A method for the biosynthesis of rebaudioside m

Abstract

The application discloses a production method for biosynthesis of rebaudioside M. Reb A / Reb D has poor solubility at low temperature, and catalytic production of Reb D / Reb M can obviously accelerate reaction efficiency in a high-temperature environment, so that the whole reaction system is carried out in a high-temperature environment, and production efficiency can be improved. In a reaction temperature environment of 70 DEG C, a wild-type first glycosyltransferase or / and a wild-type second glycosyltransferase is basically inactivated, and the catalytic enzyme combination cannot meet the demand of industrial biosynthesis of rebaudioside M. The application mutates the above wild-type catalytic enzyme, so that the catalytic enzyme can maintain high catalytic activity at 70 DEG C. Therefore, the production cost of rebaudioside M produced by using the production method of the application is obviously lower than that of the prior art, and the production method is suitable for industrial popularization.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a method for the production of rebaudioside M through biosynthesis. Background Technology

[0002] Stevia, also known as steviol glycosides, is characterized by its high sweetness (200-300 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 D (Reb D) retains the advantages of stevia's high sweetness and low calorie value while having less bitterness and licorice aftertaste. Rebaudioside M (Reb M) retains the same advantages while being virtually free of bitterness and 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 it through plant extraction is high. Furthermore, the glycosyltransferases required to catalyze the conversion of Reb D to Reb M currently have low enzyme activity, 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 enzyme 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, the present invention aims to provide an efficient production method for converting steviosides 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 protein sequence library NIH: NCBI Reference Sequence: XP_049379614.1. The wild-type glycosyltransferase sequence shown in SEQ ID NO.2 is derived from the protein sequence library NIH: GI:1777435608. The wild-type sucrose synthase sequence shown in SEQ ID NO.3 is derived from the protein sequence library NIH: WP_291511759.1. Using the Lésign platform, the sequences of the above three wild-type catalytic enzymes were designed, ultimately obtaining the computationally optimal enzyme variant.

[0006] A method for the biosynthetic production of rebaudioside M involves using a first glycosyltransferase, a second glycosyltransferase, and glucose-1,3-bisphosphate to convert steviol glycosides into rebaudioside M.

[0007] The content of rebaudioside A in the steviol glycosides 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 used for mutation: L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P;

[0009] The amino acid sequence of the second glycosyltransferase is as follows: using the wild-type glycosyltransferase described in SEQ ID NO:2 as the parent, 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 as follows: using the wild-type glycosyltransferase described in SEQ ID NO:1 as the parent, the sequence modification includes the sequence modification described in claim 1, and also includes 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: using the wild-type glycosyltransferase described in SEQ ID NO:2 as the parent, 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 sucrose synthase catalysis of nucleoside diphosphate and glycosyl donor.

[0029] Preferably, the glycosyl donor is sucrose.

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

[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 objective of this invention is to provide genetic material capable of expressing the aforementioned first and second glycosyltransferases.

[0047] To achieve the above objectives, the present invention provides the following technical solution: a recombinant genetic material of rebaudioside M, capable of expressing the DNA and / or RNA of the first and second glycosyltransferases mentioned above.

[0048] A third objective of this invention is to provide a production strain capable of expressing the aforementioned first and second glycosyltransferases.

[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-mentioned rebaudioside M.

[0050] The fourth objective of this invention is to provide genetic material capable of expressing the aforementioned first glycosyltransferase, second glycosyltransferase, and sucrose synthase.

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

[0052] The fifth objective of this invention is to provide a production strain capable of expressing the aforementioned first and second glycosyltransferases.

[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-mentioned rebaudioside M.

[0054] Compared with existing technologies, the advantages of this invention are as follows: Reb A / Reb D has poor solubility at low temperatures, and catalytic production of Reb D / Reb M at high temperatures can significantly accelerate the reaction efficiency. Therefore, the entire reaction system is carried out at high temperatures, which can improve production efficiency. At a reaction temperature of 70°C, wild-type proglycosyltransferase and / or wild-type diglycosyltransferase are essentially inactivated, and this enzyme combination for catalysis cannot meet the requirements for industrial biosynthesis of rebaudioside M. This invention mutates the above-mentioned wild-type catalytic enzymes to maintain high catalytic activity at 70°C. At the same time, the crude enzyme solution obtained from fermentation does not need to be purified and can catalyze a reaction system of 20 times its volume. The catalyzed reaction system has a high content of rebaudioside M and a very low content of impurities generated during fermentation. High-purity products can be obtained through a simple purification process, and production costs can be significantly reduced. Therefore, the production cost of rebaudioside M produced using the production method of this invention is significantly lower than that of existing technologies, making it suitable for industrial promotion. Detailed Implementation

[0055] The term "recombinant gene" refers to DNA or RNA capable of expressing the first glycosyltransferase, second glycosyltransferase, and sucrose synthase of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite triesterification, 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 sequences at 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.

