Production method for biosynthesis of rebaudioside M

Abstract

The invention discloses a production method for biosynthesis of rebaudioside M. Reb A / Reb D has poor solubility at a low temperature, and the reaction efficiency can be obviously accelerated when the Reb A / Reb D is catalytically produced in a high-temperature environment, so that the whole reaction system is carried out in the high-temperature environment, and the production efficiency can be improved. Under the reaction temperature environment of 70 DEG C, the wild type first glycosyltransferase or / and the wild type second glycosyltransferase are / is basically inactivated, and the catalytic enzyme combination cannot meet the requirement of industrial biosynthesis of rebaudioside M. The wild-type catalytic enzyme is mutated, so that the wild-type catalytic enzyme can keep high catalytic activity at 70 DEG C. Therefore, the production cost of the rebaudioside M produced by the production method disclosed by the invention is obviously lower than that of the prior art, and the method is suitable for industrial popularization.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and more particularly to a production method for biosynthesizing rebaudioside M. Background Art

[0002] Stevioside, also known as steviol glycoside, boasts high sweetness (200-300 times that of sucrose) and low caloric value (1 / 300 of sucrose). It is a natural sucrose substitute and is known as the "world's third most potent sugar source." Stevioside is extracted from the stevia leaf, and manufacturers primarily extract stevioside (STV) and rebaudioside A (Reb A). However, STV and Reb A have a distinct bitterness and licorice aftertaste, which significantly limits the use of steviol glycosides as sweeteners in food.

[0003] Research has found that rebaudioside D (Reb D) retains the advantages of stevia's high sweetness and low caloric value, while offering minimal bitterness and licorice aftertaste. Rebaudioside M (Reb M) maintains the advantages of stevia's high sweetness and low caloric value, while being virtually free of bitterness and licorice aftertaste, making it a high-quality sugar substitute. However, large-scale production of Reb M faces significant bottlenecks: Reb M accounts for less than 1% of stevia leaves, and the cost of producing Reb M through plant extraction is high. Furthermore, the catalytic activity of the glycosyltransferase required to catalyze the conversion of Reb D to Reb M is currently low, resulting in low Reb M production efficiency. This results in high costs for producing Reb M through microbial fermentation or enzymatic methods. Therefore, improving the activity of glycosyltransferases is a pressing technical challenge. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the object of the present invention is to provide a production method for efficiently converting steviol glycosides rich in rebaudioside A into rebaudioside M.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: The wild-type glycosyltransferase sequence shown in SEQ ID NO. 1 is derived from the NIH protein sequence library: NCBI Reference Sequence: XP_049379614.1. The wild-type glycosyltransferase sequence shown in SEQ ID NO. 2 is derived from the NIH protein sequence library: GI:1777435608. The wild-type sucrose synthase sequence shown in SEQ ID NO. 3 is derived from the NIH protein sequence library: WP_291511759.1. Sequence design of the three wild-type catalytic enzymes was performed using the Lésign platform, ultimately resulting in computationally optimal enzyme variants.

[0006] A method for producing rebaudioside M by biosynthesis, comprising converting steviol glycoside into rebaudioside M by using a first glycosyltransferase, a second glycosyltransferase and nucleoside diphosphate glucose.

[0007] The content of rebaudioside A in the steviol glycoside is greater than 60%;

[0008] The amino acid sequence of the first glycosyltransferase is as follows: using the wild-type mannosidase described in SEQ ID NO: 1 as the parent, the following mutation set is mutated: L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P;

[0009] The amino acid sequence of the second glycosyltransferase is as follows: the wild-type glycosyltransferase described in SEQ ID NO: 2 is used as the parent, and the following mutation set is mutated: K55N+N70T+S181P+S275R+K441E+T460K+S193R+M146R+E3V.

