WO2023006109A1 - 鼠李糖高度专一的糖基转移酶及其应用 - Google Patents
鼠李糖高度专一的糖基转移酶及其应用 Download PDFInfo
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- WO2023006109A1 WO2023006109A1 PCT/CN2022/109355 CN2022109355W WO2023006109A1 WO 2023006109 A1 WO2023006109 A1 WO 2023006109A1 CN 2022109355 W CN2022109355 W CN 2022109355W WO 2023006109 A1 WO2023006109 A1 WO 2023006109A1
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- WIPO (PCT)
- Prior art keywords
- rhamnose
- compound
- glycosyl
- seq
- ginsenoside
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Definitions
- the invention relates to the fields of biotechnology and plant biology, in particular, the invention relates to a highly specific rhamnose glycosyltransferase and its application.
- Ginsenoside is a general term for saponins isolated from plants of the genus Panax (such as ginseng, Panax notoginseng and American ginseng, etc.) and Gynostemma pentaphyllum, and is a class of triterpenoids. Ginsenosides are also known as ginsenosides, notoginseng saponins and gypenosides depending on their isolated source. Ginsenosides are the main bioactive components in these medicinal plants. Currently, about 150 saponins have been isolated. From the structural point of view, ginsenosides are mainly biologically active small molecules formed by glycosylation of saponins.
- saponins in ginsenosides mainly protopanaxadiol and protopanaxatriol of the dammarane-type tetracyclic triterpenes, and oleananoic acid.
- saponin can improve water solubility, change its subcellular location, and produce different biological activities.
- Most protopanaxadiol saponins are glycosylated at C3 and/or C20 hydroxyl, while protopanaxatriol saponins are glycosylated at C6 and/or C20 hydroxyl.
- Different types of sugar groups and different degrees of glycosylation modification have produced ginsenosides with various molecular structures.
- Ginsenosides modified by rhamnosylation have abundant biological activities.
- Rg2 extends a molecule of rhamnose at C6-O-Glc of Rh1, and Rg2 has good effects in treating depression, improving heart function, improving learning and memory ability, and resisting senile dementia;
- ginsenoside Re is at C6 of Rg1.
- -O-Glc extends a molecule of rhamnose, which may play a role in lowering blood sugar and treating diabetes by promoting the secretion of glucagon-like peptide-1 in intestinal tissue.
- Ginsenosides are prepared from the total saponins or abundant saponins of ginseng or Panax notoginseng, and rely on chemical, enzymatic and microbial fermentation hydrolysis methods. Since wild ginseng resources have been basically exhausted, ginsenoside resources are currently derived from the artificial cultivation of ginseng or Panax notoginseng, and its artificial cultivation has a long growth cycle (generally takes more than 5-7 years), and is restricted by region and often Therefore, the artificial cultivation of ginseng or Panax notoginseng has serious continuous cropping obstacles (the ginseng or Panax notoginseng plantation needs to be left fallow for more than 5-15 years to overcome the continuous cropping obstacles), so the yield, quality and Security is a challenge.
- the raw materials are cheap monosaccharides, and the preparation process is a safe and adjustable fermentation process, avoiding any external pollution (for example, the raw materials used in artificial planting of plants) Pesticides), therefore, the preparation of ginsenoside monomers by synthetic biology technology not only has a cost advantage, but also can ensure the quality and safety of the finished product.
- Synthetic biology technology is used to prepare a sufficient amount of various high-purity natural and non-natural ginsenoside monomers for activity determination and clinical trials, and to promote the development of innovative drugs for rare ginsenosides.
- UDP-glycosyltransferases that catalyze the glycosylation of ginsenosides.
- the function of UDP-glycosyltransferase is to transfer the glycosyl on the glycosyl donor (nucleoside diphosphate sugar, such as UDP-glucose, UDP-rhamnose, UDP-xylose and UDP-arabinose) to different on the sugar acceptor. From the analysis of plant genomes that have been sequenced so far, plant genomes often encode more than a hundred different glycosyltransferases.
- UDP-glycosyltransferase element UGTPg100
- Chinese researchers identified a UDP-glycosyltransferase element (UGTPg100) that can transfer a glucose group at the C6 position of protopanaxatriol.
- Chinese researchers have disclosed in the patent (PCT/CN2015/081111) glycosyltransferases (gGT29-7, etc.) that can extend the sugar chain at the C6 position of protopanaxatriol-type saponins.
- gGT29-7 can use UDP-Xyl to catalyze The C6 position of Rh1 extends a molecule of xylose to generate notoginseng saponin R2, which can use UDP-Glc to catalyze the C6 position of Rh1 to extend a molecule of glucose glycosyl to generate Rf, but basically cannot use UDP-Rha; patent (PCT/CN2015/081111 )
- the mutant gGT29-7 (N343G, A359P) of gGT29-7 disclosed by UDP-Rha has a rhamnosyl molecule extended from the C6 position of Rh1 to generate Rg2 by using UDP-Rha, but the activity is very low, only about 9% conversion rate.
- gGT29-7 (N343G, A359P) can still use UDP-glc as a donor for the transglycosylation reaction in addition to the UDP-Rha as the donor, and the catalytic efficiency is higher than that of UDP-Rha as the donor. Catalytic reactions of glycosyl donors. Therefore, the activity of gGT29-7(N343G, A359P) to catalyze UDP-Rha is low and not specific, resulting in the synthesis of a large number of by-products, which cannot meet the needs of the application.
- glycosyltransferases URT94-1 and URT94-2 that can extend UPD-rhamnose at the C6 position from ginseng, which can specifically use UDP-Rha as a glycosyl donor, Efficiently catalyze the extension of a molecule of rhamnose on the first sugar group at the C-6 position of ginsenoside Rh1, ginsenoside Rg1 or notoginseng R3 to obtain ginsenoside Rg2, ginsenoside Re or Yesanchinoside E respectively.
- URT94-1 and URT94-2 could not use UDP-glucose as the glycosyl donor to catalyze the above-mentioned saponin substrates. Therefore, these glycosyltransferases provide highly specific glycosyltransferases for the efficient preparation of saponins such as ginsenoside Rg2, ginsenoside Re and Yesanchinoside E.
