A glycosyltransferase for catalyzing the production of rebaudioside M and its recombinant gene

Abstract

This invention discloses a glycosyltransferase and its recombinant gene for catalyzing the production of rebaudioside M. Compared to the wild-type glycosyltransferase, the glycosyltransferase variant of this invention exhibits significantly improved thermostability and enzyme activity. At 70°C, the wild-type glycosyltransferase is completely inactivated, while the enzyme activity of the glycosyltransferase variant of this invention is above 50 U / mg. Simultaneously, at 70°C, the solubility of the substrate rebaudioside D increases, and the thermal motion of enzyme and substrate molecules intensifies, increasing the collision frequency and energy between them, thus further enhancing the catalytic reaction rate. Therefore, the glycosyltransferase variant of this invention is suitable as a biocatalyst for the large-scale production of the sweetener rebaudioside M with improved taste.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a glycosyltransferase for catalytic production of rebaudioside M and its recombinant gene. Background Technology

[0002] Stevia, also known as steviol glycosides, is characterized by its high sweetness (300-450 times that of sucrose) and low calorie value (1 / 300th that of sucrose). It is a natural sucrose substitute and is hailed as the "world's third largest glycogen source." Stevia is extracted from stevia leaves. The stevia extracted by manufacturers from stevia leaves is mostly composed of steviol glycosides (STV) and rebaudioside A (Reb A). However, STV and Reb A have a distinct bitter taste and a licorice-like aftertaste, which greatly limits the application of steviol glycosides as a sweetener in food.

[0003] Studies have found that Rebaudioside M (Reb M) retains the advantages of stevia's high sweetness and low calorie value while having almost no bitterness or licorice aftertaste, making it a superior sugar substitute. However, large-scale production of Reb M faces significant bottlenecks: Reb M accounts for less than 1% of stevia, and the cost of producing Reb M through plant extraction is high. Furthermore, the glycosyltransferase activity required to catalyze the conversion of RebD to Reb M is currently low, resulting in low production efficiency for Reb M. This leads to high costs for Reb M production via microbial fermentation or enzymatic catalysis. Therefore, improving the activity of glycosyltransferases is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a thermostable glycosyltransferase, the wild-type sequence of which is shown in SEQ ID NO.1. This wild-type glycosyltransferase sequence is derived from the protein sequence library GI:1777435608. Using the Lésign platform, the wild-type glycosyltransferase sequence was designed, ultimately yielding the computationally optimal enzyme variant.

[0006] A glycosyltransferase for catalyzing the production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1 and mutated using the following mutation set: K55N+N70T+S181P+S275R+K441E+T460K+S193R+M146R+E3V.

[0007] The second objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1, and is mutated using the following mutation set:

[0009] K55N+N70T+S181P+S275R+K441E+T460K+M146R+S275G+D327E+K224E.

[0010] The third objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1, and is mutated using the following mutation set:

[0012] K55N+N70T+S181P+S275R+K441E+T460K+K224E+I94F+R414H+A112S.

[0013] The fourth objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0014] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1, and is mutated using the following mutation set:

[0015] K55N+N70T+S181P+S275R+K441E+T460K+I94F+R90L+S242I+H156Y+A180V+R175H.

[0016] The fifth objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0017] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1, and is mutated using the following mutation set:

[0018] K55N+N70T+S181P+S275R+K441E+T460K+E3V+R414H+K224E+A112S+R90L+M146R+R298Q+A180V.

[0019] The sixth objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0020] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, characterized in that the glycosyltransferase uses the wild-type glycosyltransferase described in SEQ ID NO:1 as the parent, and is mutated using the following mutation set:

[0021] K55N+N70T+S181P+S275R+K441E+T460K+S193R+D100A+R298Q+K224E+T460R+R141P+R414H.

[0022] The seventh objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0023] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, characterized in that the glycosyltransferase uses the wild-type glycosyltransferase described in SEQ ID NO:1 as the parent, and is mutated using the following mutation set:

[0024] K55N+N70T+S181P+S275R+K441E+T460K+D100A+R414H+S275G+K224E+R405P+T264S+I94F+R90L.