[0010] Preferably, the amino acid sequence of the first glycosyltransferase is: based on the wild-type glycosyltransferase described in SEQ ID NO: 1 as the parent, the sequence modification includes, in addition to the sequence modification described in claim 1, the following mutation set: K135R+I48K+D411E;

[0011] or I136L+K135R+R6K+A234E;

[0012] or E391Q+I136L+I432V+I48K+G190R+N249K+S215T+E391R+G244S;

[0013] Or A122V+I48K+E391Q+S215T+G190R+L208I;

[0014] or E391Q+A122V+D411E+V7I+I136L+S215T+E391R+N383K+I432V;

[0015] or A234E+N107T+V7I+E391R+M374V+K135R+I432V+N383K;

[0016] Or A234E+K135R+M374V+R6K+E391R+N107T+E391Q+S215T.

[0017] Preferably, the amino acid sequence of the second glycosyltransferase is as follows: the wild-type glycosyltransferase described in SEQ ID NO: 2 is used as the parent, and the following mutation set is mutated: K55N+N70T+S181P+S275R+K441E+T460K+M146R+S275G+D327E+K224E;

[0018] or K55N+N70T+S181P+S275R+K441E+T460K+K224E+I94F+R414H+A112S;

[0019] Or K55N+N70T+S181P+S275R+K441E+T460K+I94F+R90L+S242I+H156Y+A180V+R175H;

[0020] or

[0021] K55N+N70T+S181P+S275R+K441E+T460K+E3V+R414H+K224E+A112S+R90L+M146R+R298Q+A180V;

[0022] or

[0023] K55N+N70T+S181P+S275R+K441E+T460K+S193R+D100A+R298Q+K224E+T460R+R141P+R414H;

[0024] or

[0025] K55N+N70T+S181P+S275R+K441E+T460K+D100A+R414H+S275G+K224E+R405P+T264S+I94F+R90L;

[0026] or

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

[0028] Preferably, the nucleoside diphosphate glucose is obtained by catalyzing nucleoside diphosphate and a glycosyl donor by sucrose synthase.

[0029] Preferably, the glycosyl donor is sucrose.

[0030] Preferably, the amino acid sequence of the sucrose synthase is as follows: the wild-type sucrose synthase described in SEQ ID NO: 3 is used as the parent, and the following mutation set is mutated:

[0031] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S187R+L176S+S526R+T727R+V126F;

[0032] or

[0033] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T191I+A164E+S652R+Q108R+D463H;

[0034] or

[0035] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T199M+E613V+A164E+A358P+D455E+V126F;

[0036] or

[0037] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S526R+S652R+H64R+D488N+I2F+D455E+A164E+V126F+H442Y;

[0038] or

[0039] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+H64R+T191I+S526R+D455E+D463H+L416W+H442Y;

[0040] or

[0041] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+L176S+L176Q+T199M+R208P+S368P+S187R+H442Y+Q108R+E414Q+H446Y;

[0042] or

[0043] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S368P+L416W+D455E+H699L+A358P+H446Y+I2F+L163R;

[0044] or

[0045] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+D488H+T727R+H442Y+V126F+T199M+E613V+A358P+S652R+A164E+S187R.

[0046] A second object of the present invention is to provide a genetic material capable of expressing the first glycosyltransferase and the second glycosyltransferase.

[0047] To achieve the above objectives, the present invention provides the following technical solution: a recombinant genetic material of rebaudioside M, which can express the DNA and / or RNA of the first glycosyltransferase and the second glycosyltransferase.

[0048] A third object of the present invention is to provide a production strain capable of expressing the first glycosyltransferase and the second glycosyltransferase.

[0049] To achieve the above objectives, the present invention provides the following technical solution: a strain for producing rebaudioside M, comprising the recombinant genetic material of the above rebaudioside M.

[0050] A fourth object of the present invention is to provide a genetic material capable of expressing the first glycosyltransferase, the second glycosyltransferase and the sucrose synthase.

