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CN-121975890-A - Application of glycosyltransferase in production of collaterals plug and collaterals plug vitamin by microorganisms

CN121975890ACN 121975890 ACN121975890 ACN 121975890ACN-121975890-A

Abstract

The invention discloses an application of UDP glucosyltransferase in producing a collaterals plug and a collaterals plug vitamin by microorganisms. The UDP-glucosyltransferase is used for catalyzing cinnamyl alcohol and UDP-glucose to generate a complex plug, is coded by OsUGT genes of Oryza sativa, and has 3.6 times of catalytic efficiency than UDP-glucosyltransferase coded by Bs-YjiC genes of bacillus subtilis. The invention can be used for large-scale industrial production of the collaterals plug and the derivative of the collaterals plug and has application value.

Inventors

  • LIU TAO
  • BI HUIPING
  • Liu Moshi

Assignees

  • 中国科学院天津工业生物技术研究所
  • 天工生物科技（天津）有限公司

Dates

Publication Date
20260505
Application Date
20241030

Claims (10)

  1. 1. The application of UDP-glucosyltransferase in preparing the collaterals plug or the collaterals plug vitamin is characterized in that the UDP-glucosyltransferase is derived from rice, and the amino acid sequence GenBank is XP_015622802.1.
  2. 2. The use according to claim 1, wherein the Gene ID of the Gene encoding UDP-glucosyltransferase is 4330775.
  3. 3. Use according to claim 2, for catalyzing the formation of a complex plug of cinnamyl alcohol and UDP-glucose.
  4. 4. A method for preparing the collaterals plug is characterized in that UDP glucosyltransferase is used for catalyzing cinnamyl alcohol and UDP-glucose to generate the collaterals plug, the UDP glucosyltransferase is from rice, the amino acid sequence GenBank of the UDP glucosyltransferase is XP_015622802.1, and preferably, the GenBank of the coding gene of the UDP glucosyltransferase is XP_015622802.1.
  5. 5. The method according to claim 3, wherein the UDP-glycosyltransferase is obtained by constructing recombinant engineering bacteria containing the coding gene, specifically by constructing prokaryotic expression vectors containing the coding gene, and respectively transferring the prokaryotic expression vectors into Escherichia coli for fermentation culture.
  6. 6. The method according to any of claims 3 to 5, wherein the concentration of cinnamyl alcohol and UDP-glucose in the reaction system during the reaction is 1:1, in particular 0.05-2mM, such as 1mM.
  7. 7. The method according to claim 6, wherein the catalytic reaction is carried out at 25-35 ℃ for 1-4 hours, such as 30 ℃ for 2 hours.
  8. 8. The method of claim 6, wherein the reaction is terminated by adding an equal volume of methanol and trifluoroacetic acid after completion of the reaction, centrifuging at 4 ℃ for 10 minutes at 12000 rpm, passing the supernatant through a 0.22 μm filter, and detecting by HPLC.
  9. 9. The method according to claim 8, further comprising the step of separating the resulting plug, in particular from 3 to 5 ℃, from 10000 to 14000 rpm, for 5 to 20 minutes, and obtaining a supernatant from the filtration membrane, for example from 4 ℃, from 12000 to rpm, and from the supernatant from the 0.22 μm filtration membrane.
  10. 10. A method for preparing the zoysin, which is characterized in that the zoysin is obtained by adding UDP-arabinose to a reaction system and simultaneously expressing an arabinosyltransferase such as SlUGT R1 on the basis of the method as claimed in claims 4 to 9.