[0025] The eighth objective of this invention is to provide another glycosyltransferase variant with an optimal reaction temperature of around 70°C and an enzyme activity of over 40 U / mg.

[0026] To achieve the above objectives, the present invention provides the following technical solution: a glycosyltransferase for catalytic production of rebaudioside M, wherein the glycosyltransferase is based on the wild-type glycosyltransferase described in SEQ ID NO:1, and is mutated using the following mutation set:

[0027] K55N+N70T+S181P+S275R+K441E+T460K+R414H+T460R+R90L+A112S+N50K+S193R+E3V+H156Y+T264S+R405P+D327E+S275G.

[0028] The ninth objective of this invention is to provide genetic material capable of expressing the above-mentioned glycosyltransferase variant.

[0029] To achieve the above objectives, the present invention provides the following technical solution: a recombinant genetic material of a glycosyltransferase, capable of expressing the DNA or RNA of the aforementioned glycosyltransferase.

[0030] The tenth objective of this invention is to provide a recombinant strain capable of expressing the above-mentioned glycosyltransferase variant.

[0031] To achieve the above objectives, the present invention provides the following technical solution: a recombinant strain of glycosyltransferase, comprising recombinant genetic material capable of expressing the above-mentioned glycosyltransferase.

[0032] Compared with the prior art, the advantages of the present invention are: the substrate Reb D and product Reb M catalyzed by the enzyme have poor solubility at low temperatures, and increasing the temperature can greatly promote dissolution, which is beneficial to the reaction.

[0033] Compared to wild-type glycosyltransferases, the glycosyltransferase variants of this invention exhibit significantly improved thermostability and enzyme activity. At 70°C, wild-type glycosyltransferases are completely inactivated, while the enzyme activities of the glycosyltransferase variants of this invention are all above 50 U / mg. Simultaneously, at 70°C, the solubility of the reaction substrate rebaudioside D increases, and the thermal motion of enzyme and substrate molecules intensifies, increasing the collision frequency and energy between them, thus further enhancing the catalytic reaction rate. Therefore, the glycosyltransferase variants of this invention are suitable as biocatalysts for the large-scale production of the flavor-enhancing sweetener rebaudioside M. Detailed Implementation

[0034] The term "recombinant gene" refers to DNA or RNA capable of expressing the glycosyltransferase of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite synthesis, TdT biosynthesis, or other suitable techniques known in the art. Once a template sequence is available, it can be amplified by PCR or other suitable techniques known in the art. With a recombinant bacterial strain, further large-scale amplification can be achieved by culturing the strain. In some embodiments, the recombinant gene may also include residual restriction enzyme sites, other accessory elements such as control elements (e.g., promoters), labeling substances (e.g., fluorescent labels), and other sequences that do not affect the expression of the target gene.

[0035] The term "clonal scar" refers to the promoter sequence of transcription, which is dependent on the initiation messenger ribonucleotide (mRNA) for protein expression, followed by the ribosome-binding site (RBS) that attracts the translation machinery, and then the signal peptide sequence that facilitates protein transport to the periplasm. Mature proteins are typically cloned after the signal peptide, cleaved from it by a signal peptidase as they cross the membrane. However, in cloning constructs after the signal peptide, restriction endonucleases often require specific sequences to cut the DNA, leaving a clonal scar following the signal peptide sequence.

[0036] The term "signal peptide" refers to a short peptide (typically 16-30 amino acids long) located at the N-terminus of most newly synthesized proteins, which are destined for the secretion pathway. It can also be called a signal sequence, targeting signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide. Signal peptides are usually cleaved from proteins by signal peptidases.

[0037] Whether it is a cloned scar, signal peptide, or other elements in a recombinant gene, it does not affect the realization of glycosyltransferase function. Therefore, if the amino acid sequence of the final protein differs from the amino acid sequence disclosed in this invention only in the amino acid sequence corresponding to the above-mentioned DNA sequence, it still falls within the protection scope of this invention.

