Multi-enzyme co-expression genetically engineered bacterium as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of genetically engineered bacteria, and particularly relates to a multienzyme co-expression genetically engineered bacterium as well as a preparation method and application thereof. According to the invention, genetic engineering bacteria capable of co-expressing sucrose synthase, beta-1, 2-glycosyltransferase and beta-1, 3-glycosyltransferase are constructed, so that fundamental transformation from a traditional complex process of three-bacterium three-enzyme step-by-step fermentation-in-vitro mixing to an innovative mode of one-bacterium three-enzyme one-step catalysis is successfully realized; the innovation of the technical route enables the production efficiency, the cost control and the process simplification to be remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering bacteria technology, specifically relating to multi-enzyme co-expression genetic engineering bacteria, their preparation methods, and applications. Background Technology

[0002] Compared to rebaudioside A (Reb A) and rebaudioside D (Reb D), rebaudioside M (Reb M) has no noticeable bitter aftertaste and a relatively better taste. As a sugar source to replace sucrose, Reb M has a more promising application prospect. Currently, the content of Reb M in dried stevia leaves is extremely low, less than 0.5% of the leaf's dry weight. Obtaining high-purity Reb M through plant extraction is difficult and has disadvantages such as multiple steps and high energy consumption. Although chemical synthesis methods have advantages such as short reaction time and high conversion efficiency, they have disadvantages such as environmental pollution, extreme reaction conditions, and difficulty in meeting food-grade standards. In comparison, the bio-enzymatic catalysis method for synthesizing steviol glycoside Reb M has advantages such as a wide range of material sources, mild reaction conditions, and low energy consumption. The technology of synthesizing steviol glycosides using enzymes or enzyme-producing microorganisms as catalysts is simple to operate, has high catalytic specificity, produces fewer byproducts, and facilitates the separation and purification of subsequent products, thus benefiting the industrial production of steviol glycosides.

[0003] The main problems with bio-enzyme catalysis are as follows: (1) The cost of producing Reb M from Reb D using bio-enzyme catalysis is high, and the content of Reb A in natural plants is very high, while the content of Reb D is very low. Converting Reb A to Reb M at low cost is a problem that urgently needs to be solved. (2) The three enzymes that realize the conversion of Reb A to Reb M are sucrose synthase, β- ... 1,2 Glycosyltransferases and β 1,3 Glycosyltransferases are produced by fermenting three enzymes separately, which is a complex process with high raw material costs. Furthermore, it is difficult to match the activity ratio of each enzyme in real time, resulting in low Reb A conversion rate and slow Reb M synthesis rate. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-enzyme co-expression genetically engineered bacterium, and to provide a method for preparing the engineered bacterium. When the engineered bacterium is applied to catalyze the production of Reb M from Reb A, the production efficiency of Reb M is significantly improved.

[0005] The technical solution of the present invention is as follows: The first objective of this invention is to provide a multi-enzyme co-expression genetically engineered bacterium, wherein the engineered bacterium can co-express sucrose synthase (SUS), β-1,2-glycosyltransferase (β-1,2-GT) and β-1,3-glycosyltransferase (β-1,3-GT).

[0006] The amino acid sequence of the sucrose synthase is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the β-1,2-glycosyltransferase is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the β-1,3-glycosyltransferase is shown in SEQ ID NO.5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6.

[0007] Three enzymes are co-expressed in the host bacteria using two or three expression vectors. Preferably, the expression vectors include pETDuet-1, pACYCDuet-1, and pRSFDuet-1. When the three enzymes are co-expressed in the host bacteria using two expression vectors, the encoding genes for sucrose synthase and β-1,2-glycosyltransferase are linked to the same expression vector, and the encoding gene for β-1,3-glycosyltransferase is linked to another expression vector.

[0008] The host bacterium is Escherichia coli (Escherichia coli) Escherichia coli ), preferred E. coli BL21(DE3).

