A method for rebaudioside m production and complex substrate conversion
By using site-directed mutagenesis-mediated catalysis of UGT11mut and UGT76G1mut enzymes and AtSUS enzyme, the problems of low yield and high cost in the synthesis of rebaudioside M were solved, achieving efficient conversion of complex substrates, increasing yield and reducing production costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI JINHE INDUSTRIAL CO LTD
- Filing Date
- 2026-02-13
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies for the synthesis of rebaudioside M suffer from low yield, high cost, and low conversion rate of complex substrates, making it difficult to meet the needs of large-scale industrial production.
Using site-directed mutagenesis-modified UGT11mut and UGT76G1mut mutant enzymes, combined with AtSUS enzymes, and employing a 1:1:2 enzyme ratio and a segmented addition strategy, sucrose, UDP, and MgCl2 were used as reaction additives to catalyze the conversion of the complex substrate RA60 into rebaudioside M.
It significantly improved the yield and conversion rate of lebodiin M, with a yield of up to 95.88 g/L and a conversion rate of up to 95%, greatly reducing production costs and breaking through the limitations of traditional methods.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a method for the production of rebaudioside M and the transformation of complex substrates. Background Technology
[0002] Rebaudioside M (Reb M) is an important natural sweetener with broad application prospects in the food, beverage, and pharmaceutical industries. It is 300-450 times sweeter than sucrose and boasts advantages such as low calories, high safety, and pure taste. With the increasing pursuit of healthy eating, its demand is growing, making it an ideal alternative to traditional sweeteners like sucrose, and its market potential is enormous.
[0003] However, the synthesis of rebaudioside M currently faces numerous challenges. In terms of yield, traditional chemical synthesis methods produce extremely low yields, typically only a few grams per liter, which is insufficient for large-scale industrial production. While biosynthetic methods have made some progress, yields are also generally low, with most reported yields between 20-50 g / L, still far from achieving efficient, low-cost large-scale production. From a cost perspective, existing synthetic processes often involve complex techniques and expensive raw materials; for example, some biosynthetic methods rely on costly substrates or coenzymes, significantly increasing production costs and limiting the widespread market application of rebaudioside M.
[0004] In recent years, with the rise and development of synthetic biology, biological methods for producing Reb M are gradually replacing traditional separation and extraction methods, becoming the mainstream approach for large-scale production. Biological production of Reb M has significant advantages such as mild reaction conditions, high specificity, and being environmentally friendly and pollution-free. Its core principle is to utilize specific glucosyltransferases to add glucose groups to the corresponding sites of steviol glycosides, thereby converting the high content of steviol glycosides in stevia leaves into Reb M, which is present in trace amounts but has higher economic value. Enzymatic catalysis, leveraging synthetic biology technology, utilizes glycosyltransferases produced by engineered bacteria fermentation to achieve efficient conversion. This process aligns better with the concept of green manufacturing, offering advantages such as mild reaction conditions, high specificity, and being environmentally friendly and pollution-free.
[0005] Patent CN114574460B describes a coupling reaction between the glycosyltransferase mutant UGT76G1-T284S / M88L / L200A and the sucrose synthase AtSuSy from Arabidopsis thaliana, achieving efficient catalytic synthesis of rebaudioside M using Reb D as a substrate. By adding Reb D as a substrate in a fed-batch manner, the reaction was carried out at 22.58 g / L (20 mM) Reb D for 7 h, resulting in the efficient synthesis of 23.37 g / L Reb M with a Reb M yield of 90.5%.
[0006] However, existing reaction systems and enzyme catalysis systems exhibit low conversion rates when dealing with complex substrates. Taking complex substrates like RA60 as an example, their composition is complex, and traditional methods typically achieve conversion efficiencies of less than 30%, resulting in significant substrate waste, further increasing production costs, and hindering the efficient synthesis of rebaudioside M from these complex substrates.
[0007] Therefore, developing a new method that can increase the yield of rebaudioside M, reduce costs, and effectively transform complex substrates is of great practical significance and market demand. Summary of the Invention
[0008] To address the above shortcomings, this invention provides a method for the production of rebaudioside M and the transformation of complex substrates.
