosynthesis of 7-polyacylated anthocyanin in Delphinium. Plant Cell, 2013. 25(10): p. 4150-65. [0361] 13. Song, X., et al., Genome-wide characterization of the cellulose synthase gene superfamily in Solanum lycopersicum. Gene, 2019. 688: p. 71-83. [0362] 14. Sainsbury, F., E. C. Thuenemann, and G. P. Lomonossoff, pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J, 2009. 7(7): p. 682-93. [0363] 15. Louveau, T., et al., Analysis of two new arabinosyltransferases belonging to the carbohydrate-active enzyme (CAZY) glycosyl transferase family 1 provides insights into disease resistance and sugar donor specificity. The Plant Cell, 2018: p. tpc.00641.2018. [0364] 16. Kite, G. C., M. J. Howes, and M. S. Simmonds, Metabolomic analysis of saponins in crude extracts of Quillaja saponaria by liquid chromatography/mass spectrometry for product authentication. Rapid Commun Mass Spectrom, 2004. 18(23): p. 2859-70. [0365] 17. Fleck, J. D., et al., Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities. Molecules, 2019. 24(1). [0366] 18. Sayama, T., et al., The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean. Plant Cell, 2012. 24(5): p. 2123-38. [0367] 19. Reed, J., et al., A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab Eng, 2017. 42: p. 185-193. [0368] 20. Kumar, S., G. Stecher, and K. Tamura, MEGA7: Molecular Evolutionary Genetics Analysis Version 70 for Bigger Datasets. Mol Biol Evol, 2016. 33(7): p. 1870-4. [0369] 21. Saitou, N. and M. Nei, The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987. 4(4): p. 406-25. [0370] 22. Stephenson, M. J., et al., Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale. Journal of visualized experiments: JoVE, 2018(138): p. 58169. [0371] 23. Guo, S., et al., Triterpenoid saponins from Quillaja saponaria. Phytochemistry, 1998. 48(1): p. 175-180. [0372] 24. Miettinen, K., et al., The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis. Nat Commun, 2017. 8: p. 14153. [0373] 25. Thimmappa, R., Geisler, K., Louveau, T., O'Maille, P., and Osbourn, A. (2014). Triterpene biosynthesis in plants. Annu. Rev. Plant Biol., 65:225-257. [0374] 26. Grabherr, M. G., et al. (2011). Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol., 29:644-652. [0375] 27. Dobin, A., et al. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29:15-21.
Tables and Sequences
[0376]
TABLE-US-00001 TABLE 1 .sup.1H, .sup.13C NMR spectral data for compounds 1 and 2 in MeOH-d4, (600, 150 MHz) QA-GlcpA-[Galp]-Rhap (1) QA-GlcpA-[Galp]-Rhap (2) Position δ.sub.H mult, δ.sub.H mult, No. δ.sub.C, Type (J in Hz) δ.sub.C, Type (J in Hz) 1 .sup. 39.4, CH.sub.2 1.71/1.13, m 39.3, CH.sub.2 1.69/1.10, m 2 .sup. 25.7, CH.sub.2 2.02/1.79, m 25.8, CH.sub.2 2.05/1.77, m 3 86.0, CH 3.85, dd, (11.9, 4.55) 85.9, CH.sup. 3.86, m 4 56.4, Cq — 56.5, Cq .sup. — 5 49.2, CH 1.35, m 49.1, CH.sup. 1.32, m 6 .sup. 21.4, CH.sub.2 1.50/0.90, m 21.5, CH.sub.2 1.47/0.90, m 7 .sup. 33.7, CH.sub.2 1.57/1.25, m 33.8, CH.sub.2 1.55/1.23, m 8 41.0, Cq — .sup. 41.1, Cq HMBC — 9 48.2, CH 1.77, m 48.2, CH.sup. 1.75, m 10 37.2, Cq — 37.1, Cq .sup. — 11 .sup. 24.6, CH.sub.2 1.93/1.93, m 24.6, CH.sub.2 1.92/1.92 12 123.3, CH 5.30, br t (3.7) 123.4, CH .sup. .sup. 5.30, bd s 13 145.3, Cq.sup. — .sup. 145.3, Cq HMBC — 14 42.8, Cq — 42.8, Cq .sup. — 15 .sup. 36.3, CH.sub.2 1.84/1.34, m 36.4, CH.sub.2 1.84/1.33, m 16 75.4, CH 4.45, m 75.5, CH.sup. .sup. 4.44, br s 17 49.7, Cq — 49.7, Cq .sup. — 18 42.2, CH 3.01, dd, (14.3, 4.4) 42.3, CH.sup. 3.01, d (14.3, 4.4) 19 .sup. 47.8, CH.sub.2 2.30, t (13.6)/1.04, m 47.9, CH.sub.2 2.29, t (13.6)/1.03, m 20 31.5, Cq — 30.9, Cq .sup. — 21 .sup. 36.7, CH.sub.2 1.96/1.16, m 36.8, CH.sub.2 195/1.14, m 22 .sup. 32.9, CH.sub.2 1.91/1.78, m 32.9, CH.sub.2 1.89/1.76, m 23 210.9, CH 9.44, s.sup. 210.8, CH .sup. 9.43, s.sup. 24 .sup. 10.9, CH.sub.3 1.15, s.sup. 10.9, CH.sub.3 1.13, s.sup. 25 .sup. 16.4, CH.sub.3 1.00, s.sup. 16.5, CH.sub.3 1.00, s.sup. 26 .sup. 17.9, CH.sub.3 0.79, s.sup. 17.9, CH.sub.3 0.80, s.sup. 27 .sup. 27.4, CH.sub.3 1.40, s.sup. 27.4, CH.sub.3 1.39, s.sup. 28 181.2, Cq.sup. — .sup. 181.0, Cq HBMC — 29 .sup. 33.6, CH.sub.3 0.88, s.sup. 33.6, CH.sub.3 0.88, s.sup. 30 .sup. 25.0, CH.sub.3 0.97, s.sup. 25.1, CH.sub.3 0.97, s.sup. GlcA-1 104.2, CH 4.46, m 104.5, CH .sup. .sup. 4.36, d (7) GlcA-2 78.4, CH 3.63, m 78.7, CH.sup. 3.65, m GlcA-3 86.0, CH 3.63, m 86.7, CH.sup. 3.67, m GlcA-4 73.2, CH 3.48, m 72.1, CH.sup. 3.55, m GlcA-5 77.0, CH 3.73, m Not detected Not detected GlcA-6 174.2, Cq.sup. — 176.3 Cq, HMBC.sup. — Gal-1 104.4, CH 4.45, m 104.0, CH .sup. .sup. 4.78, d (6) Gal-2 73.2, CH 3.48, m 73.8, CH.sup. 3.45, m Gal-3 75.2, CH 3.47, m 75.6, CH.sup. 3.43, m Gal-4 70.8, CH 3.81, m 70.9, CH.sup. 3.80, m Gal-5 77.1, CH 3.47, m 76.8, CH.sup. 3.48, m Gal-6 .sup. 62.4, CH.sub.2 3.78/3.73, m 62.3, CH.sub.2 3.75/3.72, m Rha-1 103.4, CH .sup. 5.03, d (1.8) Rha-2 72.3, CH 4.01, dd (3.4, 1.8) Rha-3 72.4, CH 3.65, m Rha-4 74.0, CH 3.40, m Rha-5 70.7, CH 3.96, m Rha-6 .sup. 18.0, CH.sub.3 .sup. 1.24, d (6.1) Xyl-1 104.9, CH .sup. .sup. 4.61, d (7.7) Xyl-2 75.5, CH.sup. 3.23, m Xyl-3 78.3, CH.sup. 3.31, m Xyl-4 71.3, CH.sup. 3.49, m Xyl-5 67.3, CH.sub.2 3.89/3.21, m
TABLE-US-00002 TABLE 2 Primers used to clone the three glycosyltransferases required for biosynthesis of the trisaccharide at C-3 of quillaic acid. Gene specific sequences are shown in black, while the attB sites required for Gateway ® cloning are shown in red. Name Sequence QsCSL1_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGAAATCCCCCTCTAACCCAAATC (SEQ ID NO.: 33) QsCSL1_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTA TCAGACCATTTTCTTGCTGATTCTAG (SEQ ID NO.: 34) Qs-3-O-GalT_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTGGAGTCTCCAGCAGATC (SEQ ID NO.: 35) Qs-3-O-GalT_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTA TCAGACACCCTGAATTCTTGATTTC (SEQ ID NO.: 36) Qs-3-O-RhaT/XylT_ GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTCTCCGGCGACGACGATG (SEQ ID NO.: 37) attB1F Qs-3-O-RhaT/XylT_ GGGGACCACTTTGTACAAGAAAGCTGGGTA TCACGATTCATGATCTTGTGCAGCC (SEQ ID NO.: 38) attB2R
TABLE-US-00003 TABLE 4 Alignment of UGT protein sequences in the region of the Family 1 UGT 44-amino acid Plant Secondary Product Glycosyltransferase (PSPG) motif. Qs_2073886_D6 (“Qs-3- O-GalT”) shares the histidine residue conserved in UGTs that transfer β-D-galactose or α-L-arabinose. Figure adapted from Louveau, Orme [15]. Accession numbers: AsUGT99A6 (AZQ26916), MtUGT73K1 (AAW56091), AtUGT78D2 (NP_197207), GmSSAT (XP_003532274), AtUGT78D3 (NP_197205), AeGaT (BADO6514), GmUGT73P2 (BAI99584). GlcT AsUGT99A6 WAPQALILSHRAAGAFVTHCGWNSTLEAVAAGLPVVTWPHFTD Q MtUGT73K1 WVPQALILDHPSIGGFLTHCGWNATVEAISSGVPMVTMPGFGD Q AtUGT78D2 WAPQVELLKHEATGVFVTHCGWNSVLESVSGGVPMICRPFFGD Q AraT/ Qs_2073886_D6 WAPQLLILDHPAIGGLLNHSGWNSVLEGATAGLPMITWPLYAE H GalT GmSSAT WVPQGLILKHDAIGGFLTHCGANSVVEAICEGVPLITMPRFGD H AtUGT78D3 WAPQVELLNHEAMGVFVSHGGWNSVLESVSAGVPMICRPIFGD H AeGaT WAPQIQVLSHDAVGVVITHGGWNSVVESIAAGVPVICRPFFGD H GmUGT73P2 WAPQLLILENPAIGGLVTHCGWNTVVESVNAGLPMATWPLFAE H (SEQ ID NOs.: 39-46)