[0051] To achieve the above objectives, the present invention provides the following technical solutions: a recombinant genetic material of rebaudioside M, DNA and / or RNA capable of expressing the above-mentioned first glycosyltransferase and second glycosyltransferase, and DNA and / or RNA capable of expressing the above-mentioned glycosyltransferase.

[0052] A fifth object of the present invention is to provide a production strain capable of expressing the first glycosyltransferase and the second glycosyltransferase.

[0053] To achieve the above objectives, the present invention provides the following technical solution: a strain for producing rebaudioside M, comprising the recombinant genetic material of the above rebaudioside M.

[0054] Compared with the prior art, the advantages of the present invention are: Reb A / Reb D have poor solubility at low temperatures, but catalyzing the production of Reb D / Reb M at high temperatures significantly accelerates reaction efficiency. Therefore, conducting the entire reaction system at high temperatures can improve production efficiency. At a reaction temperature of 70°C, the wild-type first glycosyltransferase and / or the wild-type second glycosyltransferase are essentially inactivated, making this catalytic enzyme combination incapable of meeting the requirements for industrial biosynthesis of rebaudioside M. The present invention mutates the wild-type catalytic enzymes to maintain high catalytic activity at 70°C. Furthermore, the crude enzyme solution obtained through fermentation does not require purification and can catalyze a reaction system 20 times its volume. The catalytic reaction system contains a high content of rebaudioside M, and the content of impurities generated by fermentation is very low. A high-purity product can be obtained through a simple purification process, significantly reducing production costs. Therefore, the production cost of rebaudioside M produced using the production method of the present invention is significantly lower than that of the prior art, making it suitable for industrial scale-up. DETAILED DESCRIPTION

[0055] The term "recombinant gene" refers to DNA or RNA that can express the first glycosyltransferase, the second glycosyltransferase and the sucrose synthase of the present invention. Typically, the recombinant gene is initially synthesized in vitro by the solid-phase phosphoramidite trisaccharide method or the TdT biosynthesis method or other suitable techniques known in the art. Once the template sequence is obtained, amplification can be performed by PCR or other suitable techniques known in the art. Once the recombinant strain is obtained, further large-scale amplification can be performed by culturing the strain. In certain embodiments, the recombinant gene may also include residual sequences of enzyme cleavage sites, other accessory elements, such as control elements (such as promoters, etc.), labeling substances (such as fluorescent labels, etc.), and other sequences that do not affect the expression of the target gene.

[0056] The term "cloning scar" refers to a promoter sequence that initiates transcription of messenger ribonucleotides (mRNA), followed by a ribosome-binding site (RBS) that attracts the translation machinery, followed by a signal peptide sequence that facilitates protein transport to the periplasm. Mature proteins are typically cloned after a signal peptide, from which they are cleaved by a signal peptidase as they pass through the membrane. However, when constructs are cloned after a signal peptide, restriction enzymes typically require specific sequences to cut the DNA, leaving a cloning scar after the signal peptide sequence.

[0057] The term "signal peptide" refers to a short peptide (usually 16-30 amino acids long) present at the N-terminus of most newly synthesized proteins that are destined to enter the secretory pathway. It may also be called a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide. Signal peptides are typically cleaved from proteins by a signal peptidase.

[0058] Neither the cloning scar, signal peptide nor other elements in the recombinant gene affect the realization of the function of sucrose synthase. Therefore, if the amino acid sequence of the final protein differs from the amino acid sequence disclosed in the present invention only in the amino acid sequence corresponding to the above-mentioned DNA sequence, it still falls within the scope of protection of the present invention.

[0059] The term "signal peptide cleavage site" refers to the dipeptide between which the signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, the dipeptide is Ala-Ala. The signal peptide cleavage site can be calculated using an algorithm such as SignalP4.1, which is available online at http: / / www.cbs.dtu.dk / services / SignalP / (Center for Biological Sequence Analysis, Technical University of Denmark).