[0009] the term: In this invention, the term "Rebaudioside M (Reb M)" refers to a natural sweetener derived from the biotransformation of stevia-related substrates. It is 300-450 times sweeter than sucrose, and features low calories, high safety, and pure taste. It can be applied in the food, beverage, and pharmaceutical industries and is the target product of this invention.
[0010] In this invention, the term "UGT76G1 mutant (UGT76G1mut)" refers to a mutant enzyme obtained by mutating the wild-type UGT76G1 gene at a specific site. Compared with the wild-type UGT76G1, it has superior catalytic activity in the synthesis reaction of rebaudioside M and can efficiently catalyze the conversion of intermediate products to rebaudioside M. Its amino acid sequence is shown in SEQ ID NO.3 and its nucleotide sequence is shown in SEQ ID NO.4. The UGT76G1mut is derived from stevia and belongs to the glycosyltransferase family.
[0011] In this invention, the term "UGT11 mutant (UGT11mut)" refers to a mutant enzyme obtained by mutating the wild-type UGT11 gene at a specific site. Compared with wild-type UGT11, it can more efficiently catalyze the initial transformation of complex substrates (such as RA60) and initial substrates, providing a high-quality intermediate product for the subsequent synthesis of rebaudioside M. Its amino acid sequence is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.8. The UGT11 is obtained from rice and has catalytic RD-RM function.
[0012] In this invention, the term "AtSUS" refers to a sucrose synthase derived from Arabidopsis thaliana, whose encoding gene has the amino acid sequence shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO.10. It can assist glycosyltransferases in the reaction system, promote the regeneration and utilization of glycosyl donors, and improve reaction efficiency.
[0013] The technical solution of this invention is as follows: On one hand, the present invention provides a method for the production of rebaudioside M and the transformation of complex substrates, the method comprising the following steps: S1. Add reaction additives to the substrate, add UGT11mut enzyme and AtSUS enzyme, and after reaction, obtain enzyme reaction solution; S2. Add UGT76G1mut enzyme to the enzyme reaction solution, continue the reaction, collect the reaction solution, and obtain rebaudioside M. The UGT11mut enzyme is an enzyme expressing UGT11mut, and the amino acid sequence of UGT11mut is shown in SEQ ID NO.7; the AtSUS enzyme is an enzyme expressing AtSUS, and the amino acid sequence of AtSUS is shown in SEQ ID NO.9; the UGT76G1mut enzyme is an enzyme expressing UGT76G1mut, and the amino acid sequence of UGT76G1mut is shown in SEQ ID NO.3; The weight ratio of the UGT11mut enzyme, AtSUS enzyme, and UGT76G1mut enzyme is 1:1:2.
[0014] Specifically, the amount of UGT11mut enzyme added in step S1 is 0.05%-0.15%wt of the substrate; the amount of AtSUS enzyme added in step S1 is 0.05%-0.15%wt of the substrate.
[0015] More specifically, the amount of UGT11mut enzyme added in step S1 is 0.05%-0.06%wt, 0.06%-0.07%wt, 0.07%-0.08%wt, 0.08%-0.09%wt, 0.09%-0.10%wt, 0.10%-0.11%wt, 0.11%-0.12%wt, 0.13%-0.14%wt, or 0.14%-0.15%wt of the substrate.
[0016] Preferably, the amount of UGT11mut enzyme added in step S1 is 0.08%-0.09%wt, 0.09%-0.10%wt, 0.10%-0.11%wt, or 0.11%-0.12%wt of the substrate.
[0017] More preferably, the amount of UGT11mut enzyme added in step S1 is 0.10%wt of the substrate.
[0018] More specifically, the amount of AtSUS enzyme added in step S1 is 0.05%-0.06%wt, 0.06%-0.07%wt, 0.07%-0.08%wt, 0.08%-0.09%wt, 0.09%-0.10%wt, 0.10%-0.11%wt, 0.11%-0.12%wt, 0.13%-0.14%wt, or 0.14%-0.15%wt of the substrate.
[0019] Preferably, the amount of AtSUS enzyme added in step S1 is 0.08%-0.09%wt, 0.09%-0.10%wt, 0.10%-0.11%wt, or 0.11%-0.12%wt of the substrate.
