Glycosyltransferase and application thereof

Abstract

The invention belongs to the technical field of biology, relates to a glycosyl transferase and application thereof, and provides the glycosyl transferase with a sequence as shown in SEQ ID NO.1. The invention further provides the glycosyl transferase, a mutant thereof and application of the glycosyl transferase and the mutant thereof in glycosylation reaction, and the glycosyl transferase is high in specificity, high in catalytic efficiency, good in stability and high in yield. Cascade reaction is realized by combining the glycosyl donor UDPG with sucrose synthase, the problem that the glycosyl donor UDPG is expensive is solved, and the substrate range is wide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a glycosyltransferase and an application thereof. Background Art

[0002] Glycosylation is an important biochemical process in organisms. The glycosylation process is usually carried out by enzyme catalysis. These enzymes are called glycosyltransferases. Glycosyltransferases selectively transfer sugar molecules from glycosyl donors to acceptor molecules to form glycosylated products. Glycosyltransferases are enzymes that can catalyze the formation of glycosidic bonds from activated sugar donors to acceptor molecules. Their activity and specificity are crucial to the efficiency of glycosylation reactions and the accuracy of products. Common glycosyl donors include glucose, xylulose, fructose, and UDPG. Common glycosyl acceptors include oxygen groups, carbon groups, nitrogen groups, and sulfur groups.

[0003] For example, vanillin is a widely used edible flavoring, also known as vanillin. It has a unique aroma and is widely used in food industries such as cakes, ice cream, beverages, and candies. In addition, vanillin can also be used in cosmetics, daily chemicals, and the pharmaceutical industry. Ethyl vanillin is a derivative of vanillin and has a stronger aroma than vanillin. It is a very important type of synthetic flavoring. Its aroma is 3-4 times that of vanillin and lasts for a long time. After the flavor molecules are combined with glucose and the like to form glycosides, they are non-volatile or have very low volatility, so their stability is greatly improved. Vanillin and sugar molecules form vanillin glucoside through glycosylation reaction, and ethyl vanillin and sugar molecules form ethyl vanillin glucoside through glycosylation reaction. Compared with vanillin and ethyl vanillin precursors, vanillin glucoside and ethyl vanillin glucoside have changes in solubility, stability and biological activity, and therefore have broad application value in food additives, flavor manufacturing, pharmaceutical and cosmetic industries. Vanillin glycoside and ethyl vanillin glycoside can release sweet milky and baking aromas after high-temperature heating or combustion cracking, which can be used for tobacco flavors.

[0004] However, in the prior art, the existing glycosyl donor UDPG is expensive, and the catalytic activity of glycosyltransferase is limited and the catalytic efficiency is low, which to a certain extent limits the application of glycosyltransferase in industrial production and is not conducive to large-scale industrial production. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a glycosyltransferase and an application thereof. The glycosyltransferase has strong specificity, high catalytic efficiency, good stability and a wide substrate range. The glycosyltransferase and sucrose synthase are used to achieve an enzymatically efficient catalytic glycosylation reaction. The application method has a fast conversion rate and a high yield, and is suitable for the production of glycosylation products catalyzed by various enzymatic methods.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a glycosyltransferase having:

[0008] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0009] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0010] (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2).

[0011] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the above glycosyltransferase.

[0012] In some specific embodiments of the present invention, the nucleotide sequence encoding the glycosyltransferase in the nucleic acid molecule has:

[0013] (4) the nucleotide sequence shown in SEQ ID NO: 2; or

[0014] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0015] (6) A nucleotide sequence having at least 90% homology with the nucleotide sequence shown in (4) or (5).

[0016] The present invention also provides a mutant of the above-mentioned glycosyltransferase, wherein the mutation site is at least one of H18L, P21Y, L112F, N207L, H363F, W366Q, S368L, F385K, A386D, and E387Q.

[0017] In some specific embodiments of the present invention, the mutation site of the mutant includes any of the following:

[0018] H18L; or

[0019] P21Y; or

[0020] L112F; or

[0021] N207L; or

[0022] H363F; or

[0023] W366Q; or

[0024] S368L; or

[0025] F385K; or

[0026] A386D; or

[0027] E387Q; or

[0028] H18L and L112F; or

[0029] H18L and N207L; or

[0030] L112F and H363F; or

[0031] H18L, L112F and F385K; or

[0032] L112F, N207L and F385K; or

[0033] H18L, L112F, N207L and F385K; or

[0034] H18L, L112F, F385K and E387Q; or

[0035] L112F, N207L, F385K and A386D; or

[0036] H18L, L112F, N207L, F385K and E387Q; or

[0037] L112F, N207L, F385K, A386D and E387Q; or

[0038] H18L, L112F, N207L, W366Q, F385K and E387Q; or

[0039] H18L, L112F, N207L, S368L, F385K and E387Q; or

[0040] H18L, L112F, N207L, H363F, S368L, F385K, A386D, and E387Q.