- a method for linking a rhamnose group on the first sugar group at the C-6 position of a tetracyclic triterpene (class) compound comprising: using a specific glycosyltransferase
- the specific glycosyltransferase is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide thereof.
- the use of a specific glycosyltransferase is provided for linking a rhamnosyl group (including using As a catalyst for this reaction), the specificity glycosyltransferase is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide thereof.
- the rhamnose group is provided by a glycosyl donor; preferably, the glycosyl donor is a glycosyl donor carrying a rhamnose group; more preferably,
- the glycosyl donors include (but not limited to): uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, Cytidine diphosphate (CDP)-rhamnose, thymidine diphosphate (TDP)-rhamnose.
- the tetracyclic triterpene compound is the compound of formula (I), and the compound connected to the sugar group at the C-6 position is the compound of formula (II);
- R1 and R2 are H or glycosyl
- R3 is monosaccharide glycosyl
- R4 is rhamnosyl
- described glycosyl or monosaccharide glycosyl (R3) is selected from: glucosyl, xylose group, arabinosyl or rhamnosyl;
- the compound of formula (I) is ginsenoside Rg1, and the compound of formula (II) is ginsenoside Re; when R1 and R2 are H, R3 is In the case of glucosyl, the compound of formula (I) is ginsenoside Rh1, and the compound of formula (II) is ginsenoside Rg2.
- the tetracyclic triterpene compound is the compound of formula (III), and the compound connected to the sugar group at the C-6 position is the compound of formula (IV);
- R1 is H or glycosyl
- R2, R3, R4 are monosaccharide glycosyl
- R5 is rhamnosyl
- described glycosyl (R1) or monosaccharide glycosyl (R2, R3, R4 ) is selected from: glucosyl, xylosyl, arabinosyl or rhamnosyl;
- R1 is H
- R2, R3 and R4 are glucosyl
- R5 is rhamnosyl
- the compound of formula (III) is notoginsenoside R3
- the compound of formula (IV) is Yesanchinoside E.
- group species, substrates or products are as follows:
- group species, substrates or products are as follows:
- the compounds (I) and (III) in the reaction formula include but are not limited to: S-configuration or R-configuration dammarane-type tetracyclic triterpenoids, lanolin-type tetracyclic triterpenes Cyclic triterpenes, apotirucallane-type tetracyclic triterpenes, apoturane-type tetracyclic triterpenes, cycloartane (cycloaltinane)-type tetracyclic triterpenes, cucurbitane tetracyclic Triterpenes, or neem-type tetracyclic triterpenes.
- the compound (II) or (IV) in the reaction formula includes ginsenoside Rg2, ginsenoside Re, Yesanchinoside E.
- a method for connecting a rhamnose group on the first sugar group at the C-6 position of a tetracyclic triterpene (class) compound in a cell comprising:
- glycosyltransferase is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide; there is a rhamnosyl in the host cell
- the tetracyclic triterpene compound reaction precursors include: ginsenoside Rg1, ginsenoside Rh1, notoginsenoside R3; corresponding products include: ginsenoside Re, ginsenoside Rg2, Yesanchinoside E;
- the glycosyl donors include (but not limited to): uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)- Rhamnose, cytidine diphosphate (CDP)-rhamnose, thymidine diphosphate (TDP)-rhamnose.
- UDP uridine diphosphate
- GDP guanosine diphosphate
- ADP adenosine diphosphate
- CDP cytidine diphosphate
- TDP thymidine diphosphate
- the method further includes: providing an additive for regulating enzyme activity into the reaction system.
- the additive for regulating enzyme activity is: an additive for increasing or inhibiting enzyme activity.
- the additive for regulating enzyme activity is selected from the group consisting of Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al3+, Ni 2+ , Zn 2+ , or Fe 2+ .
- the additives for regulating enzyme activity are: Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al3+, Ni 2+ , Zn 2+ , or Fe 2+ species.
- the pH of the reaction system is: pH 4.0-10.0, preferably pH 5.5-9.0.
- the temperature of the reaction system is: 10°C-105°C, preferably 20°C-50°C.
- a specific glycosyltransferase is provided, which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or its conservative Variant polypeptides; preferably, the conservative variant polypeptides include:
- polypeptide of the sequence shown by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14 undergoes one or more (such as 1-20, preferably 1-10; more preferably 1 -5; more preferably 1-3) formed by substitution, deletion or addition of amino acid residues, and have a rhamnosyl group attached to the first sugar group at the C-6 position of the tetracyclic triterpenoid compound functional peptides;
- the amino acid sequence is more than 50% (preferably more than 60%; more preferably more than 70%; more preferably more than 70%; more preferably More than 80%; More preferably more than 85%; More preferably more than 90%; More preferably more than 95%; More preferably more than 98%; More preferably more than 99%) identity, and have in tetracyclic triterpenoids A polypeptide with a rhamnosyl function connected to the first glycosyl at the C-6 position; or
- a tag sequence is added to the N or C terminus of the polypeptide shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a polypeptide formed by adding a signal peptide sequence to its N terminus.
- an isolated polynucleotide encoding said specific glycosyltransferase is provided.
- the polynucleotide encoding the specificity glycosyltransferase includes a polynucleotide selected from the group: (A) as shown in SEQ ID NO: 1, 3 or 13 Nucleotide sequence; (B) a nucleotide sequence having at least 95% identity with the sequence shown in SEQ ID NO: 1, 3 or 13; (E) the sequence shown in SEQ ID NO: 1, 3 or 13 Nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5' end and/or 3' end; (F)(A) - (E) the complementary sequence of any one of the nucleotide sequences described; (G) a fragment of 20-50 bases in length of the sequences described in (A)-(F).
- A as shown in SEQ ID NO: 1, 3 or 13 Nucleotide sequence
- B a nucleotide sequence having at least 95% identity with the sequence shown in SEQ ID NO: 1,
- the polynucleotide sequence is selected from any one of SEQ ID NO: 1, 3 or 13 or its complement.