[0020] More preferably, the amount of AtSUS enzyme added in step S1 is 0.10%wt of the substrate.
[0021] Specifically, the amount of UGT76G1mut enzyme added in step S2 is 0.1%-0.3%wt of the substrate.
[0022] More specifically, the amount of UGT76G1mut enzyme added in step S2 is 0.10%-0.15%, 0.15%-0.20%, 0.20%-0.25%, or 0.25%-0.30%wt of the substrate.
[0023] Preferably, the amount of UGT76G1mut enzyme added in step S2 is 0.15%-0.20% or 0.20%-0.25%wt of the substrate.
[0024] More preferably, the amount of UGT76G1mut enzyme added in step S2 is 0.16%-0.24%wt of the substrate.
[0025] More preferably, the amount of UGT76G1mut enzyme added in step S2 is 0.20%wt of the substrate.
[0026] Specifically, the reaction additives include any one or more of sucrose, UDP, and MgCl2.
[0027] Preferably, the reaction additive is a combination of sucrose, UDP, and MgCl2.
[0028] Specifically, the substrate is a raw material containing RA.
[0029] Preferably, the substrate is a raw material containing at least 50% RA.
[0030] Specifically, the reaction temperature in step S1 is 35-38℃, and the reaction temperature in step S2 is 40-50℃.
[0031] Preferably, the reaction temperature in step S1 is 37°C, and the reaction temperature in step S2 is 45°C.
[0032] Specifically, the reaction time in step S1 is 2-6 hours, and the reaction time in step S2 is 24-48 hours.
[0033] Preferably, the reaction time in step S1 is 3-5 hours, and the reaction time in step S2 is 24-36 hours.
[0034] More preferably, the reaction time in step S1 is 4 hours, and the reaction time in step S2 is 24 hours.
[0035] Preferably, the preparation method of the UGT11mut enzyme includes: inducing expression of UGT11mut recombinant strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain crude enzyme solution, purifying and freeze-drying it to obtain UGT11mut enzyme; Preferably, the method for preparing the AtSUS enzyme includes: inducing the expression of an AtSUS recombinant strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain a crude enzyme solution, purifying it and freeze-drying it to obtain the AtSUS enzyme; Preferably, the preparation method of the UGT76G1mu enzyme includes: inducing expression of UGT76G1mu recombinant strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain crude enzyme solution, purifying and freeze-drying it to obtain UGT76G1mut enzyme.
[0036] The beneficial effects of this invention are as follows: This invention utilizes site-directed mutagenesis-modified UGT11mut and UGT76G1mut mutant enzymes, which exhibit significantly enhanced catalytic activity and substrate binding specificity compared to wild-type enzymes. Combined with an optimized 1:1:2 enzyme ratio and a segmented addition strategy, using 75 g / L of pure RA substrate as raw material, the yield of rebaudioside M can reach 95.88 g / L, with a conversion rate exceeding 95%, far surpassing the conventional yield level of 20-50 g / L in existing technologies. This significantly improves the target product yield efficiency per unit reaction system, laying a foundation for large-scale production.
[0037] For steviol glycoside raw materials like RA60, which have complex compositions and traditional methods achieve conversion rates below 30%, this invention utilizes the UGT11mut enzyme for highly efficient catalytic conversion of multiple effective components in complex substrates. Combined with a three-enzyme synergistic reaction system, this increases the overall conversion rate of RA60 to over 90%. This technology overcomes the limitation of traditional methods that can only efficiently utilize pure substrates, enabling the direct synthesis of rebaudioside M from low-cost, complex natural substrates. This significantly reduces raw material procurement and pretreatment costs, greatly improving the economic feasibility of the process. Attached Figure Description
[0038] Figure 1 This is a schematic diagram of the reaction process of the three enzymes added in stages in the method for the production of rebaudioside M and the transformation of complex substrates according to the present invention. It clearly shows the addition of UGT76G1mut, UGT11mut and AtSUS to the reaction system at different stages, as well as the path of the substrate being gradually converted into rebaudioside M, which helps to intuitively understand the core technical process of the present invention.
[0039] Figure 2 Path diagram from RA to RM Figure 3 The composition of the complex substrate RA60. Detailed Implementation
[0040] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.