[0041] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the mutant.

[0042] The present invention also provides an expression vector having at least one of the following:

[0043] (A1), the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned glycosyltransferase;

[0044] (A2), the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned mutant;

[0045] (A3), the nucleic acid molecule having the nucleotide sequence encoding the glycosyltransferase and the gene element expressing the sucrose synthase AtSUS1;

[0046] (A4), the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned mutant and a gene element expressing sucrose synthase AtSUS1.

[0047] In some specific embodiments of the present invention, the backbone of the above expression vector is pETDuet-1.

[0048] The present invention also provides a host cell having any of the following:

[0049] (B1) the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned glycosyltransferase;

[0050] (B2) the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned mutant;

[0051] (B3) The above expression vector.

[0052] In some specific embodiments of the present invention, the host cells include animal cells, bacterial cells, and fungal cells;

[0053] The bacteria include Escherichia coli.

[0054] The present invention also provides a method for preparing glycosyltransferase, which is prepared based on any of the following:

[0055] (C1) the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned glycosyltransferase;

[0056] (C2) the above-mentioned nucleic acid molecule having a nucleotide sequence encoding the above-mentioned mutant;

[0057] (C3) the above expression vector;

[0058] (C4) The above-mentioned host cell.

[0059] The present invention also provides the use of the above glycosyltransferase or the above mutant, wherein the above glycosyltransferase or the above mutant catalyzes the transfer of glycosyl from a glycosyl donor to a glycosyl acceptor to generate a glycosylation reaction.

[0060] In some specific embodiments of the present invention, the glycosyl acceptor includes vanillin, ethyl vanillin, cinnamyl alcohol, 4-hydroxybenzyl alcohol, tyrosol or hydroquinone.

[0061] In some specific embodiments of the present invention, when the glycosyl acceptor is vanillin, the glycosylation product is vanillin glucoside; when the glycosyl acceptor is ethyl vanillin, the glycosylation product is ethyl vanillin glucoside; when the glycosyl acceptor is cinnamyl alcohol, the glycosylation product is rosin; when the glycosyl acceptor is 4-hydroxybenzyl alcohol, the glycosylation product is gastrodin; when the glycosyl acceptor is tyrosol, the glycosylation product is salidroside; when the glycosyl acceptor is hydroquinone, the glycosylation product is α-arbutin.

[0062] The present invention also provides a reaction system for glycosylation reaction catalyzed by the host cell: 0.05-0.25 g / mL of the host cell, 40-200 mM trisodium citrate, 0.01-0.5 mM magnesium ion, 100-600 g / L sucrose, 0.05-5% xylene and 20-60 g / L substrate; wherein the substrate is vanillin or ethyl vanillin, the reaction temperature is 20-40°C, and the reaction pH is 6.0-10.0.

[0063] The beneficial effects of the present invention are as follows: the glycosyltransferase UGT72E61 and its mutants have strong specificity, high catalytic efficiency, good stability, and a wide substrate range. The glycosyltransferase UGT72E61 and its mutants have strong specificity, high catalytic efficiency, good stability, and a wide substrate range. The combination of the glycosyltransferase UGT72E61 and its mutants can be used to efficiently prepare vanillin glucoside, ethyl vanillin glucoside, rosin, gastrodin, salidroside, and alpha-arbutin, thereby improving the catalytic efficiency, solving the problem of the expensive price of the glycosyl donor UDPG, providing an option for optimizing process conditions for further realizing large-scale industrial production, and being more conducive to realizing large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is the nucleic acid electrophoresis diagram of the glycosyltransferase UGT72E61 of the present invention.

[0065] Figure 2 It is the protein electrophoresis diagram of the recombinant expression bacteria V01 to V09 of the present invention.

[0066] Figure 3 It is the protein electrophoresis diagram of the recombinant expression bacteria V10-V17 of the present invention.

