WO2020097922A1 - Mutant d'udp-glucosyltransférase, utilisation correspondante et procédé de préparation de rebaudioside d - Google Patents

Mutant d'udp-glucosyltransférase, utilisation correspondante et procédé de préparation de rebaudioside d Download PDF

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Publication number
WO2020097922A1
WO2020097922A1 PCT/CN2018/115937 CN2018115937W WO2020097922A1 WO 2020097922 A1 WO2020097922 A1 WO 2020097922A1 CN 2018115937 W CN2018115937 W CN 2018115937W WO 2020097922 A1 WO2020097922 A1 WO 2020097922A1
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Prior art keywords
udp
glucosyltransferase
rebaudioside
amino acid
acid sequence
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Ceased
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PCT/CN2018/115937
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English (en)
Chinese (zh)
Inventor
傅荣昭
刘文山
刘玉凤
李振伟
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Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd
Jiangxi Bonzymes Biotechnology Co Ltd
Bontac Bio Engineering Shenzhen Co Ltd
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Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd
Jiangxi Bonzymes Biotechnology Co Ltd
Bontac Bio Engineering Shenzhen Co Ltd
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Application filed by Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd, Jiangxi Bonzymes Biotechnology Co Ltd, Bontac Bio Engineering Shenzhen Co Ltd filed Critical Jiangxi Bontac Green-Biocatalysis Ecoindustrial Park Co Ltd
Priority to CN201880037977.7A priority Critical patent/CN111344399B/zh
Priority to PCT/CN2018/115937 priority patent/WO2020097922A1/fr
Publication of WO2020097922A1 publication Critical patent/WO2020097922A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals

