Application of polyphosphate-dependent glucokinase in glycosylation modification of pentacyclic triterpenoids

Abstract

The application discloses application of polyphosphate-dependent glucokinase in glycosylation modification of pentacyclic triterpenoid compounds, a sugar group donor of the glycosylation modification is sucrose, the polyphosphate-dependent glucokinase is used for converting byproduct fructose into fructose-6-phosphate, and the polyphosphate-dependent glucokinase is CgPPGK with an amino acid sequence as shown in SEQ ID NO:1 or Asp-PPGK with an amino acid sequence as shown in SEQ ID NO:2. The polyphosphate-dependent glucokinase CgPPGK or Asp-PPGK is coupled to a traditional UDP-glucose regeneration system in the application, a coupled three-enzyme UDP-glucose regeneration system is formed, consumption of fructose is realized, more UDP-glucose is generated in the reaction, glycosylation efficiency and sucrose utilization rate are improved, and the glycosylation modification capacity for a substrate is obviously higher than that of an original traditional UDP-glucose regeneration system.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and technology. Specifically, this application relates to the application of a polyphosphate-dependent glucokinase in the glycosylation modification of pentacyclic triterpenoid compounds. Background Technology

[0002] Pentacyclic triterpenoids are a class of structurally complex and important plant natural products with various biological activities, including anti-inflammatory, antibacterial, and antitumor effects. Representative compounds include β-amyrin, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, and ursolic acid. However, most pentacyclic triterpenoids suffer from poor water solubility, poor permeability, poor bioavailability, and serious side effects. Therefore, modification is essential for their development as new drugs.

[0003] Glycosylation is an important modification of natural products. It plays a crucial role in controlling the solubility, bioavailability, and toxicity of natural products. This process is primarily achieved by glycosyltransferases, which transfer the sugar moiety from an active sugar donor to a large number of acceptors, as in natural products. Uridine diphosphate (UDP)-activated sugar donors are the largest group of nucleotide sugars, thus making UDP-dependent glycosyltransferases (UGTs) of particular interest in natural product modification. However, a problem with current UGT-mediated glycosylation processes is the high cost of UDP-sugars, which significantly limits their large-scale application. An effective approach to address this issue is to utilize sucrose as the sole consumed substrate, coupling a sucrose synthase-mediated UDP cycle system. The sucrose synthase-mediated UDP cycle system has been widely used in the glycosylation of natural products. In addition to glucose, this system has been extended to the use of other sugars, such as galactose, rhamnose, and arabinose, by coupling additional enzymes to convert UDP-glucose into the desired UDP-sugar.

[0004] However, a potential weakness of this UDP cycle system is that fructose and UDP-glucose have the same stoichiometry throughout the process. While UDP-glucose is continuously consumed, fructose accumulates as a byproduct, reducing sucrose utilization. Furthermore, the reaction catalyzed by sucrose synthase is reversible; fructose accumulation can shift the reaction equilibrium towards sucrose synthesis, further reducing UDP cycle efficiency. This problem becomes more severe with increasing UDP cycle count. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Starting with the consumption of fructose, this invention builds upon the traditional UDP loop system...

[0006] A first aspect of the present invention is to provide an application of polyphosphoric acid-dependent glucokinase CgPPGK or polyphosphoric acid-dependent glucokinase Asp-PPGK in the glycosylation modification of pentacyclic triterpenoid compounds.

[0007] A second aspect of the present invention is to provide an enzyme combination for glycosylation modification of pentacyclic triterpenoid compounds.

[0008] A third aspect of the present invention aims to provide a UDP cycling system based on polyphosphate-dependent glucokinase.

[0009] A fourth aspect of the present invention is to provide a method for glycosylation modification of pentacyclic triterpenoid compounds.

[0010] The solution of this invention embodiment is as follows:

[0011] Firstly,

[0012] This invention provides an application of polyphosphate-dependent glucokinase in the glycosylation modification of pentacyclic triterpenoid compounds; the glycosyl donor for glycosylation modification is sucrose, and the polyphosphate-dependent glucokinase is used to convert the byproduct fructose into fructose-6-phosphate. The polyphosphate-dependent glucokinase is CgPPGK (derived from Corynebacterium glutamicum ATCC13032) with the amino acid sequence shown in SEQ ID NO: 1 or Asp-PPGK (derived from Arthrobacter) with the amino acid sequence shown in SEQ ID NO: 2.

[0013] In some embodiments, the gene sequence of CgPPGK is shown in SEQ ID NO: 3; the gene sequence of Asp-PPGK is shown in SEQ ID NO: 4.

[0014] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid (OA), glycyrrhizic acid, glycyrrhetinic acid (GA), asiatic acid, or ursolic acid.

[0015] In some embodiments, the glycosylation modification system includes: glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, sucrose, uridine diphosphate, and pentacyclic triterpenoid compounds.

[0016] Secondly,

[0017] This invention also provides an enzyme combination for glycosylation modification of pentacyclic triterpenoid compounds, comprising:

[0018] (i) Glycosyltransferases;

[0019] (ii) Sucrose synthase;

[0020] (iii) Polyphosphate-dependent glucokinase; the polyphosphate-dependent glucokinase is CgPPGK with an amino acid sequence as shown in SEQ ID NO: 1 or Asp-PPGK with an amino acid sequence as shown in SEQ ID NO: 2.

[0021] In some embodiments, the glycosyltransferase is UGT73F24 with an amino acid sequence as shown in SEQ ID NO: 5 or UGT73C11 with an amino acid sequence as shown in SEQ ID NO: 6.

[0022] In some embodiments, the sucrose synthase is GuSUS1-Δ9 with the amino acid sequence shown in SEQ ID NO: 7.

[0023] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0024] In some embodiments, the gene sequence of glycosyltransferase UGT73F24 is shown in SEQ ID NO: 8; the gene sequence of glycosyltransferase UGT73C11 is shown in SEQ ID NO: 9; the gene sequence of sucrose synthase GuSUS1-Δ9 is shown in SEQ ID NO: 10; the gene sequence of polyphosphate-dependent glucokinase CgPPGK is shown in SEQ ID NO: 3; and the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK is shown in SEQ ID NO: 4.

[0025] This invention also provides a biological agent for glycosylation modification of pentacyclic triterpenoid compounds, comprising:

[0026] (i) Recombinant vectors or recombinant bacteria carrying the gene sequence of glycosyltransferase UGT73F24 or the gene sequence of glycosyltransferase UGT73C11;

[0027] (ii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of sucrose synthase GuSUS1-Δ9;

[0028] (iii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of polyphosphate-dependent glucokinase CgPPGK or the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK.

[0029] The gene sequence of the glycosyltransferase UGT73F24 is shown in SEQ ID NO: 8; the gene sequence of the glycosyltransferase UGT73C11 is shown in SEQ ID NO: 9; the gene sequence of the sucrose synthase GuSUS1-Δ9 is shown in SEQ ID NO: 10; the gene sequence of the polyphosphate-dependent glucokinase CgPPGK is shown in SEQ ID NO: 3; and the gene sequence of the polyphosphate-dependent glucokinase Asp-PPGK is shown in SEQ ID NO: 4.

[0030] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0031] Thirdly,

[0032] This invention also provides a UDP cycling system based on polyphosphate-dependent glucokinase, the UDP cycling system comprising: the above-mentioned enzyme combination, a pentacyclic triterpenoid compound, sucrose, uridine diphosphate, and Mg. 2+ And sodium polyphosphate.

[0033] Fourthly,

[0034] This invention also provides a method for glycosylation modification of pentacyclic triterpenoid compounds, comprising: catalyzing a substrate using the above-mentioned enzyme combination, wherein the substrate is a pentacyclic triterpenoid compound, and the glycosyl donor for the catalytic reaction is sucrose.

[0035] In some embodiments, the substrate includes, but is not limited to, one of β-amyrinol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0036] In some embodiments, the reaction system includes: a pentacyclic triterpenoid compound, sucrose, uridine diphosphate, glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, and Mg. 2+ And sodium polyphosphate.

[0037] In some embodiments, the pentacyclic triterpenoid compound is 0.3-1.0 mM, the sucrose is 10-100 mM, the uridine diphosphate is 0.2-1.0 mM, the glycosyltransferase is 0.15-0.20 mg / mL, the sucrose synthase is 0.15-0.20 mg / mL, the polyphosphate-dependent glucokinase is 0.40-0.50 mg / mL, and the Mg... 2+ The concentration is 10-20 mM, and the sodium polyphosphate concentration is 10 g / L.