[0067] Figure 4 It is the protein electrophoresis diagram of the recombinant expression bacteria V18-V24 of the present invention. DETAILED DESCRIPTION

[0068] The principles and features of the present invention are described below (in conjunction with the accompanying drawings). The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0069] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0070] As used herein, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0071] Herein, the mutation site is represented by the form of "XaY", where a represents the position of the amino acid in SEQ ID NO: 1, X represents the wild-type amino acid type at position a in SEQ ID NO: 1, and Y represents the mutated amino acid type at position a in SEQ ID NO: 1. For example, in the present invention, "H18L" represents that the histidine H at the 18th position corresponding to SEQ ID NO: 1 is mutated to leucine L; "H18L / N207L" represents that the histidine H at the 18th position corresponding to SEQ ID NO: 1 is mutated to leucine L, and the asparagine N at the 207th position is mutated to leucine L.

[0072] The present invention provides a glycosyltransferase UGT72E61, which is derived from a cruciferous plant (Physaria fendleri), and the amino acid sequence is shown in SEQ ID NO: 1:

[0073] MHI IKPEAAMFSSPGMGHVIPVIELANRLCVNHGFHVTVFVLETDAASAQSKFQNSTGVDIVCLPLPDISGLVDPADHVVTKIGVIMREAVPALRSKISAMHQKPTALFIDLFGTDALCLAKEFNMLTYLFIASNARYLGVSIYYPTLDKDTKEEHTVQRKPLAIPGCEPVRFEDTMDAYLVPDTPLYQDLVRHCLAYPKVDGILVNTWEEMEPKSLKSLQDPKLLGRVVSAPVYPVGPLCRPIESTKTDHPVMDWLNEQPYESVLYISFGSGGSLTAKQLTELAWGLEQSQQRFVWVVRPPVDGSSCSQYFSANGGGPKDNTPEYLPEGFVTRTCDRGFMVTSWAPQAQVLAHPAVGGYLTHCGWSSTLESVLGGVPMIAWPLFAEQNMNAALLNDELRIAVRVDDKKDRSTIEAMVRKVMSEEEGEEMRKKVKELRDTAELSLSIEGGGSAHKSLCRVKDECQQFLERIGNLARGA (SEQ ID NO:1);

[0074] Its nucleic acid sequence optimized for E. coli codons is shown in SEQ ID NO:2:

[0075]

[0076] The present invention also provides a mutant of the above-mentioned glycosyltransferase UGT72E61, wherein the mutation points are: H18L, P21Y, L112F, N207L, H363F, W366Q, S368L, F385K, A386D, and E387Q.

[0077] This application protects single point mutations and multiple point mutations:

[0078] H18L; or

[0079] P21Y; or

[0080] L112F; or

[0081] N207L; or

[0082] H363F; or

[0083] W366Q; or

[0084] S368L; or

[0085] F385K; or

[0086] A386D; or

[0087] E387Q; or

[0088] H18L / L112F; or

[0089] H18L / N207L; or

[0090] L112F / H363F; or

[0091] H18L / L112F / F385K; or

[0092] L112F / N207L / F385K; or

[0093] H18L / L112F / N207L / F385K; or

[0094] H18L / L112F / F385K / E387Q; or

[0095] L112F / N207L / F385K / A386D; or

[0096] H18L / L112F / N207L / F385K / E387Q; or

[0097] L112F / N207L / F385K / A386D / E387Q; or

[0098] H18L / L112F / N207L / W366QF385K / E387Q; or

[0099] H18L / L112F / N207L / S368L / F385K / E387Q; or

[0100] H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q.

[0101] The primers involved in this application are shown in Table 1:

[0102] Table 1 Primer list

[0103]

[0104]

[0105] Other gene or protein sequence information involved in this application is as follows:

[0106] AtSUS1 is derived from Arabidopsis thaliana

[0107] Amino acid (protein) sequence, SEQ ID NO:3:

[0108] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDVE(SEQ ID NO:3);

[0109] The nucleic acid sequence optimized for E. coli codons, SEQ ID NO:4:

[0110]

[0111] The present invention will be further described below in conjunction with the embodiments.

[0112] Example 1

[0113] 1. Cloning of glycosyltransferase and sucrose synthase genes

[0114] UGT72E61 and AtSUS1 were codon-optimized for Escherichia coli, and full-length DNA sequences were synthesized.