TABLE-US-00004 TABLE 5 Glycosyltransferases identified herein (“Qs QA-3-O-TriS sequences”): Nucleotide AA Sequence CDS - sequence - Enzyme Biological activity SEQ ID NOs SEQ ID NOs QsCSL1 QA-GlcAT 1 2 Capable of transferring D-glucuronic acid (GlcpA) at the 3-O position of quillaic acid to form 3β-{[β-D-glucopyranosiduronic acid]oxy}-quillaic acid (QA-GlcpA). QsCSLG2 25 26 Qs-3-O-GalT QA-GalT 3 4 Capable of transferring D-Galactose (Galp) via a β-1−>2 linkage to QA-GlcpA to form =>3β-{[β-D-galactopyranosyl-(1−>2)-β-D- glucopyranosiduronic acid]oxy}-quillaic acid (QA-GlcpA-Galp) Qs-3-O-RhaT/XylT QA-RhaT/XylT 5 6 Qs_0283850 27 28 DN20529_c0_g2_i8 29 30 Qs_0283870 The enzymes are capable of transferring 31 32 D-Xylose (Xylp)or L-Rhamnose via a 1,3 linkage to QA-GlcpA-Galp to form 3β-{[β-D-xylopyranosyl-(1−>3)-[β-D- galactopyranosyl-(1−>2)]-β-D- glucopyranosiduronic acid]oxy}-quillaic acid (QA-GlcpA-[Galp]-Xylp) and/or (3β-{[α-L-rhamnopyranosyl-(1−>3)-[β-D- galactopyranosyl-(1−>2)]-β-D- glucopyranosiduronic acid]oxy}-quillaic acid) (QA-GlcpA-[Galp]-Rhap) respectively
TABLE-US-00005 TABLE 6 Other GTs which may be used in QA-glycosylation (“QA-3-O-TriS sequences”) Nucleotide AA Sequence CDS - Sequence - Enzyme Activity SEQ ID NOs SEQ ID NOs GmUGT73P2 QA-GalT: a triterpene 3-O-glucuronoside- 19 20 β-1,2-D-galactosyltransferase
TABLE-US-00006 TABLE 7 Ancillary activities AA CDS - Sequence - SEQ SEQ Enzyme Activity ID NOs ID NOs AsHMGR HMG-CoA reductase (HMGR); 7 8 tHMGR HMG-CoA reductase (HMGR); 9 10 AsSQS (Avena squalene synthase (SQS) 21 22 strigosa squalene synthase) AtATR2 cytochrome P450 reductase 23 24 (Arabidopsis thaliana cytochrome P450 reductase 2)
TABLE-US-00007 TABLE 8 QA biosynthesis activities Enzyme (“QA CDS - AA Sequence - polypeptides”) Activity SEQ ID NOs SEQ ID NOs QsbAS (β-amyrin cyclisation of 2,3-oxidosqualene (OS) to a 11 12 synthase) triterpene QsCYP716-C-28 enzyme capable of oxidising β-amyrin or an 13 14 oxidised derivative thereof at the C-28 position to a carboxylic acid QsCYP716-C-16α enzyme capable of oxidising β-amyrin or an 15 16 oxidised derivative thereof at the C-16α position to an alcohol QsCYP714-C-23 enzyme capable of oxidising β-amyrin or an 17 18 oxidised derivative thereof at the C-23 position to an aldehyde
TABLE-US-00008 TABLE 9 DNA (top right) and protein (bottom left) sequence identity between the gene and protein sequences of the three UGT sequences identified in the Q. saponaria genome, the new sequence DN20529_c0_g2_i8 identified in the de novo transcriptome and Qs-3-O-RhaT/XylT. Qs_0283860 region corresponds to the genomic region of the Qs_0283860 pseudogene starting from the predicted start codon and the predicted stop codon. NA identity Qs_0283860 Qs-3-O- Protein identity Qs_0283850 region Qs_0283870 DN20529_c0_g2_i8 RhaT/XylT Qs_0283850 97% 90% 98% 92% Qs_0283860 region N/A 89% 99% 92% Qs_0283870 86% N/A 89% 97% DN20529_c0_g2_i8 98% N/A 86% 92% Qs-3-O-RhaT/XylT 90% N/A 96% 90%
TABLE-US-00009 TABLE 10 Primers used to clone the four glycosyltransferases. Gene specific sequences are shown in black, while the attB sites required for Gateway ® cloning are shown in grey. Name Sequence CslG2_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGCGACCGTCTCCTCCCT (SEQ ID NO.: 47) CslG2_attB2R GGGGACCACTTTGTAGAAGAAAGCTGGGTA TTAGGCCTTTCCCTTGCCTTT (SEQ ID NO.: 48) Qs_0283870_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTCTCCGGCGACGACGATG (SEQ ID NO.: 49) Qs_0283870_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTA TCACGATTCATGATCTTGTGCAGCC (SEQ ID NO.: 50) Qs_0283850_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTCTCCGGCGACGACGACG (SEQ ID NO.: 51) Qs_0283850_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTA TCATGCAACCTTGCCATTGTTAGCCCT (SEQ ID NO.: 52) Qs_0283860_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTCTCCGGCGACGACGAC (SEQ ID NO.: 53) Qs_0283860_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTA TCATGATTTCATTGCAGCCTTGCCA (SEQ ID NO.: 54)
TABLE-US-00010 TABLE 11 Full NMR data for quillaic acid 3-O-β-D-glucopyranosiduronic acid (QsbAS/QsCYP716-C-16α/QsCYP714-C-23/QsCYP716-C-28/QsCSL1 product) in MeOH-d.sub.4 (600, 150 MHz)
TABLE-US-00011 TABLE 12 Full NMR data for quillaic acid 3-O-β-D-glucopyranosiduronic acid (QsbAS/QsCYP716-C-16α/QsCYP714-C-23/QsCYP716-C-28/CslG2 product) in MeOH-d.sub.4 (600, 150 MHz)
TABLE-US-00012 TABLE 13 Full NMR data for quillaic acid 3-O-{-β-D-galactopyranosyl-(1.fwdarw.2)-β-D- glucopyranosiduronic acid} (QsbAS/QsCYP716-C-16α/QsCYP714-C-23/ QsCYP716-C-28/QsCSL1/Qs-3-O-GalT product) in MeOH-d.sub.4 (600, 150 MHz)
TABLE-US-00013 TABLE 14 .sup.1H, .sup.13C NMR spectral data for QA-GlcpA-[Galp]-Rhap (QsbAS/QsCYP716-C- 16α/QsCYP714-C-23/QsCYP716-C-28/QsCslG2/Qs-3-O-GalT/ Qs_0283850 product) in MeOH-d.sub.4, (400, 100 MHz)
TABLE-US-00014 TABLE 15 .sup.1H, .sup.13C NMR spectral data for QA-GlcpA-[Galp]-Xylp (QsbAS/QsCYP716-C- 16α/QsCYP714-C-23/QsCYP716-C-28/QsCslG2/Qs-3-O-GalT/ Qs_0283870 product) in MeOH-d.sub.4, (400, 100 MHz)
TABLE-US-00015 TABLE 3 Family 1 UDP-dependent glycosyltransferases (UGT) used in phylogenetic analysis. UGTs believed to be active on triterpenes are highlighted in bold. Enzyme Accession number UGT family UGT Group Plant species Reported activity Reference AtUGT79B1 Q9LVW3 UGT79 A Arabidopsis thaliana Anthocyanidin 3-O-glucoside [1,2]-xylosyltransferase Yonekura-Sakakibara et al. (2012) AtUGT79B6 Q9FN26 UGT79 A Arabidopsis thaliana Flavonol 3-O-galactoside [1,2]-glucosyltransferase Yonekura-Sakakibara et al. (2014) Cs1-6RhaT ABA18631 UGT79 A Citrus sinensis Flavonoid 7-O/3-O-glucoside [1,6]-rhamnosyltransferase Frydman et al. (2013) GmUGT79A6 BAN91401 UGT79 A Glycine max Flavonol 3-O-glucoside/galactoside [1,6]-rhamnosyltransferase Rojas Rodas et al. (2014) LeABRT2 BAU68118 UGT79 A Lobelia erinus Delphinidin 3-O-glucoside [1,6]-rhamnosyltransferase Hsu et al. (2017) GmUGT91H4 BAI99585 UGT91 A Triterpene 3-O-galactoside [1,2]-rhamnosyltransferase Shibuya et al. (2010) GmUGT91H9 NP.sub.—001348424 UGT91 A
Triterpene 3-O-galactoside [1,2]-glucosyltransferase Yano et al. (2018) In3GGT Q53UH4 UGT91 A Ipomoea nil Anthocyanidin 3-O-glucoside [1,2]-glucosyltransferase Morita et al. (2005) GjUGT94E5 F8WKW8 UGT94 A Gardenia jasminoides Apocarotenoid glucoside [1,6]-glucosyltransferase Nagatoshi et al. (2012) BpUGT94B1 Q5NTH0 UGT94 A Bellis perennis Anthocyanidin 3-O-glucoside [1,2]-glucuronosyltransferase Sawada et al. (2005) Cm1-2RhaT1 AAL06646 UGT94 A Citrus maxima Flavonoid 7-O-glucoside [1,2]-rhamnosyltransferase Frydman et al. (2013) PgUGT94Q2 AGR44632 UGT94 A
Triterpene 3-O-glucoside [1,2]-glucosyltransferase Jung et al. (2014) SIGAME18 XP_004243636 UGT94 A Solanum lycopersicum Steroidal alkaloid 3-O-glucoside [1,2]-glucosyltransferase Itkin et al. (2013) VpUGT94F1 BAI44133 UGT94 A Veronica persica Flavonoid 3-O-glucoside [1,2]-glucosyltransferase Ono et al. (2010) AtUGT89C1 AAF80123 UGT89 B Arabidopsis thaliana Flavonol 7-O-rhamnosyltransferase Yonekura-Sakakibara et al. (2007) UGT89A2-Col-0 Q9LZD8 UGT89 B Arabidopsis thaliana Dihydroxybenzoic acid xylosyltransferase Chen and Li (2017) PoUGT90A7 ACB56926 UGT90 C Pilosella officinarum Flavonol glucosyltransferase Witte et al. (2009) AcUGT73G1 AAP88406 UGT73 D Allium cepa Flavonoid glucosyltransferase Kramer et al. (2003) AtUGT73B3 AAM47999 UGT73 D Arabidopsis thaliana Flavonoid-7-O-glucosyltransferase Kim et al. (2006) AtUGT73C1 AEC09294 UGT73 D Arabidopsis thaliana Cytokinin glucosyltransferase 1 Gandia-Herrero et al. (2008) AsUGT99D1 AZQ26921 UGT99 D
Triterpene-3-O-arabinosyltransferase Louveau et al. (2018) BvUGT73C10 AFN26666 UGT73 D
Triterpene-3-O-glucosyltransferase Augustin et al. (2012) CbBet5OGT CAB56231 UGT73 D Cleretum bellidiforme Betanidin-5-O-glucosyltransferase Vogt et al. (1999) CsUGT73A20 ALO19886 UGT73 D Camellia sinensis Flavonoid 7-O/3-O-glucosyltransferase Zhou et al. (2017) CsUGT73AM3 KGN59015 UGT73 D