[0038] In some embodiments, the enzymes are added in stages: first, sucrose synthase is added, followed by polyphosphate-dependent glucokinase and glycosyltransferase 10-20 minutes later.

[0039] In some embodiments, the catalytic reaction is carried out at a pH of 8-8.5 and a temperature of 40°C.

[0040] In some embodiments, the substrate is glycyrrhetinic acid, and the glycosyltransferase in the reaction system is selected as UGT73F24.

[0041] In some embodiments, the substrate is oleanolic acid, and the glycosyltransferase in the reaction system is selected as UGT73C11.

[0042] The present invention has the following advantages and beneficial effects:

[0043] (1) The present invention relates to the application of polyphosphoric acid-dependent glucokinase in the glycosylation modification of pentacyclic triterpenoid compounds; the application of polyphosphoric acid-dependent glucokinase CgPPGK or Asp-PPGK, which originally catalyzes glucose, to the reaction catalyzing fructose expands the application scope of polyphosphoric acid-dependent glucokinase CgPPGK or Asp-PPGK.

[0044] (2) In this invention, polyphosphate-dependent glucokinase CgPPGK or Asp-PPGK is coupled to the traditional UDP-glucose regeneration system to form a UDP-glucose regeneration system with three coupled enzymes, thereby realizing the consumption of fructose and producing more UDP-glucose. Compared with the traditional UDP-glucose regeneration system, it alleviates the problem of insufficient hydrolytic kinetics of sucrose synthase, improves glycosylation efficiency and sucrose utilization, and has a significantly higher ability to modify substrate glycosylation than the original traditional UDP-glucose regeneration system.

[0045] (3) The UDP-glucose high-efficiency regeneration system constructed in this invention is non-ATP dependent and does not require the participation of ATP, thus reducing the reaction cost. Attached Figure Description

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0047] Figure 1a The purification efficiency of GuSUS1-Δ9, UGT73C11, UGT73F24, and CgPPGK enzymes was analyzed by SDS-PAGE.

[0048] Figure 1b The purification efficiency of GuSUS1-Δ9, UGT73F24, and Asp-PPGK enzymes was analyzed by SDS-PAGE.

[0049] Figure 2a Comparison of the catalytic activities of CgPPGK on glucose and fructose;

[0050] Figure 2b A comparison of the catalytic activities of Asp-PPGK on glucose and fructose;

[0051] Figure 3a Optimization of temperature and pH for CgPPGK catalysis of fructose;

[0052] Figure 3b Optimization of temperature and pH for Asp-PPGK catalysis of fructose;

[0053] Figure 4a HPLC analysis chromatogram of fructose catalyzed by CgPPGK;

[0054] Figure 4b This is the HPLC analysis chromatogram of fructose catalyzed by Asp-PPGK.

[0055] Figure 5 The second-order mass spectra of fructose catalyzed by CgPPGK and Asp-PPGK are shown.

[0056] Figure 6a This is a schematic diagram of the construction of a UDP-glucose efficient regeneration cycle system using three coupled enzymes (GuSUS1-Δ9, UGT73F24 and CgPPGK) with GA as a glycosylation substrate.

[0057] Figure 6b This is a schematic diagram illustrating the construction of a highly efficient UDP-glucose regeneration cycle pathway using three coupled enzymes (GuSUS1-Δ9, UGT73F24, and Asp-PPGK) with GA as the glycosylation substrate.

[0058] Figure 7a The change in the concentration of 3-O-glucose-glycyrrhetinic acid (GA-3-O-Glc) over time in a UDP-glucose high-efficiency regeneration cycle system using GA as a substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK).

[0059] Figure 7b The change in GA conversion rate over time in a GA-based UDP-glucose high-efficiency regeneration cycle system with three coupled enzymes (GuSUS1-Δ9, UGT73F24 and Asp-PPGK) as substrates.

[0060] Figure 8 To optimize the pH of a high-efficiency regeneration cycle system for UDP-glucose using GA as a substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK);

[0061] Figure 9To optimize the temperature of a high-efficiency regeneration cycle system for UDP-glucose using GA as a substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK);

[0062] Figure 10 A high-efficiency UDP-glucose regeneration cycle system using GA as a substrate and coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK) Mg 2+ Concentration optimization;

[0063] Figure 11 A schematic diagram illustrating the construction of a highly efficient UDP-glucose regeneration cycle system using OA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK).

[0064] Figure 12 The change in the concentration of 3-O-glucose-oleanolic acid (OA-3-O-Glc) over time in a highly efficient regeneration cycle system of OA-based coupled triple enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK) UDP-glucose.

[0065] Figure 13 To optimize the pH of a high-efficiency regeneration cycle system for OA-based coupled triple enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK) UDP-glucose;

[0066] Figure 14 To optimize the temperature of the UDP-glucose high-efficiency regeneration cycle system using OA as a substrate coupled with three enzymes (GuSUS1-Δ9, UGT73C11 and CgPPGK);

[0067] Figure 15 A high-efficiency UDP-glucose regeneration cycle system using OA as a substrate and coupled with three enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK) Mg 2+ Concentration optimization. Detailed Implementation

[0068] The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0069] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0070] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are standard procedures widely used in the field or performed according to the manufacturer's recommendations.

[0071] Materials: Primer Synthesis: All primers used in this invention were synthesized and prepared by Genewiz Biotechnology Co., Ltd. FastPfu high-fidelity polymerase and Taqmix DNA polymerase were purchased from TransGen Biotech Ltd.; Primestar DNA polymerase was purchased from Takara Bio Inc., Japan; the plasmid miniprep kit was purchased from Tiangen Biotech Ltd., and the agarose gel extraction kit was purchased from Thermo Fisher Scientific Ltd.

[0072] Firstly,

[0073] This invention provides an application of polyphosphate-dependent glucokinase in the glycosylation modification of pentacyclic triterpenoid compounds; the glycosyl donor for glycosylation modification is sucrose, and the polyphosphate-dependent glucokinase is used to convert the byproduct fructose into fructose-6-phosphate. The polyphosphate-dependent glucokinase is CgPPGK (derived from Corynebacterium glutamicum ATCC13032) with the amino acid sequence shown in SEQ ID NO: 1 or Asp-PPGK (derived from Arthrobacter) with the amino acid sequence shown in SEQ ID NO: 2.

[0074] MTETGFGIDIGGSGIKGARVNLKTGEFIDERIKIATPKPATPEAVAEVVAEIISQAEWEGPVG

[0075] ITLPSVVRGQIALSAANIDKSWIGTDVHELFDRHLNGREITVLNDADAAGIAEATFGNPAA

[0076] REGAVILLTLGTGIGSAFLVDGQLFPNTELGHMIVDGEEAEHLAAASVKENEDLSWKKW

[0077] AKHLNKVLSEYEKLFSPSVFIIGGGISRKHEKWLPLMELDTDIVPAELRNRAGIVGAAMAVNQHLTP (SEQ ID NO: 1)

[0078] MAKKDEKSHKNAPLIGIDIGGTGIKGGIVDLKKGKLLGERFRVPTPQPATPESVAEAVAL

[0079] VVAELSARPEAPAAGSPVGVTFPGIIQHGVVHSAANVDKSWLNTDIDALLTARLGRPVE

[0080] VINDADAAGLAEARYGAGAGVKGTVLVITLGTGIGSAFIFDGKLVPNAELGHLEIDGHD

[0081] AETKASAVARERDGLSWDEYSVLLQRYFSHVEFLFSPELFIVGGGISKRADEYLPNLRLRTPIVPAVLRNEAGIVGAAIEIALQHKLAK(SEQ ID NO:2)

[0082] In some embodiments, the gene sequence of CgPPGK is shown as SEQ ID NO: 3; the gene sequence of Asp-PPGK is shown as SEQ ID NO: 4.