[0060] The term "promoter" refers to a region of DNA that initiates transcription (writing to mRNA) of a specific gene. A promoter is typically located near the transcription start site of a gene, on the same strand and upstream of the DNA (toward the 5' region of the sense strand). A promoter can be inducible, meaning that expression of a gene operably linked to the promoter can be turned on by the presence of an inducer substance. Alternatively, a promoter can be constitutive, meaning that it is not regulated by any inducer substance.

[0061] The abbreviation "RBS" refers to the ribosome-binding site, or the binding site of the ribosome. This is the sequence of nucleotides upstream of the start codon of the mRNA transcript that is responsible for recruiting the ribosome during the initiation of protein translation.

[0062] The term "expression" refers to the process of transcribing DNA into messenger RNA (mRNA) and then translating it into protein. To achieve the smooth expression and screening of sucrose synthase, the aforementioned signal peptide, promoter, and RBS may be introduced into the recombinant gene, resulting in the possibility that some corresponding peptide segments will remain on the expressed first glycosyltransferase, second glycosyltransferase, and sucrose synthase proteins. These peptide segments do not affect the functions of the first glycosyltransferase, second glycosyltransferase, and sucrose synthase. Therefore, even if the product contains the aforementioned peptide segments, as long as the amino acid sequence of the main part is the same as that of the present invention, the product will still be an infringing product.

[0063] The term "expression vector" has the ability to incorporate and express heterologous polynucleic acid fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selecting an appropriate expression vector is within the knowledge of a skilled artisan.

[0064] The term "chassis cell" refers to a suitable host vector for expressing the DNA of the present invention. The host may include any organism capable of containing and expressing the nucleic acid or gene disclosed herein, but is not limited thereto. Chassis cells can be prokaryotes or eukaryotes, unicellular or multicellular, including mammalian cells, plant cells, fungi, etc. According to the prior art, 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, Candida, Rhodotorula, Bacillus, Escherichia, Salmonella, Clostridium, Streptomyces, Staphylococcus, Neisseria, and Shigella. The present invention only lists the types of chassis cells and does not constitute a limitation on the types of chassis cells. The chassis cells are preferably E. 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. + etc. The above E. coli strains are all commercially available strains.

[0065] The term "stevioside" is intended to include all components having a steviol glycoside structure.

[0066] The term "stevioside" refers to one of the components of steviol glycosides.

[0067] The present invention will be further described in detail below with reference to the embodiments.

[0068] Example 1:

[0069] Proteins are the material foundation of life and a crucial component of human cells and tissues. All vital components of the human body require the participation of proteins, playing a crucial role in the vital processes of cells and organisms. It can be said that without proteins, there would be no life. The human body contains numerous types of proteins, each with a distinct function. Some form tissues, others provide energy, participate in metabolism and transport, promote growth and development, and regulate immune function. Different proteins have diverse responsibilities and roles, and their functions are determined by their structure. A protein's 3D structure is, in turn, determined by its amino acid sequence. Therefore, protein design relies on the correspondence between structure and sequence. To design a protein with a specific function, a sequence must be designed that matches that function. Understanding and designing proteins is crucial for driving innovation and progress in biology and medicine.

[0070] Designing protein sequences for specific functions is a complex undertaking. The final structure and function of the designed sequence are difficult to predict. Furthermore, the sample space for protein sequences of fixed length is enormous. To accomplish this, the Liwen Institute has developed Lésign, a protein design platform based on deep learning algorithms. This platform implements functions such as protein structure prediction, sequence design, and result evaluation. Interfaces enable collaboration between various functional modules, forming a computational pipeline that integrates prediction, design, and evaluation.

[0071] Using the Lésign platform, sequence design was performed on the wild-type first glycosyltransferase (amino acid sequence shown in SEQ ID NO.1), the wild-type second glycosyltransferase (amino acid sequence shown in SEQ ID NO.2), and the wild-type sucrose synthase (amino acid sequence shown in SEQ ID NO.3), and ultimately the optimal enzyme variants at the computational level were obtained.