[0115] 2. Construction of recombinant expression vector

[0116] Using pETDuet-1 as the expression vector, the In-Fusion strategy was used to construct a recombinant expression plasmid. PCR amplification primers containing homology arms were designed (see Table 1) to clone UGT72E61 and the linearized pETDuet-1 vector. According to the instructions of the kit, the DNA fragment and the linearized vector fragment were subjected to homologous recombination reaction. After reacting at 50 degrees for 1 hour, the competent cells of E. coli DH5α were transformed and coated with Amp-resistant plates and cultured overnight at 37 degrees. Positive clones were screened by colony PCR, and positive clones were picked in 5 mL of LB medium containing Amp. After sequencing verification, the correct recombinant plasmid pETDuet-1-UGT72E61 was extracted.

[0117] Using pETDuet-1 as the expression vector, the In-Fusion strategy was used to construct a recombinant expression plasmid. PCR amplification primers containing homology arms were designed (see Table 1) to clone AtSUS1 and the linearized pETDuet-1-UGT72E61 recombinant vector. According to the instructions of the kit, the DNA fragment and the linearized vector fragment were subjected to homologous recombination reaction. After reacting at 50 degrees for 1 hour, the competent cells of E. coli DH5α were transformed, and the Amp-resistant plates were coated and cultured at 37 degrees overnight. Positive clones were screened by colony PCR, and the positive clones were picked in 5 mL of LB medium containing Amp. After sequencing verification, the correct recombinant plasmid pETDuet-1-UGT72E61-AtSUS1 was extracted.

[0118] 3. Construction of UGT72E61 mutant

[0119] Take H18L single point mutation as an example:

[0120] Using pETDuet-1-UGT72E61 as a template, mutant primers were designed (see Table 1) and PCR amplification was performed. The PCR product was digested with restriction endonuclease Dpn I for 2 h to remove the template; the target band was recovered after agarose gel electrophoresis, eluted with ddH2O during recovery, and then transformed into E. coli DH5α competent cells, coated with Amp-resistant plates and cultured at 37°C overnight; colony PCR was used to screen positive clones, and positive clones were picked in 5 mL of LB medium containing Amp, sequenced, and the correct plasmid pETDuet-1-UGT72E61 (H18L) was extracted.

[0121] The remaining plasmid construction methods were the same as those for the H18L single point mutation. The primers are shown in Table 1. The plasmids obtained were as follows:

[0122] pETDuet-1-UGT72E61(P21Y)

[0123] pETDuet-1-UGT72E61(L112F)

[0124] pETDuet-1-UGT72E61(N207L)

[0125] pETDuet-1-UGT72E61(H363F)

[0126] pETDuet-1-UGT72E61(W366Q)

[0127] pETDuet-1-UGT72E61(S368L)

[0128] pETDuet-1-UGT72E61(F385K)

[0129] pETDuet-1-UGT72E61(A386D)

[0130] pETDuet-1-UGT72E61(E387Q)

[0131] pETDuet-1-UGT72E61(H18L / L112F)

[0132] pETDuet-1-UGT72E61(H18L / N207L)

[0133] pETDuet-1-UGT72E61(L112F / H363F)

[0134] pETDuet-1-UGT72E61(H18L / L112F / F385K)

[0135] pETDuet-1-UGT72E61(L112F / N207L / F385K)

[0136] pETDuet-1-UGT72E61(H18L / L112F / N207L / F385K)

[0137] pETDuet-1-UGT72E61(H18L / L112F / F385K / E387Q)

[0138] pETDuet-1-UGT72E61(L112F / N207L / F385K / A386D)

[0139] pETDuet-1-UGT72E61(H18L / L112F / N207L / F385K / E387Q)

[0140] pETDuet-1-UGT72E61(L112F / N207L / F385K / A386D / E387Q)

[0141] pETDuet-1-UGT72E61(H18L / L112F / N207L / W366QF385K / E387Q)

[0142] pETDuet-1-UGT72E61(H18L / L112F / N207L / S368L / F385K / E387Q)

[0143] pETDuet-1-UGT72E61(H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q).

[0144] Using pETDuet-1-UGT72E61-AtSUS1 as a template, mutant primers were designed (primers are shown in Table 1) and PCR amplification was performed. The PCR product was digested with restriction endonuclease Dpn I for 2 h to remove the template; the target band was recovered after agarose gel electrophoresis, eluted with ddH2O during recovery, and then transformed into E. coli DH5α competent cells, coated with Amp-resistant plates and cultured at 37°C overnight; colony PCR was used to screen positive clones, and positive clones were selected and placed in 5 mL of LB medium containing Amp for sequencing verification, and the correct plasmid pETDuet-1-UGT72E61(H18L)-AtSUS1 was extracted.