[0083] ATGACTGAGACTGGATTTGGAATTGATATCGGTGGCTCCGGCATCAAAGGCGCCCGC

[0084] GTTAACCTTAAGACCGGTGAGTTTATTGATGAACGCATAAAAATCGCCACCCCTAAG

[0085] CCAGCAACCCCAGAGGCTGTCGCCGAAGTAGTCGCAGAGATTATTTCTCAAGCCGAA

[0086] TGGGAGGGTCCGGTCGGAATTACCCTGCCGTCGGTCGTTCGCGGGCAGATCGCGCTA

[0087] TCCGCAGCCAACATTGACAAGTCCTGGATCGGCACCGATGTGCACGAACTTTTTGAC

[0088] CGCCACCTAAATGGCCGAGAGATCACCGTTCTCAATGACGCAGACGCCGCCGGCATC

[0089] GCCGAAGCAACCTTTGGCAACCCTGCCGCACGCGAAGGCGCAGTCATCCTGCTGACC

[0090] CTTGGTACAGGTATTGGATCCGCATTCCTTGTGGATGGCCAACTGTTCCCCAACACA

[0091] GAACTCGGTCACATGATCGTTGACGGCGAGGAAGCAGAACACCTTGCAGCAGCATC

[0092] CGTCAAAGAAAACGAAGATCTGTCATGGAAGAAATGGGCGAAGCACCTGAACAAGG

[0093] TGCTGAGCGAATACGAGAAACTTTTCTCCCCATCCGTCTTCATCATCGGTGGCGGAA

[0094] TTTCCAGAAAGCACGAAAAGTGGCTTCCATTGATGGAGCTAGACACTGACATTGTCCCAGCTGAGCTGCGCAATCGAGCCGGAATCGTAGGAGCTGCCATGGCAGTAAACCAACACCTCACCCCA(SEQ ID NO:3)

[0095] (SEQ ID NO: 4)

[0096] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid (OA), glycyrrhizic acid, glycyrrhetinic acid (GA), asiatic acid, or ursolic acid.

[0097] In some embodiments, the glycosylation modification system includes: glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, sucrose, uridine diphosphate, and pentacyclic triterpenoid compounds.

[0098] Secondly,

[0099] This invention also provides an enzyme combination for glycosylation modification of pentacyclic triterpenoid compounds, comprising:

[0100] (i) Glycosyltransferases;

[0101] (ii) Sucrose synthase;

[0102] (iii) Polyphosphate-dependent glucokinase; the polyphosphate-dependent glucokinase is CgPPGK with an amino acid sequence as shown in SEQ ID NO: 1 or Asp-PPGK with an amino acid sequence as shown in SEQ ID NO: 2.

[0103] In some embodiments, the glycosyltransferase is UGT73F24 with an amino acid sequence as shown in SEQ ID NO: 5 or UGT73C11 with an amino acid sequence as shown in SEQ ID NO: 6.

[0104] MDVAEEQPLKIYFIPYLAAGHMIPLCDIATLFASRGHHVTIITTPSNAQTLRESHHFRVQTIQFPSQEVGLPAGVQNLTAVTNLDDSYKIYHATMLLRKHIEDFVERDPPDCIVADFLFP WVDDVATKLHIPRLVFNGFTLFTICAMESHKAHPLPVDAASGSFVIPDFPHHVTINSTPPKRTKEFVDPLLTEAFKSHGFLINSFVELDGEECVEHYERITGGHKAWHLGPAFLVHRTAQ DRGEKSVVSTQECLSWLDSKRDNSVLYICFGTICYFPDKQLYEIASAIEASGHEFIWVVPEKRGNADESEEEKEKWLPKGFEERNNGKKGMIIRGWAPQVAILGHPAVGGFLTHCGWNST VEAVSAGVPMITWPVHSDQYFNEKLITQVRGIGVEVGAEEWIVTAFRETEKLVGRDRIERAVRRVMDGGDEAVQIRRRARELGEMARQAVQEGGSSHTNLTALINDLKRWRDSKQLN(SEQ ID NO:5)

[0105] MVSEITHKSYPLHFVLFPFMAQGHMIPMVDIARLLAQRGVKITIVTTPHNAARFENVLSRAIESGLPISIVQVKLPSQEAGLPEGNETFDSLVSMELLVPFFKAVNMLEEPVQKLFEEMSPQPSCIISDFCLPYTSKIAKKFNIPKILFHGMCCFCLLCMHVLRKNREILENLKSDKEHFVVPYFPDRVEFTRPQVPMATYVPGEWHEIKEDIVEADKTSYGVIVNTYQELEPAYANDYKEARSGKAWTIGPVSLCNKVGADKAERGNKADIDQDECLKWLDSKEEGSVLYVCLGSICSLP

[0106] LSQLKELGLGLEESQRPFIWVVRGWEKNKELLEWFSESGFEERVKDRGLLIKGWSPQML

[0107] ILAHHSVGGFLTHCGWNSTLEGITSGIPLLTWPLFGDQFCNQKLVVQVLKVGVSAGVEE

[0108] VTNWGEEEKIGVLVDKEGVKKAVEELMGESDDAKERRKRVKELGQLAQKAVEEGGSSHSNITSLLEDIMQLAQSNN(SEQ ID NO:6)

[0109] In some embodiments, the sucrose synthase is GuSUS1-Δ9 with the amino acid sequence shown in SEQ ID NO: 7. (SEQ ID NO: 7)

[0110] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0111] In some embodiments, the gene sequence of glycosyltransferase UGT73F24 is shown in SEQ ID NO: 8; the gene sequence of glycosyltransferase UGT73C11 is shown in SEQ ID NO: 9; the gene sequence of sucrose synthase GuSUS1-Δ9 is shown in SEQ ID NO: 10; the gene sequence of polyphosphate-dependent glucokinase CgPPGK is shown in SEQ ID NO: 3; and the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK is shown in SEQ ID NO: 4.

[0112] ATGGACGTTGCTGAAGAACAGCCACTCAAAATTTACTTCATTCCATACTTAGCAGCT

[0113] GGTCACATGATCCCTCTATGCGACATAGCCACTCTCTTCGCCTCACGTGGCCACCACG

[0114] TGACCATCATCACCACTCCCTCCAACGCCCAAACCCTCCGCGAATCCCACCACTTCC

[0115] GCGTCCAAACCATCCAATTCCCCTCCCAAGAAGTGGGCCTCCCCGCCGGCGTCCAAA

[0116] ACCTCACCGCCGTCACGAACCTCGACGACTCCTATAAGATCTACCACGCCACCATGC

[0117] TTCTCCGCAAACACATCGAGGACTTCGTGGAGCGGGACCCACCAGACTGCATCGTCG

[0118] CCGACTTCCTATTCCCCTGGGTGGATGACGTGGCAACCAAGCTTCACATCCCAAGAC

[0119] TCGTCTTCAACGGCTTCACCTTATTTACCATCTGTGCCATGGAATCCCACAAGGCACA

[0120] CCCTCTCCCAGTCGATGCCGCCTCGGGTTCTTTTGTGATTCCCGATTTCCCTCACCAT

[0121] GTCACTATCAATTCAACACCCCCAAAGCGCACCAAGGAATTCGTAGACCCTCTGCTC

[0122] ACGGAAGCGTTCAAGAGCCACGGCTTCCTCATCAACAGCTTCGTGGAGCTCGACGGA

[0123] GAAGAGTGCGTCGAGCACTACGAGAGAATCACCGGTGGTCACAAGGCTTGGCATCT

[0124] TGGCCCTGCCTTTCTCGTTCACAGGACCGCTCAAGATAGGGGAGAGAAGAGCGTGGT

[0125] GAGCACGCAGGAGTGCCTGAGTTGGCTCGACTCGAAGCGAGACAACTCAGTGCTCT

[0126] ACATATGCTTTGGAACCATTTGCTATTTCCCAGACAAGCAGCTTTACGAGATCGCAA

[0127] GCGCGATTGAAGCGTCGGGTCACGAATTCATATGGGTTGTTCCCGAGAAGAGAG

[0128] GGAATGCTGATGAGAGCGAGGAGGAGAAAGAAAAGTGGCTGCCAAAGGGATTTGA

[0129] AGAGAGGAATAATGGAAAGAAGGGGATGATTATAAGGGGGTGGGCCCCGCAGGTG

[0130] GCGATCCTGGGCCACCCTGCTGTGGGCGGGTTTCTAACGCATTGCGGGTGGAACTCC

[0131] ACTGTGGAGGCCGTTAGCGCGGGGGTTCCGATGATAACGTGGCCGGTACATAGCGAT

[0132] CAATACTTCAACGAGAAGCTGATAACTCAGGTGAGGGGGATTGGGGTGGAGGTGGG

[0133] TGCGGAGGAGTGGATCGTCACTGCATTTCGGGAGACGGAGAAGCTGGTGGGAAGGG

[0134] ATCGCATAGAGAGGGCTGTAAGGAGGGTGATGGACGGTGGTGATGAGGCGGTACAG

[0135] ATCAGACGGCGCGCTCGAGAGCTTGGGGAAATGGCTAGACAAGCTGTTCAGGAAGG

[0136] GGGCTCGTCTCACACTAATTTGACGGCCTTGATTAATGATCTTAAGCGATGGAGAGACTCTAAGCAGCTTAAC(SEQ ID NO:8)