[0072] 1. Preparation of crude enzyme solutions of the first glycosyltransferase and the second glycosyltransferase

[0073] 1.1. Construction of recombinant cells:

[0074] The nucleotide sequences of the target genes were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the nucleotide sequences for the first and second glycosyltransferases were inserted into expression vectors. Specifically, they were inserted into the plasmid pET28a(+) to obtain the corresponding plasmids. The synthesized plasmids were then transformed into chassis cells (E. coli BL21(DE3)) to construct an E. coli strain containing the plasmids. Numerous other plasmids and chassis cells are available in the prior art; this example provides only one specific approach.

[0075] 1.2. Expression and purification of the first glycosyltransferase and the second glycosyltransferase:

[0076] The recombinant bacteria were inoculated into shake flasks at 1% for overnight culture, and then inoculated into fermenters containing liquid TB medium at 1% for 24 h. 600 The value is about 6.0, and then IPTG is added to a final concentration of 100 μM to induce expression. After overnight culture, the cells are collected by centrifugation and rinsed with 0.1M PB buffer (pH 7.0). Next, the cells are disrupted by ultrasound, and then centrifuged at 12000 rpm for 1 hour. The supernatant is taken as the crude enzyme solution. There are many other methods available in the prior art for inducing expression of recombinant bacteria. This example only provides a specific solution.

[0077] 2. Determination of biocatalytic conversion rate:

[0078] 2.1. Catalytic Reaction System: Conversion Rate Determination The total reaction system was 1000 L, which contained 50 L of crude enzyme solution, 100 kg of stevioside, 170 kg of sucrose, 0.5 kg of uridine diphosphate glucose (UDP-G) or adenosine diphosphate glucose (ADP-G) or guanosine diphosphate glucose (GDP-G) or cytidine diphosphate glucose (CDP-G), citric acid-sodium citrate (0.1 M) to adjust the pH to 6.0 ± 0.5, and water was added to make up to 1000 L.

[0079] 2.2 Method for determining RM conversion at 70°C:

[0080] The reaction was carried out according to the catalytic reaction system of 2.1, with the reaction temperature being 70°C, the reaction time being 1 h, and the reaction condition being 1000 rpm / min.

[0081] The RM content in the steviol glycosides before and after the reaction was determined by HPLC using an LC-2030C HT system (SHIMADZU, Japan). The mobile phase ratio was: 25% CH3CN (0-3 min), 25%-50% CH3CN (3-8 min), 50%-100% CH3CN (8-12 min), 100% CH3CN (12-15 min), and 25% CH3CN (acetonitrile containing 0.1% HCOOH) for 15-20 min, combined with a separate mobile phase of H2O (containing 0.1% HCOOH). The flow rate was 1 ml / min, the column was 210 nm, and the column temperature was 30°C. The peaks were obtained, and the RM concentrations were calculated based on the peak areas.

[0082] RM conversion rate = (RM molar amount in the solution after reaction - RM molar amount in steviol glycoside before reaction) / total molar amount of steviol glycoside * 100%.

[0083] GLF-1: wild-type first glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO.1;

[0084] GLF-2: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0085] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P;

[0086] GLF-3: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0087] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+K135R+I48K+D411E;

[0088] GLF-4: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0089] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+I136L+K135R+R6K+A234E;

[0090] GLF-5: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0091] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+E391Q+I136L+I432V+I48K+G190R+N249K+S215T+E391R+G244S;

[0092] GLF-6: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0093] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A122V+I48K+E391Q+S215T+G190R+L208I;

[0094] GLF-7: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0095] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+E391Q+A122V+D411E+V7I+I136L+S215T+E391R+N383K+I432V;

[0096] GLF-8: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0097] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A234E+N107T+V7I+E391R+M374V+K135R+I432V+N383K;

