Glycosyl transferase mutant and application thereof in salidroside production

Abstract

The invention discloses a glycosyl transferase mutant and application thereof in production of salidroside, and relates to the technical field of biology. The glycosyl transferase mutant is any one of A1) to A2): A1) a protein obtained by performing amino acid mutation on at least one of the 41st site, the 114 site, the 272th site and the 340th site of an amino acid sequence of wild-type glycosyl transferase; a2) is a fusion protein obtained by connecting a label to the N end or / and C end of the protein shown in A1), and the amino acid sequence of the wild type glycosyltransferase is shown in SEQ ID NO: 1. The wild type glycosyltransferase can efficiently catalyze tyrosol to generate salidroside.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a glycosyltransferase mutant and its application in the production of rhodioloside. Background Technology

[0002] Rhodioloside is a glycosylated product of tyrosol, the main active ingredient in Rhodiola rosea. Rhodioloside possesses functions such as anti-hypoxia, anti-cold, anti-fatigue, anti-microwave radiation, antiviral, and anti-tumor effects. It also enhances concentration, improves work efficiency, delays aging, and prevents age-related diseases. It has significant application value, particularly in military medicine, aerospace medicine, sports medicine, and health care medicine, and is a promising environmental adaptation drug that has attracted considerable attention in recent years.

[0003] In related technologies, rhodioloside is generally obtained by extraction from Rhodiola rosea plants, which suffers from high extraction costs and low yields. Another method involves synthesizing rhodioloside through fermentation using engineered bacteria, but this method suffers from low synthesis efficiency and is difficult to industrialize. Therefore, there is a need to provide a method for the efficient synthesis of rhodioloside. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a glycosyltransferase mutant.

[0005] The present invention also provides biological materials related to the above-mentioned glycosyltransferase mutants.

[0006] This invention also provides applications of the above-mentioned biomaterials.

[0007] The present invention also provides applications of the above-mentioned glycosyltransferase mutant.

[0008] This invention also provides a method for preparing rhodioloside.

[0009] The present invention also provides an enzyme preparation comprising the above-mentioned glycosyltransferase mutant.

[0010] According to a first aspect of the present invention, the glycosyltransferase mutant is any one of A1) to A2):

[0011] A1) A protein obtained by mutating an amino acid at at least one of the following positions: position 41, position 114, position 272, and position 340 of the amino acid sequence of the wild-type glycosyltransferase. A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in A1); The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO: 1.

[0012] The glycosyltransferase mutant according to embodiments of the present invention has at least the following beneficial effects: The glycosyltransferase mutant in this example can efficiently catalyze the conversion of tyrosol to rhodioloside, exhibiting higher catalytic activity than the wild-type glycosyltransferase. Microorganisms expressing the glycosyltransferase mutant show a significantly enhanced ability to convert tyrosol to rhodioloside, demonstrating promising application potential.

[0013] According to some embodiments of the present invention, the glycosyltransferase mutant is a protein obtained by mutating an amino acid at position 41 of the amino acid sequence of the wild-type glycosyltransferase.

[0014] According to some embodiments of the present invention, the glycosyltransferase mutant is a protein obtained by mutating an amino acid at position 114 of the amino acid sequence of the wild-type glycosyltransferase.

[0015] According to some embodiments of the present invention, the glycosyltransferase mutant is a protein obtained by mutating an amino acid at position 272 of the amino acid sequence of the wild-type glycosyltransferase.

[0016] According to some embodiments of the present invention, the glycosyltransferase mutant is a protein obtained by mutating an amino acid at position 340 of the amino acid sequence of the wild-type glycosyltransferase.

[0017] According to some embodiments of the present invention, the glycosyltransferase mutant is a protein obtained by mutating amino acids at positions 41, 114, 272, and 340 of the amino acid sequence of the wild-type glycosyltransferase.

[0018] According to some embodiments of the present invention, the amino acid mutation performed at position 41 is Y41H.

[0019] According to some embodiments of the present invention, the amino acid mutation performed at position 114 is G114A.