[0137] ATGGTTTCCGAAATCACCCACAAATCTTACCCGCTGCACTTTGTTCTGTTCCCGTTTA

[0138] TGGCGCAAGGTCATATGATTCCGATGGTGGACATCGCACGTCTGCTGGCACAGCGTG

[0139] GCGTAAAAATTACCATCGTTACGACCCCGCATAACGCGGCGCGTTTCGAGAACGTTC

[0140] TGTCTCGTGCTATCGAATCTGGTCTGCCGATCAGCATTGTTCAGGTTAAACTGCCAAG

[0141] CCAAGAAGCGGGTCTGCCTGAGGGTAACGAGACCTTCGACTCCCTGGTAAGCATGG

[0142] AGCTGCTGGTCCCGTTCTTCAAAGCTGTGAACATGCTGGAAGAACCGGTGCAAAAGC

[0143] TGTTTGAAGAAATGTCCCCTCAGCCGTCCTGTATCATTTCCGACTTCTGTCTGCCGTA

[0144] CACCTCCAAAATTGCGAAAAAATTCAACATTCCGAAGATCCTGTTCCACGGCATGTG

[0145] CTGTTTCTGTCTGCTGTGCATGCACGTGCTGCGCAAAAACCGCGAAATTCTGGAAAA

[0146] CCTGAAATCTGACAAGGAACACTTCGTGGTTCCGTACTTCCCGGATCGCGTTGAATT

[0147] CACCCGTCCGCAAGTGCCGATGGCAACTTACGTACCAGGCGAGTGGCACGAAATCA

[0148] AGGAAGATATTGTAGAGGCGGATAAAACCAGCTATGGTGTGATCGTAAATACCTAC

[0149] CAGGAACTGGAACCGGCTTACGCAAACGACTACAAAGAAGCGCGTTCCGGTAAAGC

[0150] GTGGACCATTGGTCCGGTTTCCCTGTGTAACAAAGTAGGTGCCGATAAGGCGGAACG

[0151] TGGTAACAAGGCCGACATTGACCAGGATGAATGTCTGAAATGGCTGGACTCTAAAG

[0152] AGGAAGGCTCTGTACTGTACGTTTGTCTGGGCTCTATCTGCTCCCTGCCGCTGTCTCA

[0153] GCTGAAAGAACTGGGCCTGGGCCTGGAAGAAAGCCAGCGCCCATTCATCTGGGTGG

[0154] TTCGTGGTTGGGAAAAAAACAAAGAACTGCTGGAATGGTTCTCTGAAAGCGGTTTCG

[0155] AGGAACGTGTGAAAGATCGCGGTCTGCTGATCAAAGGTTGGTCTCCGCAGATGCTGA

[0156] TCCTGGCTCACCATAGCGTGGGCGGTTTCCTGACTCATTGTGGCTGGAATTCTACCCT

[0157] GGAGGGCATCACGTCCGGTATTCCGCTGCTGACTTGGCCACTGTTCGGTGACCAGTT

[0158] CTGTAACCAGAAGCTGGTTGTGCAGGGTTCTGAAAGTTGGCGTTAGCGCCGGCGTCGA

[0159] AGAAGTGACCAACTGGGGTGAAGAAGAAGAAAAAATCGGCGTACTGGTGGACAAGGAG

[0160] GGCGTGAAGAAGGCGGTAGAAGAACTGATGGGTGAATCCGACGACGCAAAAGAAC

[0161] GTCGTAAACGTGTTAAAGAACTGGGCCAAGCTGGCCCAGAAAGCAGTAGAAGAAGGC

[0162] GGTTCTTCTCATTCTAACATCACCAGCCTGCTGGAAGACATCATGCAGCTGGCCCAGTCTAACAAC(SEQ ID NO:9)