[0098] GLF-9: Using the wild-type first glycosyltransferase described in SEQ ID NO: 1 as the parent, the following mutation sets were mutated:

[0099] L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A234E+K135R+M374V+R6K+E391R+N107T+E391Q+S215T;

[0100] ORM-1: wild-type second glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO. 2;

[0101] ORM-2: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0102] K55N+N70T+S181P+S275R+K441E+T460K+S193R+M146R+E3V;

[0103] ORM-3: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0104] K55N+N70T+S181P+S275R+K441E+T460K+M146R+S275G+D327E+K224E;

[0105] ORM-4: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0106] K55N+N70T+S181P+S275R+K441E+T460K+K224E+I94F+R414H+A112S;

[0107] ORM-5: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0108] K55N+N70T+S181P+S275R+K441E+T460K+I94F+R90L+S242I+H156Y+A180V+R175H;

[0109] ORM-6: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0110] K55N+N70T+S181P+S275R+K441E+T460K+E3V+R414H+K224E+A112S+R90L+M146R+R298Q+A180V;

[0111] ORM-7: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0112] K55N+N70T+S181P+S275R+K441E+T460K+S193R+D100A+R298Q+K224E+T460R+R141P+R414H;

[0113] ORM-8: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0114] K55N+N70T+S181P+S275R+K441E+T460K+D100A+R414H+S275G+K224E+R405P+T264S+I94F+R90L;

[0115] ORM-9: Using the wild-type second glycosyltransferase described in SEQ ID NO: 2 as the parent, the following mutation sets were mutated:

[0116] K55N+N70T+S181P+S275R+K441E+T460K+R414H+T460R+R90L+A112S+N50K+S193R+E3V+H156Y+T264S+R405P+D327E+S275G.

[0117] According to the above-mentioned methods 1 and 2, the crude enzyme solutions of the first glycosyltransferase and the second glycosyltransferase were prepared, and the corresponding biocatalytic conversion rate determination method was used to determine the RM conversion rates of the production strains of the wild-type first glycosyltransferase, the wild-type second glycosyltransferase, the mutated first glycosyltransferase, and the mutated second glycosyltransferase. The specific determination results are shown in Tables 1 to 4.

[0118] Table 1 RM conversion rate catalyzed by wild-type first glycosyltransferase and wild-type second glycosyltransferase

[0119]

[0120]

[0121] Table 2 RM conversion rate catalyzed by the mutated first glycosyltransferase and the mutated second glycosyltransferase

[0122]

[0123] Table 3 RM conversion rate catalyzed by the mutated first glycosyltransferase and the mutated second glycosyltransferase

[0124]

[0125]

[0126] Table 4 RM conversion rate catalyzed by the mutated first glycosyltransferase and the mutated second glycosyltransferase

[0127]

[0128] As can be seen from Tables 1 to 4, under a reaction temperature environment of 70°C, the wild-type first glycosyltransferase and / or the wild-type second glycosyltransferase are essentially inactivated, and this catalytic enzyme combination cannot meet the needs of industrial biosynthesis of rebaudioside M. The conversion rate of RM by the combination of different mutated first glycosyltransferases and mutated second glycosyltransferases is all above 90%, which can achieve efficient industrial production of rebaudioside M. At the same time, the crude enzyme solution obtained by fermentation does not need to be purified to catalyze a reaction system 20 times its volume. The content of rebaudioside M in the catalyzed reaction system is high, and the content of impurities produced by fermentation is very low. A high-purity product can be obtained through a simple purification process, and the production cost can be greatly reduced.

[0129] Example 2:

[0130] The difference from Example 1 is that the constructed recombinant plasmid also contains the gene corresponding to the glycosyltransferase.