[0041] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.
[0042] Example 1 Enzyme Sequence Information 1. UGT76G1 mutant and its encoding gene The amino acid sequence of UGT76G1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.
[0043] SEQ ID NO.1: MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLHHHHHH 。
[0044] SEQ ID NO.2:
[0045] The UGT76G1 mutant (UGT76G1mut) is obtained by mutating position 83 (G83Q) of the UGT76G1 gene. Its amino acid sequence is shown in SEQ ID NO.3 and its nucleotide sequence is shown in SEQ ID NO.4. It was synthesized by Suzhou Genewise Biotechnology Co., Ltd.
[0046] SEQ ID NO.3: MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHQPLAGMRIPIINEHGADELRRELELLMLASEEDE EVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELET VIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHG AIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAAIRRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLHHHHHH .
[0047] SEQ ID NO.4:
[0048] 2. UGT11 mutant and its encoding gene The amino acid sequence of UGT11 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.
[0049] SEQ ID NO.5: MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFL GTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLL STLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAV GAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH .
[0050] SEQ ID NO.6: The UGT11 mutant (UGT11mut) is obtained by mutating position 158 (S158F) of the UGT11 gene. Its amino acid sequence is shown in SEQ ID NO.7 and its nucleotide sequence is shown in SEQ ID NO.8. It was synthesized by Suzhou Genewise Biotechnology Co., Ltd.
[0051] SEQ ID NO.7: MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFL GTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLL STLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAV GAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH .
[0052] SEQ ID NO.8: 3. AtSUS and Its Encoding Gene The amino acid sequence of AtSUS is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.
[0053] SEQ ID NO.9: MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH
[0054] SEQ ID NO.10:
[0055] Example 2 Construction and purification of recombinant Escherichia coli and preparation of enzyme solution 1. Plasmid construction Escherichia coli BL21(DE3) (Catalog No.: B528414) and DH5α competent cells (Catalog No.: A338951) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid pET-30a(+) (Catalog No.: B540185) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; all gene and primer synthesis and sequencing services were provided by Suzhou Genewise Biotech Co., Ltd.; 2×Phanta Flash Master Mix high-fidelity DNA polymerase (Catalog No.: P510-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases NdeI (Code No. 1621) and XhoI (Code No. 1635) were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.
[0056] The genes encoding UGT76G1 and its mutants, UGT11 and its mutants, and AtSUS were synthesized by Suzhou Genewise Biotechnology Co., Ltd. Primers used for PCR amplification of UGT76G1, UGT76G1mut, UGT11, UGT11mut, and AtSUS are shown in Table 1. Table 1
[0057] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; bold slashes indicate the correct font. CATATG This is the NdeI restriction site. CTCGAG This is the XhoI restriction site.
[0058] The PCR amplification program was to amplify the target fragment using 2×Phanta Flash Master Mix high-fidelity DNA polymerase. The specific reaction system and amplification program are shown in Table 2.
[0059] Table 2
[0060] The amplified DNA fragments or plasmids are digested with restriction endonucleases. Different restriction enzymes require different digestion times, and the appropriate digestion time must be selected according to the specific requirements of the restriction enzyme. This invention uses the Fastdigest series of restriction endonucleases, and the reaction system is shown in Table 3.
[0061] Table 3
[0062] After purification following enzyme digestion, the T4 DNA ligase, purified DNA fragment, and vector plasmid fragment were mixed and incubated in a metal bath at 22°C for 2.5 hours to complete the ligation reaction. The ligation reaction system is shown in Table 4.
[0063] Table 4
[0064] (3) Transformation verification The above enzyme-linked products were transformed (using conventional chemical transformation) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, the positive plasmids pET30a(+)-UGT76G1, pET30a(+)-UGT11, and pET30a(+)-AtSUS were returned.
[0065] Based on the above plasmids, PCR amplification was performed using the amplification primers in Table 1. The PC products purified by gel excision were transformed (using conventional chemical transformation) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, the positive plasmids pET30a(+)-UGT76G1mut and pET30a(+)-UGT11mut were returned.