[0163] CACAGTCTCCGTGAGAGGCTCGATGAAACCTTGACTGCTAATAGAAATGAAATTTTTGG

[0164] CCCTTCTCTCAAGGATCGAAGCCAAGGGCAAGGGGATCCTGCAACACCACCAGGTCA

[0165] TTGCTGAGTTTGAGGAAATTCCTGAGGAGAATAGACATAAGCTGATGGATGGGGCATT

[0166] TGGAGAAGTCTTGAGATCCACACAGGAAGCCATAGTTTTACCACCATGGGTTGCTCTG

[0167] GCTGTTCGTCCAAGGCCTGGTGTTTGGGAGTACCTGAGAGTGAATGTGCACGCTCTTG

[0168] TTGTCGAAGAGTTGCAACCTGCTGAGTTTCTCCGCTTCAAGGAGGAACTTGTTGATGG

[0169] AAGTTCTAATGGCAACTTTGTGCTTGAGTTGGACTTTGAACCATTTACTGCATCCTTCC

[0170] CCCGCCCAACTCTCAACAAGTCAATTGGAAATGGTGTGCAATTCCTCAACCGTCACCT

[0171] TTCTGCAAAACTCTTCCATGACAAGGAGAGCTTGCATCCACTTCTGGAATTCCTCAGA

[0172] CTTCACAGCTACAAGGGAAAGACATTGATGTTGAATGACAGAATTCAAACCCCGGATT

[0173] CTCTTCAACATGTTCTGAGGAAAGCTGAAGAGTATCTTGGAACACTTTCTCCTGAGAC

[0174] ACCCTACTCAGTATTTGAGCACAAGTTCCAGGAGATCGGTTTGGAGAGAGGGTGGGG

[0175] TGACACCGCGGAGCGTGTCCTCGAGTCCATCCAACTCCTCTTGGATCTTCTTGAGGCT

[0176] CCTGACCCTTGCACCCTTGAGACTTTCCTTGGAAGGATCCCCATGGTCTTTAATGTTGT

[0177] GATCCTTTCGCCCCACGGTTACTTTGCCCAAGATAATGTCTTGGGATACCCTGATACCG

[0178] GTGGCCAGGTTGTTTACATCTTGGATCAAGTTCGCGCCTTGGAGAATGAGATGCTCCA

[0179] TCGCATTAAGCAACAAGGCTTGGATATCGTCCCTCGCATTCTCATTATCACCCGTCTTC

[0180] TCCCCGATGCAGTAGGAACTACCTGTGGCCAACGACTCGAGAAGGTCTTTGGAACCG

[0181] AGCATTGCCACATCTTCGAGTTCCCTTCAGAAACGAGAAGGGAATGGTTCGCAAGT

[0182] GGATCTCAAGATTCGAAGTCTGGCCATACCTAGAAACTTACACTGAGGATGTTGCCCA

[0183] TGAACTTGCCAAAGAGTTGCAAGGCAAGCCAGATCTGATTGTTGGAAACTACAGTGGA

[0184] TGGAAACATTGTTGCCTCTTTGTTGGCACATAAATTAGGTGTCACTCAGTGTACCATTG

[0185] CTCATGCACTTGGAAGACCAAGTACCCTGAATCTGACATTACTGGAAAAAATTCGA

[0186] AGAGAAATATCACTTCTCTTGCCAATTCACAGCTGATCTCTTTGCTATGAACCACACAG

[0187] ACTTCATCATCACCAGTACCTTCCAAGAGATTGCTGGAAGCAAGGACACTGTTGGACA

[0188] GTATGAGAGTCACACTGCCTTCACCCTTCCTGGACTCTACCGTGTCGTGCACGGTAT

[0189] TGATGTCTTTGATCCAAAATTCAACATTGTATCTCCCGGAGCTGATCAGACCATCTAC

[0190] TTCCCCTACACCGACACCAGCCGCAGGCTGACATCCTTCCACCCCGAAATCGAAGA

[0191] GCTTCTTTACAGCTCAGTGGAGAATGAAGAGCACATATGTGTATTGAAGGACCGCAA

[0192] CAAGCCAATTATCTTCACCATGGCGAGGTTGGACCGTGTGAAGAACATCACTGGACT

[0193] TGTCGAGTGGTACGGCAAGAACGCCAAGCTCCGTGAGCTGGTGAACCTTGTGGTTG

[0194] TTGCCGGAGACAGGAGGAAGGAGTCCAAGGACTTGGAAGAGAAGGCCGAGATGAA

[0195] GAAGATGTACGGCCTGATTGAGACCTACAAGCTGAATGGCCAATTCAGGTGGATCTC

[0196] CTCTCAGATGAACCGGGTGAGGAACGGGGAGCTGTACCGTGTCATCTGCGACACAA

[0197] AGGGAGCTTTCGTGCAGCCTGCTGTCTATGAGGCCTTTGGATTGACAGTTGTTGAGG

[0198] CCATGACTTGTGGGTTGCCAACATTTGCAACATGCAATGGTGGCCCTGCTGAGATCA

[0199] TTGTTCATGGCAAGTCTGGTTTCCACATTGACCCTTACCACGGCGCGGCCGCCGCCG

[0200] ATCTCCTTGTTGAATTCTTTGAGAAGTGCAAGGCTGACCCATCTCACTGGGACAACA

[0201] TCTCCCATGGTGGTCTCCAACGTATTGAAGAGAAGTATACATGGCAAATTTACTCTGA

[0202] GAGGCTTCTCACTCTCACTGGTGTCTATGGCTTCTGGAAGCATGTGTCTAACCTTGA

[0203] CCGCCGCGAGAGCCGCCGTTATCTTGAGATGTTCTATGCTCTCAAGTACCGCAAATTGGCTGAGTCTGTGCCCCTAGCTGTTGAGGAG (SEQ ID NO: 10)

[0204] This invention also provides a biological agent for glycosylation modification of pentacyclic triterpenoid compounds, comprising:

[0205] (i) Recombinant vectors or recombinant bacteria carrying the gene sequence of glycosyltransferase UGT73F24 or the gene sequence of glycosyltransferase UGT73C11;

[0206] (ii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of sucrose synthase GuSUS1-Δ9;

[0207] (iii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of polyphosphate-dependent glucokinase CgPPGK or the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK.

[0208] The gene sequence of glycosyltransferase UGT73F24 is shown in SEQ ID NO: 8; the gene sequence of glycosyltransferase UGT73C11 is shown in SEQ ID NO: 9; the gene sequence of sucrose synthase GuSUS1-Δ9 is shown in SEQ ID NO: 10; the gene sequence of polyphosphate-dependent glucokinase CgPPGK is shown in SEQ ID NO: 3; and the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK is shown in SEQ ID NO: 4.

[0209] In some embodiments, the pentacyclic triterpenoid compound includes, but is not limited to, one of β-amyrinol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0210] Thirdly,

[0211] This invention also provides a UDP cycling system based on polyphosphate-dependent glucokinase, the UDP cycling system comprising: the above-mentioned enzyme combination, a pentacyclic triterpenoid compound, sucrose, uridine diphosphate, and Mg. 2+ And sodium polyphosphate.

[0212] Fourthly,

[0213] This invention also provides a method for glycosylation modification of pentacyclic triterpenoid compounds, comprising: catalyzing a substrate using the above-mentioned enzyme combination, wherein the substrate is a pentacyclic triterpenoid compound and the glycosyl donor for the catalytic reaction is sucrose.

[0214] In some embodiments, the substrate includes, but is not limited to, one of β-amyrin alcohol, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

[0215] In some embodiments, the reaction system includes: a pentacyclic triterpenoid compound, sucrose, uridine diphosphate, glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, and Mg. 2+ And sodium polyphosphate.

[0216] In some embodiments, the pentacyclic triterpenoid compound is 0.3-1.0 mM, sucrose is 10-100 mM, uridine diphosphate is 0.2-1.0 mM, glycosyltransferase is 0.15-0.20 mg / mL, sucrase synthase is 0.15-0.20 mg / mL, polyphosphate-dependent glucokinase is 0.40-0.50 mg / mL, and Mg... 2+ The concentration is 10-20 mM, and the concentration of sodium polyphosphate is 10 g / L.

[0217] As a specific example, in the reaction system, the pentacyclic triterpenoid compound was 1.0 mM, sucrose was 100 mM, uridine diphosphate was 1.0 mM, glycosyltransferase was 0.15 mg / mL, sucrase synthase was 0.15 mg / mL, polyphosphate-dependent glucokinase was 0.40 mg / mL, and Mg... 2+ The concentration is 10 mM, and the sodium polyphosphate concentration is 10 g / L.

[0218] As a specific example, the concentrations were: pentacyclic triterpenoids 1.0 mM, sucrose 100 mM, uridine diphosphate 0.8 mM, glycosyltransferase 0.18 mg / mL, sucrase synthase 0.18 mg / mL, polyphosphate-dependent glucokinase 0.45 mg / mL, and Mg... 2+ The concentration is 10 mM, and the sodium polyphosphate concentration is 10 g / L.

[0219] As a specific example, the concentrations were: pentacyclic triterpenoids 1.0 mM, sucrose 100 mM, uridine diphosphate 0.2 mM, glycosyltransferase 0.15 mg / mL, sucrase synthase 0.15 mg / mL, polyphosphate-dependent glucokinase 0.40 mg / mL, and Mg... 2+ The concentration is 10 mM, and the sodium polyphosphate concentration is 10 g / L.

[0220] As a specific example, the concentrations were: pentacyclic triterpenoids 1.0 mM, sucrose 80 mM, uridine diphosphate 0.8 mM, glycosyltransferase 0.18 mg / mL, sucrase synthase 0.18 mg / mL, polyphosphate-dependent glucokinase 0.45 mg / mL, and Mg... 2+ The concentration is 10 mM, and the sodium polyphosphate concentration is 10 g / L.

[0221] In some embodiments, the enzymes are added in stages: first, sucrose synthase is added, followed by polyphosphate-dependent glucokinase and glycosyltransferase 10-20 minutes later.

[0222] In some embodiments, the catalytic reaction is carried out at a pH of 8-8.5 and a temperature of 40°C.

[0223] In some embodiments, the substrate is glycyrrhetinic acid, and the glycosyltransferase in the reaction system is selected as UGT73F24.

[0224] In some embodiments, the substrate is oleanolic acid, and the glycosyltransferase used in the reaction system is UGT73C11. Example 1: Plasmid Construction and Engineered Bacteria Construction

[0225] pET-28a and pET-32a can be purchased commercially and are stored in the laboratory in the early stages. The construction method of pET-28a-GuSUS1-Δ9 is described in reference [1]; it is constructed and stored in the laboratory in the early stages.

[0226] The construction method of pET-28a-UGT73C11 is described in reference [2]; the laboratory early construction was preserved.

[0227] The construction method of pET-32a-UGT73F24 is described in reference [3]; the laboratory early construction was preserved.

[0228] Literature [1]: Zhang L, Gao Y, Liu

[0229] https: / / doi.org / 10.1021 / acs.jafc.9b05178.

[0230] Literature [2] Liu X, Zhang L, Feng

[0231] Literature [3] Zhang, L, Ren, S, Liu,

[0232] Construction of pET-28a-CgPPGK: The target gene fragment was cloned from the bacterial genome, and the vector fragment was cloned from the pET-28a plasmid. The primers used are shown in Table 1. The target gene fragment was cloned using Primestar DNA polymerase. The PCR reaction conditions were: 98℃ pre-denaturation for 10 s, Tm-5℃ annealing for 10 s, 72℃ extension, 32 cycles, 72℃ extension for 7 min, and storage at 16℃. Then, high-fidelity Fast Pfu polymerase was used for PCR amplification. The PCR reaction conditions were: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 20 s, Tm-5℃ annealing for 20 s, 72℃ extension, 32 cycles, 72℃ extension for 7 min, and storage at 16℃. Following the instructions of the ThermoFisher agarose gel extraction kit, the cloned target gene fragment and corresponding vector fragment were recovered. The target gene fragment and the corresponding vector were ligated using the Gibson Assembly method. After incubation at 50°C for 1 hour, the ligation product was transformed into *E. coli* DH5α competent cells (purchased from Bomeide). The constructed vector was sequenced using Genewiz. The correctly sequenced vector was then transformed into *E. coli* BL21 competent cells (purchased from Bomeide) for subsequent expression.