- nucleic acid construct which comprises the polynucleotide, or expresses the specificity glycosyltransferase; preferably, the nucleic acid construct It is an expression vector or a homologous recombination vector.
- a recombinant host cell which expresses the specific glycosyltransferase, or contains the polynucleotide, or contains the nucleic acid construct; preferably, The recombinant host cell also contains a tetracyclic triterpene compound reaction precursor or a construct for expressing/forming it; preferably, a glycosyl donor carrying a rhamnose group is also present in the recombinant host cell or a glycosyl donor carrying a rhamnose group is introduced Glycosyl donors for rhamnose groups (including constructs/precursors capable of forming such glycosyl donors);
- the tetracyclic triterpenoid reaction precursors include: ginsenoside Rg1, ginsenoside Rh1, notoginsenoside R3; corresponding products include: ginsenoside Re, ginsenoside Rg2, Yesanchinoside E.
- the glycosyl donors include (but are not limited to): uridine diphosphate (UDP)-rhamnose, uridine diphosphate (UDP)-rhamnose, guanosine diphosphate Phosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, cytidine diphosphate (CDP)-rhamnose, thymidine diphosphate (TDP)-rhamnose.
- UDP uridine diphosphate
- UDP uridine diphosphate
- UDP uridine diphosphate
- GDP guanosine diphosphate Phosphate
- ADP adenosine diphosphate
- CDP cytidine diphosphate
- TDP thymidine diphosphate
- the host cell is a prokaryotic or eukaryotic cell.
- the host cell is a eukaryotic cell, such as a yeast cell or a plant cell. In one or more embodiments, the host cell is a Saccharomyces cerevisiae cell. In one or more embodiments, the host cells are ginseng cells or notoginseng cells.
- the host cell is a prokaryotic cell, such as E. coli.
- the host cell is not a cell that naturally produces the product formed after the specific glycosyltransferase treatment of the present invention; for example, it is not a cell that naturally produces the compound of formula (II), (IV) .
- the host cell is not a cell that naturally produces one or more of the following substances: ginsenoside Rh1, ginsenoside Rg1, notoginsenoside R3, ginsenoside Rg2, ginsenoside Re, Yesanchinoside E.
- the host cell further has a characteristic selected from the group consisting of:
- protopanaxatriol saponins include ginsenoside Rh1, ginsenoside Rg1, notoginsenoside R3, ginsenoside Rg2, ginsenoside Re, Yesanchinoside E.
- key genes in the ginsenoside Rh1 synthetic metabolic pathway include (but are not limited to): dammarenediol synthase gene, cytochrome P450 CYP716A47 gene and P450 CYP716A47 reductase gene and tetracyclic Glycosyltransferase UGTPg100 (Genbank accession number AKQ76388.1) of triterpene C6, or a combination thereof.
- key genes in the ginsenoside Rg1 synthetic metabolic pathway include (but are not limited to): dammarenediol synthase gene, cytochrome P450 CYP716A47 gene and P450 CYP716A47 reductase gene and tetracyclic Triterpene C20 and C6 glycosyltransferases UGTPg1 and UGTPg100 (Genbank accession number AKQ76388.1), or a combination thereof.
- the key genes in the ginsenoside Rg2 synthetic metabolic pathway include (but are not limited to): dammarenediol synthase gene, cytochrome P450 CYP716A47 gene and P450 CYP716A47 reductase gene and tetracyclic Glycosyltransferase UGTPg100 (Genbank accession number AKQ76388.1) of triterpene C6 and glycosyltransferase URT94-1 and URT94-2 that catalyze C6 glycosyl extension in the present invention, or a combination thereof.
- key genes in the synthetic metabolic pathway of ginsenoside Re include (but are not limited to): dammarenediol synthase gene, cytochrome P450 CYP716A47 gene and reductase gene of P450 CYP716A47 and tetracyclic Triterpene C20 and C6 glycosyltransferases UGTPg1 and UGTPg100 (Genbank accession number AKQ76388.1), as well as glycosyltransferases URT94-1 and URT94-2 that catalyze C6 glycosyl extension herein, or combinations thereof.
- Another aspect of the present invention also provides the use of the host cell of the present invention in the preparation of glycosyltransferases, catalytic reagents, or compounds of formula (II), (IV).
- the present invention also provides a method for producing a glycosyltransferase or a compound of formula (II) or (IV), comprising incubating the host cell described in the present invention.
- Another aspect of the present invention also provides the use of the host cell of the present invention for preparing enzyme catalytic reagents, or producing glycosyltransferases, or as catalytic cells, or producing compounds of formula (II) and (IV).
- the present invention also provides a method for producing a transgenic plant, comprising the step of: regenerating the host cell of the present invention into a plant, wherein the host cell is a plant cell.
- the host cell is a ginseng cell.
- the host cell is a Panax notoginseng cell.
- a test kit for glycosyltransfer which includes: the specificity glycosyltransferase, which can be used in C-6 of tetracyclic triterpene (class) Rhamnosyl is connected to the first glycosyl at the position, and the specificity glycosyltransferase is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or Its conservative variant polypeptide.
- kit for glycosyl transfer comprising: the isolated polynucleotide.
- a kit for glycosyl transfer which includes: the nucleic acid construct (construct).
- kits for glycosyl transfer which includes: said recombinant host cell.
- the kit also includes: a glycosyl donor carrying a rhamnose group; more preferably, the glycosyl donor includes (but not limited to): uridine diphosphate ( UDP)-Rhamnose, Guanosine Diphosphate (GDP)-Rhamnose, Adenosine Diphosphate (ADP)-Rhamnose, Cytidine Diphosphate (CDP)-Rhamnose, Thymidine Diphosphate (TDP) -D.
- UDP uridine diphosphate
- GDP Guanosine Diphosphate
- ADP Adenosine Diphosphate
- CDP Cytidine Diphosphate
- TDP Thymidine Diphosphate
- the kit further includes: a tetracyclic triterpene compound reaction precursor.