Triterpene-3-O-glucosyltranferase Zhong et al. (2017) GmUGT73F2 BAM29362 UGT73 D
Triterpene 22-O-arabinoside [1,3]-glucosyltransferase Sayama et al. (2012) GmUGT73F4 BAM29363 UGT73 D
Triterpene 22-O-arabinoside [1,3]-xylosyltransferase Sayama et al. (2012) GmUGT73P2 (GmSGT2) BAI99584 UGT73 D
Triterpene 3-O-glucoronide [1,2]-galactosyltransferase Shibuya et al. (2010) GuUGAT ANJ03631 UGT73 D
Triterpene 3-O-glucoronosyltransferase/Triterpene 3-O-glucuronide Xu et al. (2016) [1,2]-glucuronosyltransferase MtUGT73F3 ACT34898 UGT73 D
Triterpene 28-O-glucosyltransferase Naoumkina et al. (2010) SIUGT73L4 ADQ37966 UGT73 D Solanum lycopersicum Steroidal alkaloid 3-O-glucoside [1,3]-xylosyltransferase Itkin et al. (2013) StSGT3 ABB84472 UGT73 D Solanum tuberosum Steroidal alkaloid 3-O-glucoside/galactoside [1,2]-rhamnosyltransferase McCue et al. (2007) CsUGT707B1 CCG85331 UGT707 E Crocus sativus Flavonol 3-O-glucoside [1,2]-glucosyltransferase Trapero et al. (2012) AtUGT71B6 NP_188815 UGT71 E Arabidopsis thaliana Abscisate β-glucosyltransferase Priest et al. (2006) AtUGT71C1 NP_180536 UGT71 E Arabidopsis thaliana UDP-glucosyl transferase 71C1 Lim et al. (2008) OsUGT707A3 BAC83989 UGT71 E Oryza sativa Flavonoid 3-O-glycosyltransferase Ko et al. (2008) AtUGT72B1 Q9M156 UGT72 E Arabidopsis thaliana UDP-glycosyltransferase 72B1 Brazier-Hicks et al. (2007) AtUGT72E2 AED98252 UGT72 E Arabidopsis thaliana Hydroxycinnamate 4- β-glucosyltransferase Lanot et al. (2006) MtUGT71G1 AAW56092 UGT71 E
Triterpenoid-O-glucosyltransferase Achnine et al. (2005) PgUGTPg1 AIE12479 UGT71 E
Protopanaxadiol-20-O-glucosyltransferase Yan et al. (2014) ScUGT5 BAJ11653 UGT88 E Sinningia cardinalis 3-Deoxyanthocyanidin 5-O-glucosyltransferase Nakatsuka and Nishihara (2010) AtUGT78D1 Q9S9P6 UGT78 F Arabidopsis thaliana Flavonol 3-O-glucosyltransferase Jones et al. (2003) Fh3GT1 ADK75021 UGT78 F Freesia hybrid cultivar Anthocyanidin 3-O-glucosyltransferase Sun et al. (2016) VmUF3GaT BAA36972 UGT78 F Vigna mungo Flavonoid 3-O-galactosyltransferase Mato et al. (1998) VvGT1 AAB81683 UGT78 F Vitis vinifera Anthocyanidin 3-O-glucosyltransferase Ford et al. (1998) AtUGT85A1 AAF18537 UGT85 G Arabidopsis thaliana Cytokinin-O-glucosyltransferase 2 Hou et al. (2004) PdUGT85A19 ABV68925 UGT85 G Prunus dulcis Cyanohydrin glucoside [1,6]-glucosyltransferase Franks et al. (2008) SbUGT85B1 AAF17077 UGT85 G Sorghum bicolor Cyanohydrin glycosyltransferase UGT85B1 Hansen et al. (2003) AtUGT76D1 AEC07843 UGT76 H Arabidopsis thaliana Flavonoid-7-O-glucosyltransferase Lim et al. (2004) SrUGT76G1 AAR06912 UGT76 H Stevia rebaudiana Diterpenoid 13-O-glucoside [1,3]-glucosyltransferase Richman et al. (2005) AtUGT83A1 Q9SGA8 UGT83 I Arabidopsis thaliana Unknown Ross et al. (2001) AtUGT87A1 O64732 UGT87 J Arabidopsis thaliana Unknown Ross et al. (2001) AtUGT87A2 NP_001077979 UGT87 J Arabidopsis thaliana Unknown Wang et al. (2012) AtUGT86A1 Q9SJL0 UGT86 K Arabidopsis thaliana Unknown Ross et al. (2001) AtUGT74E2 NP_172059 UGT74 L Arabidopsis thaliana Auxin (IBA) glycosyltransferase Tognetti et al. (2010) AsUGT74H5 ACD03250 UGT74 L Avena strigosa N-Methylanthranilate O-glucosyltransferase Owatworakit et al. (2012) PgUGT74A1 AGR44631 UGT74 L
Triterpene-3-O-glucosyltransferase Jung et al. (2014) SgUGT74AC1 AEM42999 UGT74 L
Triterpene (PPD)-3-O-glucosyltransferase Dai et al. (2015) VhUGT74M1 ABK76266 UGT74 L
Triterpene carboxylic acid 28-O-glucosyltransferase Meesapyodsuk et al. (2007) ZmIAGT AAA59054 UGT74 L Zea mays Auxin glucosyltransferase Szerszen et al. (1994) AtUGT75C1 Q0WW21 UGT75 L Arabidopsis thaliana Anthocyanin 5-O-glucosyltransferase Yamazaki et al (1999) GjUGT75L6 F8WKW0 UGT75 L Gardenia jasminoides Apocarotenoid glucosyltransferase Nagatoshi et al. (2012) Via5GT AHL68667 UGT75 L Vitis amurensis Rupr. Anthocyanin 5-O-glucosyltransferase He et al. (2015) cv. ‘Zuoshanyi AtUGT84A1 Q5XF20 UGT84 L Arabidopsis thaliana Hydroxycinnamate glucosyltransferase 2 Milkowski et al. (2000) GtUF6CGT1 BAQ19550 UGT84 L Gentiana triflora Flavonoid 6-C-glucosyltransferase Sasaki et al. (2015) CuLGT BAA93039 UGT84 L
Triterpene (limonoid)-17-O-glucosyltransferase Kita et al. (2000) AtUGT92A1 Q9LXV0 UGT92 M Arabidopsis thaliana Unknown Ross et al. (2001) CcDOPA5GT BAD91804 UGT92 M Celosia cristata Cyclo-DOPA 5-O-glucosyltransferase Sasaki et al. (2005) MjcDOPA5GT BAD91803 UGT92 M Mirabilis jalapa Cyclo-DOPA 5-O-glucosyltransferase Sasaki et al. (2005) AtUGT82A1 Q9LHJ2 UGT82 N Arabidopsis thaliana Unknown Ross et al. (2001) SIGAME17 XP_004243637 UGT93 O Solanum lycopersicum Steroidal alkaloid 3-O-galactoside [1,4]-glucosyltransferase Itkin etal. (2013) ZmcisZog1 AAK53551 UGT93 O Zea mays cis-zeatin O-glucosyltransferase Martin et al. (2001) OsUGT709A4 BAC80066 UGT709A4 P Oryza sativa Isoflavonoid-7-O-glucosyltransferase Ko et al. (2008)
REFERENCES FOR TABLE 3
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TABLE-US-00016 Sequences SEQ ID NO: 1-Q. saponaria quillaic acid 3-O-glucuronosyltransferase (cellulose synthase-like enzyme QsCSL1) coding sequence (2142 bp) ATGAAATCCCCCTCTAACCCAAATCAGAAACCCATCCTCCACACTTGTACAATTCAGCAGCCTCGT GCTACCCTTAACAAAATTCATAGTCTTATTCATTTCTCAGCCATACTTGTCCTATTTTATTACCGG ATAACCCGTCTATTCTTCACCGACGATTTCAAGGTACCCAAGTTACTATGGACTCTAATGACAATC TCCGAGTTCATTCTTGCCTTCATTTGGGTTCTCATCCAACCTTTCCGGTGGCGACCGGTGTCCCGT TCCGTCATACCAGAGAATATGCCGAAGGACATCAGTTTGCCGGCGGTGGACGTGTTTGTATGCACA GCTGACCCTCAAAAAGAACCCACAGTGGAGGTGATGAACACAATTTTATCAGCCATGGCTTTAGAC TACCCGGCGGAGAAGCTCGCCGTGTATCTTTCCGATGATGGGGGTTCTGCTGTCACCTTATATGCT ATAAAAGAAGCTTGTTGTTTTGCTAAGATGTGGCTTCCGTTTTGTAACAAGTATGGGATCAAATCA AGGTGTCCCGAGGCTTATTTTTCAAAGCTTGCCGCTGACGAGTGGCTTCACCGGAGTGTGGAATTC GTGGCAGAAGAAAAGGAGGTCAAGGCTAATTATGAAGAGTTCAAGAGAAATGTGCAGAAATTTGGT GAGCAACAAGAAAACAGTCGTGTTGTGCATGATCGTCACCCTCATGTTGAGATTATACACAATAAT TGGAATAACGAAGACCAAGCTCATGAGATGCCACTCCTTGTTTATGTCTCTCGTGAAAGAAGACCA TCTCACCATCCTCGATTCAAAGCTGGAGCTCTTAACACCCTTCTTCGAGTTTCTGGCATCATCAGC AACAGCCCCTACATACTGGTTCTAGACTGTGACATGTACTGCAATGACCCAACCTCAGCTAGACAA GCAATGTGCTTCCATCTTGATCCCCAACTGTCTAAAAATCTTGCTTTTGTACAATTCCCTCAAATA TTCTATAACGCTAGTAAGAATGACGTCTATGATGCCCAAGTCAGGGCGGCATACCAGACAAAGTGG CAGGGTATGGATGGACTTCAAGGACCAATTTTTTCTGGCACTGGCTTTTACTTAAAGAGGAAGGCA ATGTATGGAAACCCTGATCAAGATGATAATTGTCTACTCAAGCCATATAAGAAATTTGGCATGTCT GGAGAATTTGTAGAATCACTTAAGGTCCTTAACGAACAAGATGGTACCCAGAAGAAATTATTGGAT GGATTTTTACAAGAGGCCAAACTATTGGCCTCGTGTGCCTATGAAACAAAGACAAGTTGGGGTAAA GAGATTGGATTCTCATATGACTGTTTAATAGAGAGCACTTTCACTGGTTATCTTTTGCACTGCAGA GGGTGGATATCTGTTTATCTTTATCCCAAGAGACCATGTTTTTTAGGATGCTGTCCTACTGATATG AAGGATGCCATGGTTCAATATACCAAGTGGATGTCTGAGCTATTTTCAATTGCTATCTCAAGATTC AATCCTCTGCTCTATGGGGTGGCAAGAATGTCCATTCTTCAAAGCCTGTGTTATGGATCCTTTACA CTGGCGCCTATTTTGTCATTTCCTTTGTTCTTATATGGAACGGTTCCTCAATTATGCCTCTTGAAA GGCATATCTTTGTTTCCAAAGGTTTCGGACCCATGGTTTGCTGTGTTTGCAGCTATCTTTGTATCC TCCCTGTGTCAACACTGGTTCGAGGTCCTCTCTTGTGATGGTACATTTACGACTTGGTGTAATGAA CAGCGGAGTTGGCTTATAAAGTCGGTTTCCGGTAGTTTGTTTGGAGTTGTGGGCGCAATCTTGCAG CGGCTAGGCTTGAAGACAAAGTTTAGTTTATCAAACAAAGCCATGGACAAAGAAAAGCTGGAGAAA TATGAAAAGGGTAAATTTAATTTCCAAGGGGCTGCCATGTTCATGGTTCCTGTGTCTATTTTAGTC ATACTGAACACATTTTGCTTCCTCGGTGGGTTTTGGAAAGTGATCATAATGAAGAATATCCTGGAC ATGTTTGGACAACTTTCTCTCTCTGCCTACGTTCTGGTTCTCAGTTGTCCAGTTCTTGAAGGGATG TTAACTAGAATCAGCAAGAAAATGGTCTGA SEQ ID NO: 2-Q. saponaria quillaic acid 3-O-glucuronosyltransferase (cellulose synthase-like enzyme QsCSL1) translated