[0145] The construction methods of the remaining plasmids were the same as pETDuet-1-UGT72E61(H18L)-AtSUS1. The primers are shown in Table 5. The plasmids obtained were as follows:

[0146] pETDuet-1-UGT72E61(P21Y)-AtSUS1

[0147] pETDuet-1-UGT72E61(L112F)-AtSUS1

[0148] pETDuet-1-UGT72E61(N207L)-AtSUS1

[0149] pETDuet-1-UGT72E61(H363F)-AtSUS1

[0150] pETDuet-1-UGT72E61(W366Q)-AtSUS1

[0151] pETDuet-1-UGT72E61(S368L)-AtSUS1

[0152] pETDuet-1-UGT72E61(F385K)-AtSUS1

[0153] pETDuet-1-UGT72E61(A386D)-AtSUS1

[0154] pETDuet-1-UGT72E61(E387Q)-AtSUS1

[0155] pETDuet-1-UGT72E61(H18L / L112F)-AtSUS1

[0156] pETDuet-1-UGT72E61(H18L / N207L)-AtSUS1

[0157] pETDuet-1-UGT72E61(L112F / H363F)-AtSUS1

[0158] pETDuet-1-UGT72E61(H18L / L112F / F385K)-AtSUS1

[0159] pETDuet-1-UGT72E61(L112F / N207L / F385K)-AtSUS1

[0160] pETDuet-1-UGT72E61(H18L / L112F / N207L / F385K)-AtSUS1

[0161] pETDuet-1-UGT72E61(H18L / L112F / F385K / E387Q)-AtSUS1

[0162] pETDuet-1-UGT72E61(L112F / N207L / F385K / A386D)-AtSUS1

[0163] pETDuet-1-UGT72E61(H18L / L112F / N207L / F385K / E387Q)-AtSUS1

[0164] pETDuet-1-UGT72E61(L112F / N207L / F385K / A386D / E387Q)-AtSUS1

[0165] pETDuet-1-UGT72E61(H18L / L112F / N207L / W366QF385K / E387Q)-AtSUS1

[0166] pETDuet-1-UGT72E61(H18L / L112F / N207L / S368L / F385K / E387Q)-AtSUS1

[0167] pETDuet-1-UGT72E61(H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q)-AtSUS1.

[0168] 4. Construction of recombinant expression bacteria

[0169] (1) Construction of recombinant bacteria G01-24

[0170] Take the recombinant expression bacteria G01 as an example:

[0171] The recombinant plasmid pETDuet-1-UGT72E61 was transferred into E. coli BL21 (DE3) to obtain the recombinant strain G01, which was frozen in a -80°C refrigerator. The frozen strain G01 was inoculated into 5-10 mL of LB medium containing Amp resistance and cultured at 37°C overnight to prepare the fermentation seed liquid; the seed liquid was inoculated into 50 mL of TB medium according to a 1% inoculum amount, and cultured at 200 rpm and 37°C to OD600-0.6-0.8, 0.1 mM IPTG was added, and the expression was induced at 22°C for 24 h, and then the bacteria were collected by centrifugation at 12,000 rpm and 4°C, and stored at -20°C for later use.

[0172] The construction and preparation methods of the remaining recombinant expression bacteria containing the UGT72E61 mutant were the same as the recombinant expression bacteria G01, and recombinant expression bacteria G02-24 were obtained, as shown in Table 2 below.

[0173] Table 2 Recombinant bacteria G01-24

[0174]

[0175]

[0176] (2) Construction of recombinant bacteria V01-V24

[0177] Take the recombinant expression bacteria V01 as an example:

[0178] The recombinant plasmid pETDuet-1-UGT72E61-AtSUS1 was transferred into E. coli BL21 (DE3) to obtain recombinant strains V01-V24, which were frozen in a -80°C refrigerator. The frozen strains V01-V24 were inoculated into 5-10 mL of LB medium containing Amp resistance and cultured at 37°C overnight to prepare fermentation seed liquid; the seed liquid was inoculated into 50 mL of TB medium according to the inoculum amount of 1%, and cultured at 200 rpm and 37°C to OD600-0.6-0.8, and 0.1 mM IPTG was added to induce expression at 22°C for 24 hours, and then centrifuged at 12,000 rpm and 4°C to collect the bacteria, and stored at -20°C for later use.