[0020] According to some embodiments of the present invention, the amino acid mutation performed at position 272 is S272T.

[0021] According to some embodiments of the present invention, the amino acid mutation performed at position 340 is A340T.

[0022] According to some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, purification, and detection of the glycosyltransferase mutant. It is understood that the glycosyltransferase mutant of the present invention may contain one or more tags; multiple tags may comprise a combination of multiple identical tags, or a combination of multiple different tags. For example: tags facilitating the dissolution of the glycosyltransferase mutant include, but are not limited to, nus tags or maltose-binding protein tags; tags facilitating the purification of the glycosyltransferase mutant include, but are not limited to, strep tags, His tags, GST tags, pelB signal tags, or ompA signal tags; tags facilitating the detection of the glycosyltransferase mutant include, but are not limited to, horseradish peroxidase (HRP) tags, β-galactosidase tags, luciferase tags, green fluorescent protein (GFP) tags, HcRed tags, DsRed tags, or cyan fluorescent protein (CFP) tags.

[0023] According to a second aspect of the present invention, a biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the glycosyltransferase mutant described in the first aspect embodiment; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0024] According to some embodiments of the present invention, the nucleic acid molecule in B1) is any of the following: The nucleotide sequence of the nucleic acid molecule described in B1-1) is shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15; B1-2) has more than 75% identity with the nucleotide sequence described in B1-1) and encodes a cDNA molecule or DNA molecule of the glycosyltransferase mutant described in the first aspect embodiment; B1-3) hybridizes under stringent conditions with any of the defined nucleotide sequences in B1-1)-B1-2) and encodes a cDNA molecule or DNA molecule of the glycosyltransferase mutant described in the first aspect embodiment.

[0025] According to some embodiments of the present invention, the identity described in B1-2) can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0026] According to some embodiments of the present invention, the stringent conditions may be hybridization and washing twice at 68°C in a solution of 2×SSC and 0.1% SDS, each time for 5 min; or hybridization and washing twice at 68°C in a solution of 0.5×SSC and 0.1% SDS, each time for 15 min.

[0027] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the glycosyltransferase mutant in a host cell. This DNA may include not only a promoter for initiating transcription of the glycosyltransferase mutant gene, but also a terminator for terminating transcription of the glycosyltransferase mutant gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0028] According to some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector. For example, it may be the expression vector pZBK.

[0029] According to some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a DNA molecule encoding the glycosyltransferase mutant into the multiple cloning site of the vector.

[0030] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The bacteria may be *Escherichia coli*. The recombinant biological cells do not contain reproductive material.

[0031] According to some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell. Specifically, it can be recombinant Escherichia coli obtained by introducing the recombinant expression vector into Escherichia coli capable of synthesizing tyrosol.

[0032] Application of the biomaterials described in the second aspect of the present invention, according to a third aspect embodiment, in any one of C1) to C3): C1) Preparation of the glycosyltransferase mutant described in the first aspect embodiment; C2) Preparation of rhodioloside; C3) Prepare products that generate rhodioloside.

[0033] According to some embodiments of the present invention, the preparation of rhodioloside includes catalytically generating rhodioloside from tyrosol.

[0034] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0035] The application of the glycosyltransferase mutant as described in the first aspect embodiment according to the fourth aspect embodiment of the present invention in the preparation of products containing rhodioloside or in the preparation of products generating rhodioloside.

[0036] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0037] A method for preparing rhodioloside according to a fifth aspect embodiment of the present invention includes the following steps: The glycosyltransferase mutant described in the first aspect embodiment or recombinant cells expressing the glycosyltransferase mutant are used to catalyze the production of rhodioloside from tyrosol.

[0038] According to some embodiments of the present invention, the glycosyltransferase mutant or recombinant cells expressing the glycosyltransferase mutant use tyrosol as a substrate to catalyze the addition of a glucose group to the hydroxyl group of tyrosol to generate rhodioloside.

[0039] According to some embodiments of the present invention, the catalytic temperature is 25°C-35°C. For example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0040] According to some embodiments of the present invention, the recombinant cells of the glycosyltransferase mutant are recombinant Escherichia coli.