Description

Application of glycosyltransferase in production of collaterals plug and collaterals plug vitamin by microorganisms Technical Field The invention relates to the field of genetic engineering, in particular to application of glycosyltransferase in producing a collaterals plug and a collaterals plug vitamin by microorganisms. Background The zoyside (cinnamyl- (6 '-O-alpha-L-arabinopyranosyl) O-beta-D-glucopyranoside) has a structure shown in the following formula I, and is cinnamyl-glucose-arabinoside with the English name Rosavin, the chemical name trans-cinnamyl- (6' -O-beta-D-arabinpyranosyl) -O-beta-D-glucopyranoside, the CAS number is 84954-92-7 molecular formula C 20H28O10 and the molecular weight 428.18. The zoysia japonica is an important and unique secondary metabolite and main active ingredient of Rhodiola rosea (Rhodiola rosea l.) of the family Rhodiola, and is an important marker commonly used for evaluating the quality of Rhodiola rosea extracts. The research shows that the zoysia japonica has a plurality of beneficial pharmacological effects, such as intelligence improvement, anticancer, immunity enhancement, depression resistance, ultraviolet radiation resistance and the like. Currently, the production of zoysia japonica is mainly performed by means of plant extraction. While rhodiola rosea grows in alpine regions, the wild resources are rare. In recent years, the rhodiola rosea resource is seriously damaged due to over exploitation, and the rhodiola rosea resource belongs to the national secondary important protection of wild plants. The rhodiola rosea is not planted in a large scale artificially until now due to the restriction of growth conditions, diseases and insect pests, etc., and the accumulation period of active ingredients in the rhodiola rosea is long, and enough active ingredients can be obtained only after 5-7 years of growth. In vivo, the precursor substances of the zoysin are cinnamyl alcohol and zoysin. The cinnamyl alcohol has the following characteristics that the English name is Cinnamic alcohol, the chemical name is 3-phenyl-2-propene-1-ol, the molecular formula is C 9H10 O, the molecular weight is 134.18, the CAS number is 104-54-1, and the structural formula is as follows: The collaterals plug is characterized by having the following chemical name of Rosin, (2R, 3S,4S,5R, 6R) -2- (hydroxyymethyl) -6- [ (E) -3-phenyl-2-enoxy ] oxane-3,4,5-triol or trans-cinnamy-O-beta-D-glucopyranoside, molecular formula of C 15H20O6, molecular weight of 296.32, CAS number of 85026-55-7, structural formula In 2006 Patov et al reported chemical synthesis (Kishida M,Akita H.Synthesis of Rosavin and its analogues based on a Mizoroki-Heck type reaction[J].Tetrahedron Asymmetry,2005,16(15):26252630). of zoysin but the chemical synthesis steps are more, the yield is low, the cost is high, and the environmental pollution is serious. In 2023, li, L et al reported that a recombinant E.coli producing the zoysin and its use in (Li,L.,et al.(2024)."High-level production of Rhodiola rosea characteristic component rosavin from D-glucose and L-arabinose in engineered Escherichia coli."Metab Eng 82:274-285.), shake flask fermentation gave a yield of 1203.7mg/L, which still had room for further improvement. Disclosure of Invention The invention aims to provide UDP glucosyltransferase capable of catalyzing cinnamyl alcohol to generate a collaterals plug. The present invention finds that the efficiency of catalyzing the formation of a plug by UDP-glucosyltransferase encoding OsUGT13 gene from Oryza sativa is highest and the conversion rate of catalyzing the formation of a plug by cinnamyl alcohol is 3.6 times that of the enzyme encoding Bs-YjiC gene from Bacillus subtilis by studying glycosyltransferases Bs-YjiC, osUGT13, blUGT1, slUGT5, hoUGT A1, srUGT G1, csUGT85K11, vvUGT A26, vvGT, adGT and PpGT1 from Bacillus subtilis、Oryza sativa、Bacillus licheniformis、Solanum lycopersicum、Human olfactory、Stevia rebaudiana、Camellia sinensis、Vitis vinifera、Vitis vinifera、Actinidia deliciosa and Paenibacillus polymyxa. Therefore, the invention can be directly used for preparing the collaterals plug and the collaterals plug vitamin and has application value. Accordingly, the present invention has been made. The invention provides an application of UDP (user datagram protocol) glucosyltransferase in preparing a collaterals plug or a collaterals plug vitamin, wherein the UDP glucosyltransferase is derived from rice, and the amino acid sequence GenBank number of the UDP glucosyltransferase is XP_015622802.1. Specifically, the GenBank number of the coding gene of the UDP-glucosyltransferase is XP_015622802.1. In a specific embodiment, it is used to catalyze the formation of a complex plug of cinnamyl alcohol and UDP-glucose. The invention provides a method for preparing a collaterals plug, which adopts UDP glucosyltransferase to catalyze cinnamyl alcohol and UDP-glucose to generate the collaterals plug, wherein the UDP glucosyl