[0179] The construction and preparation methods of the remaining recombinant expression bacteria containing the UGT76AN70 mutant were the same as the recombinant expression bacteria V01, and the recombinant expression bacteria V02-24 were obtained, as shown in Table 3 below.

[0180] Table 3 Recombinant expression bacteria V01-24

[0181]

[0182]

[0183] Example 2 Single enzyme activity

[0184] The G01-G24 bacteria obtained in Example 1 were resuspended in 100 mM pH 7.0 PBS buffer, and a final concentration of 1 mM PMSF and 1 mg / mL lysozyme were added, and stirred on ice for 30 min; the cells were ultrasonically disrupted, the supernatant was collected by centrifugation, and it was incubated with a Ni-NTA affinity column for 40-60 min, and the effluent was removed; then the impurities and the target protein were eluted with 100 mM pH 7.0 PBS buffer containing different concentrations of imidazole (20 mM, 250 mM); the target protein was replaced with buffer, dialyzed, desalted and concentrated with an ultrafiltration tube to obtain glycosyltransferase UGT72E61 or its mutant, which was packaged and frozen at -80°C for use.

[0185] (1) Test of single enzyme activity of vanillin to produce vanillin glucoside

[0186] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM vanillin, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction, the reaction temperature was 37°C, the reaction time was 30 min, and each group of reaction conditions was repeated 3 times; after the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of vanillin and vanillin glucoside in the reaction sample and calculate the conversion rate.

[0187] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 4.

[0188] Table 4

[0189]

[0190] It can be seen from Table 4 that the single enzyme activity of the glycosyltransferase UGT72E61 mutant of G02-24 is higher than that of the glycosyltransferase UGT72E61 of G01, among which G24 (UGT72E61 (H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q)) has the best single enzyme activity, and the vanillin conversion rate can reach 98.8%.

[0191] (2) Activity test of single enzyme from ethyl vanillin to vanillin glucoside

[0192] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM ethyl vanillin, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction at 37°C for 30 min. Each set of reaction conditions was repeated 3 times. After the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of ethyl vanillin and ethyl vanillin glucoside in the reaction sample and calculate the conversion rate.

[0193] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 5.

[0194] Table 5

[0195]

[0196]

[0197] It can be seen from Table 5 that the single enzyme activity of the glycosyltransferase UGT72E61 mutant of G02-24 is higher than that of the glycosyltransferase UGT72E61 of G01, among which G24 (UGT72E61 (H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q)) has the best single enzyme activity, and the conversion rate of ethyl vanillin can reach 96.17%.

[0198] (3) Activity test of single enzyme for cinnamyl alcohol production

[0199] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM cinnamyl alcohol, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction at 37°C for 30 min. Each set of reaction conditions was repeated 3 times. After the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of cinnamyl alcohol and glycosyltransferase in the reaction sample and calculate the conversion rate.

[0200] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 6.

[0201] Table 6

[0202]

[0203]

[0204] Table 6 shows that in the production of cinnamyl alcohol by glycosyltransferase UGT72E61 or its mutants, G24 (UGT72E61 (H18L / L112F / N207L / H363F / S368L / F385K / A386D / E387Q)) has the best single enzyme activity, and the conversion rate of cinnamyl alcohol can reach 89.17%.

[0205] (4) Single enzyme activity test of 4-hydroxybenzyl alcohol to produce gastrodin

[0206] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM 4-hydroxybenzyl alcohol, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction, the reaction temperature was 37°C, the reaction time was 30 min, and each group of reaction conditions was repeated 3 times; after the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of 4-hydroxybenzyl alcohol and gastrodin in the reaction sample and calculate the conversion rate.

[0207] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 7.

[0208] Table 7

[0209]

[0210]

[0211] It can be seen from Table 7 that in the production of gastrodin from 4-hydroxybenzyl alcohol catalyzed by glycosyltransferase UGT72E61 or its mutants, the single enzyme activity of the recombinant bacteria G24 is the best.

[0212] (5) Activity test of single enzyme to generate salidroside from tyrosol

[0213] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM tyrosol, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction, the reaction temperature was 37°C, the reaction time was 30 min, and each group of reaction conditions was repeated 3 times; after the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of tyrosol and salidroside in the reaction sample and calculate the conversion rate.