[0041] According to some embodiments of the present invention, the culture rotation speed of the recombinant cells is 100 rpm to 300 rpm. For example, it can be 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm, or 300 rpm.

[0042] According to some embodiments of the present invention, the initial seeding amount of the recombinant cells is OD. 600 The range is 0.05-0.5. For example, it can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5.

[0043] According to some embodiments of the present invention, the catalytic time is 40 h to 100 h. For example, it can be 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h, 75 h, 80 h, 85 h, 90 h, 95 h or 100 h.

[0044] An enzyme preparation according to a sixth aspect of the present invention includes the glycosyltransferase mutant described in the first aspect embodiment.

[0045] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0046] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0047] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0048] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0049] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0050] Example 1 The amino acid sequence of the wild-type glycosyltransferase UGT85A1 is shown in SEQ ID NO: 1. A biotechnology company was commissioned to synthesize the gene sequence encoding the wild-type glycosyltransferase UGT85A1 (as shown in SEQ ID NO: 2), and this gene sequence was ligated into the expression vector pZBK (Biotechnology Biofuels 2019, 12:94) to obtain the expression vector pZBK-UGT85A1.

[0051] Using the primers in Table 1, reverse PCR was performed with expression vector pZBK-UGT85A1 as a template to obtain point mutation genes, resulting in the expression vectors pZBK-UGT85A1(Y41H) encoding the glycosyltransferase mutant Y41H, pZBK-UGT85A1(G114A) encoding the glycosyltransferase mutant G114A, pZBK-UGT85A1(S272T) encoding the glycosyltransferase mutant S272T, pZBK-UGT85A1(A340T) encoding the glycosyltransferase mutant A340T, and pZBK-UGT85A1(Y41H / G114A / S272T / A340T) encoding the glycosyltransferase mutants Y41H / G114A / S272T / A340T.

[0052] Table 1

[0053] Note: F represents the upstream primer, and R represents the downstream primer.

[0054] The nucleic acid sequences of the genes encoding glycosyltransferase mutants Y41H, G114A, S272T, A340T, and Y41H / G114A / S272T / A340T are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, respectively.

[0055] The expression vectors pZBK-UGT85A1, pZBK-UGT85A1(Y41H), pZBK-UGT85A1(G114A), pZBK-UGT85A1(S272T), pZBK-UGT85A1(A340T), and pZBK-UGT85A1(Y41H / G114A / S272T / A340T) were transformed into tyrosol-producing bacteria. E. coliTYR-14B1(PpurR::P37) (pZEA(U)-SpyTag-aro10-linker-yahK) (Advanced Biotechnology (2024) 2:15) was used to obtain recombinant Escherichia coli.

[0056] Detection example The recombinant *E. coli* constructed in Example 1 was streaked on LB agar plates overnight. Single colonies were picked and inoculated into test tubes containing 5 mL of LB medium, and cultured overnight at 30°C and 220 rpm to obtain the fermentation broth. The fermentation broth was then inoculated into shake flasks containing 50 mL of M9YGT medium to allow it to begin OD. 600 The value was 0.1. Fermentation was carried out at 30℃ and 220 rpm for 72 h, and samples were taken for OD analysis. 600 The concentration of rhodioloside was determined by HPLC.

[0057] M9YGT medium: yeast extract 2.5 g / L, Na2HPO4·12H2O 17.48 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 2.5 g / L, MgSO4 0.6 g / L, CaCl2 0.011 g / L, glucose 20 g / L, trace element solution 1 (v / v)%. Trace element solution: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2.2 g / L, MnSO4·4H2O 0.58 g / L, CuSO4·5H2O 1 g / L, (NH4)6Mo7O 24 ·4H2O 0.1 g / L, Na2B4O7·10H2O 0.2 g / L and 35% HCl 10 mL / L.