- Figure 2 shows the expression of glycosyltransferases URT94-1 and URT94-2 in Escherichia coli by Western Blot.
- "1" represents the lysate supernatant of the empty vector pET28a E. coli recombinant;
- Marker represents the protein molecular weight marker;
- “2” represents the lysate supernatant of the glycosyltransferase BL21-URT94-1 E.
- FIG. 3 a diagram shows the TLC spectrum of the transglycosylation reaction catalyzed by glycosyltransferases URT94-1 and URT94-2 with protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor .
- "1" represents the lysate supernatant of the pet28a empty vector recombinant as the enzyme solution
- "2”, “3", "4", "5" respectively represent BL21-URT94-1, BL21-URT94-2 , BL21-gGT29-7 (N343G, A359P) and the lysate supernatant of BL21-gGT29-7 were used as the enzyme solution.
- Figure b shows that the glycosyltransferases URT94-1 and URT94-2 catalyze protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor The HPLC profile of the transglycosylation reaction.
- FIG. 4 a diagram shows the TLC spectrum of the transglycosylation reaction catalyzed by glycosyltransferases URT94-1 and URT94-2 with protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor .
- "1" represents the lysate supernatant of the pet28a empty vector recombinant as the enzyme solution
- "2”, “3", "4", "5" respectively represent BL21-gGT29-7, BL21-gGT29-7( N343G, A359P), the lysate supernatant of BL21-URT94-1 and BL21-URT94-2 was used as enzyme solution.
- Figure b shows that glycosyltransferases URT94-1 and URT94-2 catalyze protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor The HPLC profile of the transglycosylation reaction.
- Fig. 6 TLC spectra of glycosyltransferases URT94-1 and URT94-2 catalyzing the transglycosylation reaction with protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Glc as the glycosyl donor.
- "1" represents the lysate supernatant of the pet28a empty vector recombinant as the enzyme solution;
- "2”, "3", "4", "5" respectively represent BL21-gGT29-7, BL21-gGT29-7( N343G, A359P), the lysate supernatant of BL21-URT94-1 and BL21-URT94-2 was used as enzyme solution.
- Arrows indicate the migration positions of saponin standards.
- the inventors provided a specific glycosyltransferase for the first time, which can catalyze rhamnosylation at a specific position of a substrate and improve catalytic activity.
- the specificity glycosyltransferase of the present invention can specifically and efficiently catalyze the C-6 hydroxyl glycosylation of the first sugar group of the tetracyclic triterpenoid substrate to extend the rhamnose group .
- isolated polypeptide or “active polypeptide” means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
- Those skilled in the art can purify the polypeptides using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the polypeptide can also be further analyzed by amino acid sequence.
- active polypeptide As used herein, the terms “active polypeptide”, “polypeptide of the present invention and derivative polypeptide thereof”, “enzyme of the present invention”, and “glycosyltransferase” are used interchangeably, including URT94-1 (SEQ ID NO: 2) , URT94-2 (SEQ ID NO: 4) polypeptide or derivative polypeptide thereof; meanwhile, they may also refer to mutants of glycosyltransferases, including URT94-1m (SEQ ID NO: 14).
- the term “conservatively variant polypeptide” refers to a polypeptide that substantially maintains the same biological function or activity of the polypeptide.
- the “conservative variant polypeptide” may be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may be It may also not be encoded by the genetic code, or (ii) have a substituent group in one or more amino acid residues, or (iii) the mature polypeptide is combined with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol).
- an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence or protein sequence used to purify the polypeptide, or with an antigen IgG Fragment-forming fusion proteins.
- mutant refers to a peptide or polypeptide whose amino acid sequence is altered by insertion, deletion or substitution of one or more amino acids but retains at least one biological activity compared to a reference sequence .
- the mutant described in any embodiment herein comprises at least 50%, 60% or 70%, preferably at least 80%, preferably at least 85% of the reference sequence (SEQ ID NO: 2, 4 or 14 as described herein). , preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity and retain the biological activity (eg as a glycosyltransferase) amino acid sequence of the reference sequence.
- Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
- Mutants also include amino acid sequences having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of a reference sequence, while still retaining the biological activity of the reference sequence.
- the plurality of mutations generally refers to within 1-20, such as 1-15, 1-10, 1-8, 1-5 or 1-3.
- Substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
- amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains, which families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains, chain amino acids (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acid), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), with Amino acids with ⁇ -branched side chains (eg threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Therefore, substitution of one or several positions in a polypeptide of the invention
- URT94-1m (SEQ ID NO: 14), it is a mutant of URT94-1.
- the conservative variant polypeptide of URT94-1m may also be included, but the conservative variant polypeptide is conserved at the amino acid residue corresponding to the 55th position of SEQ ID NO:14.
- Active polypeptide its coding gene, vector and host
- the inventors discovered a new type of specific glycosyltransferase by mining genome and transcriptome information, combined with a large amount of research and experimental work, which can specifically and efficiently convert the tetracyclic triterpenoid substrate C
- the first sugar group of -6 is transferred to a sugar group to extend the sugar chain, and the reaction product has good application value in the fields of medicine and the like.
- sequence of the specific glycosyltransferase described in the present invention is preferably a polypeptide as shown in SEQ ID NO: 2, 4 or 14.
- the polypeptide also includes "conservative variant polypeptides" having the same function as the indicated polypeptide, SEQ ID NO: 2, 4 or 14 sequence.
- the invention also includes fragments, derivatives and analogs of said polypeptides.
- fragment refers to a polypeptide that substantially retains the same biological function or activity of the polypeptide.
- the "conservative variant polypeptide” refers to a polypeptide that basically maintains the same biological function or activity of the polypeptide.
- the “conservative variant polypeptide” may be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may be It may also not be encoded by the genetic code, or (ii) have a substituent group in one or more amino acid residues, or (iii) the mature polypeptide is combined with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol).
- an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence or protein sequence used to purify the polypeptide, or with an antigen IgG Fragment-forming fusion proteins.