nucleotide sequence (713 aa): MKSPSNPNQKPILHTCTIQQPRATLNKIHSLIHFSAILVLFYYRITRLFFTDDFKVPKLLWTLMTI SEFILAFIWVLIQPFRWRPVSRSVIPENMPKDISLPAVDVFVCTADPQKEPTVEVMNTILSAMALD YPAEKLAVYLSDDGGSAVTLYAIKEACCFAKMWLPFCNKYGIKSRCPEAYFSKLAADEWLHRSVEF VAEEKEVKANYEEFKRNVQKFGEQQENSRVVHDRHPHVEIIHNNWNNEDQAHEMPLLVYVSRERRP SHHPRFKAGALNTLLRVSGIISNSPYILVLDCDMYCNDPTSARQAMCFHLDPQLSKNLAFVQFPQI FYNASKNDVYDAQVRAAYQTKWQGMDGLQGPIFSGTGFYLKRKAMYGNPDQDDNCLLKPYKKFGMS GEFVESLKVLNEQDGTQKKLLDGFLQEAKLLASCAYETKTSWGKEIGFSYDCLIESTFTGYLLHCR GWISVYLYPKRPCFLGCCPTDMKDAMVQYTKWMSELFSIAISRFNPLLYGVARMSILQSLCYGSFT LAPILSFPLFLYGTVPQLCLLKGISLFPKVSDPWFAVFAAIFVSSLCQHWFEVLSCDGTFTTWCNE QRSWLIKSVSGSLFGVVGAILQRLGLKTKFSLSNKAMDKEKLEKYEKGKFNFQGAAMFMVPVSILV ILNTFCFLGGFWKVIIMKNILDMFGQLSLSAYVLVLSCPVLEGMLTRISKKMV* SEQ ID NO: 3-Q. saponaria QA-GlcpA β-1,2-D-galactosyltransferase (Qs-3-O-GalT) coding sequence (1479 bp) ATGGTGGAGTCTCCAGCAGATCATGATGTGCTCAAAATCATTGTCCTTCCATGGGTAACCTCAGGT CACATGATTCCCATGGTAGATGCAGCCAGACTATTTGCTATGCATGGTGCAGATGTTACCATCATC ACCACCCCAGCTAATGCCCTTACATTCCAGAAATCCGTCGACCGTGATTTCAATTCCGGTCGTTTA ATCAGAACTCACACCCTTAAATTCCCTGCAGCAGAAGTTGGTGTACCTGAAGGAGTTGAAAACTTC AACAATACTTCCCCTGAAATGACCTCCAAAGTCTACCTTGGAGTCTCAATGCTCCGAGAACCAACC CAACAATTGATTGAGGATCTGCGTCCAGATTGTCTTATCACTGATATGTTCTATCCTTGGGCTGTG GATGTTGCTGACAAATTAGGCATTCCAAGGCTAATTTTTCAAGGTCCTGGAAGTTTTGGTTTGTCA GCTATGCATTCTATCAAACAGTATGAGCCCTTTAAGTCAGTAACTTCAGATACTGAGACATTCCCA CTACCTGGATTGCCGCATAAGGTAGAGATGACAAGGTTGCAGATACCAAAATGGGTTCGTGAGCCA AATGGGTACACTCAATTGATGGGCAGGGTAAAAGATTCGGAGAGAAGAAGCTATGGGTCATTGGTG AATAGCTTTTATGACTTCGAAGGCCCTTATGAAGAGCACTATAGGAAGGCAACAGGACAGAGGGTT TGGAGCATTGGACCAGTTTCAGTTTGGGTGAACCAAGATGCTGCAGATAAGGTTGGAAGAGGACAG GATCTTGTTGCTGAAGACCAAAACAGCTGGTTGAATTGGCTCAATTCCAAAGAGAAAAACTCTGTT CTGTATGTAAGTTTTGGGAGCATGGCCAAGTTCCCATCTGCTCAGCTTCTTGAAATAGCTCATGGG CTTGAAGCTTCAGGTCATAGTTTCATCTGGGTTGTCAGAAAAGTTGACGGGGATGATGATGTAGAC GTGTGGCTTCCAGATTTTGAGAAGAAAATGAAAGAGAACAACAAGGGTTTCATCATAAGGAATTGG GCACCACAATTGCTCATATTGGACCATCCAGCAATTGGAGGTTTGCTGAATCACAGTGGATGGAAT TCAGTACTGGAAGGTGCTACAGCAGGCTTGCCAATGATCACTTGGCCTCTGTATGCCGAGCATTTT TACAATGAAAGGTTGGTTCTAGATGTGTTGAAAATTGGAGTACCAGTTGGGGTGAAGGAGTGGAAG AACTTGCATGAGGTGGGTGAGTTGGTGAGAAGGGATGCAATTGCCAAGGCAATTAAATTGTTAATG GGTAGTGGAGAAGAAGCTGAGGTAATGAGGAAAAAAGCCAAAGAGCTTGGTGTTGGAGCAAAGAAA GGTATTCAGGTTGGAGGTTCTTCTCATACCAATTTGATAGCAGTGATTGATGAGTTAAAGTCACTA AAGAAATCAAGAATTCAGGGTGTCTGA SEQ ID NO: 4-Q. saponaria QA-GlcpA β-1,2-D-galactosyltransferase (Qs-3-O-GalT) translated nucleotide sequence (492 aa): MVESPADHDVLKIIVLPWVTSGHMIPMVDAARLFAMHGADVTIITTPANALTFQKSVDRDFNSGRL IRTHTLKFPAAEVGVPEGVENFNNTSPEMTSKVYLGVSMLREPTQQLIEDLRPDCLITDMFYPWAV DVADKLGIPRLIFQGPGSFGLSAMHSIKQYEPFKSVTSDTETFPLPGLPHKVEMTRLQIPKWVREP NGYTQLMGRVKDSERRSYGSLVNSFYDFEGPYEEHYRKATGQRVWSIGPVSVWVNQDAADKVGRGQ DLVAEDQNSWLNWLNSKEKNSVLYVSFGSMAKFPSAQLLEIAHGLEASGHSFIWVVRKVDGDDDVD VWLPDFEKKMKENNKGFIIRNWAPQLLILDHPAIGGLLNHSGWNSVLEGATAGLPMITWPLYAEHF YNERLVLDVLKIGVPVGVKEWKNLHEVGELVRRDAIAKAIKLLMGSGEEAEVMRKKAKELGVGAKK GIQVGGSSHTNLIAVIDELKSLKKSRIQGV* SEQ ID NO: 5-Q. saponaria QA-GlcpA-Galp dual β-1,3-D-xylosyltransferase/α-1,3-L- rhamnosyltransferase (Qs-3-O-RhaT/XylT) coding sequence (1515 bp) ATGGTCTCCGGCGACGACGATGTTTCTCGTCGGCCACTGAAAGTTTACTTCATTGCACACCCCTCA CCTGGCCATATTGCCCCTCTGACCAAAATAGCCCATCTCTTCGCTGCCCTCGGTGAGCACGTGACT ATTCTCACTACTCCCGCCAATGTCCACTTCCATGAGAAATCCATCGACAAAGGAAAGGCTTCCGGC TATCATGTTAACATCCACACCGTTAAATTTCCTTCTAAAGAGGTCGGTCTCCCTGACGGCATCGAA AACTTCTCTTACGCCTCCGATGTTGAAACAGCAGCTAAAATTTGGGCTGGATTCGCCATGCTACAA ACTGAAATGGAGCAATATATGGAGCTTAACCCACCCGATTGCATCGTTGCCGACATGTTCACCTCC TGGACCTCCGACTTTGCTATCAAATTGGGAATCACAAGAATCGTTTTCAACGTCTATTGTATTTTC ACACGCTGTTTGGAAGAAGCCATCCGATCACCGGACTCGCCACACTTGAACAAAGAAATCTCTGAT AATGAACCGTTTGTTATCCCGGGTCTACCAGACCCCATAACAATTACCCGAGCTCAACTGCCCGAC GGTACCTTTTCTCCCATGAAAGAACTAGCTAGAACAGCTGAGTTGAAGAGCTTTGGAATGGTGATC AACGGGTTTTCCGAACTCGAAACCGATTACATCGAGCATTACAAGAAAATCATGGGTCACAAACGG ATTTGGCATGTCGGACCCCTTCAGCTAATCCACCGTAACGATGAAGACAAAATTCAGAGGAGCCAC AAGACAGCGGTGCTGAGTGATAACGATAACGAGTTAGTGAGTTGGCTTAACTCGAAGAAACCCGAC TCAGTTATTTACATTTGCTTCGGTAGTGCAACTCGTTTCTCTAATCACCAGCTCTATGAAATCGCC TGTGGATTAGAAGCTTCCGGGCACCCATTTTTGTGGGGCCTACTTTGGGTGCCAGAAGATGAAGAT AACGATGACGTGGGCAACAAATGGTTGCCAGCTTTCGAAGAAAGAATTAAAAAGGAAAATAAGGGA ATGATTTTAAGGGGGTGGGCTCCACAGATGTTAATCTTAAACCACCCGGCGATCGGTGGTTTCATG ACGCATTGTGGTTGGAATGCGGTGGTGGAAGCACTTTCATTCGGTGTTCCGACTATTACGCTTCCA GTTTTCTCGGAGCAGTTTTATACTGAGAGACTGATATCACAAGTGCTCAAGACTGGTGTGGAGGTT GGTGCAGAGAAGTGGACCTATGCATTTGATGCGGGGAAATATCCGGTGAGTAGGGAAAAGATAGCG ACGGCGGTGAAGAAGATATTAGACGATGGAGAAGAGGCAGAAGGAATGAGAAAGCGGGCCAGGGAG ATGAAAGAAAAAGCCCAAAAAAGTGTTGAAGAAGGTGGATCCTCTTATAATAATTTAACGGCTATG ATTGAAGATCTTAAAGAATTTAGGGCTAACAATGGCAAGGCTGCACAAGATCATGAATCGTGA SEQ ID NO: 6-Q. saponaria QA-GlcpA-Galp dual β-1,3-D-xylosyltransferase/α-1,3-L- rhamnosyltransferase (Qs-3-O-RhaT/XylT) translated nucleotide sequence (504 aa): MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASG YHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTS WTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPD GTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSH KTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDED NDDVGNKWLPAFEERIKKENKGMILRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLP VFSEQFYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRARE MKEKAQKSVEEGGSSYNNLTAMIEDLKEFRANNGKAAQDHES* Part 2-Other biosynthetic enzymes: SEQ ID NO: 7-AsHMGR (Avena strigosa HMG-CoA reductase) coding sequence (1689bp): The full-length HMGR sequence is provided below. The 5’ region (underlined) can be removed to generate a truncated feedback-insensitive form (tHMGR). The sequence for tHMGR is also given separately below. ATGGCTGTGGAGGTTCACCGCCGGGCTCCCGCGCCCCATGGCCGGGGCACCGGGGAGAAGGGCCGC GTGCAGGCCGGGGACGCGCTGCCGCTGCCGATCCGCCACACCAACCTCATCTTCTCGGCGCTCTTC GCCGCCTCCCTCGCATACCTCATGCGCCGCTGGAGGGAGAAGATCCGCAACTCCACGCCGCTCCAC GTCGTGGGGCTCACCGAGATCTTCGCCATCTGCGGCCTCGTCGCCTCCCTCATCTACCTCCTCAGC TTCTTCGGCATCGCCTTCGTGCAGTCCGTCGTATCCAACAGCGACGACGAGGACGAGGACTTCCTC ATCGCGGCTGCAGCATCCCAGGCCCCCCCGCCGCCCTCCTCCAAGCCCGCGCCGCAGCAGTGCGCC CTGCTGCAGAGCGCCGGAGTCGCGCCCGAGAAAATGCCCGAGGAGGACGAGGAAATCGTCGCCGGG GTCGTCGCAGGGAAGATCCCCTCCTACGTGCTCGAGACCAGGCTAGGCGACTGCCGCAGGGCAGCC GGGATCCGCCGCGAGGCGCTGCGCCGGATCACCGGCAGGGAGATCGACGGCCTTCCCCTCGACGGC TTCGACTACGACTCGATTCTCGGACAGTGCTGCGAGATGCCCGTCGGGTACGTGCAGCTGCCGGTC GGCGTCGCGGGGCCGCTCGTCCTCGACGGCCGCCGCATATACGTCCCGATGGCCACCACGGAGGGC TGCCTAATCGCCAGCACCAACCGCGGATGCAAGGCCATTGCCGAGTCCGGAGGCGCATCCAGCGTC GTGTACCGCGACGGGATGACCCGCGCCCCCGTAGCCCGCTTCCCCTCCGCACGACGCGCCGCAGAG CTCAAGGGCTTCCTGGAGAATCCGGCCAACTACGACACCCTGTCCGTGGTCTTTAACAGATCAAGC AGATTTGCAAGGCTGCAGGGGGTCAAGTGCGCCATGGCTGGGAGGAACTTGTACATGAGGTTCACC TGCAGCACCGGGGATGCCATGGGGATGAACATGGTCTCCAAGGGCGTCCAAAATGTGCTCGACTAT CTGCAGGAGGACTTCCCTGACATGGACGTTGTCAGCATCTCAGGCAACTTTTGTTCCGACAAGAAA TCAGCTGCTGTAAACTGGATTGAAGGCCGTGGAAAGTCCGTGGTTTGTGAGGCAGTAATCAGAGAG GAAGTTGTCCACAAGGTTCTCAAGACCAACGTTCAGTCACTCGTGGAGTTGAATGTGATCAAGAAC CTTGCTGGCTCAGCAGTTGCTGGTGCTCTTGGGGGTTTCAACGCCCACGCAAGCAACATCGTAACG GCTATCTTCATTGCCACTGGTCAGGATCCTGCACAGAATGTGGAGAGCTCACAGTGTATCACTATG TTGGAAGCTGTAAATGATGGCAGAGACCTTCACATCTCCGTTACAATGCCATCTATCGAGGTGGGC ACAGTTGGTGGAGGCACGCAGCTGGCCTCACAGTCGGCCTGCTTGGACCTACTGGGCGTCAAAGGC GCCAACAGGGAATCTCCGGGGTCGAACGCTAGGCTGCTGGCCACGGTGGTGGCTGGTGCCGTCCTA GCTGGGGAGCTGTCCCTCATCTCCGCCCAAGCTGCCGGCCATCTGGTCCAGAGCCACATGAAATAC AACAGATCCAGC AAGGACATGTCCAAGATCGCCTGCTGA SEQ ID NO: 8-AsHMGR (Avena strigosa HMG-CoA reductase) translated nucleotide sequence (562 