Definitions

  • the invention relates to the technical field of biological enzymes, in particular to a UDP-glucosyltransferase mutant artificially obtained by a method of genetic site-directed mutation and a method for catalytically preparing rebaudioside D.
  • Glucosyltransferase is an enzyme that transfers only glucosyl groups in an enzyme reaction. The mechanism of this enzyme is to catalyze the transfer of glucose residues from sugar donors to glycosyl acceptor molecules, thereby regulating the activity of the acceptor molecules.
  • UDP-glucosyltransferase UDP-glucosyltransferase, UGT for short
  • UDP-glucose is used as a glycosyl donor in almost all organisms.
  • UDP-glucose is the abbreviation of uridine diphosphate glucose, or UDP-glucose or UDPG. It is a vitamin composed of uridine diphosphate and glucose. It can be regarded as "active glucose” and is widely distributed. In the cells of plants, animals and microorganisms, it is used as a glucose-based donor in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides. It is the most common type of sugar-based donor.
  • UDP-glucosyltransferase is increasingly used in the field of biocatalytic preparation of stevioside.
  • UDP-glucosyltransferases There are many types of UDP-glucosyltransferases.
  • most of the enzymes used in the biological enzymatic preparation of stevioside are wild enzymes derived from plant cells.
  • Such wild enzymes often have the disadvantages of low enzyme activity and poor stability.
  • the cost of preparing steviol glycosides for industrial mass production is relatively high. Therefore, it is necessary to transform wild enzymes of UDP-glucosyltransferase to obtain engineered enzymes with higher enzyme activity and better stability, so as to better serve industrialized mass production.
  • the purpose of the present invention is to solve the technical problems of low enzyme activity and poor stability of wild-type UDP-glucosyltransferase, and to develop a UDP-glucosyltransferase with higher enzyme activity and stability.
  • the present invention provides a UDP-glucosyltransferase mutant, which is a protein of (a), (b) or (c) as follows:
  • amino acid sequence of the protein defined in (a) has more than 90% homology and has a higher catalytic rebaudioside A than the UDP-glucosyltransferase parent whose amino acid sequence is shown in SEQ ID NO: 2 Enzymatic protein converted to rebaudioside D.
  • the UDP-glucosyltransferase mutant provided by the present invention has a mutation at at least one of the following positions: No. 148th, No. 244, No. 251, No. 252, No. 255, No. 368, No. 426 and No. 432.
  • the UDP-glucosyltransferase mutant provided by the present invention has at least one of the following mutations: A69D, M148L, F244P , L251S, H252P, R255G, M368L, S426R, K432N.
  • the mutations of the UDP-glucosyltransferase mutant provided by the present invention are A69D, M148L, F244P, R255G, M368L, S426R, K432N or L251S / H252P.
  • the present invention also provides a biological material, including a recombinant vector, a recombinant cell or a recombinant microorganism, the biological material contains the gene sequence encoding the above UDP-glucosyltransferase mutant provided by the present invention, under suitable conditions, The biological material can express and secrete the UDP-glucosyltransferase mutant provided by the present invention.
  • the present invention also provides the use of the above UDP-glucosyltransferase mutant, which is used to apply the above-mentioned UDP-glucosyltransferase mutant provided by the present invention in the preparation of stevioside, in particular, the present invention provides The above UDP-glucosyltransferase mutant is used in the preparation of rebaudioside D.
  • the use of the above UDP-glucosyltransferase mutant provided by the present invention is: in the presence of UDPG, the above UDP-glucosyltransferase mutant is used to catalyze the conversion of rebaudioside A to rebaudioside D .
  • the present invention also provides a method for preparing rebaudioside D.
  • the method includes: in the presence of sucrose, UDP and sucrose synthase, using UDP-glucosyltransferase to catalyze the conversion of rebaudioside A to Rebaudioside D, wherein UDP-glucosyltransferase is the parent of UDP-glucosyltransferase whose amino acid sequence is shown in SEQ ID NO: 2 or the above-mentioned mutant of UDP-glucosyltransferase provided by the present invention, sucrose Synthetase is derived from the AtSUS gene of Arabidopsis thaliana, which has been included in the NCBI database, and its accession number is NP_197583.
  • UDP is the English abbreviation of Uridine Diphosphate, and is one of the substances for synthesizing UDPG.
  • the catalytic reaction process is performed in an aqueous environment, the pH value of the catalytic reaction is 4-9, and the temperature of the catalytic reaction is 4-45 ° C.
  • the pH value of the catalytic reaction is 7-8, and the temperature of the catalytic reaction is 35-45 ° C.
  • the final concentration of rebaudioside A in the reaction system is 10-50 g / L.
  • the UDP-glucosyltransferase mutants provided by the present invention have significantly improved enzyme activity and pH stability, especially the enzyme activity has been increased by at least 200% .
  • the method for preparing rebaudioside D developed by using the UDP-glucosyltransferase mutant provided by the present invention can greatly reduce the use of enzymes while ensuring the conversion rate of substrate rebaudioside A as high as 97% or more the amount.
  • the PCR amplification product was treated with Dpn I enzyme digestion template and then transformed into E. coli Rosetta competent cell DE3. After Amp screening, colonies were picked and sequenced to determine the target mutant.
  • the mutations of the UDP-glucosyltransferase mutant prepared in this section are A69D, M148L, and F244P, respectively , R255G, M368L, S426R, K432N or L251S / H252P.
  • the recombinant E. coli expression strains containing the UDP-glucosyltransferase parent and mutant prepared in parts 1 and 2 were inoculated into 4mL liquid LB medium at a rate of 1%, and cultured at 37 ° C with shaking (200rpm) overnight. The overnight culture was transferred to a large volume of liquid LB medium at 1% inoculation volume, and the culture was shaken (200 rpm) at 37 ° C until the OD600 value reached 0.6-0.8, and the final concentration of 0.1 mM to 1 mM IPTG was added to shake culture at 20 to 37 ° C for 12 to 16h.
  • the cells were collected by centrifugation, and the cells were resuspended with 50 mM phosphate buffer (pH 7.4).
  • the cells were disrupted by ultrasound in an ice bath.
  • the disrupted solution was centrifuged and the supernatant was collected to obtain a parent containing UDP-glucosyltransferase. Or the crude enzyme solution of the mutant.
  • the prepared crude enzyme solution of the parent or mutant of UDP-glucosyltransferase and 10 g of AtSUS sucrose synthase (from Arabidopsis thaliana) were adjusted to pH 7-8.
  • the reaction was carried out at 37 ° C and 200 rpm for 10 hours, and the conversion rate was measured. After the reaction, the reaction solution was separated, purified, and dried to obtain the rebaudioside D product.
  • Table 3 shows the amount of UDP-glucosyltransferase added and the conversion rate of each reaction.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Mutant d'UDP-glucosyltransférase, utilisation correspondante et procédé de préparation de rebaudioside D. une série de mutants comprenant SEQ ID NO : 3 sont obtenus en soumettant des séquences de gènes d' UDP-glucosyltransférase de type sauvage à une mutagenèse dirigée, l'activité enzymatique et la stabilité de pH de ces mutants étant améliorées par rapport au parent de type sauvage.
PCT/CN2018/115937 2018-11-16 2018-11-16 Mutant d'udp-glucosyltransférase, utilisation correspondante et procédé de préparation de rebaudioside d Ceased WO2020097922A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201880037977.7A CN111344399B (zh) 2018-11-16 2018-11-16 一种udp-葡萄糖基转移酶突变体、其应用及其制备莱鲍迪苷d的方法
PCT/CN2018/115937 WO2020097922A1 (fr) 2018-11-16 2018-11-16 Mutant d'udp-glucosyltransférase, utilisation correspondante et procédé de préparation de rebaudioside d