- the "conservative variant polypeptide” may include (but not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1 - 10) amino acid deletions, insertions and/or substitutions, and addition or deletion of one or several (such as within 50, within 20 or within 10, preferably 5) at the C-terminal and/or N-terminal within) amino acids.
- substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
- adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
- the invention also provides analogs of said polypeptides.
- the difference between these analogues and the natural polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both.
- These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
- the amino terminal or carboxyl terminal of URT94-1 (SEQ ID NO: 2), URT94-2 (SEQ ID NO: 4) or URT94-1m (SEQ ID NO: 14) or its conservative variant polypeptide of the present invention may also contain One or more polypeptide fragments as protein tags.
- Any suitable label can be used in the present invention.
- the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE, and Ty1. These tags can be used to purify proteins.
- a signal peptide sequence can also be added to the amino terminus of the polypeptide of the present invention in order to secrete and express the translated protein (such as secreted out of the cell).
- the signal peptide can be cleaved during the secretion of the polypeptide from the cell.
- the active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
- the polypeptides of the present invention may be natural purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.
- the polynucleotides encoding the specific glycosyltransferases and other enzymes of the invention may be in the form of DNA or RNA.
- Forms of DNA include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be either the coding strand or the non-coding strand.
- polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
- the present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or polypeptide coding sequences of the present invention, and methods for producing the polypeptides of the present invention through recombinant techniques.
- the present invention relates to nucleic acid constructs comprising the polynucleotides described herein, and one or more regulatory sequences or sequences required for genomic homologous recombination operably linked to these sequences.
- the polynucleotides of the invention can be manipulated in a variety of ways to ensure expression of the polypeptide or protein. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
- the nucleic acid construct is a vector.
- the vector can be a cloning vector, an expression vector, or a gene knock-in vector.
- the polynucleotides of the invention can be cloned into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses and cosmids.
- Cloning vectors can be used to provide coding sequences for proteins or polypeptides of the invention.
- Expression vectors can be provided to cells as bacterial or viral vectors. Expression of a polynucleotide of the invention is typically achieved by operably linking the polynucleotide of the invention to a promoter, and incorporating the construct into an expression vector.
- the vector may be suitable for replication and integration in eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence.
- Knock-in vectors are used to integrate the polynucleotide sequences described herein into regions of interest in the genome.
- the gene knock-in vector can also contain the 5' homology arm and the 3' homology arm required for homologous recombination in the genome.
- a nucleic acid construct herein comprises a 5' homology arm, a polynucleotide sequence described herein, and a 3' homology arm.
- CRISPR/Cas9 technology can be used to homologously recombine the polynucleotide sequence into the position of interest.
- CRISPR/Cas9 technology guides Cas9 nuclease to modify the genome at the insertion position by designing a guide RNA for the target gene, resulting in an increase in the efficiency of homologous recombination in the gene modification region, and homologous recombination of the target fragment contained in the gene knock-in vector to the target site.
- the steps of CRISPR/Cas9 technology and the reagents used, such as Cas9 nuclease, are well known in the art.
- nucleic acid constructs can be constructed. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.
- promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.
- the present invention also provides host cells for the biosynthesis of the desired product.
- the host cells may be prokaryotic cells, such as but not limited to Escherichia coli, yeast, Streptomyces; more preferably Escherichia coli cells.
- a cell host is a production tool. Those skilled in the art can transform various host cells through some technical means, so as to realize the biosynthesis of the present invention.
- the host cells and production methods thus constituted should also be included in the present invention. middle.
- the polynucleotide sequences of the invention can be used to express or produce the polypeptides described herein by conventional recombinant DNA techniques. Generally, the steps are as follows: (1) transform or transduce a suitable polynucleotide (or variant) with the polynucleotide (or variant) encoding the specificity glycosyltransferase of the present invention, or an expression vector containing the polynucleotide (2) host cells cultured in a suitable medium; (3) separating and purifying proteins from the medium or cells.
- Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.
- the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells animal cells, etc.
- Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.
- Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
- the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
- the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art.
- Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- the present inventors are committed to the research of glycosyltransferases, but in the previous work, the first glycosyltransferase that can efficiently utilize the rhamnosyl donor and is specific at the C-6 position of the tetracyclic triterpene (class) compound has not been obtained yet.
- An enzyme that attaches a rhamnosyl group to a sugar group Among the existing enzymes, some basically cannot use rhamnosyl donors (such as UDP-Rha); some have very low activity and cannot fully meet the needs of applications.
- the specificity glycosyltransferase that the inventor screens and obtains from ginseng that can extend rhamnose at the C6 position can efficiently catalyze the production of protopanaxatriol saponins (protopanaxatriol type saponins) / Protopanaxatriol saponins): Ginsenoside Rh1, ginsenoside Rg1, and notoginsenoside R3 have a molecule of rhamnose extended on the first sugar group at the C-6 position; thereby obtaining ginsenoside Rg2, ginsenoside Re or Yesanchinoside e.
- the glycosyltransferase is a highly specific glycosyltransferase provided for the efficient preparation of ginsenoside Rg2 or ginsenoside Re or Yesanchinoside E.
- the protopanaxatriol saponins include ginsenoside Rh1 and ginsenoside Rg1.
- the active polypeptide of the present invention has glycosyltransferase activity and can catalyze one or more of the following reactions:
- R1 and R2 are H or sugar groups
- R3 and R4 are monosaccharide sugar groups
- substituted compounds of R1-R4 are as follows:
- the compound of formula (I) is ginsenoside Rg1, and when R4 is rhamnosyl, the compound of formula (II) is notoginsenoside Re or when R1 and R2 are H, and when R3 is a glucose group, the compound of formula (I) is ginsenoside Rh1, and when R4 is rhamnosyl, the compound of formula (II) is notoginsenoside Rg2 .
- R1 is H or glycosyl
- R2, R3, R4 and R5 are monosaccharide glycosyl
- the polypeptide is selected from SEQ ID NO: 2, 4 or 14 or its derivative polypeptides.
- substituted compounds of R1-R5 are as follows:
- the present invention also provides a method of constructing a transgenic plant comprising regenerating a host cell comprising a polypeptide or polynucleotide described herein into a plant, the host cell being a plant cell.