[0038] The term "signal peptide cleavage site" refers to a dipeptide between which a signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, the dipeptide is Ala-Ala. Signal peptide cleavage sites can be calculated using algorithms such as SignalP4.1, which can be found at http: / /

[0039] The service is available online at / / www.cbs.dtu.dk / services / SignalP / (Center for Biological Sequence Analysis, Technical University of Denmark).

[0040] The term "promoter" refers to a region of DNA that initiates the transcription (writing to mRNA) of a specific gene. Promoters are typically located near the transcription start site of a gene, on the same strand of the DNA and upstream of it (pointing to the 5' region of the sense strand). Promoters can be inducible, meaning that the expression of a gene operatively linked to the promoter can be activated in the presence of an inducing agent. Alternatively, promoters can be constitutive, meaning they are not regulated by any inducing agent.

[0041] The abbreviation "RBS" stands for ribosome-binding site, or ribosome binding site. This is the sequence of nucleotides upstream of the start codon in mRNA transcripts, responsible for recruiting ribosomes during the initiation of protein translation.

[0042] The term "expression" refers to the process of DNA being transcribed into messenger RNA (mRNA) and then translated into protein. To achieve successful expression and screening of glycosyltransferases, the aforementioned signal peptide, promoter, and RBS may be introduced into the recombinant gene. Therefore, a portion of the corresponding peptide may remain on the expressed glycosyltransferase protein. This portion of the peptide does not affect the function of the glycosyltransferase; therefore, even if the product contains additional peptides, as long as the amino acid sequence of the main component is identical to the sequence of this invention, the product is still an infringing product.

[0043] The term "expression vector" refers to the ability to incorporate and express heterologous polynucleotide fragments into host cells. Many prokaryotic and eukaryotic expression vectors are commercially available. Choosing a suitable expression vector is within the knowledge of a technician.

[0044] The term "chassis cell" refers to a suitable host vector for expressing DNA containing the DNA of the present invention. The host can be any organism capable of containing and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. Chassis cells can be prokaryotes or eukaryotes, single-celled or multicellular, including mammalian cells, plant cells, fungi, etc. According to existing technology, those skilled in the art can achieve heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters through a limited number of experiments. Chassis cells can be selected from at least one of *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Candida*, *Rhodotorula*, *Bacillus*, *Escherichia coli*, *Salmonella*, *Clostridium*, *Streptomyces*, *Staphylococcus*, *Neisseria*, and *Shigella*. This invention merely lists types of chassis cells and does not constitute a limitation on the types of chassis cells. Chassis cells are preferably *Escherichia coli*, and suitable *E. coli* strains (including many others) include BL21(DE3), C600, DH5αF′, 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue, and Y1089. + The above E. coli strains are all commercially available strains.

[0045] The term "identity" means that residues in two sequences are identical when aligned to the maximum correspondence, as measured using sequence comparison or analysis algorithms such as those described herein. For example, if corresponding fragments of two sequences have the same residues at 5 out of 10 positions when correctly aligned, the two sequences are said to have 50% identity. Most bioinformatics programs report the percentage identity of aligned sequence regions, which are typically not the entire molecule. If the alignment is long enough and contains a sufficient number of identical residues, an expected value can be calculated, indicating that the level of identity in the alignment is unlikely to occur randomly.

[0046] The present invention will be further described in detail below with reference to the embodiments.

[0047] Example 1

[0048] Proteins are the material basis of life and essential components of human cells and tissues. All vital components of the human body require protein participation, playing a crucial role in cellular and biological life activities. It can be said that without protein, there is no life. There are many types of proteins in the human body, each with different functions. Some constitute human tissues, some provide energy, some participate in metabolism and transport, and some promote growth and development and regulate immune function. Different proteins perform different duties and roles, and their functions are determined by their structure. The 3D structure of a protein is determined by its amino acid sequence. Therefore, protein design depends on the correspondence between structure and sequence; designing proteins with specific functions requires designing sequences that conform to that functional structure. Understanding and designing proteins is of great significance for promoting innovation and progress in biology and medicine.