[0233] Table 1 Primer sequences

[0234]

[0235] pET-28a-Asp-PPGK was synthesized and constructed by Anshengda Company using conventional techniques in the field. The primer sequences involved are shown in Table 2.

[0236] Table 2 Primer sequences

[0237]

[0238]

[0239] Example 2 Protein Expression and Purification

[0240] Add kanamycin or ampicillin to 400 mL of LB medium at a final concentration of 50 μg / mL or 100 μg / mL, with an inoculum size of 1%. Incubate at 37°C for 3-4 h until OD600 = 0.8. Add IPTG to a final concentration of 0.1 mM and induce overnight at 16°C for 16 h. Centrifuge at 8000 rpm to collect the bacteria. Resuspend the cells in 50 mM PB (pH 7.5). Hypolyze at 1200 bar using ultra-high pressure. Separate the supernatant and precipitate at 12000 rpm. Discard the precipitate and filter the supernatant through a 0.45 μm water film. Purify using an AKTA system and a HisTrap HP affinity chromatography column. Loading buffer is 50 mM PBS (pH 7.5, 0.15 M NaCl), and elution buffer is 50 mM PBS (pH 7.5, 0.15 M NaCl, 200 mM imidazole). Collect the target protein fraction and analyze by SDS-PAGE (12%).

[0241] Figure 1a The purification efficiency of GuSUS1-Δ9, UGT73C11, UGT73F24, and CgPPGK enzymes was analyzed by SDS-PAGE. Figure 1a In the middle, lane 1 is GuSUS1-Δ9, lane 2 is UGT73C11, lane 3 is UGT73F24, and lane 4 is CgPPGK. Figure 1a It can be seen that the four proteins were successfully purified.

[0242] Figure 1b The purification efficiency of GuSUS1-Δ9, UGT73F24, and Asp-PPGK enzymes was analyzed by SDS-PAGE. Figure 1b In the middle, lane 1 is UGT73F24, lane 2 is GuSUS1-Δ9, and lane 3 is Asp-PPGK. (The sentence appears to be incomplete and requires further context.) Figure 1b As can be seen, the three proteins were successfully purified.

[0243] Example 3: Enzyme Characteristics and Activity Analysis

[0244] (1) Enzyme activity assay of two glycosyltransferases

[0245] The enzyme activity assay of UGT73F24 was performed in a 200 μL reaction system containing 4 mM uridine diphosphate glucose (UDPG), 500 μM glycyrrhetinic acid (GA), 50 mM PB (pH 7.5), and 20 μg UGT73F24, at 45 °C for 10 min. After the reaction was complete, the mixture was boiled for 3 min, and 200 μL of methanol was added to stop the reaction. The mixture was filtered through a 0.22 μm organic filter membrane, and the final product was detected by high performance liquid chromatography (HPLC). The detection method was as follows: Instrument parameters: Shimadzu LC-10A, column: ASC Accurasil (4.6 × 250 mm, 5 μm), detector: SPD-10AVP deuterium lamp detector, detection wavelength: 254 nm, mobile phase A: methanol, mobile phase B: 6‰ acetic acid solution, injection volume: 10 μL, flow rate: 1 mL / min, column temperature: 40 °C, workstation: LCsolution. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of 3-O-glucose-glycyrrhetinic acid (GA-3-O-Glc) per minute. Specific enzyme activity data are shown in Table 3.

[0246] The enzyme activity assay of UGT73C11 was performed in a 200 μL reaction system containing 4 mM UDPG, 500 μM oleanolic acid (OA), 50 mM Tris-HCl buffer (pH 8.5), and 20 μg UGT73C11, and incubated at 40 °C for 10 min. After the reaction was complete, the mixture was boiled for 3 min, and 200 μL of methanol was added to stop the reaction. The mixture was then filtered through a 0.22 μm organic filter membrane. The final product was detected by ultra-high performance liquid chromatography (UPLC) under the following conditions and methods: mobile phase A: 100% acetonitrile, mobile phase B: 0.1% phosphoric acid, gradient elution: 0-0.02 min 80% B, 3 min 65% B, 4 min 50% B, 8 min 35% B, 12 min 15% B, 15 min 5% B, 16-20 min 80% B, flow rate 0.3 mL / min, UV detector (203 nm), column oven temperature: 35℃, HPLC column: InfinityLab Poroshell 120EC-C18 column (2.1 × 100 mm, 2.7 μm). One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of 3-O-glucose-oleanolic acid (OA-3-O-Glc) per minute. Specific enzyme activity data are shown in Table 3.

[0247] (2) Assay of sucrose synthase activity

[0248] The enzyme activity assay of GuSUS1-Δ9 was mainly performed by measuring the fructose produced from sucrose hydrolysis. This assay was conducted in a 100 μL reaction system containing 50 mM sodium citrate buffer (pH 5.5), 50 mM sucrose, 50 mM UDP, and 20 μg GuSUS1-Δ9, and was carried out at 50 °C for 10 min. After the reaction, the mixture was boiled for 3 min, and 100 μL of acetonitrile was added to terminate the reaction. After filtration through a 0.22 μm organic filter membrane, the final product was detected by high-performance liquid chromatography (HPLC). The detection conditions and methods were as follows: mobile phase A: 100% acetonitrile, mobile phase B: water, mobile phase A:mobile phase B = 7:3, flow rate 1 mL / min, detector: differential refractive index detector, column oven temperature: 40 °C, HPLC column: Arcus BP-NH2 column (4.6 × 250 mm, 5 μm). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of fructose per minute. The data for enzyme activity determination are shown in Table 3.

[0249] (3) Characterization of polyphosphate-dependent glucokinase CgPPGK derived from Corynebacterium glutamicum

[0250] The enzyme activity assay of CgPPGK catalyzing glucose and fructose was characterized by measuring the reduction in substrate content. This assay was performed in a 100 μL reaction system comprising 50 mM PB (pH 8), 50 mM glucose or fructose, 10 mM MgCl2, 10 g / L sodium polyphosphate (catalog number: 68915-31-1; manufacturer: Aladdin), and 100 μg CgPPGK, and was carried out at 40 °C for 10 min. After boiling for 3 min, the reaction was terminated by adding 100 μL acetonitrile. The mixture was filtered through a 0.22 μm organic filter membrane, and the final product was detected by high-performance liquid chromatography (HPLC). The detection conditions and methods are as follows: Mobile phase A: 100% acetonitrile, mobile phase B: water, mobile phase A:mobile phase B = 7:3, flow rate: 1 ml / min, detector: differential refractive index detector, column oven temperature: 40℃, liquid chromatography column: Arcus BP-NH2 column (4.6 × 250 mm, 5 μm). One unit of enzyme activity is defined as the amount of enzyme required to reduce 1 μmol of glucose or fructose per minute. Specific enzyme activity data are shown in Table 3. A comparison of the catalytic activities of CgPPGK on glucose and fructose is shown in [Table 3]. Figure 2a ,pass Figure 2a It can be seen that the specific enzyme activity of CgPPGK catalyzing glucose is about 3 times that of fructose.

[0251] The optimal pH conditions for CgPPGK-catalyzed fructose determination were as follows: reaction at 40℃ for 10 min, with buffer pH variations of sodium citrate buffer (pH 5.0-6.0), PB buffer (pH 6.0-8.0), and Tris-HCl buffer (pH 8.5-9.0). The optimal temperature was determined at 50 mM PB (pH 8), within a range of 20℃-50℃. The optimal temperature and pH were ultimately characterized by fructose conversion. Figure 3a It can be seen that the optimal temperature for CgPPGK to catalyze fructose is 40℃, and the optimal pH is 8.

[0252] HPLC and LC-MS / MS analysis of fructose-6-phosphate, a product of CgPPGK catalysis of fructose.