aa): MAVEVHRRAPAPHGRGTGEKGRVQAGDALPLPIRHTNLIFSALFAASLAYLMRRWREKIRNSTPLH VVGLTEIFAICGLVASLIYLLSFFGIAFVQSVVSNSDDEDEDFLIAAAASQAPPPPSSKPAPQQCA LLQSAGVAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDG FDYDSILGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSV VYRDGMTRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFT CSTGDAMGMNMVSKGVQNVLDYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSVVCEAVIRE EVVHKVLKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVESSQCITM LEAVNDGRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVL AGELSLISAQAAGHLVQSHMKYNRSSKDMSKIAC* SEQ ID NO: 9-AstHMGR (Avena strigosa truncated HMG-CoA reductase) coding sequence (1275 bp): ATGGCGCCCGAGAAAATGCCCGAGGAGGACGAGGAAATCGTCGCCGGGGTCGTCGCAGGGAAGATC CCCTCCTACGTGCTCGAGACCAGGCTAGGCGACTGCCGCAGGGCAGCCGGGATCCGCCGCGAGGCG CTGCGCCGGATCACCGGCAGGGAGATCGACGGCCTTCCCCTCGACGGCTTCGACTACGACTCGATT CTCGGACAGTGCTGCGAGATGCCCGTCGGGTACGTGCAGCTGCCGGTCGGCGTCGCGGGGCCGCTC GTCCTCGACGGCCGCCGCATATACGTCCCGATGGCCACCACGGAGGGCTGCCTAATCGCCAGCACC AACCGCGGATGCAAGGCCATTGCCGAGTCCGGAGGCGCATCCAGCGTCGTGTACCGCGACGGGATG ACCCGCGCCCCCGTAGCCCGCTTCCCCTCCGCACGACGCGCCGCAGAGCTCAAGGGCTTCCTGGAG AATCCGGCCAACTACGACACCCTGTCCGTGGTCTTTAACAGATCAAGCAGATTTGCAAGGCTGCAG GGGGTCAAGTGCGCCATGGCTGGGAGGAACTTGTACATGAGGTTCACCTGCAGCACCGGGGATGCC ATGGGGATGAACATGGTCTCCAAGGGCGTCCAAAATGTGCTCGACTATCTGCAGGAGGACTTCCCT GACATGGACGTTGTCAGCATCTCAGGCAACTTTTGTTCCGACAAGAAATCAGCTGCTGTAAACTGG ATTGAAGGCCGTGGAAAGTCCGTGGTTTGTGAGGCAGTAATCAGAGAGGAAGTTGTCCACAAGGTT CTCAAGACCAACGTTCAGTCACTCGTGGAGTTGAATGTGATCAAGAACCTTGCTGGCTCAGCAGTT GCTGGTGCTCTTGGGGGTTTCAACGCCCACGCAAGCAACATCGTAACGGCTATCTTCATTGCCACT GGTCAGGATCCTGCACAGAATGTGGAGAGCTCACAGTGTATCACTATGTTGGAAGCTGTAAATGAT GGCAGAGACCTTCACATCTCCGTTACAATGCCATCTATCGAGGTGGGCACAGTTGGTGGAGGCACG CAGCTGGCCTCACAGTCGGCCTGCTTGGACCTACTGGGCGTCAAAGGCGCCAACAGGGAATCTCCG GGGTCGAACGCTAGGCTGCTGGCCACGGTGGTGGCTGGTGCCGTCCTAGCTGGGGAGCTGTCCCTC ATCTCCGCCC AAGCTGCCGGCCATCTGGTCCAGAGCCACATGAAATAC AACAGATCCAGCAAGGAC ATGTCCAAGATCGCCTGCTGA SEQ ID NO: 10-AstHMGR (Avena strigosa truncated HMG-CoA reductase) translated nucleotide sequence (424 aa): MAPEKMPEEDEEIVAGVVAGKIPSYVLETRLGDCRRAAGIRREALRRITGREIDGLPLDGFDYDSI LGQCCEMPVGYVQLPVGVAGPLVLDGRRIYVPMATTEGCLIASTNRGCKAIAESGGASSVVYRDGM TRAPVARFPSARRAAELKGFLENPANYDTLSVVFNRSSRFARLQGVKCAMAGRNLYMRFTCSTGDA MGMNMVSKGVQNVLDYLQEDFPDMDVVSISGNFCSDKKSAAVNWIEGRGKSWCEAVIREEVVHKV LKTNVQSLVELNVIKNLAGSAVAGALGGFNAHASNIVTAIFIATGQDPAQNVESSQCITMLEAVND GRDLHISVTMPSIEVGTVGGGTQLASQSACLDLLGVKGANRESPGSNARLLATVVAGAVLAGELSL ISAQAAGHLVQSHMKYNRSSKDMSKIAC SEQ ID NO: 11-Q. saponaria β-amyrin synthase, QsbAS (OQHZ-2074321) coding sequence (2277 bp): ATGTGGAGGCTGAAGATAGCAGAAGGTGGTTCCGATCCATATCTGTTCAGCACAAACAACTTCGTG GGTCGCCAGACATGGGAGTTCGAACCGGAGGCCGGCACACCTGAGGAGCGAGCAGAGGTCGAAGCT GCCCGCCAAAACTTTTACAACAACCGTTACCAGGTCAAGCCCTGTGACGACCTCCTTTGGAGATAT CAGTTCCTGAGAGAGAAGAATTTCAAACAAACAATACCGCCTGTCAAGGTTGAAGATGGCCAAGAA ATTACTTATGAGATGGCCACAACCTCAATGCAGAGGGCGGCCCGTCACCTATCAGCCTTGCAGGCC AGCGATGGCCATTGGCCAGCTCAAATTGCTGGCCCCTTGTTCTTCATGCCACCCTTGGTCTTTTGT GTGTACATTACTGGGCATCTTAATACAGTATTCCCATCTGAACATCGCAAAGAAATCCTTCGTTAC ATGTACTATCACCAGAACGAAGATGGTGGGTGGGGACTGCACATAGAGGGTCACAGCACCATGTTT TGCACAGCACTCAACTACATTTGTATGCGTATCCTTGGGGAAGGACCAGAGGGGGGTCAAGACAAT GCTTGTGCCAGAGCACGAATGTGGATTCTTGATCATGGTGGTGTAACACATATTCCATCTTGGGGA AAGACCTGGCTTTCGATACTTGGTCTATTTGAGTGGTCTGGAAGCAATCCAATGCCTCCAGAGTTT TGGATCCTTCCTTCATTTCTTCCTATGCATCCAGCAAAAATGTGGTGCTATTGCCGGATGGTTTAC ATGCCCATGTCTTATTTATATGGGAAAAGGTTTGTTGGCCCAATCACGCCTCTCATTGTTCAGTTA AGAGAGGAAATACACACTCAAAATTACCATGAAATCAACTGGAAGTCAGTCCGCCATCTATGTGCA AAGGAGGATATCTACTATCCCCATCCACTCATCCAAGATTTGATTTGGGACAGTTTGTACATACTA ACGGAGCCTCTTCTCACTCGCTGGCCCTTGAACAAGTTGGTGCGGGAGAGGGCTCTCCAAGTAACA ATGAAGCATATCCACTATGAAGATGAAAATAGTCGATACATAACCATTGGATGTGTGGAAAAGGTG TTATGTATGCTTGCTTGTTGGGTTGATGATCCAAATGGAGATGCTTTCAAGAAGCACCTTGCTCGA GTCCCAGATTACGTATGGGTCTCTGAAGATGGAATTACTATGCAGAGTTTTGGTAGTCAAGAATGG GATGCTGGCTTTGCCGTCCAGGCTCTGCTTGCTTCTAATCTTACCGAGGAACTTGGCCCTGCTCTT GCCAAAGGACATGACTTCATAAAGCAATCTCAGGTTAAGGACAATCCTTCAGGTGACTTCAAAAGC ATGTATCGTCACATTTCTAGAGGATCATGGACCTTCTCTGACCAAGATCATGGATGGCAAGTTTCT GATTGCACTGCAGAAGGTCTGAAGTGTTGCCTGCTTTTGTCGATGTTGCCACCAGAAATTGTTGGT GAAAAAATGGAACCACAAAGGCTATTTGATTCTGTCAATGTGCTGCTCTCTCTACAGAGCAAAAAA GGTGGTTTAGCTGCCTGGGAGCCAGCAGGGGCGCAAGATTGGTTGGAATTACTCAATCCCACAGAA TTTTTTGCGGACATTGTCGTTGAGCATGAATATGTTGAATGTACTGGATCAGCAATTCAGGCATTA GTTTTGTTCAAGAAGCTGTATCCGGGGCACAGGAAAAAAGAGATTGACAGTTTCATTACAAATGCT GTCCGGTTCCTTGAGAATACACAAACGGCAGATGGCTCTTGGTATGGAAACTGGGGAGTTTGCTTC ACCTATGGTTGTTGGTTCGCACTGGGAGGGCTAGCAGCAGCTGGCAAGACTTACAACAACTGTCCT GCAATACGCAAAGCTGTTAATTTCCTACTTACAACACAAAGAGAAGACGGTGGTTGGGGAGAAAGC TATCTTTCAAGCCCAAAAAAGATATATGTACCCCTGGAAGGAAGCCGATCAAATGTGGTACATACT GCATGGGCTATGATGGGTCTAATTCATGCTGGGCAGGCTGAAAGAGACTCAACTCCTCTTCATCGT GCAGCAAAGTTGATCATCAATTATCAACTAGAAAATGGCGATTGGCCGCAACAGGAAATCACTGGA GTATTCATGAAAAACTGCATGTTACATTACCCTATGTACAGAAACATCTACCCAATGTGGGCTCTT GCAGAATACCGGAGGCGGGTTCCATTGCCTTAA SEQ ID NO: 12-QsbAS (OQHZ-2074321) translated nucleotide sequence (758 aa): MWRLKIAEGGSDPYLFSTNNFVGRQTWEFEPEAGTPEERAEVEAARQNFYNNRYQVKPCDDLLWRY QFLREKNFKQTIPPVKVEDGQEITYEMATTSMQRAARHLSALQASDGHWPAQIAGPLFFMPPLVFC VYITGHLNTVFPSEHRKEILRYMYYHQNEDGGWGLHIEGHSTMFCTALNYICMRILGEGPEGGQDN ACARARMWILDHGGVTHIPSWGKTWLSILGLFEWSGSNPMPPEFWILPSFLPMHPAKMWCYCRMVY MPMSYLYGKRFVGPITPLIVQLREEIHTQNYHEINWKSVRHLCAKEDIYYPHPLIQDLIWDSLYIL TEPLLTRWPLNKLVRERALQVTMKHIHYEDENSRYITIGCVEKVLCMLACWVDDPNGDAFKKHLAR VPDYVWVSEDGITMQSFGSQEWDAGFAVQALLASNLTEELGPALAKGHDFIKQSQVKDNPSGDFKS MYRHISRGSWTFSDQDHGWQVSDCTAEGLKCCLLLSMLPPEIVGEKMEPQRLFDSVNVLLSLQSKK GGLAAWEPAGAQDWLELLNPTEFFADIVVEHEYVECTGSAIQALVLFKKLYPGHRKKEIDSFITNA VRFLENTQTADGSWYGNWGVCFTYGCWFALGGLAAAGKTYNNCPAIRKAVNFLLTTQREDGGWGES YLSSPKKIYVPLEGSRSNVVHTAWAMMGLIHAGQAERDSTPLHRAAKLIINYQLENGDWPQQEITG VFMKNCMLHYPMYRNIYPMWALAEYRRRVPLP* SEQ ID NO: 13-QsCYP716-C-28 (OQHZ-2073932) (C-28 oxidase, named previously as CYP716A224 [24]) coding sequence (1443 bp): ATGGAGCACTTGTATCTCTCCCTTGTGCTCCTGTTTGTTTCCTCAATCTCCCTCTCCCTCTTCTTC CTGTTCTACAAACACAAATCTATGTTCACCGGGGCCAACCTACCACCTGGTAAAATCGGTTACCCA TTGATCGGAGAGAGCTTGGAGTTCTTGTCCACGGGATGGAAGGGCCACCCGGAGAAATTCATCTTC GATCGCATGAGCAAGTACTCATCCCAAATCTTCAAGACCTCGATTTTAGGGGAACCAACGGCGGTG TTCCCGGGAGCCGTATGCAACAAGTTCCTCTTCTCCAACGAGAACAAGCTGGTGAATGCATGGTGG CCTGCCTCCGTGGACAAGATCTTTCCTTCCTCACTCCAGACATCCTCCAAAGAAGAGGCCAAGAAG ATGAGGAAGTTGCTTCCTCAGTTTCTCAAGCCCGAAGCTCTGCACCGCTACATTGGTATTATGGAT TCTATTGCCCAGAGACACTTTGCCGATAGCTGGGAAAACAAAAACCAAGTCATTGTCTTTCCTCTA GCAAAGAGGTATACTTTCTGGCTGGCTTGCCGTTTGTTCATTAGCGTCGAGGATCCGACCCACGTA TCCAGATTTGCTGACCCGTTCCAACTTTTGGCCGCCGGAATCATATCAATCCCAATCGACTTGCCA GGGACACCGTTCCGCAAGGCAATCAATGCGTCCCAGTTCATCAGGAAGGAATTGTTGGCCATCATC AGGCAGAGAAAGATCGATTTGGGTGAAGGGAAGGCATCTCCGACGCAGGACATACTGTCTCACATG TTGCTCACATGCGACGAGAACGGACAATACATGAATGAATTGGACATTGCCGACAAGATTCTTGGC TTGTTGGTCGGCGGACATGACACTGCCAGTGCCGCTTGCACTTTCATTGTCAAGTTCCTCGCTGAG CTTCCCCACATTTATGAACAAGTCTACAAGGAGCAAATGGAGATTGCAAAATCAAAAGTGCCAGGA GAGTTGTTGAATTGGGAGGACATCCAAAAGATGAAATATTCGTGGAACGTAGCTTGTGAAGTGATG AGACTTGCCCCTCCACTCCAAGGAGCTTTCAGGGAAGCCATTACTGACTTCGTCTTCAACGGTTTC TCCATTCCAAAAGGCTGGAAGTTGTACTGGAGCGCAAATTCCACCCACAAAAGTCCGGATTATTTC CCTGAGCCCGACAAGTTCGACCCAACTAGATTCGAAGGAAATGGACCTGCGCCTTACACCTTTGTT CCATTTGGGGGAGGACCCAGGATGTGCCCGGGCAAAGAGTATGCCCGATTGGAAATACTTGTGTTC ATGCATAACTTGGTGAAGAGGTTCAAGTGGGAGAAATTGGTTCCTGATGAAAAGATTGTGGTTGAT CCAATGCCCATTCCAGCAAAGGGTCTTCCTGTTCGCCTTTATCCTCACAAAGCTTGA SEQ ID NO: 14-QsCYP716-C-28 (OQHZ-2073932) translated nucleotide sequence (480 