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CN115433249A (zh) * 2022-08-23 2022-12-06 江南大学 一种新型甜菊糖苷衍生物莱鲍迪苷l2的制备方法
CN115449514A (zh) * 2021-06-08 2022-12-09 弈柯莱生物科技(上海)股份有限公司 一种β-1,2-糖基转移酶及其应用
CN116064452A (zh) * 2022-10-25 2023-05-05 皖西学院 一种糖基转移酶突变体及其在合成淫羊藿次甙d2中的应用
CN116218806A (zh) * 2022-10-21 2023-06-06 山东大学 一种N-糖基转移酶突变体AaFQ及其应用
CN116334019A (zh) * 2022-08-05 2023-06-27 北京理工大学 一种udp-糖基转移酶及其在合成黄酮糖苷衍生物中的应用
CN116622664A (zh) * 2023-06-27 2023-08-22 湖南师范大学 一种生物催化生成c-糖苷的方法
WO2025001471A1 (fr) * 2023-06-30 2025-01-02 东台市浩瑞生物科技有限公司 Mutant de la glycosyltransférase ugtsl2, mutant de la glycosyltransférase et procédé de synthèse du rébaudioside m2 avec ceux-ci
CN119464245A (zh) * 2025-01-13 2025-02-18 浙江正硕生物科技有限公司 一种糖基转移酶突变体及编码基因和催化Reb D的用途
CN120758477A (zh) * 2025-09-10 2025-10-10 山东三元生物科技股份有限公司 一种糖基转移酶突变体及其在合成莱鲍迪苷m中的应用
CN120813686A (zh) * 2025-05-07 2025-10-17 邦泰生物工程(深圳)有限公司 一种udp-葡萄糖基转移酶突变体及其制备莱鲍迪苷d的方法

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JP2024520118A (ja) * 2021-06-01 2024-05-21 エーバイオケム バイオテクノロジー（グループ）カンパニー，リミティド 糖転移酵素及びその使用
CN115478060B (zh) * 2021-06-16 2023-11-28 弈柯莱生物科技(集团)股份有限公司 一种糖基转移酶及其应用
CN115678867B (zh) * 2021-07-27 2023-12-01 弈柯莱生物科技(集团)股份有限公司 一种蔗糖合成酶及其应用
CN116949002B (zh) * 2023-04-04 2026-03-27 南京师范大学 葡萄糖基转移酶突变体及其编码基因、重组载体、重组菌株和酶制剂以及它们的应用
CN117431229B (zh) * 2023-11-03 2024-07-09 铭诚惠众(江苏)药物研究有限公司 一种糖基转移酶ugt91c1突变体及其应用
CN118240791B (zh) * 2024-03-22 2025-06-24 百开盛(上海)生物科技有限公司 糖基转移酶突变体及其在Reb D合成中的应用
CN118460497B (zh) * 2024-07-04 2024-12-20 青岛奔月生物技术有限公司 Udp-葡萄糖基转移酶突变体及其应用
CN119265154B (zh) * 2024-12-10 2025-05-13 诸城市浩天药业有限公司 一种糖基转移酶ugt76g4突变体及其应用
CN119265152B (zh) * 2024-12-10 2025-03-18 诸城市浩天药业有限公司 一种糖基转移酶ugt91c1突变体及其应用
CN119391668B (zh) * 2025-01-06 2025-04-04 内蒙古金达威药业有限公司 Udp-葡萄糖基转移酶突变体及其应用和甜菊糖苷莱鲍迪苷d的制备方法
CN120485149B (zh) * 2025-05-21 2026-03-20 深圳瑞德林生物技术有限公司 酶的突变体、糖基转移酶的突变体及其在制备红景天苷中的应用

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CN115449514A (zh) * 2021-06-08 2022-12-09 弈柯莱生物科技(上海)股份有限公司 一种β-1,2-糖基转移酶及其应用
CN116334019A (zh) * 2022-08-05 2023-06-27 北京理工大学 一种udp-糖基转移酶及其在合成黄酮糖苷衍生物中的应用
CN115433249B (zh) * 2022-08-23 2025-08-15 桂林莱茵生物科技股份有限公司 一种新型甜菊糖苷衍生物莱鲍迪苷l2的制备方法
CN115433249A (zh) * 2022-08-23 2022-12-06 江南大学 一种新型甜菊糖苷衍生物莱鲍迪苷l2的制备方法
CN116218806A (zh) * 2022-10-21 2023-06-06 山东大学 一种N-糖基转移酶突变体AaFQ及其应用
CN116218806B (zh) * 2022-10-21 2024-05-10 山东大学 一种N-糖基转移酶突变体AaFQ及其应用
CN116064452A (zh) * 2022-10-25 2023-05-05 皖西学院 一种糖基转移酶突变体及其在合成淫羊藿次甙d2中的应用
CN116622664A (zh) * 2023-06-27 2023-08-22 湖南师范大学 一种生物催化生成c-糖苷的方法
WO2025001471A1 (fr) * 2023-06-30 2025-01-02 东台市浩瑞生物科技有限公司 Mutant de la glycosyltransférase ugtsl2, mutant de la glycosyltransférase et procédé de synthèse du rébaudioside m2 avec ceux-ci
CN119464245A (zh) * 2025-01-13 2025-02-18 浙江正硕生物科技有限公司 一种糖基转移酶突变体及编码基因和催化Reb D的用途
CN120813686A (zh) * 2025-05-07 2025-10-17 邦泰生物工程(深圳)有限公司 一种udp-葡萄糖基转移酶突变体及其制备莱鲍迪苷d的方法
CN120758477A (zh) * 2025-09-10 2025-10-10 山东三元生物科技股份有限公司 一种糖基转移酶突变体及其在合成莱鲍迪苷m中的应用

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