[0056] 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.

[0057] 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.

[0058] Whether it is a cloned scar, signal peptide, or other elements in a recombinant gene, none of them affect the realization of sucrose synthase 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.

[0059] 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 SignalP 4.1, which are 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 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.

[0061] 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.

[0062] 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 sucrase synthase, the aforementioned signal peptide, promoter, and RBS may be introduced into the recombinant gene. Therefore, some corresponding peptide segments may remain on the expressed first glycosyltransferase, second glycosyltransferase, and sucrase synthase proteins. These peptide segments do not affect the function of the first glycosyltransferase, second glycosyltransferase, and sucrase synthase. Therefore, even if the product contains additional peptide segments, 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.

[0063] 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.

[0064] 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.

[0065] The term "stevioside" refers to all components that have 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 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.

[0070] 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.

[0071] Using the Lésign platform, sequences of wild-type first glycosyltransferase (amino acid sequence as shown in SEQ ID NO.1), wild-type second glycosyltransferase (amino acid sequence as shown in SEQ ID NO.2), and wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO.3) were designed, and the computationally optimal enzyme variants were finally obtained.

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

[0073] 1.1 Construction of recombinant cells:

[0074] The nucleotide sequences of the target gene were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the nucleotide sequences of the first and second 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.

[0075] 1.2 Expression and purification of first and second glycosyltransferases:

[0076] 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. IPTG was then added to a final concentration of 100 μM for induction of expression. After overnight culture, the bacterial cells were collected by centrifugation and washed with 0.1 M PB buffer (pH 7.0). Next, the bacterial cells were sonicated and then centrifuged at 12000 rpm for 1 hour. The supernatant was collected as the crude enzyme solution. Many other methods for inducing recombinant bacterial expression exist in the prior art; this embodiment only provides one specific method.

[0077] 2. Biocatalytic conversion rate determination:

[0078] 2.1 Catalytic reaction system: The total reaction system for conversion determination is 1000L, which includes 50L crude enzyme solution, 100kg steviol glycosides, 170kg sucrose, 0.5kg uridine diphosphate glucose (UDP-G), adenosine diphosphate glucose (ADP-G), guanosine diphosphate glucose (GDP-G), or cytidine diphosphate glucose (CDP-G), and the pH is adjusted to 6.0±0.5 with citric acid-sodium citrate (0.1M). Water is added to make up to 1000L.

[0079] 2.2 Method for determining RM conversion rate at 70℃:

[0080] The reaction was carried out according to the catalytic reaction system in 2.1. The reaction temperature was 70℃, the reaction time was 1h, and the reaction condition was 1000rpm / min.

[0081] The content of RM in steviol glycosides before and after the reaction 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. Liquid chromatography peak chromatograms were obtained, and the concentration of RM was calculated based on the peak area.

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

[0083] GLF-1: Wild-type first glycosyltransferase, amino acid sequence as 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was mutated:

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

[0100] ORM-1: Wild-type diglycosyltransferase, amino acid sequence as shown in SEQ ID NO.2;

[0101] ORM-2: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0103] ORM-3: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0105] ORM-4: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0107] ORM-5: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0109] ORM-6: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0111] ORM-7: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0113] ORM-8: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0115] ORM-9: Using the wild-type diglycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set was mutated:

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

[0117] The crude enzyme solutions of the first and second glycosyltransferases were prepared according to Method 1 and Method 2 above, and the RM conversion rates of the production strains of wild-type first glycosyltransferase, wild-type second glycosyltransferase, mutant first glycosyltransferase and mutant second glycosyltransferase were determined according to the corresponding biocatalytic conversion rate determination methods. The specific results are shown in Tables 1 to 4.

[0118] Table 1. RM conversion rates catalyzed by wild-type proglycosyltransferase and wild-type diglycosyltransferase.

[0119]

[0120]

[0121] Table 2. RM conversion rates catalyzed by the mutant first glycosyltransferase and the mutant second glycosyltransferase.