aa): MEHLYLSLVLLFVSSISLSLFFLFYKHKSMFTGANLPPGKIGYPLIGESLEFLSTGWKGHPEKFIF DRMSKYSSQIFKTSILGEPTAVFPGAVCNKFLFSNENKLVNAWWPASVDKIFPSSLQTSSKEEAKK MRKLLPQFLKPEALHRYIGIMDSIAQRHFADSWENKNQVIVFPLAKRYTFWLACRLFISVEDPTHV SRFADPFQLLAAGIISIPIDLPGTPFRKAINASQFIRKELLAIIRQRKIDLGEGKASPTQDILSHM LLTCDENGQYMNELDIADKILGLLVGGHDTASAACTFIVKFLAELPHIYEQVYKEQMEIAKSKVPG ELLNWEDIQKMKYSWNVACEVMRLAPPLQGAFREAITDFVFNGFSIPKGWKLYWSANSTHKSPDYF PEPDKFDPTRFEGNGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKLVPDEKIVVD PMPIPAKGLPVRLYPHKA* SEQ ID NO: 15-QsCYP716-C-16a (OQHZ-2012090) (C-16α oxidase) coding sequence (1506 bp/1443 bp): Long and short isoforms as described herein are distinguished by the presence of the first 63 nucleotides, underlined in the sequences below (21 amino acids). ATGATATATAATAATGATAGTAATGATAATGAATTAGTAATCAGCTCAGTTCAGCAACCATCCATG GATCCTTTCTTCATTTTTGGCTTACTTCTCTTGGCTCTCTTTCTCTCTGTTTCTTTTCTTCTCTAC CTTTCCCGTAGAGCCTATGCTTCTCTCCCCAACCCTCCGCCGGGGAAGCTCGGCTTCCCCGTCGTC GGCGAGAGTCTCGAATTTCTCTCCACCCGACGCAAAGGTGTTCCTGAGAAATTCGTCTTCGACAGA ATGGCCAAATACTGTCGGGATGTCTTTAAGACATCAATATTGGGAGCAACCACCGCCGTCATGTGC GGCACCGCCGGTAACAAATTCTTGTTCTCCAACGAGAAAAAACACGTCACTGGTTGGTGGCCGAAA TCTGTAGAGCTGATTTTCCCAACCTCACTTGAGAAATCATCCAACGAAGAATCCATCATGATGAAA CAATTCCTTCCCAACTTCTTGAAACCAGAACCTTTGCAGAAGTACATACCCGTTATGGACATAATT ACCCAAAGACACTTCAATACAAGCTGGGAAGGACGCAACGTGGTCAAAGTGTTTCCTACGGCTGCC GAATTCACCACGTTGCTGGCTTGTCGGGTATTCCTCAGTGTTGAGGATCCCATTGAAGTAGCCAAG ATTTCAGAGCCATTTGAAATCTTAGCTGCTGGGTTTCTTTCAATACCCATAAATCTTCCGGGTACC AAATTAAATAAAGCGGTTAAGGCAGCGGATCAGATTAGAGACGCAATTGTACAGATTTTGAAACGG AGAAGGGTTGAAATTGCGGAGAATAAAGCAAATGGAATGCAAGATATAGCGTCCATGTTGTTGACG ACACCAACTAATGCTGGGTTTTATATGACCGAGGCTCACATTTCTGAGAAAATTTTGGGTATGATT GTTGGTGGCCGTGATACTGCTAGTACTGTTATCACCTTCATCATCAAGTATTTGGCAGAGAATCCT GAAATTTATAATAAGGTCTATGAGGAGCAAATGGAAGTGGTAAAGTCAAAGAAACCAGGTGAGTTG CTGAACTGGGAAGATGTGCAGAAAATGAAGTACTCTTGGTGCGTAGCATGTGAAGCTATGCGACTT GCTCCTCCTGTTCAAGGTGGTTTCAAGGTGGCCATTAATGACTTTGTGTATTCTGGGTTCAACATT CGCAAGGGTTGGAAGTTATATTGGAGTGCCATTGCAACACACATGAATCCAGAATATTTCCCAGAA CCTGAGAAATTCAACCCCTCAAGGTTTGAAGGGAAGGGACCAGTACCTTACAGCTTCGTACCCTTC GGAGGCGGACCTCGGATGTGTCCCGGGAAAGAGTATTCCCGGCTGGAAACACTTGTTTTCATGCAT CATTTGGTGACGAGGTACAATTGGGAGAAAGTGTATCCCACAGAGAAGATAACAGTGGATCCAATG CCATTCCCTGTCAACGGCCTCCCCATTCGCCTTATTCCTCACAAGCACCAATGA SEQ ID NO: 16-QsCYP716-C-16α translated nucleotide sequence (501 aa/480 aa): MIYNNDSNDNELVISSVQQPSMDPFFIFGLLLLALFLSVSFLLYLSRRAYASLPNPPPGKLGFPVV GESLEFLSTRRKGVPEKFVFDRMAKYCRDVFKTSILGATTAVMCGTAGNKFLFSNEKKHVTGWWPK SVELIFPTSLEKSSNEESIMMKQFLPNFLKPEPLQKYIPVMDIITQRHFNTSWEGRNVVKVFPTAA EFTTLLACRVFLSVEDPIEVAKISEPFEILAAGFLSIPINLPGTKLNKAVKAADQIRDAIVQILKR RRVEIAENKANGMQDIASMLLTTPTNAGFYMTEAHISEKILGMIVGGRDTASTVITFIIKYLAENP EIYNKVYEEQMEVVKSKKPGELLNWEDVQKMKYSWCVACEAMRLAPPVQGGFKVAINDFVYSGFNI RKGWKLYWSAIATHMNPEYFPEPEKFNPSRFEGKGPVPYSFVPFGGGPRMCPGKEYSRLETLVFMH HLVTRYNWEKVYPTEKITVDPMPFPVNGLPIRLIPHKHQ* SEQ ID NO: 17-QsCYP714-C-23 (C-23 oxidase) coding sequence (1524 bp): ATGTGGTTCACAGTAGGATTGGTCTTGGTTTTCGCCCTATTCATACGTCTCTACAGCAGTCTGTGG TTGAAGCCTCGTGCAACTCGGATTAAGCTTAGCAATCAAGGAATTAAAGGTCCAAAACCAGCATTT CTTCTGGGTAATGTTGCAGAGATGAGAAGATTTCAATCTAAGCTTCCAAAATCTGAACTCAAACAA GGCCAAGTTTCTCATGATTGGGCTTCTAAATCTCTGTTTCCATTTTTCAGTCTTTGGTCCCAGAAA TACGGAAATACGTTCGTGTTCTCATTGGGGAACATACAGGTGCTCTATGTTTCTGATCATGAGTTG GTGAAAGAAATTAATCAGAATACCTCTTTAGATTTGGGCAAACCCAAGTACCTGCAGAAGGAGCGT GGCCCTTTGCTGGGACAAGGTATTTTGACCTCCAATGGACAGCTTTGGGCGTACCAGAGAAAAATC ATGACTCCTGAACTCTACAAGGAGAAAATCAAGGGCATGTGCGAGTTGATGGTGGAATCTGTAGCT TGGTTGGTTGAGGAATGGGGAACGAAGATCCAAGCTGAGGGTGGGGCAGCAGACATTAGAATAGAC GAGGATCTTAGAAGCTTCTCTGGTGATGTAATTTCAAAAGCTTGTTTTGGGAGCTGCTATGCCGGA GGGAGGGAAATCTTTCTTAGGCTCAGAGCTCTTCAACACCAAATTGCTTCCAAAGCCTTACTCATG GGCTTCCCTGGATTAAAGTACCTGCCCATTAAGAGCAACAGAGAGATATGGAGATTGGAGAAGGAG ATCTTCCAGCTGATTATGAAGCTGGCTGAAGATAGAAAAAAAGAACAACATGAGAGAGACCTATTA CAGATTATAATTGAGGGAGCTAAAAGTAGTGATCTGAGTTCGGAAGCAATGGCAAAATTCATTGTG GACAACTGCAAGAATGTCTACTTGGCTGGCCATGAAACTACTGCAATGTCTGCTGGTTGGACTTTG CTTCTCTTGGCTAATCATCCTGAGTGGCAAGCCCGTGTCCGTGATGAGATTTTACAAGTCACCGAG GGCCGCAATCCTGATTTTGACATGCTGCACAAGATGAAACTGTTAACAATGGTAATTCAGGAGGCA CTGCGACTCTACCCAACAGTCATATTCATGTCAAGAGAAGCATTGGAAGATATTAATGTTGGAAAC ATCCAAGTTCCAAAAGGTGTTAACATATGGATACCTGTGGTAAATCTTCAAAGGGACACAACGGTA TGGGGTGCAGACGCAAACGAGTTTAATCCTGAAAGGTTTGCCAATGGAGTTAACAATTCATGCAAG GTTCCACAACTTTACCTACCATTTGGAGCTGGACCTCGCATTTGTCCTGGAATTAATCTGGCCATG ACTGAGATCAAGATACTTCTGTGTATCCTGCTCACCAAGTTTTCGTTTTCAGTTTCACCCAACTAT CGCCACTCACCGGTGTTTAAATTGGTGCTTGAGCCTGAAAATGGAATCAATGTCATCATGAAGAAG CTCTAA SEQ ID NO: 18-QsCYP714-C-23 translated nucleotide sequence (507 aa): MWFTVGLVLVFALFIRLYSSLWLKPRATRIKLSNQGIKGPKPAFLLGNVAEMRRFQSKLPKSELKQ GQVSHDWASKSLFPFFSLWSQKYGNTFVFSLGNIQVLYVSDHELVKEINQNTSLDLGKPKYLQKER GPLLGQGILTSNGQLWAYQRKIMTPELYKEKIKGMCELMVESVAWLVEEWGTKIQAEGGAADIRID EDLRSFSGDVISKACFGSCYAGGREIFLRLRALQHQIASKALLMGFPGLKYLPIKSNREIWRLEKE IFQLIMKLAEDRKKEQHERDLLQIIIEGAKSSDLSSEAMAKFIVDNCKNVYLAGHETTAMSAGWTL LLLANHPEWQARVRDEILQVTEGRNPDFDMLHKMKLLTMVIQEALRLYPTVIFMSREALEDINVGN IQVPKGVNIWIPVVNLQRDTTVWGADANEFNPERFANGVNNSCKVPQLYLPFGAGPRICPGINLAM TEIKILLCILLTKFSFSVSPNYRHSPVFKLVLEPENGINVIMKKL* SEQ ID NO: 19-GmSGT2 (GmUGT73P2) (Glycine max (soybean) β-D- galactosyltransferase) coding sequence (1488bp): ATGTGGTTCACAGTAGGATTGGTCTTGGTTTTCGCCCTATTCATACGTCTCTACAGCAGTCTGTGG TTGAAGCCTCGTGCAACTCGGATTAAGCTTAGCAATCAAGGAATTAAAGGTCCAAAACCAGCATTT CTTCTGGGTAATGTTGCAGAGATGAGAAGATTTCAATCTAAGCTTCCAAAATCTGAACTCAAACAA GGCCAAGTTTCTCATGATTGGGCTTCTAAATCTCTGTTTCCATTTTTCAGTCTTTGGTCCCAGAAA TACGGAAATACGTTCGTGTTCTCATTGGGGAACATACAGGTGCTCTATGTTTCTGATCATGAGTTG GTGAAAGAAATTAATCAGAATACCTCTTTAGATTTGGGCAAACCCAAGTACCTGCAGAAGGAGCGT GGCCCTTTGCTGGGACAAGGTATTTTGACCTCCAATGGACAGCTTTGGGCGTACCAGAGAAAAATC ATGACTCCTGAACTCTACAAGGAGAAAATCAAGGGCATGTGCGAGTTGATGGTGGAATCTGTAGCT TGGTTGGTTGAGGAATGGGGAACGAAGATCCAAGCTGAGGGTGGGGCAGCAGACATTAGAATAGAC GAGGATCTTAGAAGCTTCTCTGGTGATGTAATTTCAAAAGCTTGTTTTGGGAGCTGCTATGCCGGA GGGAGGGAAATCTTTCTTAGGCTCAGAGCTCTTCAACACCAAATTGCTTCCAAAGCCTTACTCATG GGCTTCCCTGGATTAAAGTACCTGCCCATTAAGAGCAACAGAGAGATATGGAGATTGGAGAAGGAG ATCTTCCAGCTGATTATGAAGCTGGCTGAAGATAGAAAAAAAGAACAACATGAGAGAGACCTATTA CAGATTATAATTGAGGGAGCTAAAAGTAGTGATCTGAGTTCGGAAGCAATGGCAAAATTCATTGTG GACAACTGCAAGAATGTCTACTTGGCTGGCCATGAAACTACTGCAATGTCTGCTGGTTGGACTTTG CTTCTCTTGGCTAATCATCCTGAGTGGCAAGCCCGTGTCCGTGATGAGATTTTACAAGTCACCGAG GGCCGCAATCCTGATTTTGACATGCTGCACAAGATGAAACTGTTAACAATGGTAATTCAGGAGGCA CTGCGACTCTACCCAACAGTCATATTCATGTCAAGAGAAGCATTGGAAGATATTAATGTTGGAAAC ATCCAAGTTCCAAAAGGTGTTAACATATGGATACCTGTGGTAAATCTTCAAAGGGACACAACGGTA TGGGGTGCAGACGCAAACGAGTTTAATCCTGAAAGGTTTGCCAATGGAGTTAACAATTCATGCAAG GTTCCACAACTTTACCTACCATTTGGAGCTGGACCTCGCATTTGTCCTGGAATTAATCTGGCCATG ACTGAGATCAAGATACTTCTGTGTATCCTGCTCACCAAGTTTTCGTTTTCAGTTTCACCCAACTAT CGCCACTCACCGGTGTTTAAATTGGTGCTTGAGCCTGAAAATGGAATCAATGTCATCATGAAGAAG CTCTAA SEQ ID NO: 20-GmSGT2 (GmUGT73P2) (Glycine max (soybean) β-D- galactosyltransferase) translated nucleotide sequence (495 aa): MEKKKGELKSIFLPFLSTSHIIPLVDMARLFALHDVDVTIITTAHNATVFQKSIDLDASRGRPIRT HVVNFPAAQVGLPVGIEAFNVDTPREMTPRIYMGLSLLQQVFEKLFHDLQPDFIVTDMFHPWSVDA AAKLGIPRIMFHGASYLARSAAHSVEQYAPHLEAKFDTDKFVLPGLPDNLEMTRLQLPDWLRSPNQ YTELMRTIKQSEKKSYGSLFNSFYDLESAYYEHYKSIMGTKSWGIGPVSLWANQDAQDKAARGYAK EEEEKEGWLKWLNSKAESSVLYVSFGSINKFPYSQLVEIARALEDSGHDFIWVVRKNDGGEGDNFL EEFEKRMKESNKGYLIWGWAPQLLILENPAIGGLVTHCGWNTVVESVNAGLPMATWPLFAEHFFNE KLVVDVLKIGVPVGAKEWRNWNEFGSEVVKREEIGNAIASLMSEEEEDGGMRKRAKELSVAAKSAI KVGGSSHNNMKELIRELKEIKLSKEAQETAPNP* SEQ ID NO: 21-AsSQS (Avena strigosa squalene synthase) coding sequence (1212 bp): ATGGGGGCGCTGTCGCGGCCGGAGGAGGTGGTGGCGCTGGTCAAGCTGAGGGTGGCGGCGGGGCAG