[0253] High-performance liquid chromatography (HPLC) was used to determine the product of CgPPGK-catalyzed fructose. The detection conditions and methods were as follows: mobile phase A: H₂O, mobile phase B: 5 mM H₂SO₄, and the detection method was isostatic elution. The flow rate was 0.35 mL / min, the detector was a differential detector, the column temperature was 55℃, and the HPLC column was an Aminex HPX-87H column (300 × 7.8 mm). Figure 4a It can be seen that the peak position of the standard fructose-6-phosphate corresponds to the peak of the reaction product.

[0254] To further confirm that the product of CgPPGK catalysis of fructose is fructose-6-phosphate, the reaction product was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mobile phase was water (A) and acetonitrile (B). The gradient elution program was as follows: 0 min, 50% A; 2.5–5 min, 80% A; column: 120 HILIC-Z (150 mm × 2.0 mm, 5 μm); flow rate: 0.6 mL / min. The reaction product was separated by the above-described LC method and detected by tandem mass spectrometry in multiple reaction ion negative mode. Figure 5 The LC-MS / MS results of the CgPPGK-catalyzed fructose products show that the mass / charge ratio of the precursor ion is 259 in the negative mode, and the mass / charge ratios of the daughter ions produced by the precursor ion are 79, 97, 139, and 169, which are the same as those of the product standard.

[0255] (4) Characterization of Asp-PPGK, a polyphosphate-dependent glucokinase derived from Arthrobacterium.

[0256] The enzyme activity assay of Asp-PPGK catalyzing glucose and fructose was characterized by measuring the reduction in substrate content. This assay was performed in a 100 μL reaction system, comprising 50 mM Tris-HCl buffer (pH 8.5), 50 mM glucose or fructose, 10 g / L sodium polyphosphate (catalog number: 68915-31-1; manufacturer: Aladdin), 10 mM MgCl2, and 100 μg Asp-PPGK, and reacted at 30 °C for 10 min. After boiling for 3 min, the reaction was terminated by adding 100 μL acetonitrile. The mixture was filtered through a 0.22 μm organic filter membrane, and the final product was detected by high-performance liquid chromatography (HPLC). The detection conditions and methods are as follows: Mobile phase A: 100% acetonitrile, mobile phase B: water, mobile phase A:mobile phase B = 7:3, flow rate: 1 ml / min, detector: differential refractive index detector, column oven temperature: 40℃, liquid chromatography column: Arcus BP-NH2 column (4.6×250 mm, 5 μm). One unit of enzyme activity is defined as the amount of enzyme required to reduce 1 μmol of glucose or fructose per minute. Specific enzyme activity data are shown in Table 3. A comparison of the catalytic activities of Asp-PPGK on glucose and fructose is shown in [Table 3]. Figure 2b ,from Figure 2b It can be seen that the specific enzyme activity of Asp-PPGK catalyzing glucose is about twice that of fructose.

[0257] The optimal pH conditions for determining fructose catalysis using Asp-PPGK were as follows: reaction at 30℃ for 10 min, with buffer pH variations of sodium citrate buffer (pH 5.0-6.0), PB buffer (pH 6.0-8.0), and Tris-HCl buffer (pH 8.5-9.0). The optimal temperature was determined at 50 mM PB (pH 8), within a range of 20℃-50℃. The optimal temperature and pH were ultimately characterized by fructose conversion. Figure 3b It can be seen that the optimal temperature for Asp-PPGK to catalyze fructose is 30℃, and the optimal pH is 8.5.

[0258] HPLC and LC-MS / MS analysis of fructose-6-phosphate, the product of Asp-PPGK catalysis of fructose.

[0259] High-performance liquid chromatography (HPLC) was used to determine the product of Asp-PPGK-catalyzed fructose. The detection conditions and methods were as follows: mobile phase A: H₂O, mobile phase B: 5 mM H₂SO₄, and the detection method was iso-concentration elution. The flow rate was 0.35 mL / min, the detector was a differential detector, the column temperature was 55℃, and the HPLC column was an Aminex HPX-87H column (300 × 7.8 mm). Figure 4b It can be seen that the peak position of the standard fructose-6-phosphate corresponds to the peak of the reaction product.

[0260] To further confirm that the product of Asp-PPGK catalysis of fructose is fructose-6-phosphate, the reaction product was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mobile phase was water (A) and acetonitrile (B). The gradient elution program was as follows: 0 min, 50% A; 2.5–5 min, 80% A; column: 120 HILIC-Z (150 mm × 2.0 mm, 5 μm); flow rate: 0.6 mL / min. The reaction product was separated by the above-described LC method and detected by tandem mass spectrometry in multiple reaction ion negative mode. Figure 5 The LC-MS / MS results of the fructose product catalyzed by Asp-PPGK show that the mass / charge ratio of the precursor ion is 259 in the negative mode, and the mass / charge ratios of the daughter ions produced by the precursor ion are 79, 97, 139, and 169, which are the same as those of the product standard.

[0261] Table 3. Sources and specific enzyme activities of GuSUS1-Δ9, UGT73C11, UGT73F24, CgPPGK, and Asp-PPGK

[0262]

[0263]

[0264] Example 4: Construction of a high-efficiency UDP-glucose regeneration system using GA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK).

[0265] Construction of a high-efficiency UDP-glucose regeneration system coupled with GuSUS1-Δ9, UGT73F24, and CgPPGK as follows: Figure 6a We name this system UDP-3E, while the traditional UDP-glucose high-efficiency regeneration system is named UDP-2E. The UDP-3E reaction system is 200 μL and includes 1 mM GA, 100 mM sucrose, 1 mM UDP, PB (50 mM, pH 6.5), 10 g / L sodium polyphosphate (catalog number: 68915-31-1; manufacturer: Aladdin), 10 mM MgCl2, 0.15 mg / mL UGT73F24, 0.15 mg / mL GuSUS1-Δ9, and 0.40 mg / mL CgPPGK. The UDP-2E system does not contain CgPPGK, sodium polyphosphate, or MgCl2. The reaction is carried out at 40℃ with periodic sampling. The reaction products are detected by HPLC. Figure 7a It can be seen that the reaction in the UDP-2E system stopped after 8 hours, while the UDP-3E reaction continued. Ultimately, the concentration of the glycosylated product GA-3-O-Glc of the generated GA was 2.11 times higher than that in UDP-2E.

[0266] Example 5: Construction of a high-efficiency UDP-glucose regeneration system using GA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and Asp-PPGK).

[0267] The construction of a high-efficiency UDP-glucose regeneration system coupled with GuSUS1-Δ9, UGT73F24, and Asp-PPGK is as follows: Figure 6b We name this system UDP-3E, while the traditional UDP-glucose high-efficiency regeneration system is named UDP-2E. The UDP-3E reaction system is 200 μL and includes 1 mM GA, 100 mM sucrose, 1 mM UDP, PB (50 mM, pH 6.5), 10 g / L sodium polyphosphate (catalog number: 68915-31-1; manufacturer: Aladdin), 10 mM MgCl2, 0.15 mg / mL UGT73F24, 0.15 mg / mL GuSUS1-Δ9, and 0.40 mg / mL Asp-PPGK. The UDP-2E system does not contain Asp-PPGK, sodium polyphosphate, or MgCl2. The reaction is carried out at 40℃ with periodic sampling. The reaction products are detected by HPLC. Figure 7b It can be seen that after 4 hours of reaction, the conversion rate of GA in the UDP-2E system was 48.96%, while the conversion rate of GA in the UDP-3E reaction system was 73.42%, which is 0.5 times higher than that of the UDP-2E system.

[0268] Example 6: Optimization of a high-efficiency UDP-glucose regeneration system using GA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73F24, and CgPPGK).

[0269] The reaction system consisted of 200 μL of GA, 100 mM sucrose, 1 mM UDP, PB (50 mM, pH 6.5), 10 g / L sodium polyphosphate, 10 mM MgCl2, 0.15 mg / mL UGT73F24, 0.15 mg / mL GuSUS1-Δ9, and 0.40 mg / mL CgPPGK.

[0270] Figure 8 The relative conversion of substrate GA was shown after 2 hours of reaction at a temperature of 40℃, a magnesium ion concentration of 10 mM, and pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 (buffer solutions and pH variations: sodium citrate buffer (pH 5.0-6.0), PB buffer (pH 6.0-8.0), and Tris-HCl buffer (pH 8.5-9.0)). The substrate conversion was determined by HPLC. Figure 8It can be seen that the optimal pH for the UDP-3E reaction system is 8-8.5.