[0122]

[0123] Table 3. RM conversion rates catalyzed by the mutant first glycosyltransferase and the mutant second glycosyltransferase.

[0124]

[0125]

[0126] Table 4. RM conversion rates catalyzed by the mutant first glycosyltransferase and the mutant second glycosyltransferase.

[0127]

[0128] As shown in Tables 1-4, at a reaction temperature of 70℃, the wild-type I-glycosyltransferase and / or wild-type II-glycosyltransferase are essentially inactivated. Therefore, this enzyme combination cannot meet the requirements for the industrial biosynthesis of rebaudioside M. Combinations of different mutant I-glycosyltransferases and mutant II-glycosyltransferases all achieved a conversion rate of over 90% for RM, enabling the efficient industrial production of rebaudioside M. Furthermore, the crude enzyme solution obtained from fermentation does not require purification and can catalyze a reaction system 20 times its volume. The catalyzed reaction system contains a high content of rebaudioside M and very low levels of impurities generated during fermentation. A high-purity product can be obtained through a simple purification process, significantly reducing production costs.

[0129] Example 2:

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

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

[0132] ZTM-1: Wild-type sucrose synthase, amino acid sequence as 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was 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 set was 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 rates catalyzed by wild-type proglycosyltransferase, wild-type diglycosyltransferase, and wild-type sucrose synthase.

[0150]

[0151] Table 6. RM conversion rates catalyzed by mutant first glycosyltransferase, mutant second glycosyltransferase, and mutant sucrose synthase.

[0152]

[0153] Table 7. RM conversion rates catalyzed by mutant first glycosyltransferase, mutant second glycosyltransferase, and mutant sucrose synthase.

[0154]

[0155]

[0156] Table 8. RM conversion rates catalyzed by mutant first glycosyltransferase, mutant second glycosyltransferase, and mutant sucrose synthase.

[0157]

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

[0159] 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 method for the biosynthetic production of rebaudioside M, comprising converting steviol glycosides into rebaudioside M using a first glycosyltransferase, a second glycosyltransferase, and nucleoside diphosphate glucose, characterized in that, The amino acid sequence of the first glycosyltransferase is as follows: using the wild-type glycosyltransferase described in SEQ ID NO:1 as the parent, the following mutation set is mutated: L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+K135R+I48K+D411E; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+I136L+K135R+R6K+A234E; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+E391Q+I136L+I432V+I48K+G190R+N249K+S215T+E391R+G244S; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A122V+I48K+E391Q+S215T+G190R+L208I; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+E391Q+A122V+D411E+V7I+I136L+S215T+E391R+N383K+I432V; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A234E+N107T+V7I+E391R+M374V+K135R+I432V+N383K; Or L41P+I136F+N149H+G244D+A373G+E391K+I48E+A122I+N191P+A234E+K135R+M374V+R6K+E391R+N107T+E391Q+S215T; The amino acid sequence of the second glycosyltransferase is as follows: using the wild-type glycosyltransferase described in SEQ ID NO:2 as the parent, the following mutation set is mutated: K55N+N70T+S181P+S275R+K441E+T460K+S193R+M146R+E3V; Or 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.

2. The method for producing rebaudioside M by biosynthesis according to claim 1, characterized in that, The nucleoside diphosphate glucose is obtained by sucrose synthase catalysis of nucleoside diphosphate and glycosyl donor.

3. The method for producing rebaudioside M by biosynthesis according to claim 2, characterized in that, The amino acid sequence of the sucrose synthase is as follows: using the wild-type sucrose synthase described in SEQ ID NO:3 as the parent, the following mutation set was 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.

4. A recombinant genetic material for producing rebaudioside M, characterized in that, This includes the DNA and / or RNA corresponding to the first glycosyltransferase and the DNA and / or RNA corresponding to the second glycosyltransferase described in the method for producing rebaudioside M according to claim 1.

5. A production strain for producing rebaudioside M, characterized in that, It contains the recombinant genetic material for producing rebodiin M as described in claim 4.

6. A recombinant genetic material for producing rebaudioside M, characterized in that, Includes the DNA and / or RNA corresponding to the first glycosyltransferase and the DNA and / or RNA corresponding to the second glycosyltransferase in the biosynthetic production method of rebaudioside M according to claim 1; And the DNA and / or RNA corresponding to any of the sucrose synthases described in the production method of biosynthetic rebaudioside M according to claim 3.

7. A production strain for producing rebaudioside M, characterized in that, It contains the recombinant genetic material for producing rebodiin M as described in claim 6.

Images