[0214] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 8.

[0215] Table 8

[0216]

[0217]

[0218] It can be seen from Table 8 that in the production of salidroside from tyrosol catalyzed by glycosyltransferase UGT72E61 or its mutants, the single enzyme activity of the recombinant bacteria G24 is the best.

[0219] (6) Test of single enzyme activity of hydroquinone to generate α-arbutin

[0220] Enzyme activity test system: 100 μL volume including 20 mM pH 8.0 Tris-HCl buffer, 0.5 mM hydroquinone, 1 mM UDPG and 2 μg glycosyltransferase UGT72E61 or its mutant was placed in a constant temperature incubator for reaction, the reaction temperature was 37°C, the reaction time was 30 min, and each group of reaction conditions was repeated 3 times; after the reaction, an equal volume of methanol was added for quenching, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant after filtration through a 0.22 μm filter membrane was used for HPLC analysis to determine the amount of hydroquinone and α-arbutin in the reaction sample and calculate the conversion rate.

[0221] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 9.

[0222] Table 9

[0223]

[0224]

[0225] It can be seen from Table 9 that in the process of glycosyltransferase UGT72E61 or its mutant catalyzing the production of α-arbutin from hydroquinone, the single enzyme activity of the recombinant bacteria G24 is the best.

[0226] Example 3 Whole-cell biotransformation reaction experimental verification

[0227] The V01-24 cells were first resuspended and washed twice with 100 mM Tris-HCl buffer at pH 9.0 to obtain wet cells for whole-cell catalytic reactions.

[0228] (1) Vanillin generates vanillin glycoside

[0229] The biotransformation reaction system is:

[0230] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L vanillin, reaction temperature was 25°C, reaction time was 24h, each group was paralleled 3 times. After the reaction, an equal volume of methanol was added to quench, centrifuged at 12,000rpm for 10min to remove the precipitate, and the supernatant was filtered through a 0.22μm filter membrane for HPLC analysis to determine the amount of vanillin and glucosyl vanillin in the reaction sample and calculate the conversion rate.

[0231] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 10.

[0232] Table 10

[0233]

[0234] As can be seen from Table 10, compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant had a higher conversion rate of vanillin glucoside.

[0235] (2) Ethyl vanillin generates ethyl vanillin glucoside

[0236] The biotransformation reaction system is:

[0237] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L ethyl vanillin, reaction temperature was 25°C, reaction time was 24h, each group was paralleled 3 times. After the reaction, an equal volume of methanol was added to quench, centrifuged at 12,000rpm for 10min to remove the precipitate, and the supernatant was filtered through a 0.22μm filter membrane for HPLC analysis to determine the amount of ethyl vanillin and ethyl vanillin glucoside in the reaction sample and calculate the conversion rate.

[0238] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 11.

[0239] Table 11

[0240]

[0241]

[0242] As can be seen from Table 11, compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant had a higher conversion rate of ethyl vanillin glucoside.

[0243] (3) Cinnamyl alcohol forms a network plug

[0244] The biotransformation reaction system is:

[0245] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L cinnamyl alcohol, reaction temperature was 25°C, reaction time was 24h, and each group was paralleled 3 times. After the reaction, an equal volume of methanol was added to quench, centrifuged at 12,000rpm for 10min to remove the precipitate, and the supernatant was filtered through a 0.22μm filter membrane for HPLC analysis to determine the amount of cinnamyl alcohol and chelating agent in the reaction sample and calculate the conversion rate.

[0246] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 12.

[0247] Table 12

[0248]

[0249]

[0250] It can be seen from Table 12 that compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant have a higher conversion rate of network plugs.

[0251] (4) 4-Hydroxybenzyl alcohol generates gastrodin

[0252] The biotransformation reaction system is:

[0253] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L 4-hydroxybenzyl alcohol, the reaction temperature was 25°C, the reaction time was 24h, and each group was paralleled 3 times. After the reaction was completed, an equal volume of methanol was added to quench, and the precipitate was removed by centrifugation at 12,000rpm for 10min. The supernatant was filtered through a 0.22μm filter membrane and used for HPLC analysis to determine the amount of 4-hydroxybenzyl alcohol and gastrodin in the reaction sample and calculate the conversion rate.

[0254] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 13.