[0066] 2. Construction of recombinant strains 2.1 Construction of mutant strains The constructed plasmid was transformed (using conventional chemical transformation) into competent Escherichia coli BL21(DE3) cells and cultured overnight. Validation primers were designed: upstream primer test-pET-F and downstream primer test-pET-R. Single colonies grown in the screening plate (50 µg / mL kanamycin) were validated by colony PCR, and the corresponding recombinant strains UGT76G1mut and UGT11mut were constructed.
[0067] Upstream primer test-pET-F (SEQ ID NO.21): 5'-TTGTGAGCGGATAACAATTCCCCTCT -3'; Downstream primer test-pET-R (SEQ ID NO.22): 5'-TTAGCAGCCGGATCTCAGTGGTGGTG-3'.
[0068] 2.2 Construction of non-mutant strains Positive plasmids pET30a(+)-UGT76G1, pET30a(+)-UGT11, and pET30a(+)-AtSUS were transformed (using conventional chemical transformation) into competent Escherichia coli BL21(DE3) cells and cultured overnight. Validation primers were designed: upstream primer test-pET-F and downstream primer test-pET-R. Single colonies grown in the screening plate (50 µg / mL kanamycin) were validated by colony PCR to construct the corresponding recombinant strains UGT76G1, UGT11, and AtSUS.
[0069] 3. Preparation of recombinant Escherichia coli induced expression and purified enzyme solution 1. Induced expression Single colonies of the recombinant strain were inoculated into 2YT medium containing 50 µg / mL kanamycin (2YT medium formulation: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) and cultured at 37°C and 220 rpm for 6 h. The inoculum was then transferred at a 1% inoculum to 150 mL of 2YT medium containing 50 µg / mL kanamycin and cultured at 37°C and 220 rpm until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.3 mM, and the mixture was incubated at 16°C for 20 h to induce expression. The mixture was then centrifuged at 4°C and 6000 rpm for 15 min, the supernatant was discarded, and the precipitate was kept for later use.
[0070] 2. Preparation of crude enzyme solution A 10% w / v recombinant bacterial culture was prepared using Tris (50 mM, pH 7.5) solution. The bacterial cells were homogenized by high pressure (900 bar) to release the target protein. The culture was centrifuged at 4°C and 6000 rpm for 15 min. The supernatant enzyme solution was collected to obtain the crude enzyme solution for later use.
[0071] 3. Protein purification (1) Nickel gravity column pretreatment: HisSep Ni-NTAAgarose Resin was loaded into a suitable purification column by gravity, the column was rinsed with 2 column volumes of deionized water, and the column was equilibrated with 2 column volumes of Tris. (2) Sample loading: Add the crude enzyme solution prepared above to the purification column. Pay attention to controlling the sample loading speed to ensure that the target protein is in full contact with Ni2+ in order to improve the purification yield. (3) Washing: Washing a 2-cylinder volume of Wash Buffer; (4) Elution: Elute with 30 mL of Elution Buffer and collect the eluent, which is the target protein solution; (5) Column regeneration: one column volume of 1M imidazole washing, followed by two column volumes of deionized water rinsing. (6) Storage: Finally, store the resin in deionized water at 4°C; (7) Ultrafiltration: Add 50mM Tris buffer (pH 7.5) to the ultrafiltration tube, centrifuge at 5000 rpm and 4℃ for 10 min, and discard the centrifuged liquid; add an appropriate amount of the eluted target protein solution, centrifuge at 5000 rpm and 4℃ to a volume of about 1 mL; add an equal volume of 50mM Tris buffer (pH 7.5), centrifuge at 5000 rpm and 4℃ to a volume of about 1 mL, and repeat this step once to remove the high concentration of imidazole and obtain the protein purification solution.
[0072] Using the above method, purified solutions of UGT76G1, UGT76G1mut, UGT11, UGT11mut and AtSUS proteins were prepared respectively. After lyophilization, they became UGT76G1 enzyme, UGT76G1mut enzyme, UGT11 enzyme, UGT11mut enzyme and AtSUS enzyme.