ATCAAGCGCCAGATCCCGGCCGAGGAACACTGGGCCTTCGCCTACGACATGCTCCAGAAGGTCTCC CGCAGCTTCGCGCTCGTCATCCAGCAGCTCGGACCCGAACTCCGCAATGCCGTGTGCATCTTCTAC CTCGTGCTCCGGGCCCTGGACACCGTCGAGGACGACACCAGCATCCCCAACGACGTGAAGCTGCCC ATCCTTCGGGATTTCTACCGCCATGTCTACAACCCCGACTGGCGTTATTCATGTGGAACAAACCAC TACAAGGTGCTGATGGATAAGTTCAGACTCGTCTCCACGGCTTTCCTGGAGCTAGGCGAAGGATAT CAAAAGGCAATTGAAGAAATCACTAGGCGAATGGGAGCAGGAATGGCAAAATTTATATGCCAGGAG GTTGAAACGATTGATGACTATAATGAGTACTGCCACTATGTAGCAGGGCTAGTAGGCTATGGACTT TCCAGGCTCTTTCATGCTGCTGGGACAGAAGATCTGGCTTCAGATCAACTTTCGAATTCAATGGGT TTGTTTCTTCAGAAAACCAATATAATAAGGGATTATTTGGAGGATATAAATGAGATACCAAAGTGC CGTATGTTTTGGCCTCGAGAAATATGGAGTAAATATGCAGATAAACTTGAGGACCTCAAGTATGAG GAAAATTCAGAAAAAGCAGTGCAATGCTTGAATGATATGGTGACTAATGCTTTGGTCCACGCCGAA GACTGTCTTCAATACATGTCTGCGTTGAAGGATAATACTAATTTTCGGTTTTGTGCAATACCTCAG ATAATGGCAATTGGGACATGTGCTATTTGCTACAATAATGTGAAAGTCTTTAGAGGAGTTGTTAAG ATGAGGCGTGGGCTCACTGCACGAATAATTGATGAGACAAAATCAATGTCAGATGTCTATTCTGCT TTCTATGAGTTCTCTTCATTGCTAGAGTCAAAGATTGACGATAACGACCCAAGTTCTGCACTAACA CGGAAGCGTGTAGAGGCAATAAAGAGGACTTGCAAGTCATCCGGTTTACTAAAGAGAAGGGGATAC GACCTGGAAAAGTCAAAGTATAGGCATATGTTGATCATGCTTGCACTTCTGTTGGTGGCTATTATC TTCGGTGTACTGTACGCCAAGTGA SEQ ID NO: 22-AsSQS (Avena strigosa squalene synthase) translated nucleotide sequence (403 aa): MGALSRPEEVVALVKLRVAAGQIKRQIPAEEHWAFAYDMLQKVSRSFALVIQQLGPELRNAVCIFY LVLRALDTVEDDTSIPNDVKLPILRDFYRHVYNPDWRYSCGTNHYKVLMDKFRLVSTAFLELGEGY QKAIEEITRRMGAGMAKFICQEVETIDDYNEYCHYVAGLVGYGLSRLFHAAGTEDLASDQLSNSMG LFLQKTNIIRDYLEDINEIPKCRMFWPREIWSKYADKLEDLKYEENSEKAVQCLNDMVTNALVHAE DCLQYMSALKDNTNFRFCAIPQIMAIGTCAICYNNVKVFRGVVKMRRGLTARIIDETKSMSDVYSA FYEFSSLLESKIDDNDPSSALTRKRVEAIKRTCKSSGLLKRRGYDLEKSKYRHMLIMLALLLVAII FGVLYAK* SEQ ID NO: 23-AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2) coding sequence (2325 bp): ATGAAAAACATGATGAATTATAAATTAAAACTCTGTTCTGTCTCAAAAAACTCAAAAGGAGTCTCT CTCTCACCTACACCACACCTAACCAAACCCCCTACGATTCACACAGAGAGAGATCTTCTTCTTCCT TCTTCTTCCTTCTTCTTTCTTCTTCTTTCTTCTTCTAGCTACAACATCTACAACGCCATGTCCTCT TCTTCTTCTTCGTCAACCTCCATGATCGATCTCATGGCAGCAATCATCAAAGGAGAGCCTGTAATT GTCTCCGACCCAGCTAATGCCTCCGCTTACGAGTCCGTAGCTGCTGAATTATCCTCTATGCTTATA GAGAATCGTCAATTCGCCATGATTGTTACCACTTCCATTGCTGTTCTTATTGGTTGCATCGTTATG CTCGTTTGGAGGAGATCCGGTTCTGGGAATTCAAAACGTGTCGAGCCTCTTAAGCCTTTGGTTATT AAGCCTCGTGAGGAAGAGATTGATGATGGGCGTAAGAAAGTTACCATCTTTTTCGGTACACAAACT GGTACTGCTGAAGGTTTTGCAAAGGCTTTAGGAGAAGAAGCTAAAGCAAGATATGAAAAGACCAGA TTCAAAATCGTTGATTTGGATGATTACGCGGCTGATGATGATGAGTATGAGGAGAAATTGAAGAAA GAGGATGTGGCTTTCTTCTTCTTAGCCACATATGGAGATGGTGAGCCTACCGACAATGCAGCGAGA TTCTACAAATGGTTCACCGAGGGGAATGACAGAGGAGAATGGCTTAAGAACTTGAAGTATGGAGTG TTTGGATTAGGAAACAGACAATATGAGCATTTTAATAAGGTTGCCAAAGTTGTAGATGACATTCTT GTCGAACAAGGTGCACAGCGTCTTGTACAAGTTGGTCTTGGAGATGATGACCAGTGTATTGAAGAT GACTTTACCGCTTGGCGAGAAGCATTGTGGCCCGAGCTTGATACAATACTGAGGGAAGAAGGGGAT ACAGCTGTTGCCACACCATACACTGCAGCTGTGTTAGAATACAGAGTTTCTATTCACGACTCTGAA GATGCCAAATTCAATGATATAAACATGGCAAATGGGAATGGTTACACTGTGTTTGATGCTCAACAT CCTTACAAAGCAAATGTCGCTGTTAAAAGGGAGCTTCATACTCCCGAGTCTGATCGTTCTTGTATC CATTTGGAATTTGACATTGCTGGAAGTGGACTTACGTATGAAACTGGAGATCATGTTGGTGTACTT TGTGATAACTTAAGTGAAACTGTAGATGAAGCTCTTAGATTGCTGGATATGTCACCTGATACTTAT TTCTCACTTCACGCTGAAAAAGAAGACGGCACACCAATCAGCAGCTCACTGCCTCCTCCCTTCCCA CCTTGCAACTTGAGAACAGCGCTTACACGATATGCATGTCTTTTGAGTTCTCCAAAGAAGTCTGCT TTAGTTGCGTTGGCTGCTCATGCATCTGATCCTACCGAAGCAGAACGATTAAAACACCTTGCTTCA CCTGCTGGAAAGGATGAATATTCAAAGTGGGTAGTAGAGAGTCAAAGAAGTCTACTTGAGGTGATG GCCGAGTTTCCTTCAGCCAAGCCACCACTTGGTGTCTTCTTCGCTGGAGTTGCTCCAAGGTTGCAG CCTAGGTTCTATTCGATATCATCATCGCCCAAGATTGCTGAAACTAGAATTCACGTCACATGTGCA CTGGTTTATGAGAAAATGCCAACTGGCAGGATTCATAAGGGAGTGTGTTCCACTTGGATGAAGAAT GCTGTGCCTTACGAGAAGAGTGAAAACTGTTCCTCGGCGCCGATATTTGTTAGGCAATCCAACTTC AAGCTTCCTTCTGATTCTAAGGTACCGATCATCATGATCGGTCCAGGGACTGGATTAGCTCCATTC AGAGGATTCCTTCAGGAAAGACTAGCGTTGGTAGAATCTGGTGTTGAACTTGGGCCATCAGTTTTG TTCTTTGGATGCAGAAACCGTAGAATGGATTTCATCTACGAGGAAGAGCTCCAGCGATTTGTTGAG AGTGGTGCTCTCGCAGAGCTAAGTGTCGCCTTCTCTCGTGAAGGACCCACCAAAGAATACGTACAG CACAAGATGATGGACAAGGCTTCTGATATCTGGAATATGATCTCTCAAGGAGCTTATTTATATGTT TGTGGTGACGCCAAAGGCATGGCAAGAGATGTTCACAGATCTCTCCACACAATAGCTCAAGAACAG GGGTCAATGGATTCAACTAAAGCAGAGGGCTTCGTGAAGAATCTGCAAACGAGTGGAAGATATCTT AGAGATGTATGGTAA SEQ ID NO: 24-AtATR2 (Arabidopsis thaliana cytochrome P450 reductase 2) translated nucleotide sequence (774 aa): MKNMMNYKLKLCSVSKNSKGVSLSPTPHLTKPPTIHTERDLLLPSSSFFFLLLSSSSYNIYNAMSS SSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIVM LVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTR FKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGV FGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGD TAVATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCI HLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFP PCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVM AEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKN AVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVL FFGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYV CGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW* SEQ ID NO: 25-Q. saponaria quillaic acid 3-O-glucuronosyltransferase (cellulose synthase-like enzyme QsCslG2) coding sequence (2124 bp): ATGGCGACCGTCTCCTCCCTCCACACTTGCACTGTACAGCAACCCCGTGCAGCCATTAATCGAATT CACATTTTCTTACACTTTATTGCCATACTTTTCCTCTTTTACTACCGGGTCACCGGTCTTTTCTAT GACAATGCAGTACCCACTTTAGCTTGGTCTCTAATGACCTTAGCTGAGTTGATTTTCGCCTTCGTT TGGGTGCTCAGCCAAGCCTTCCGGTGGCGCCCGGTGTTGCGTTCAGTTATTCCTGAGAGGATTCCC AAAGATGTACGATTGCCCGCGGTGGATATCTTAATTTGTACGGCTGACCCATTAAAGGAACCGACG GTGGAGGTGATGAACACTGTCTTGTCCGCCATGGCATTGGACTATCCTGCGGAGAATCTGGCTGTA TATCTTTCTGATGACGGGGGTTCTCCGGTCACCTTATTTGCTATGAAGCAAGTGGGTCCGTTTGCT AAGCTGTGGCTTCCGTTTTGCAACAAGTACGGAATCAAAACAAGGCATCCTGAGTCTTTTTTCTCG GCATTTGCGGATGACGAAAGGCTTCACCGGAGTGATGAATTCAGGGCAGAGGAGGAGGCGATCAAG GACAAATATGAAGAATTTAAGAGAACTATAGAGAAATATGGTGGAGAAGGAAAAAATAGTCATGTT GTACAAGACCGGCCTCCTCATGTGGAGATTATACATGACACTAGGAAGATTAGAGAGAACAGTGAA GACCAAGCTGTGCCTCTTCTTGTCTACGTCTCTCGTGAGAAAAGACCATCCTACAATTCTCGGTTC AAAGCAGGAGCTCTGAACACCCTTCTTCGAGTTTCTGGGGTAATCAGCAATAGCCCATATGTATTG GTGTTAGACTGTGACATGTACTGCAATGATCCAACATCAGCTAGACAAGCAATGTGCTTCCATCTT GATCCACAAATGTCTCGCACTCTCTCTTTTGTACAATTCCCCCAGGTTTTCTACAATGTTAGTAAA AATGATATCTATGATGGCCAAGCTAGGGCAGCCTTTAAGACAAAGTGGCAAGGTATGGATGGACTA CGTGGGCCACTGCTTTCTGGTACTGGCTTTTATTTGAAGAGGAAGTCCTTGTATGGAAGTCCAAAC CAAGAAGATGATTGTTTACTTGAGCCCCATAAGAATTTTGGAAAGTGTGACAAGCTCATAGAATCA GTAAAGGTCATTTATGAACGTGATGTTTCAATAAAGGCAGATTCATCAGATGCCATTTTGCAAGAT GCCAAACAATTAGCATCTTGTCCCTATGAAACAAACACAAGCTGGGGCAAAGAGGTTGGGTTCTCG TATGACTGCTTATTAGAGAGTACATTCACAGGTTATCTGTTGCACTGCAGAGGGTGGACATCAGTT TATCTTTATCCAAAGAAGCCATGTTTCTTAGGGTGTACTCCAGTTGATATGAAGGAAGCCATGGTT CAGTATACGAAGTGGATTTCTGAATTATTTTTACTTGCTATCTCAAGATTCAACCCTCTGACATTT GGGATATCCAGAATGTCCATTCTCCAGAGCATGTGTTACGGATACCTTACAATCATGCCCATTTTA TCTGTTGCTATGATCTTCTATGCCACAGTTCCTCAATTGTGCCTCTTGAGAGGCGTACCTCTGTTT CCCAAGGTTTCAGACCCATGGTTTGCAGTGTTCCTAGCAATATTTGTGTCCTCCCTCTGTCAGCAC TTAATTGAAGTCCTCACGAGTGATGGCACGCTCAAGACTTGGTGGAATGAACAAAGAAATTGGGTG ATAAAGTCTGGTTCCGGTAGCGTATTTGGAGCTCTGAGTGGAATATTGAAGTGGTTTGGCATGAAG ATTAAATTTGGTTTATCAAACAAAGCCGTGGACAAAGAAAAGCTTGAGAAATATGAAAAGGGTAAG TTTGATTTCCAAGGGGCTGCCATGTTTATGGTTCCCTTAACTATATCAGTCATCTTGAACACATTA TGCCTTATCGGTGGTTTATGGAGAGTAATCACACTTAAAAACTTCGAAGAGATGTCAGGGCAGTTC ATCATCTCCTTGTACTTTCTAGCTCTCAGCTATCCAATTCTTGAAGGGTTACTAAGAAAAGGCAAG GGAAAGGCCTAA SEQ ID NO: 26-Q. saponaria quillaic acid 3-O-glucuronosyltransferase (cellulose synthase-like enzyme QsCslG2) translated nucleotide sequence (707 aa): MATVSSLHTCTVQQPRAAINRIHIFLHFIAILFLFYYRVTGLFYDNAVPTLAWSLMTLAELIFAFV