- Methods and reagents for regenerating plant cells are well known in the art.
- the glycosyltransferases of the present invention are particularly capable of converting ginsenoside Rh1 into ginsenoside Rg2 with other activities, respectively.
- the glycosyltransferases of the present invention are particularly capable of converting ginsenoside Rg1 into ginsenoside Re with other activities, respectively.
- the active polypeptide or glycosyltransferase involved in the present invention can be used to artificially synthesize known ginsenosides and new ginsenosides and derivatives thereof, and can convert Rh1 into active ginsenoside Rg2, and convert Rg1 into active ginsenoside Saponin Re.
- the present invention also provides a method for constructing a transgenic plant, comprising transforming a plant with the polynucleotide or nucleic acid construct described herein, and obtaining the expression of the polypeptide described herein, comprising the polynucleotide or comprising the polynucleotide in the progeny of the plant through hybridization and screening.
- Transgenic positive plants of the nucleic acid construct Methods for transforming plants with nucleic acids and for crossing plants and selecting transgene-positive plants are well known in the art.
- the present invention also provides a kit for biosynthesizing the target product or its intermediate, including: a novel specificity glycosyltransferase or its conservative variant polypeptide shown in SEQ ID NO: 2, 4 or 14 ; Preferably it also includes a glycosyl donor; Preferably it also includes a host cell. More preferably, the kit also includes an instruction manual describing the method for biosynthesis.
- the specific glycosyltransferase of the present invention can specifically and efficiently transfer the first sugar group of C-6 of the tetracyclic triterpenoid substrate into a sugar group to extend the sugar chain;
- Rh1 can be efficiently converted into active ginsenoside Rg2 by the glycosyltransferase of the present invention
- Rg1 can be efficiently converted into active ginsenoside Re by the glycosyltransferase of the present invention
- Rg2 has the activity of preventing and treating neurodegenerative diseases
- Re has the activity of lowering blood sugar and treating diabetes. Therefore, the glycosyltransferase of the present invention has wide application value.
- URT94-1 and URT94-2 use UDP-rhamnose as the glycosyl donor to catalyze the C6 sugar chain extension activity of Rh1 at least 5 times higher.
- SEQ ID NO: 2 (URT94-1 protein)
- SEQ ID NO: 4 (URT94-2 protein)
- SEQ ID NO: 14 (URT94-1m1 protein)
- AxyPrep DNA Gel Extraction Kit (AXYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment.
- the DNA fragment was ligated with the commercially available cloning vector pMD18T plasmid with rTaq DNA polymerase from Treasure Bioengineering Co., Ltd. to obtain recombinant plasmids URT94-1-pMD18T and URT94-2-pMD18T.
- the ligation product was transformed into commercially available E. coli Top10 competent cells, and the transformed E. coli liquid was spread on an LB plate supplemented with 100 ug/mL of ampicillin, and the recombinant clone was further verified by PCR and enzyme digestion.
- URT94-1 and URT94-2 are glycosyltransferase genes, and their ORFs encode the PSPG box, a conserved functional domain of the glycosyltransferase family 1.
- the present inventors expressed and analyzed the transglycosylation reaction of URT94-1 and URT94-2 respectively.
- the glycosyltransferase (respectively SEQ ID NO: 2 or 4) encoded by the two nucleic acid sequences (respectively SEQ ID NO: i, 3) can catalyze the C6 position of Rh1 to extend a rhamnosyl to generate Rg2,
- the catalytic activity is at least 5 times higher, neither of which can catalyze the extension of a glucose group at the C6 position of Rh1 Generate Rf.
- the forward primer includes two parts, and the 5'-3' end contains the pET28a homology arm in sequence
- the sequence 20bp of the sequence and the starting sequence 20bp of coding URT94-1, the reverse primer includes two parts, the sequence 20bp of the homology arm of pET28a and the end sequence 20bp of coding URT94-1 (SEQ ID NO : 9-SEQ ID NO: 10, see Table 1), utilize above-mentioned primer to amplify the gene (containing pET28a homologous arm) of coding URT94-1 by PCR method.
- the high-fidelity DNA polymerase PrimeSTAR of Bao Biological Engineering Co., Ltd. was selected as the DNA polymerase, and the PCR program was set by referring to its manual: 94°C for 2min; 94°C for 15s, 57°C for 30s, 68°C for 1.5min, a total of 33 cycles; 68°C for 10min ; Keep warm at 16°C.
- the PCR product was detected by agarose gel electrophoresis, and a band consistent with the size of the target DNA was excised under ultraviolet light. Then AxyPrep DNA Gel Extraction Kit (AXYGEN Company) was used to recover DNA fragments from the agarose gel.
- the plasmid pET28a was double-digested with FD restriction endonuclease NcoI and SalI from Thermo Company, 37°C for 50 min, and then the linear plasmid pET28a was recovered from the agarose gel using AxyPrep DNA Gel Extraction Kit (AXYGEN Company).
- AXYGEN Company AxyPrep DNA Gel Extraction Kit
- Use the recombinase of Shanghai Yisheng Biotechnology Co., Ltd. to carry out homologous recombination between the enzyme-cut linear plasmid and the two UGTs obtained above, such as URT94-1, and transform the ligated product into E.coli BL21 (DE3) competent cells, and coat On LB plates supplemented with 50 ⁇ g/mL kanamycin (Kana).
- the positive transformants were verified by colony PCR and sequenced to further verify whether the recombinant expression plasmid was successfully constructed. Positive transformants were called Escherichi
- Embodiment 3 expression of ginseng glycosyltransferase URT94s in Escherichia coli
- the supernatant of the cell lysates of recombinant Escherichia coli BL21-URT94-1 and BL21-URT94-2 in Example 4 was used as the crude enzyme solution to carry out the transglycosylation reaction, and the cell lysates of recombinant Escherichia coli transformed with the empty vector pET28a were used as a control.
- Ginseng glycosyltransferase gGT29-7 and gGT29-7 (N343G, A359P) derived from the patent PCT/CN2015/081111 were selected as positive controls.