[0073] Example 3: A method for the production of rebaudioside M and the transformation of complex substrates. 1. Experimental Preparation 1.1 Substrate preparation Prepare substrate powder RA and complex substrate RA60 for the synthesis of rebaudioside M. The substrate powder RA was purchased from Xinghua Green, catalog number M-RA97-2508002; the complex substrate RA60 was purchased from Shengxiangyuan, catalog number RA60-20250808, and their purity was ensured to meet the experimental requirements by high performance liquid chromatography (HPLC) analysis.
[0074] 1.2 Preparation of reaction vessels and instruments A reaction vessel is used as the reaction container, and it undergoes rigorous cleaning and sterilization before use to avoid interference from impurities. A high-precision temperature control device (accuracy up to ±0.1℃), a pH adjustment system (capable of precisely adjusting the pH value to ±0.01), and a stirring device (with adjustable stirring speed) are prepared to ensure precise control of the reaction system conditions. Simultaneously, analytical instruments such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) are provided for qualitative and quantitative analysis of the substrates and products during the reaction process.
[0075] 1.3 High Performance Liquid Chromatography (HPLC) Analysis Conditions The elution methods for high performance liquid chromatography (HPLC) are shown in Table 5, and the experimental conditions are shown in Table 6.
[0076] Table 5. Elution methods for liquid chromatography
[0077] Table 6. Experimental conditions for liquid chromatography
[0078] 1.4 Mass Spectrometer (MS) Fractionation Conditions Mass spectrometry conditions were as follows: electrospray ionization (ESI) source; positive ion sensitivity mode; capillary voltage (3 kV (+)); cone voltage (40 V); dry gas (N2, purity >99.999%); desolvation gas flow rate (900 L / h); desolvation gas temperature (500 ℃); cone backflush gas flow rate (50 L / h); ion source temperature (120 ℃); full scan detection molecular weight range (50~1200 Da); collision energy (6 eV); Reference Scan Frequency (20 s) was used to lock the mass to ensure an error <10 ppm.
[0079] 2. Operating Procedures (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 37℃, and the pH value to 7.0. Add substrate powder RA or complex substrate RA60 to make the final concentration of substrate powder RA or complex substrate RA60 in the reaction system reach 75 g / L. At the same time, add 20% wt sucrose, 0.5% wt UDP and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA or complex substrate RA60 in the reaction system). After stirring thoroughly, add 0.1% wt UGT11mut enzyme and 0.1% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA or complex substrate RA60 in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH value to 7 to ensure that the enzyme and substrate are in full contact. The reaction is carried out for 4 hours, and samples are taken at regular intervals during the period. The changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0080] (2) After 4 hours of reaction, UGT76G1mut enzyme was added to the reaction system at a ratio of 0.2%wt of the substrate powder RA or complex substrate RA60. Simultaneously, the reaction temperature was increased to 45℃ and the pH was adjusted to 7.5. The reaction was continued with stirring. The reaction was continued for 24 hours, and samples were taken periodically. The reaction solution was analyzed using HPLC and MS to monitor the conversion of the intermediate product to the target product.
[0081] 3. Experimental Results Quantitative analysis of the supernatant after the reaction was performed by HPLC. The results showed that, under the optimized reaction system of the three-enzyme segmented addition of the present invention, with substrate powder RA as the substrate, the yield of rebaudioside M reached 95.88 g / L (74.25 mM), and the conversion rate reached more than 95%. Compared with the traditional synthesis method, the yield was significantly improved.
[0082] Experiments were conducted using the complex substrate RA60, and the catalytic reaction was carried out using the technology of this invention. After the reaction was completed, the reaction products were analyzed by HPLC and MS, and the conversion rate of RA60 was calculated.
[0083] The complex substrate RA60 feedstock contains 75 g / L of RA. 0.9 0.53 = 35.78 g / L (37 mM); STV is 75 0.9 0.14 = 9.45 g / L (11.74 mM); it will also contain about 5 mM of RA and RD, and finally generate 64.57 g / L (50.02 mM). Experimental results show that the conversion rate of RA60 by the technology of this invention reaches more than 90%, while the conversion rate of RA60 in the traditional reaction system is usually less than 30%.