WVLSQAFRWRPVLRSVIPERIPKDVRLPAVDILICTADPLKEPTVEVMNTVLSAMALDYPAENLAV YLSDDGGSPVTLFAMKQVGPFAKLWLPFCNKYGIKTRHPESFFSAFADDERLHRSDEFRAEEEAIK DKYEEFKRTIEKYGGEGKNSHVVQDRPPHVEIIHDTRKIRENSEDQAVPLLVYVSREKRPSYNSRF KAGALNTLLRVSGVISNSPYVLVLDCDMYCNDPTSARQAMCFHLDPQMSRTLSFVQFPQVFYNVSK NDIYDGQARAAFKTKWQGMDGLRGPLLSGTGFYLKRKSLYGSPNQEDDCLLEPHKNFGKCDKLIES VKVIYERDVSIKADSSDAILQDAKQLASCPYETNTSWGKEVGFSYDCLLESTFTGYLLHCRGWTSV YLYPKKPCFLGCTPVDMKEAMVQYTKWISELFLLAISRFNPLTFGISRMSILQSMCYGYLTIMPIL SVAMIFYATVPQLCLLRGVPLFPKVSDPWFAVFLAIFVSSLCQHLIEVLTSDGTLKTWWNEQRNWV IKSGSGSVFGALSGILKWFGMKIKFGLSNKAVDKEKLEKYEKGKFDFQGAAMFMVPLTISVILNTL CLIGGLWRVITLKNFEEMSGQFIISLYFLALSYPILEGLLRKGKGKA SEQ ID NO: 27-Q. saponaria QA-GlcpA-Galp α-1,3-L-rhamnosyltransferase (Qs0283850) coding sequence (1485 bp): ATGGTCTCCGGCGACGACGACGTTTCTCGTCGGCCACTGAAAGTTTACTTTATTGCACACCCCTCA CCTGGCCATATTGCCCCTCTAACCAAAATAGCCCAACTCTTTGCTGCACGTGGTGAGCACGTGACT ATTCTTACTACTCCCGCCAATGTCCACTTCCATGAGAAATCCATCGACAAAGGAAAGACTTCCGGC TATCATGTTAACATCCACGCCGTTAAATTTCCTTCTAAAGAGGTCGGTCTCCCCGACGGCATCGAA AACTTCTCTCACGCCTCCGATAATGAAACAGCAGCCAAAATTTGGGCCGGATTCTCCATGCTTCAA ACTGAAATGGAGCAATATATGGAACAAAACCCACCCGATTGCATTGTTGCCGACATGTTCAACCGC TGGACTTCCGACTTCGCTATCAAATTGGGAATCCCGAGAATAGTTTTCAACGTCTACTGTATTTTC ACACGCTGTTTGGAAGAAGCAATCAGATCACCTGACTCGCCACACTTGAAACTAAACTCCGATAAT GAACAGTTTATTATTCCGGGTCTACCCGACCCCATAACAATTACCCGAGCTCAACTGCCCGACGGT GCCTTTTCTGTCGTCAAAGAACAAGTTAGTGAAGCTGAGTTGAAAAGCTTCGGAATGGTGATCAAC GGGTTTTCCGAACTCGAAACCGAATACATCGAGTATTACAAGAATATCATGGGTCGAAAACGGATT TGGCATGTCGGACCCCTTCAGCTCATTTACCAAAACGATGACCCCAAAGTTCAGAGGAGCCAGAAG ACAGCGGTCGTGAGTGACAACGAGTTAGTGAGTTGGCTTGACTCGAAGAAACCCGACTCAGTGATT TACATTTCCTTCGGTAGTGCAATTCGTTTCTCTAATAAGCAGCTCTATGAAATAGCATGTGGATTA GAAGCTTCCGGCTACCCATTTTTGTGGGCCTTACTTTGGGTGCCAGAAGATGACGACGACGTGGGC AACAAATGGTTGCCTGATTTCGAAGAAAGAATAAAAAGAGAAAATAAGGGAATAATTTTCAGGGGG TGGGCCCCACAGATGTTAATCTTAAACCACCCGGCGATCGGTGGTTTCATGACGCATTGTGGTTGG AATGCGGTGGTGGAAGCGCTTTCTTTCGGTGTTCCGACTATTACGCTTCCGGTTTTCTCGGAGCAG TTTTATACTGAGAGACTGATATCACAAGTGCTCAAGACTGGTGTCGAGGTCGGTGCAGAGAAGTGG ACCTATGCATTTGATGCGGGGAAATATCCGGTGAGTCGGGAAAAGATAGCGACGGCGGTGAAGAAG ATATTAGACTGTGGAGAAGAGGCAGAAGGAATGAGAAAGCGGGCCAGGGAGATGAAAGAAAAAGCC CAAAAAAGTGTTGAAGAAGGTGGGTCCTCTTATAATAATTTAACGGCTATGATTGAAGATCTTAAA GAATTTAGGGCTAACAATGGCAAGGTTGCATGA SEQ ID NO: 28-Q. saponaria QA-GlpA-Galp α-1,3-L-rhamnosyltransferase (Qs_0283850) translated nucleotide sequence (494 aa): MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKGKTSG YHVNIHAVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNR WTSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDG AFSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQK TAVVSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVG NKWLPDFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQ FYTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDCGEEAEGMRKRAREMKEKA QKSVEEGGSSYNNLTAMIEDLKEFRANNGKVA SEQ ID NO: 29 -Q. saponaria QA-GlcpA-Galp α-1,3-L-rhamnosyltransferase (TRINITY_DN20529_c0_g2_i8) coding sequence (1491 bp): ATGGTCTCCGGCGACGATACCGTTTCACGGCCACTGATAGTTTACTTTATTGCACACCCCTCACCT GGCCATATTGCCCCTCTAACCAAAATAGCCCAACTCTTCGCTGCACGTGGTGAGCACGTCACTATT CTTACTACTCCCGCCAATGTCCACTTCCATGAGAAATCCATCGACAAAAGAAAGAATTCCGGCTAT CATGTTAACATCCACACCGTTAAATTTCCTTCTAAAGAGGTCGGTCTCCCTGACGGCATCGAAAAC TTCTCTCACGCCTCCGATAATGAAACAGCAGCCAAAATTTGGGCCGGATTCTCCATGCTTCAAACT GAAATGGAGCAATATATGGAACAAAACCCACCCGATTGCATCGTTGCCGACATGTTCAACCGCTGG ACTTCCGACTTCGCTATCAAATTGGGAATCCCGAGAATAGTTTTCAACGTCTACTGTATTTTCACA CGCTGTTTGGAAGAAGCAATCAGATCACCTGACTCGCCACACTTGAAACTAAACTCCGATAATGAA CAGTTTATTATTCCCGGTCTACCCGACCCCATAACAATTACCCGAGCTCAACTCCCCGACGGTGCC TTTTCTGTCGTCAAAGAACAAGTTAGTGAAGCTGAGTTGAAAAGCTTCGGAATGGTGATCAACGGG TTTTCCGAACTCGAAACTGAATACATCGAGTATTACAAGAATATCATGGGTCGCAAACGGATTTGG CATGTCGGACCCCTTCAGCTAATTTACCAAAACGACGACCCCAAAGTTCAGAGGAGCCAGAAGACA GCGGTCTTGAGTGACAACGAGTTAGTGAGTTGGCTTGACTCGAAGAAACCCGACTCAGTGATTTAC ATTTCCTTCGGTAGTGCAATTCGTTTCTCTAATAAGCAGCTCTATGAAATCGCATGTGGATTAGAA GCTTCCGGCTACCCATTTTTGTGGGCCTTACTTTGGGTGCCAGAAGATGATGACGACGTGGGCAAC AAATGGTTGCCGGGTTTCGAAGAAAGAATAAAAAGAGAAAATAAGGGAATAATTTTCAGGGGGTGG GCCCCACAGATGTTAATCTTAAACCACCCGGCGATCGGTGGTTTCATGACGCATTGTGGTTGGAAT GCGGTGGTGGAAGCACTTTCATTCGGTGTTCCGACTATTACGCTTCCAGTTTTCTCGGAGCAGTTT TATACTGAGAGACTGATATCACAAGTGCTCAAGACTGGTGTGGAGGTTGGTGCAGAGAAGTGGACC TATGCATTTGATGCGGGGAAATATCCGGTGAGTAGGGAAAAGATAGCGACGGCGGTGAAGAAGATA TTAGACGATGGAGAAGAGGCAGAAGGAATGAGAAAGCGGGCCAGGGAGATGAAAGAAAAAGCCCAA AAAAGTGTTGAAGAAGGTGGATCCTCTTATAATAATTTAACGGCTATGATTGAAGATCTTAAAGAA TTTAGGGCTAACAATGGCAAGGCTGCAATGAAATCATGA SEQ ID NO: 30-Q. saponaria QA-GlcpA-Galp α-1,3-L-rhamnosyltransferase (TRINITY_DN20529_c0_g2_i8) translated nucleotide sequence (496 aa): MVSGDDTVSRPLIVYFIAHPSPGHIAPLTKIAQLFAARGEHVTILTTPANVHFHEKSIDKRKNSGY HVNIHTVKFPSKEVGLPDGIENFSHASDNETAAKIWAGFSMLQTEMEQYMEQNPPDCIVADMFNRW TSDFAIKLGIPRIVFNVYCIFTRCLEEAIRSPDSPHLKLNSDNEQFIIPGLPDPITITRAQLPDGA FSVVKEQVSEAELKSFGMVINGFSELETEYIEYYKNIMGRKRIWHVGPLQLIYQNDDPKVQRSQKT AVLSDNELVSWLDSKKPDSVIYISFGSAIRFSNKQLYEIACGLEASGYPFLWALLWVPEDDDDVGN KWLPGFEERIKRENKGIIFRGWAPQMLILNHPAIGGFMTHCGWNAVVEALSFGVPTITLPVFSEQF YTERLISQVLKTGVEVGAEKWTYAFDAGKYPVSREKIATAVKKILDDGEEAEGMRKRAREMKEKAQ KSVEEGGSSYNNLTAMIEDLKEFRANNGKAAMKS SEQ ID NO: 31-Q. saponaria QA-GlcpA-Galp β-1,3-D-xylosyltransferase (Qs_0283870) coding sequence (1515 bp): ATGGTCTCCGGCGACGACGATGTTTCTCGTCGGCCACTGAAAGTTTACTTCATTGCACACCCCTCA CCTGGCCATATTGCCCCTCTGACCAAAATAGCCCATCTCTTCGCTGCCCTCGGTGAGCACGTGACT ATTCTCACTACTCCCGCCAATGTCCACTTCCATGAGAAATCCATCGACAAAGGAAAGGCTTCCGGC TATCATGTTAACATCCACACCGTTAAATTTCCTTCTAAAGAGGTCGGTCTCCCTGACGGCATCGAA AACTTCTCTTACGCCTCCGATGTTGAAACAGCAGCTAAAATTTGGGCTGGATTCGCCATGCTACAA ACTGAAATGGAGCAATATATGGAGCTTAACCCACCCGATTGCATCGTTGCCGACATGTTCACCTCC TGGACCTCCGACTTTGCTATCAAATTGGGAATCACAAGAATCGTTTTCAACGTCTATTGTATTTTC ACACGCTGTTTGGAAGAAGCCATCCGATCACCGGACTCGCCACACTTGAACAAAGAAATCTCTGAT AATGAACCGTTTGTTATCCCGGGTCTACCAGACCCCATAACAATTACCCGAGCTCAACTGCCCGAC GGTACCTTTTCTCCCATGAAAGAACTAGCTAGAACAGCTGAGTTGAAGAGCTTTGGAATGGTGATC AACGGGTTTTCCGAACTCGAAACCGATTACATCGAGCATTACAAGAAAATCATGGGTCACAAACGG ATTTGGCATGTCGGACCCCTTCAGCTAATCCACCGTAACGATGAAGACAAAATTCAGAGGAGCCAC AAGACAGCGGTGCTGAGTGATAACGATAACGAGTTAGTGAGTTGGCTTAACTCGAAGAAACCCGAC TCAGTTATTTACATTTGCTTCGGTAGTGCAACTCGTTTCTCTAATCACCAGCTCTATGAAATCGCC TGTGGATTAGAAGCTTCCGGGCACCCATTTTTGTGGGGCCTACTTTGGGTGCCAGAAGATGAAGAT AACGATGACGTGGGCAACAAATGGTTGCCAGCTTTCGAAGAAAGAATTAAAAAGGAAAATAAGGGA ATGATTTTAAGGGGGTGGGCTCCACAGATGTTAATCTTGAATCACCCGGCGATCGGTGGTTTCATG ACGCATTGTGGTTGGAATGCGGCGGTGGAGGCGCTTTCTTCCGGTGTTCCGATTATTACATTTCCG GTTTTCTCGGATCAGTTTTATAATGAAAGGCTGATATCACAAGTGCATAAGTGTGGGGTGGGGGTT GGTACGGAGGCGTGGAGCTATGCATTCGATGCCGGGAAGAATCCGGTGGGTCGGGAAAAGATAATG ACGGCGGTGAAGAAGATATTAGACGGTGGAGAAGAGGCGGAAGGAATGAGAAAGAGGGCCCGGGAG CTGAAAGAAATAGCTAAAAGAAGTGTGGAAGAAGGTGGGTCCTCTTATAATAATTTAACGGCTATG ATTCAAGATCTGAAAGAATTTAGAGCTAACAATGGCAAGGCTGCACAAGATCATGAATCGTGA SEQ ID NO: 32-Q. saponaria QA-GlcpA-Galp β-1,3-D-xylosyltransferase (Qs_0283870) translated nucleotide sequence (504 aa): MVSGDDDVSRRPLKVYFIAHPSPGHIAPLTKIAHLFAALGEHVTILTTPANVHFHEKSIDKGKASG YHVNIHTVKFPSKEVGLPDGIENFSYASDVETAAKIWAGFAMLQTEMEQYMELNPPDCIVADMFTS WTSDFAIKLGITRIVFNVYCIFTRCLEEAIRSPDSPHLNKEISDNEPFVIPGLPDPITITRAQLPD GTFSPMKELARTAELKSFGMVINGFSELETDYIEHYKKIMGHKRIWHVGPLQLIHRNDEDKIQRSH KTAVLSDNDNELVSWLNSKKPDSVIYICFGSATRFSNHQLYEIACGLEASGHPFLWGLLWVPEDED NDDVGNKWLPAFEERIKKENKGMILRGWAPQML1LNHPAIGGFMTHCGWNAAVEALSSGVP11TFP VFSDQFYNERLISQVHKCGVGVGTEAWSYAFDAGKNPVGREKIMTAVKKILDGGEEAEGMRKRARE LKEIAKRSVEEGGSSYNNLTAMIQDLKEFRANNGKAAQDHES