[0271] Figure 9 The relative conversion of substrate GA (detected by HPLC) after 2 hours of reaction at pH 6.5, magnesium ion concentration of 10 mM, and temperatures of 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃ is presented. Figure 9 It can be seen that the optimal temperature for the UDP-3E reaction system is 40℃.

[0272] Figure 10 The relative conversion of substrate GA (detected by HPLC) after 2 hours of reaction is shown under the conditions of pH 6.5, temperature 40℃, and magnesium ion concentrations of 0 mM, 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM. Figure 10 It can be seen that the optimal magnesium ion concentration for the UDP-3E reaction system is 10-20 mM. Example 7: Construction of a high-efficiency UDP-glucose regeneration system using OA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK).

[0273] The UDP-glucose high-efficiency regeneration system UDP-3E, coupled with GuSUS1-Δ9, UGT73C11, and CgPPGK, was developed in 200 μL volume. The reaction mixture included 1 mM OA, 100 mM sucrose, 200 μM UDP, 50 mM PB (pH 6.5), 10 g / L sodium polyphosphate, 10 mM MgCl2, 0.15 mg / mL UGT73C11, 0.15 mg / mL GuSUS1-Δ9, and 0.40 mg / mL CgPPGK. The UDP-2E system, however, did not contain CgPPGK, sodium polyphosphate, or MgCl2. The reaction was carried out at 40 °C with periodic sampling. The reaction products were analyzed by UPLC. Figure 12 It can be seen that the glycosylation product OA-3-O-Glc generated by the UDP-3E reaction was increased by 0.30 times.

[0274] Example 8: Optimization of a high-efficiency UDP-glucose regeneration system using OA as a glycosylation substrate coupled with three enzymes (GuSUS1-Δ9, UGT73C11, and CgPPGK).

[0275] The reaction system consisted of 200 μL of OA, 100 mM sucrose, 200 μM UDP, PB (50 mM, pH 6.5), 10 g / L sodium polyphosphate, 10 mM MgCl2, 0.15 mg / mL UGT73C11, 0.15 mg / mL GuSUS1-Δ9, and 0.40 mg / mL MgPPGK.

[0276] Figure 13 The relative conversion of substrate OA was shown after 2 hours of reaction at a temperature of 40℃, a magnesium ion concentration of 10 mM, and pH values ​​of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5 (buffer solutions and pH variations: sodium citrate buffer (pH 5.0-6.0), PB buffer (pH 6.0-8.0), and Tris-HCl buffer (pH 8.5-9.0)). The reaction substrate was detected by UPLC. Figure 8 It can be seen that the optimal pH for the UDP-3E reaction system is 8-8.5.

[0277] Figure 14 The relative conversion of substrate OA (response substrate was detected by UPLC) was shown under the following conditions: pH 6.5, magnesium ion concentration 10 mM, and temperatures of 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, after a reaction time of 2 hours. Figure 9 It can be seen that the optimal temperature for the UDP-3E reaction system is 40℃.

[0278] Figure 15 The relative conversion of substrate OA (response substrate was detected by UPLC) after 2 hours of reaction at pH 6.5, temperature 40℃, and magnesium ion concentrations of 0 mM, 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM. Figure 10 It can be seen that the optimal magnesium ion concentration in the UDP-3E reaction system is 10-20 mM.

[0279] It should be noted that in Examples 4-8, the enzymes in the reaction system were added in stages: first, sucrose synthase was added, and then polyphosphate-dependent glucokinase and glycosyltransferase were added 10 minutes later.

[0280] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0281] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a polyphosphate-dependent glucokinase in the glycosylation modification of pentacyclic triterpenoid compounds, characterized in that, The glycosylation-modified glycosyl donor is sucrose, and the polyphosphate-dependent glucokinase is used to convert the byproduct fructose into fructose-6-phosphate. The polyphosphate-dependent glucokinase is CgPPGK with an amino acid sequence as shown in SEQ ID NO: 1 or Asp-PPGK with an amino acid sequence as shown in SEQ ID NO:

2. The pentacyclic triterpenoid compound is one of β-amyrin, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

2. The application according to claim 1, characterized in that, The gene sequence of CgPPGK is shown in SEQ ID NO: 3; the gene sequence of Asp-PPGK is shown in SEQ ID NO:

4.

3. The application according to any one of claims 1 or 2, characterized in that, The glycosylation modification system includes: glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, sucrose, uridine diphosphate, and pentacyclic triterpenoid compounds.

4. An enzyme assembly for glycosylation modification of pentacyclic triterpenoid compounds, characterized in that, include: (i) Glycosyltransferases; (ii) Sucrose synthase; (iii) Polyphosphate-dependent glucokinase; the polyphosphate-dependent glucokinase is CgPPGK with the amino acid sequence shown in SEQ ID NO: 1 or Asp-PPGK with the amino acid sequence shown in SEQ ID NO: 2; The pentacyclic triterpenoid compound is one of β-amyrin, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

5. The enzyme combination for glycosylation modification of pentacyclic triterpenoid compounds according to claim 4, characterized in that, The glycosyltransferase is UGT73F24 with an amino acid sequence as shown in SEQ ID NO: 5 or UGT73C11 with an amino acid sequence as shown in SEQ ID NO:

6.

6. The enzyme combination for glycosylation modification of pentacyclic triterpenoid compounds according to claim 4, characterized in that, The sucrose synthase has the amino acid sequence GuSUS1-Δ9 as shown in SEQ ID NO:

7.

7. A biological agent for glycosylation modification of pentacyclic triterpenoid compounds, comprising: (i) A recombinant vector or recombinant bacteria carrying the gene sequence of glycosyltransferase UGT73F24 or the gene sequence of glycosyltransferase UGT73C11; (ii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of sucrose synthase GuSUS1-Δ9; (iii) Recombinant vectors or recombinant bacteria loaded with the gene sequence of polyphosphate-dependent glucokinase CgPPGK or the gene sequence of polyphosphate-dependent glucokinase Asp-PPGK. The gene sequence of the glycosyltransferase UGT73F24 is shown in SEQ ID NO: 8; the gene sequence of the glycosyltransferase UGT73C11 is shown in SEQ ID NO: 9; the gene sequence of the sucrose synthase GuSUS1-Δ9 is shown in SEQ ID NO: 10; the gene sequence of the polyphosphate-dependent glucokinase CgPPGK is shown in SEQ ID NO: 3; and the gene sequence of the polyphosphate-dependent glucokinase Asp-PPGK is shown in SEQ ID NO:

4. The pentacyclic triterpenoid compound is one of β-amyrin, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

8. A UDP cycling system based on polyphosphate-dependent glucokinase, characterized in that, The UDP cycle system comprises: the enzyme combination according to any one of claims 4-6, a pentacyclic triterpenoid compound, sucrose, uridine diphosphate, and Mg. 2+ And sodium polyphosphate.

9. A method for glycosylation modification of pentacyclic triterpenoid compounds, characterized in that, The process includes the following steps: using the enzyme combination according to any one of claims 4-6 to catalyze a substrate reaction, wherein the substrate is a pentacyclic triterpenoid compound, and the glycosyl donor for the catalytic reaction is sucrose; The substrate is one of β-amyrin, oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, asiatic acid, or ursolic acid.

10. The method according to claim 9, characterized in that, The reaction system includes: pentacyclic triterpenoids, sucrose, uridine diphosphate, glycosyltransferase, sucrose synthase, polyphosphate-dependent glucokinase, and Mg. 2+ And sodium polyphosphate.

11. The method according to claim 10, characterized in that, The pentacyclic triterpenoid compound is 0.3-1.0 mM, the sucrose is 10-100 mM, the uridine diphosphate is 0.2-1.0 mM, the glycosyltransferase is 0.15-0.20 mg / mL, the sucrose synthase is 0.15-0.20 mg / mL, the polyphosphate-dependent glucokinase is 0.40-0.50 mg / mL, and the Mg... 2+ The concentration is 10-20 mM, and the sodium polyphosphate concentration is 10 g / L.

12. The method according to claim 10 or 11, characterized in that, The enzymes are added in stages: first, the sucrose synthase is added, and then the polyphosphate-dependent glucokinase and the glycosyltransferase are added 10-20 minutes later. And / or, the catalytic reaction is carried out at a pH of 8-8.5 and a temperature of 40 °C.

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