[0084] Example 4: A method for the production of rebaudioside M and the transformation of complex substrates. (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 37℃, and the pH to 7.0. Add substrate powder RA to achieve a final concentration of 75 g / L in the reaction system. Simultaneously add 20% wt sucrose, 0.5% wt UDP, and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA in the reaction system). After thorough mixing, add 0.15% wt UGT11mut enzyme and 0.15% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH to 7 to ensure sufficient contact between the enzyme and the substrate. The reaction proceeds for 4 hours, during which samples are taken periodically, and the changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0085] (2) After 4 hours of reaction, UGT76G1mut enzyme was added to the reaction system at a ratio of 0.3% wt of the substrate powder RA. Simultaneously, the reaction temperature was increased to 45°C and the pH was adjusted to 7.5. The reaction was continued with stirring. The reaction was carried out for 24 hours, and samples were taken periodically. The reaction solution was analyzed using HPLC and MS to monitor the conversion of the intermediate product to the target product.
[0086] Comparative Example 1: A method for the production of rebaudioside M and the transformation of complex substrates. (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 37℃, and the pH to 7.0. Add substrate powder RA to achieve a final concentration of 75 g / L in the reaction system. Simultaneously add 20% wt sucrose, 0.5% wt UDP, and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA in the reaction system). After thorough mixing, add 0.1% wt UGT76G1mut enzyme and 0.1% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH to 7 to ensure sufficient contact between the enzyme and the substrate. The reaction proceeds for 4 hours, during which samples are taken periodically, and the changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0087] (2) After 4 hours of reaction, UGT11mut enzyme was added to the reaction system. The addition ratio of UGT76G1mut enzyme was 0.2% wt of the substrate powder RA in the reaction system. At the same time, the reaction temperature was raised to 45℃ and the pH was adjusted to 7.5. The reaction was stirred continuously. The reaction continued for 24 hours. Samples were taken at regular intervals, and the reaction solution was analyzed by HPLC and MS to monitor the conversion of intermediate products to target products.
[0088] Comparative Example 2: A method for the production of rebaudioside M and the transformation of complex substrates. (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 37℃, and the pH value to 7.0. Add substrate powder RA to make the final concentration of substrate in the reaction system reach 75 g / L, and add 20% wt sucrose, 0.5% wt UDP and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA in the reaction system). After stirring thoroughly, add 0.1% wt UGT11 enzyme and 0.1% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH value to 7 to ensure that the enzyme and substrate are in full contact. The reaction is carried out for 4 hours, and samples are taken at regular intervals during the period. The changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0089] (2) After 4 hours of reaction, UGT76G1 enzyme was added to the reaction system at a ratio of 0.2% wt% of the substrate powder RA. Simultaneously, the reaction temperature was increased to 45°C and the pH was adjusted to 7.5. The reaction was continued with stirring. The reaction continued for 24 hours, and samples were taken periodically. The reaction solution was analyzed using HPLC and MS to monitor the conversion of the intermediate product to the target product.
[0090] Comparative Example 3: A method for the production of rebaudioside M and the transformation of complex substrates. (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 45℃, and the pH value to 7.0. Add substrate powder RA to make the final concentration of substrate in the reaction system reach 75 g / L, and add 20% wt sucrose, 0.5% wt UDP and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA in the reaction system). After stirring thoroughly, add 0.1% wt UGT11 enzyme and 0.1% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH value to 7 to ensure that the enzyme and substrate are in full contact. The reaction is carried out for 4 hours, and samples are taken at regular intervals during the period. The changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0091] (2) After 4 hours of reaction, UGT76G1mut enzyme was added to the reaction system at a ratio of 0.2%wt of the substrate powder RA. Simultaneously, the reaction temperature was increased to 55℃ and the pH was adjusted to 7.5. The reaction was continued with stirring. The reaction was carried out for 24 hours, and samples were taken periodically. The reaction solution was analyzed using HPLC and MS to monitor the conversion of the intermediate product to the target product.