[0131] The catalytic reaction system is as follows: the total reaction system for conversion determination is 1000 L, which includes 50 L of crude enzyme solution, 100 kg of stevioside, 170 kg of sucrose, 0.5 kg of uridine diphosphate (UDP) or adenosine diphosphate (ADP) or guanosine diphosphate (GDP) or cytidine diphosphate (CDP), citric acid-sodium citrate (0.1 M) to adjust the pH to 6.0±0.5, and add water to make up to 1000 L.

[0132] ZTM-1: wild-type sucrose synthase, the amino acid sequence of which is shown in SEQ ID NO.3;

[0133] ZTM-2: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0134] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S187R+L176S+S526R+T727R+V126F;

[0135] ZTM-3: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0136] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T191I+A164E+S652R+Q108R+D463H;

[0137] ZTM-4: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0138] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T199M+E613V+A164E+A358P+D455E+V126F;

[0139] ZTM-5: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0140] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S526R+S652R+H64R+D488N+I2F+D455E+A164E+V126F+H442Y;

[0141] ZTM-6: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0142] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+H64R+T191I+S526R+D455E+D463H+L416W+H442Y;

[0143] ZTM-7: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0144] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+L176S+L176Q+T199M+R208P+S368P+S187R+H442Y+Q108R+E414Q+H446Y;

[0145] ZTM-8: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0146] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S368P+L416W+D455E+H699L+A358P+H446Y+I2F+L163R;

[0147] ZTM-9: Using the wild-type sucrose synthase described in SEQ ID NO: 3 as the parent, the following mutation sets were mutated:

[0148] S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+D488H+T727R+H442Y+V126F+T199M+E613V+A358P+S652R+A164E+S187R.

[0149] Table 5 RM conversion rate catalyzed by wild-type first glycosyltransferase, wild-type second glycosyltransferase and wild-type sucrose synthase

[0150]

[0151] Table 6 RM conversion rate catalyzed by the mutated first glycosyltransferase, the mutated second glycosyltransferase and the mutated sucrose synthase

[0152]

[0153] Table 7 RM conversion rate catalyzed by the mutated first glycosyltransferase, the mutated second glycosyltransferase and the mutated sucrose synthase

[0154]

[0155]

[0156] Table 8 RM conversion rate catalyzed by the mutated first glycosyltransferase, the mutated second glycosyltransferase and the mutated sucrose synthase

[0157]

[0158] As shown in Tables 5 to 8, at a reaction temperature of 70°C, the wild-type first glycosyltransferase and / or the wild-type second glycosyltransferase and / or sucrose synthase are essentially inactivated, and this catalytic enzyme combination cannot meet the requirements for industrial biosynthesis of rebaudioside M. The conversion rate of RM in the reaction system containing the mutated glycosyltransferase and a small amount of nucleoside diphosphate is essentially equivalent to that in the reaction system containing a large amount of nucleoside diphosphate glucose, demonstrating that the mutated sucrose synthase of the present invention can effectively recycle and provide active glycosyl groups for rebaudioside A and rebaudioside D. Therefore, the production cost of the production strain of the mutated sucrose synthase can be further significantly reduced.

[0159] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for ordinary researchers in this field, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing rebaudioside M by biosynthesis, comprising converting steviol glycoside into rebaudioside M using a first glycosyltransferase, a second glycosyltransferase, and nucleoside diphosphate glucose, wherein: The content of rebaudioside A in the steviol glycoside is greater than 60%; The amino acid sequence of the first glycosyltransferase is as follows: using the wild-type mannosidase described in SEQ ID NO: 1 as the parent, the following mutation set is mutated: L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P; The amino acid sequence of the second glycosyltransferase is as follows: the wild-type glycosyltransferase described in SEQ ID NO: 2 is used as the parent, and the following mutation set is mutated: K55N+N70T+S181P+S275R+K441E+T460K+S193R+M146R+E3V.