[0049] Designing protein sequences for a specific function is extremely difficult, as the final structure and function of the designed sequence are unpredictable. Furthermore, the sample space for fixed-length protein sequences is enormous. To address these challenges, Lésign, a protein design platform based on deep learning algorithms, was developed. This platform enables protein structure prediction, sequence design, and result evaluation. The various functional modules collaborate through interfaces, forming a comprehensive computational pipeline integrating prediction, design, and evaluation.

[0050] Using the Lésign platform, sequence design was performed on wild-type glycosyltransferases (amino acid sequences as shown in SEQ ID NO.1), ultimately yielding computationally optimal enzyme variants.

[0051] 1. Preparation of glycosyltransferases

[0052] 1.1 Construction of recombinant cells:

[0053] The nucleotide sequences of the target genes were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the nucleotide sequences of the glycosyltransferases were inserted into the expression vector. Specifically, they were inserted into plasmid pET28a(+) to obtain the corresponding plasmid. The synthesized plasmid was then transformed into chassis cells (E. coli BL21(DE3)), thereby constructing an E. coli strain containing the plasmid. Many other plasmids and chassis cells are available in the prior art; this embodiment only provides one specific method.

[0054] 1.2 Expression and purification of glycosyltransferases:

[0055] The recombinant bacteria were inoculated at 1% into shake flasks and cultured overnight. Then, they were inoculated at 1% into fermenters containing liquid TB medium and cultured until OD reached [value missing]. 600 The pH was set to approximately 6.0, then IPTG was added to a concentration of 100 μM, and the temperature was lowered to 16 degrees Celsius for overnight incubation. After overnight incubation, the bacterial cells were collected by centrifugation at 4000g for 10 min and washed with 0.1M PB buffer (pH 7.0). Next, the bacterial cells were sonicated and then centrifuged at 12000 rpm for 1 h. The supernatant was collected as the crude enzyme solution. Many other methods for inducing recombinant bacterial expression are available in the prior art; this embodiment only provides one specific method.

[0056] 1.3. The crude enzyme solution is purified by passing it through a nickel column. Different elution conditions are used to elute the crude enzyme solution to obtain the target protein. According to existing technology, those skilled in the art can achieve the purification of glycosyltransferases by adjusting parameters through a limited number of experiments; specific details are not described here. Many other available glycosyltransferase purification methods exist in the prior art; this embodiment only provides one specific method.

[0057] Wild-type glycosyltransferase (ORM, SEQ ID NO:1) and glycosyltransferase variants were heterologously expressed and purified according to the above-described method for preparing glycosyltransferases. The prepared glycosyltransferases and mutants were verified by gel electrophoresis (SDS-PAGE) to confirm that their sizes were consistent with expectations.

[0058] 2. Enzyme activity assay:

[0059] 3.1 Catalytic reaction system: The total reaction system for enzyme activity assay is 0.5 mL, which includes substrate concentration RD 10 mM, glycosyltransferase or variant concentration 0.002 mg / mL, uridine diphosphate glucose (UDP-G) or adenosine diphosphate glucose (ADP-G) or guanosine diphosphate glucose (GDP-G) or cytidine diphosphate glucose (CDP-G) 1 mM, magnesium chloride 3 mM, and glycine-sodium hydroxide buffer (0.1 M) at pH 9.0 to a final volume of 0.5 mL.

[0060] 3.2 Method for determining the optimal reaction temperature:

[0061] The reaction was carried out according to the catalytic reaction system in 3.1. The reaction temperatures of wild-type glycosyltransferase were 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃, and the reaction temperatures of glycosyltransferase variants were 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃, respectively. The reaction time was 1 h for all reactions and the reaction condition was 1000 rpm / min.