- the in vitro transglycosylation test was carried out according to the reaction system presented in Table 2, and reacted overnight at 35°C.
- TLC thin-layer chromatography
- HPLC high-performance liquid chromatography
- protopanaxatriol-type ginsenoside Rh1 is used as the glycosyl acceptor
- UDP-Rha is used as the glycosyl donor
- BL21-URT94-1 and BL21-URT94-2 catalyze it to generate Rg2, and they
- the catalytic efficiencies of the enzymes are significantly better than those of the previously disclosed glycosyltransferase gGT29-7 (N343G, A359P).
- the results of HPLC were consistent with the results of TLC.
- URT94-1 can catalyze the C6-O-Glc of Rh1 to extend a molecule of rhamnose to generate ginsenoside Rg2.
- Example 5 In vitro transglycosylation activity and product identification of glycosyltransferase URT94s using protopanaxatriol-type saponin Rg1 as a substrate
- the supernatant of the cell lysates of recombinant Escherichia coli BL21-URT94-1 and BL21-RT94-2 in Example 4 was used as the crude enzyme solution to carry out the transglycosylation reaction, and the cell lysates of recombinant Escherichia coli transformed with the empty vector pET28a were used as a control.
- Ginseng glycosyltransferase gGT29-7 and gGT29-7 (N343G, A359P) derived from the patent PCT/CN2015/081111 were selected as positive controls. According to the reaction system presented in Table 3, in vitro transglycosylation test was carried out at 35°C overnight.
- TLC thin-layer chromatography
- HPLC high-performance liquid chromatography
- URT94-1 can catalyze the C6-O-Glc of Rg1 to extend a molecule of rhamnose to generate ginsenoside Re.
- Example 6 Glycosyltransferase URT94s uses protopanaxatriol-type saponin Rh1/Rg1 as substrate and UDP-Glc as sugar donor for in vitro transglycosylation activity and product identification
- Protopanaxatriol-type ginsenoside Rh1 was used as the glycosyl acceptor, and UDP-Glc was used as the glycosyl donor.
- URT94-1 and URT94-2 could not catalyze it to generate Rf, and the results of HPLC were consistent with those of TLC. Therefore, unlike gGT29-7 and gGT29-7 (N343G, A359P), the glycosyltransferase URT94-1 and URT94-2 of the present invention cannot catalyze the C6-O-Glc of Rh1 to extend a molecule of glucose to generate ginsenoside Rf , as shown in Figure 5.
- Protopanaxatriol-type ginsenoside Rg1 was used as the glycosyl acceptor, and UDP-Glc was used as the glycosyl donor.
- URT94-1 and URT94-2 could not catalyze it to generate C20-O-Glc-Rf, and the results of HPLC were consistent with the results of TLC unanimous. Therefore, unlike gGT29-7 and gGT29-7 (N343G, A359P), the glycosyltransferases URT94-1 and URT94-2 of the present invention cannot catalyze C6-O-Glc of Rg1 to extend a molecule of glucose to generate ginsenoside C20 -O-Glc-Rf, as shown in Figure 6. It shows that URT94-1 and URT94-2 are UDP-rhamnose highly specific glycosyltransferases.
- glycosyltransferase gGT29-7 derived from the patent PCT/CN2015/081111 can extend a molecule of glucose at C6, gGT29-7 (N343G, A359P), can extend a molecule of glucose at C6 or a molecule of rhamnose at C6.
- Glycosyltransferases gGT29-7, gGT29-7 (N343G, A359P) and glycosyltransferases URT94-1 and URT94-2 of the present invention were expressed according to the method of Example 4 and crude enzymes were prepared liquid.
- URT94-1 and URT94-2 use UDP-rhamnose as the glycosyl
- the activity of the donor to catalyze the extension of sugar chains at the C6 position of Rh1 and/or Rg1 was improved.
- URT94-1 and URT94-2 of the present invention can specifically and efficiently add mouse Lysyl to extend the sugar chain.
- the inventors used a random mutation method to construct a mutant library for URT94-1.
- URT94-1_Pet28a-F 5'-ctttaagaaggagatataccatggataccaatgaaaaacca-3' (SEQ ID NO: 9)
- URT94-1_Pet28a- R 5'-ctcgagtgcggccgcaagcttggggcatcgcttcccctggcctg-3' (SEQ ID NO: 10) was used for error-prone PCR.
- the error-prone PCR selected Stratagene company GeneMorph II Random Mutagenesis Kit random mutation kit.
- the PCR program is: 95°C for 2min; 95°C for 10s, 55°C for 15s, 72°C for 2min, a total of 28 cycles; 72°C for 10min to 10°C, and the amount of template used is 50ng.
- the PCR product was recovered by agarose gel electrophoresis to obtain the rhamnosyltransferase URT94-1 error-prone PCR product.
- the above PCR product was connected to the pET28a plasmid (one-step cloning kit, purchased from Shanghai Yisheng), and the connected product was transformed into Escherichia coli BL21 competent cells prepared in the laboratory, and the transformed Escherichia coli liquid was coated on the added Kanamycin 100ug/mL on the LB plate, and further verified recombinant clones by PCR. Several clones were selected to extract recombinant plasmids and then sequenced.
- the enzyme activity assay reaction system in Table 3 was used to compare the enzyme activity. After analyzing and screening a large number of mutants, the inventor obtained a mutant with particularly high enzyme activity, named URT94-1m1 (nucleic acid sequence such as SEQ ID NO: 13; protein sequence such as SEQ ID NO: 14), corresponding to wild-type URT94-1, its 55th position is mutated from L to M (L55M).
- URT94-1m1 nucleic acid sequence such as SEQ ID NO: 13; protein sequence such as SEQ ID NO: 14
- Table 5 shows the activity comparison results of rhamnosyltransferase URT94-1 and its mutant URT94-1m1.
- the inventors used Western blot to detect protein expression, and the results are shown in Figure 8, the mutant URT94-1m1 can be highly expressed.