2. The method for producing rebaudioside M by biosynthesis according to claim 1, wherein: The amino acid sequence of the first glycosyltransferase is: based on the wild-type glycosyltransferase described in SEQ ID NO: 1 as the parent, the sequence modification includes, in addition to the sequence modification described in claim 1, the following mutation set: K135R+I48K+D411E; or I136L+K135R+R6K+A234E; or E391Q+I136L+I432V+I48K+G190R+N249K+S215T+E391R+G244S; Or A122V+I48K+E391Q+S215T+G190R+L208I; or E391Q+A122V+D411E+V7I+I136L+S215T+E391R+N383K+I432V; or A234E+N107T+V7I+E391R+M374V+K135R+I432V+N383K; Or A234E+K135R+M374V+R6K+E391R+N107T+E391Q+S215T.

3. The method for producing biosynthetic rebaudioside M according to claim 1, wherein: The amino acid sequence of the second glycosyltransferase is as follows: the wild-type glycosyltransferase described in SEQ ID NO: 2 is used as the parent, and the following mutation set is mutated: K55N+N70T+S181P+S275R+K441E+T460K+M146R+S275G+D327E+K224E; or K55N+N70T+S181P+S275R+K441E+T460K+K224E+I94F+R414H+A112S; or K55N+N70T+S181P+S275R+K441E+T460K+I94F+R90L+S242I+H156Y+A180V+R175H; or K55N+N70T+S181P+S275R+K441E+T460K+E3V+R414H+K224E+A112S+R90L+M146R+R298Q+A180V; or K55N+N70T+S181P+S275R+K441E+T460K+S193R+D100A+R298Q+K224E+T460R+R141P+R414H; or K55N+N70T+S181P+S275R+K441E+T460K+D100A+R414H+S275G+K224E+R405P+T264S+I94F+R90L; or K55N+N70T+S181P+S275R+K441E+T460K+R414H+T460R+R90L+A112S+N50K+S193R+E3V+H156Y+T264S+R405P+D327E+S275G.

4. The method for producing rebaudioside M by biosynthesis according to claim 1, wherein: The nucleoside diphosphate glucose is obtained by catalyzing nucleoside diphosphate and a glycosyl donor by sucrose synthase.

5. The method for producing biosynthetic rebaudioside M according to claim 4, wherein: The glycosyl donor is sucrose.

6. The method for producing biosynthetic rebaudioside M according to claim 4 or 5, characterized in that: The amino acid sequence of the sucrose synthase is as follows: the wild-type sucrose synthase described in SEQ ID NO: 3 is used as the parent, and the following mutation set is mutated: S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S187R+L176S+S526R+T727R+V126F; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T191I+A164E+S652R+Q108R+D463H; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+T199M+E613V+A164E+A358P+D455E+V126F; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S526R+S652R+H64R+D488N+I2F+D455E+A164E+V126F+H442Y; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+H64R+T191I+S526R+D455E+D463H+L416W+H442Y; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+L176S+L176Q+T199M+R208P+S368P+S187R+H442Y+Q108R+E414Q+H446Y; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+S368P+L416W+D455E+H699L+A358P+H446Y+I2F+L163R; or S49P+T103P+G112R+L163P+L176R+A608P+D53R+G189P+V281W+D488H+T727R+H442Y+V126F+T199M+E613V+A358P+S652R+A164E+S187R.

7. A recombinant genetic material of rebaudioside M, characterized in that: DNA and / or RNA capable of expressing the first glycosyltransferase and the second glycosyltransferase according to any one of claims 1 to 3.

8. A strain for producing rebaudioside M, characterized in that: A recombinant genetic material comprising rebaudioside M as claimed in claim 7.

9. A recombinant genetic material of rebaudioside M, characterized in that: DNA or / and RNA capable of expressing the first glycosyltransferase and the second glycosyltransferase according to any one of claims 1 to 3, and DNA or / and RNA capable of expressing the glycosyltransferase according to claim 6.

10. A strain for producing rebaudioside M, characterized in that: A recombinant genetic material comprising the rebaudioside M as claimed in claim 9.

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