[0092] Comparative Example 4: A method for the production of rebaudioside M and the transformation of complex substrates. (1) Add 50 mM pH=7.5 Tris-HCl reaction buffer to the reaction vessel, adjust the temperature to 37℃, and the pH to 7.0. Add substrate powder RA to achieve a final concentration of 75 g / L in the reaction system. Simultaneously add 20% wt sucrose, 0.5% wt UDP, and 0.5% wt MgCl2 (the above addition ratios are relative to the substrate powder RA in the reaction system). After thorough mixing, add 0.2% wt UGT11mut enzyme and 0.1% wt AtSUS enzyme (the above addition ratios are relative to the substrate powder RA in the reaction system). Turn on the stirrer, set the stirring speed to 220 rpm, raise the reaction temperature to 37℃, and adjust the pH to 7 to ensure sufficient contact between the enzyme and the substrate. The reaction proceeds for 4 hours, during which samples are taken periodically, and the changes in the content of substrate and intermediate products in the reaction solution are analyzed by HPLC.
[0093] (2) After 4 hours of reaction, UGT76G1mut enzyme was added to the reaction system at a ratio of 0.1% wt% of the substrate powder RA. Simultaneously, the reaction temperature was increased to 45°C and the pH was adjusted to 7.5. The reaction was continued with stirring. The reaction was carried out for 24 hours, and samples were taken periodically. The reaction solution was analyzed using HPLC and MS to monitor the conversion of the intermediate product to the target product.
[0094] The yields of rebaudioside M and the conversion rates of substrate powder RA for Comparative Examples 1-4 are shown in Table 7. Table 7
[0095] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.
Claims
1. A method for the production of rebaudioside M and the transformation of complex substrates, characterized in that, The method includes the following steps: S1. Add reaction additives to the substrate, add UGT11mut enzyme and AtSUS enzyme, and after reaction, obtain enzyme reaction solution; S2. Add UGT76G1mut enzyme to the enzyme reaction solution, continue the reaction, collect the reaction solution, and obtain rebaudioside M. The UGT11mut enzyme is an enzyme expressing UGT11mut, and the amino acid sequence of UGT11mut is shown in SEQ ID NO.7; the AtSUS enzyme is an enzyme expressing AtSUS, and the amino acid sequence of AtSUS is shown in SEQ ID NO.9; the UGT76G1mut enzyme is an enzyme expressing UGT76G1mut, and the amino acid sequence of UGT76G1mut is shown in SEQ ID NO.3; The weight ratio of the UGT11mut enzyme, AtSUS enzyme, and UGT76G1mut enzyme is 1:1:
2.
2. The method according to claim 1, characterized in that, The amount of UGT11mut enzyme added in step S1 is 0.05%-0.15%wt of the substrate; the amount of AtSUS enzyme added in step S1 is 0.05%-0.15%wt of the substrate.
3. The method according to claim 2, characterized in that, The amount of UGT11mut enzyme added in step S1 is 0.08%-0.12%wt of the substrate; the amount of AtSUS enzyme added in step S1 is 0.08%-0.12%wt of the substrate.
4. The method according to claim 1, characterized in that, The amount of UGT76G1mut enzyme added in step S2 is 0.1%-0.3%wt of the substrate.
5. The method according to claim 4, characterized in that, The amount of UGT76G1mut enzyme added in step S2 is 0.16%-0.24%wt of the substrate.
6. The method according to claim 1, characterized in that, The reaction additives include any one or more of sucrose, UDP, and MgCl2.
7. The method according to claim 1, characterized in that, The substrate is a raw material containing RA.
8. The method according to claim 1, characterized in that, The reaction temperature in step S1 is 35-38℃, and the reaction temperature in step S2 is 40-50℃.
9. The method according to claim 1, characterized in that, The reaction time in step S1 is 2-6 hours, and the reaction time in step S2 is 24-48 hours.
10. The method according to claim 1, characterized in that, The preparation method of the UGT11mut enzyme includes: inducing expression of UGT11mut recombinant strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain crude enzyme solution, purifying and freeze-drying it to obtain UGT11mut enzyme; The method for preparing the AtSUS enzyme includes: inducing the expression of a recombinant AtSUS strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain a crude enzyme solution, purifying it and freeze-drying it to obtain the AtSUS enzyme; The preparation method of the UGT76G1mu enzyme includes: inducing expression of UGT76G1mu recombinant strain, collecting the precipitate, resuspending and breaking it, collecting the supernatant to obtain crude enzyme solution, purifying and freeze-drying it to obtain UGT76G1mut enzyme.