[0255] Table 13

[0256]

[0257]

[0258] It can be seen from Table 13 that compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant have a higher gastrodin conversion rate.

[0259] (5) Tyrosol produces salidroside

[0260] The biotransformation reaction system is:

[0261] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L tyrosol, reaction temperature was 25°C, reaction time was 24h, each group was paralleled 3 times. After the reaction, an equal volume of methanol was added to quench, centrifuged at 12,000rpm for 10min to remove the precipitate, and the supernatant was filtered through a 0.22μm filter membrane for HPLC analysis to determine the amount of tyrosol and salidroside in the reaction sample and calculate the conversion rate.

[0262] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8min 60% A, 8-9min 15% A, 9-14min 15% A, 14-15min 60% A, 15-23min 60% A, detection wavelength is 280nm, column temperature is 40°C. The detection results are shown in Table 14.

[0263] Table 14

[0264]

[0265]

[0266] It can be seen from Table 14 that compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant have a higher conversion rate of salidroside.

[0267] (6) Hydroquinone generates α-arbutin

[0268] The biotransformation reaction system is:

[0269] 0.1g / mL wet bacteria (V01-V24), 60mM trisodium citrate, 100mM sodium phosphate buffer at pH 8.0, 0.1mM MgCl2, 1% (v / v) xylene, 400g / L sucrose and 40g / L hydroquinone, reaction temperature was 25°C, reaction time was 24h, each group was paralleled 3 times. After the reaction, an equal volume of methanol was added to quench, centrifuged at 12,000rpm for 10min to remove the precipitate, and the supernatant was filtered through a 0.22μm filter membrane for HPLC analysis to determine the amount of hydroquinone and α-arbutin in the reaction sample and calculate the conversion rate.

[0270] HPLC detection conditions are: phosphoric acid water (pH 2.3, A)-methanol (B), 0-8 min 60% A, 8-9 min 15% A, 9-14 min 15% A, 14-15 min 60% A, 15-23 min 60% A, detection wavelength 280 nm, column temperature 40° C. The detection results are shown in Table 15.

[0271] Table 15

[0272]

[0273] It can be seen from Table 15 that compared with the recombinant expression bacteria V01 expressing UGT72E61, the recombinant expression bacteria V02-24 expressing the UGT72E61 mutant have a higher conversion rate of α-arbutin.

[0274] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A glycosyltransferase, characterized in that The glycosyltransferase has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in the sequence shown in (1), and having the same or similar function as (1); or (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2).

2. A nucleic acid molecule, characterized in that Having a nucleotide sequence encoding the glycosyltransferase according to claim 1, the nucleotide sequence having (4) the nucleotide sequence shown in SEQ ID NO: 2; or (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or (6) A nucleotide sequence having at least 90% homology with the nucleotide sequence shown in (4) or (5).

3. The mutant of glycosyltransferase according to claim 1, characterized in that The mutation site is at least one of H18L, P21Y, L112F, N207L, H363F, W366Q, S368L, F385K, A386D, and E387Q.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule has a nucleotide sequence encoding the mutant according to claim 4.

5. An expression vector, characterized in that Have any of the following: (A1) The nucleic acid molecule according to claim 2; (A2) the nucleic acid molecule according to claim 4; (A3) The nucleic acid molecule according to claim 2 and the gene element expressing sucrose synthase AtSUS1; (A4) The nucleic acid molecule according to claim 4 and the gene element expressing sucrose synthase AtSUS1.

6. A host cell, excluding a totipotent cell, characterized in that Have any of the following: (B1) the nucleic acid molecule according to claim 2; (B2) the nucleic acid molecule according to claim 4; (B3) The expression vector according to claim 5.

7. A method for preparing a glycosyltransferase, characterized in that: Prepared based on any of the following: (C1) the nucleic acid molecule according to claim 2; (C2) the nucleic acid molecule according to claim 4; (C3) The expression vector according to claim 5; (C4) The host cell according to claim 6.

8. Use of the glycosyltransferase according to claim 1 or the mutant according to claim 3, characterized in that: The glycosyltransferase according to claim 1 or the mutant according to claim 3 catalyzes the transfer of a glycosyl from a glycosyl donor to a glycosyl acceptor to produce a glycosylation reaction.

9. The use according to claim 8, characterized in that: The glycosyl acceptor includes vanillin, ethyl vanillin, cinnamyl alcohol, 4-hydroxybenzyl alcohol, tyrosol or hydroquinone.

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