[0062] After the reaction, the concentration of RM in the post-reaction solution was determined by HPLC: LC-2030C HT system (SHIMADZU, Japan); mobile phase ratio: 0-3 min 25% CH3CN, 3-8 min 25%-50% CH3CN, 8-12 min 50%-100% CH3CN, 12-15 min 100% CH3CN, 15-20 min 25% CH3CN (acetonitrile containing 0.1% HCOOH), combined with another mobile phase H2O (containing 0.1% HCOOH). Flow rate: 1 mL / min; 210 nm; Shim-pack GIST C18 column (250 × 4.6 mm, 5 μm); column temperature: 30 °C. The HPLC peak chromatogram was obtained, and the concentration of RM was calculated based on the peak area.

[0063] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μM Reb M per minute. The enzyme activities of wild-type glycosyltransferases and glycosyltransferase variants are shown in Tables 1–9.

[0064] Table 1 Enzyme activity of wild-type glycosyltransferases at different reaction temperatures

[0065]

[0066]

[0067] Table 2 Enzyme activity of glycosyltransferase variant ORM-2 at different reaction temperatures

[0068]

[0069] Table 3 Enzyme activity of glycosyltransferase variant ORM-3 at different reaction temperatures

[0070]

[0071] Table 4 Enzyme activity of glycosyltransferase variant ORM-4 at different reaction temperatures

[0072]

[0073] Table 5 Enzyme activity of glycosyltransferase variant ORM-5 at different reaction temperatures.

[0074]

[0075] Table 6 Enzyme activity of glycosyltransferase variant ORM-6 at different reaction temperatures.

[0076]

[0077] Table 7 Enzyme activity of glycosyltransferase variant ORM-7 at different reaction temperatures.

[0078]

[0079] Table 8 Enzyme activity of glycosyltransferase variant ORM-8 at different reaction temperatures

[0080]

[0081]

[0082] Table 9 Enzyme activity of glycosyltransferase variant ORM-9 at different reaction temperatures.

[0083]

[0084] As shown in Tables 1-9, the optimal reaction temperature for wild-type glycosyltransferases is around 30°C, exhibiting poor thermal stability. At 50°C, the enzyme activity is only about 60% of its maximum value, and it is completely inactivated at 60°C. Even at the optimal reaction temperature, the activity of wild-type glycosyltransferases is low, only 0.24 U / mg, far below the activity required for industrial production. The optimal reaction temperature for the glycosyltransferase variants is around 70°C, with excellent thermal stability. At a reaction temperature of around 70°C, the enzyme activity of the glycosyltransferase variants of this invention is consistently above 50 U / mg, demonstrating a significant improvement in activity and making them suitable for industrial production.

[0085] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A glycosyltransferase for catalyzing the production of rebaudioside M, characterized in that The glycosyltransferase is mutated from the wild-type glycosyltransferase set forth in SEQ ID NO: 1 with the following mutation set: K55N + N70T + S181P + S275R + K441E + T460K + S193R + M146R + E3V.

2. A glycosyltransferase for catalyzing the production of rebaudioside M, characterized in that The glycosyltransferase is mutated from the wild-type glycosyltransferase set forth in SEQ ID NO: 1 with the following mutation set: K55N + N70T + S181P + S275R + K441E + T460K + K224E + I94F + R414H + A112S.

3. A glycosyltransferase for catalyzing the production of rebaudioside M, characterized in that The glycosyltransferase is mutated from the wild-type glycosyltransferase set forth in SEQ ID NO: 1 with the following mutation set: K55N + N70T + S181P + S275R + K441E + T460K + I94F + R90L + S242I + H156Y + A180V + R175H.

4. A glycosyltransferase for catalyzing the production of rebaudioside M, characterized in that The glycosyltransferase is mutated from the wild-type glycosyltransferase set forth in SEQ ID NO: 1 with the following mutation set: K55N + N70T + S181P + S275R + K441E + T460K + E3V + R414H + K224E + A112S + R90L + M146R + R298Q + A180V.

5. A recombinant genetic material of a glycosyltransferase, characterized in that DNA or RNA capable of expressing the glycosyltransferase according to any one of claims 1 to 4.

6. A recombinant strain of glycosyltransferase, characterized in that Recombinant genetic material comprising the glycosyltransferase according to claim 5.

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