[0058] Chromatographic conditions: Instrument: LC-20A (Shimadzu). Column: Inertsil ODS-SP C18 reversed-phase column (5 mm, 4.6 × 50 mm); Mobile phase: Phase A: 0.1% formic acid, Phase B: 0.1% formic acid + acetonitrile; Gradient elution: 0-8 min, Phase B concentration increases from 10% to 40%; 8-12 min, Phase B concentration increases to 90%, held for 3 min; Phase B concentration rapidly decreases to 10% and held for 5 min. Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 280 nm.

[0059] Table 2

[0060] The results are shown in Table 2. Compared with the wild-type glycosyltransferase, the rhodioloside production of the glycosyltransferase mutants Y41H, G114A, S272T, A340T, and Y41H / G114A / S272T / A340T increased by more than 5 times. Among them, the rhodioloside production of the glycosyltransferase mutants Y41H / G114A / S272T / A340T was 25 times that of the wild-type glycosyltransferase.

[0061] In addition to the expression vectors and tyrosol-producing strains described in this embodiment, any expression vector and tyrosol-producing strain can be used.

[0062] In addition to the methods mentioned in this embodiment, rhodioloside can also be produced using a whole-cell catalytic method.

[0063] In addition to the above embodiments, glycosyltransferase genes can also be integrated into the Escherichia coli chromosome to construct corresponding recombinant Escherichia coli.

[0064] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that, The glycosyltransferase mutant is any one of A1) to A2): A1) A protein obtained by mutating an amino acid at at least one of the following positions: position 41, position 114, position 272, and position 340 of the amino acid sequence of the wild-type glycosyltransferase. A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in A1); The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO:

1.

2. The glycosyltransferase mutant according to claim 1, characterized in that, The glycosyltransferase mutant is a protein obtained by mutating an amino acid at position 41 of the amino acid sequence of the wild-type glycosyltransferase, a protein obtained by mutating an amino acid at position 114 of the amino acid sequence of the wild-type glycosyltransferase, a protein obtained by mutating an amino acid at position 272 of the amino acid sequence of the wild-type glycosyltransferase, a protein obtained by mutating an amino acid at position 340 of the amino acid sequence of the wild-type glycosyltransferase, or a protein obtained by mutating an amino acid at positions 41, 114, 272, or 340 of the amino acid sequence of the wild-type glycosyltransferase; preferably, the amino acid mutation at position 41 is Y41H; and / or, the amino acid mutation at position 114 is G114A; and / or, the amino acid mutation at position 272 is S272T; and / or, the amino acid mutation at position 340 is A340T.

3. A biomaterial, characterized in that, The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the glycosyltransferase mutant of claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that, B1) The nucleic acid molecule is any one of the following: The nucleotide sequence of the nucleic acid molecule described in B1-1) is shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15; B1-2) has more than 75% identity with the nucleotide sequence described in B1-1) and encodes a cDNA molecule or DNA molecule of the glycosyltransferase mutant described in the first aspect embodiment; B1-3) hybridizes under stringent conditions with any of the defined nucleotide sequences in B1-1)-B1-2) and encodes a cDNA molecule or DNA molecule of the glycosyltransferase mutant described in the first aspect embodiment.

5. The use of the biomaterial according to claim 3 or 4 in any one of C1) to C3): C1) Prepare the glycosyltransferase mutant according to claim 1 or 2; C2) Preparation of rhodioloside; C3) Prepare products that generate rhodioloside.

6. The application according to claim 5, characterized in that, The product includes at least one of reagents and reagent kits.

7. The use of the glycosyltransferase mutant according to claim 1 or 2 in the preparation of products containing rhodioloside or in the preparation of products that generate rhodioloside.

8. A method for preparing rhodioloside, characterized in that, Includes the following steps: The glycosyltransferase mutant of claim 1 or 2 or recombinant cells expressing the glycosyltransferase mutant are used to catalyze the production of rhodioloside from tyrosol.

9. The method according to claim 8, characterized in that, The catalytic temperature is 25℃-35℃.

10. An enzyme preparation, characterized in that, Including the glycosyltransferase mutant as described in claim 1 or 2.

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