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Abstract
Description
底物 | R1 | R2 | R3 | R4 | 产物 |
人参皂苷Rg1 | H | Glc | Glc | Rha | 人参皂苷Re |
人参皂苷Rh1 | H | H | Glc | Rha | 人参皂苷Rg2 |
底物 | R1 | R2 | R3 | R4 | R5 | 产物 |
三七皂苷R3 | H | Glc | Glc | Glc | Rha | Yesanchinoside E |
底物 | R1 | R2 | R3 | R4 | 产物 |
Rg1 | H | Glc | Glc | Rha | 人参皂苷Re |
Rh1 | H | H | Glc | Rha | 人参皂苷Rg2 |
底物 | R1 | R2 | R3 | R4 | R5 | 产物 |
三七皂苷R3 | H | Glc | Glc | Glc | Rha | Yesanchinoside E |
序列编号 | 序列名称 | 突变位点 | Rg2转化率(%) | Re转化率(%) |
SEQ ID NO:2 | PgURT94 | - | 75% | 62% |
SEQ ID NO:14 | PgURT94m1 | L55M | 92% | 99% |
Claims (15)
- 一种在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基的方法,包括:以专一性糖基转移酶进行转移,所述专一性糖基转移酶是具有SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列的多肽,或其保守性变异多肽。
- 如权利要求1所述的方法,其特征在于,所述的鼠李糖基由糖基供体提供;较佳地,所述糖基供体是携带鼠李糖基团的糖基供体;更佳地,所述的糖基供体包括选自:尿苷二磷酸-鼠李糖,鸟苷二磷酸-鼠李糖,腺苷二磷酸-鼠李糖,胞苷二磷酸-鼠李糖,胸苷二磷酸-鼠李糖,或其组合。
- 专一性糖基转移酶的用途,用于在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基,所述专一性糖基转移酶是具有SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列的多肽,或其保守性变异多肽。
- 如权利要求5所述的用途,其特征在于,所述的鼠李糖基由糖基供体提供;较佳地,所述糖基供体是携带鼠李糖基团的糖基供体;更佳地,所述的糖基供体包括 选自:尿苷二磷酸-鼠李糖,鸟苷二磷酸-鼠李糖,腺苷二磷酸-鼠李糖,胞苷二磷酸-鼠李糖,胸苷二磷酸-鼠李糖,或其组合。
- 一种胞内在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基的方法,包括:(a)在宿主细胞内引入四环三萜化合物反应前体或表达/形成其的构建体,以及引入专一性糖基转移酶或表达其的构建体,获得重组的宿主细胞;所述专一性糖基转移酶是具有SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列的多肽,或其保守性变异多肽;所述宿主细胞内存在携带鼠李糖基团的糖基供体或引入有携带鼠李糖基团的糖基供体;(b)培养(a)的重组的宿主细胞,获得C-6位的第一个糖基上连接鼠李糖基的四环三萜化合物产物。
- 如权利要求9所述的方法,其特性在于,所述四环三萜化合物反应前体包括:人参皂苷Rg1,人参皂苷Rh1,三七皂苷R3;相应的产物包括:人参皂苷Re,人参 皂苷Rg2,Yesanchinoside E;较佳地,所述的糖基供体包括选自:尿苷二磷酸-鼠李糖,鸟苷二磷酸-鼠李糖,腺苷二磷酸-鼠李糖,胞苷二磷酸-鼠李糖,胸苷二磷酸-鼠李糖,或其组合。
- 一种专一性糖基转移酶,其是具有SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列的多肽,或其保守性变异多肽;较佳地,所述保守性变异多肽包括:(1)由SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示序列的多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的,且具有在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基功能的多肽;(2)氨基酸序列与SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示序列的多肽有50%以上相同性,且具有在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基功能的多肽;或(3)在SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示序列的多肽的N或C末端添加标签序列,或在其N末端添加信号肽序列后形成的多肽。
- 分离的多核苷酸,所述多核苷酸编码权利要求11所述的专一性糖基转移酶。
- 一种核酸构建物,其包含权利要求11所述的多核苷酸,或表达权利要求11所述的专一性糖基转移酶;较佳地,所述核酸构建物是表达载体或同源重组载体。
- 一种重组宿主细胞,其表达权利要求11所述的专一性糖基转移酶,或含有权利要求12所述的多核苷酸,或含有权利要求13所述的核酸构建物;较佳地,该重组宿主细胞中还含有四环三萜化合物反应前体或表达/形成其的构建体;较佳地,该重组宿主细胞中还存在携带鼠李糖基团的糖基供体或引入有携带鼠李糖基团的糖基供体;较佳地,所述四环三萜化合物反应前体包括:人参皂苷Rg1,人参皂苷Rh1,三七皂苷R3;相应的产物包括:人参皂苷Re,人参皂苷Rg2,Yesanchinoside E;较佳地,所述的糖基供体包括选自:尿苷二磷酸-鼠李糖,尿苷二磷酸-鼠李糖,鸟苷二磷酸-鼠李糖,腺苷二磷酸-鼠李糖,胞苷二磷酸鼠李糖,胸苷二磷酸-鼠李糖,或其组合。
- 一种用于糖基转移的试剂盒,其特征在于,其中包括:权利要求7所述的专一性糖基转移酶,该酶能在四环三萜化合物的C-6位的第一个糖基上连接鼠李糖基,所述专一性糖基转移酶是具有SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列的多肽,或其保守性变异多肽;或权利要求12所述分离的多核苷酸;或权利要求13所述的核酸构建物;或权利要求14所述的重组宿主细胞;较佳地,其中还包括:携带鼠李糖基团的糖基供体;更佳地,所述的糖基供体包括:尿苷二磷酸-鼠李糖,鸟苷二磷酸-鼠李糖,腺苷二磷酸-鼠李糖,胞苷二磷酸-鼠李糖,胸苷二磷酸-鼠李糖;较佳地,其中还包括:四环三萜化合物反应前体。
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CN116286712B (zh) * | 2023-05-11 | 2023-08-25 | 中国中医科学院中药研究所 | 鼠李糖基转移酶突变体、编码基因、制备方法及应用 |
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