[0009] A second objective of this invention is to provide a method for preparing the multi-enzyme co-expression genetically engineered bacteria, comprising the following steps: (1) Construct a first expression vector containing the gene encoding β-1,3-glycosyltransferase; (2) Construct a second expression vector containing genes encoding sucrose synthase and β-1,2-glycosyltransferase; (3) The vectors constructed in steps (1) and (2) are transformed into the host bacteria to obtain the multi-enzyme co-expression genetically engineered bacteria.

[0010] A third objective of this invention is to provide a method for synthesizing rebaudioside M using the multi-enzyme co-expression genetically engineered bacteria, comprising the following steps: (1) Culture the multi-enzyme co-expression genetically engineered bacteria to obtain whole-cell crude enzyme solution; (2) Using rebaudine A, sucrose, and ADP as substrates, the reaction is carried out under the catalysis of the whole cell crude enzyme solution to generate rebaudine M.

[0011] In step (2), the reaction conditions are: pH 5.5-7.5 and temperature 55-70℃.

[0012] The fourth objective of this invention is to provide the application of the multi-enzyme co-expression genetically engineered bacteria, or the method for synthesizing rebaudioside M using the engineered bacteria, in the catalytic production of rebaudioside M from rebaudioside A.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention has successfully transformed the traditional complex process of "three bacteria and three enzymes stepwise fermentation-in vitro mixing" into an innovative model of "one bacteria and three enzymes one-step catalysis" by constructing genetically engineered bacteria capable of co-expressing sucrose synthase, β-1,2-glycosyltransferase and β-1,3-glycosyltransferase. This technological innovation has brought about significant improvements in production efficiency, cost control and process simplification.

[0014] 1. Significantly improved production efficiency and catalytic performance Ultra-high conversion rate and yield: In a 1mL small-scale test system, the optimal engineered bacteria... E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS catalyzed 10 g / L Reb A, and after 1 hour of reaction, the Reb M production was 10.56 g / L, with a Reb A conversion rate of 80.40%. In contrast, the traditional three-enzyme mixed method (control group) produced 1.80 g / L of Reb M with a conversion rate of 39.38%, showing a significant difference. Good stability in scale-up production: In a 100mL scale-up system, using a high concentration of Reb A of 100g / L as substrate, after 10 hours of reaction, the Reb M production of the optimal strain reached 74.98g / L, with a conversion rate as high as 99.75%, which is much higher than the Reb M production of 51.60g / L in the control group. This proves that the technical solution of the present invention has good potential for large-scale application. Increased reaction throughput: The co-expression system enabled the three enzymes to form a "substrate channel effect" in the bacteria. The residual amount of intermediate product Reb D (8.93 g / L) was significantly lower than that of the control group (18.94 g / L), indicating that the reaction process was smoother and the overall catalytic efficiency was higher.

[0015] 2. Production costs have been significantly reduced. This invention simplifies the production process from the source, reducing production costs by more than 40%. Traditional processes require fermenting three enzyme-producing bacteria separately, while this invention only requires fermenting one co-expression bacteria, reducing the number of fermentation batches by 2 / 3 and directly saving on the corresponding costs of culture medium, sterilization, energy consumption, and labor. It also avoids the complex processes and related costs of fermenting, extracting, purifying, transporting, and storing the three enzymes separately.

[0016] 3. Extreme simplification of process flow Simplified operation: There is no need to process the three enzymes separately and optimize their mixing ratio. Simply cultivate one engineered bacteria and obtain its crude enzyme solution for use in catalytic reactions, which greatly reduces the technical threshold and operational complexity. Easier quality control: Fermentation with a single strain is easier to control in terms of quality and ensure batch-to-batch consistency, avoiding performance fluctuations caused by the difficulty in accurately matching the proportions when mixing multiple enzymes.

[0017] 4. The advanced nature of the technology itself and its synergistic effect Breakthrough in technical bottlenecks: Successfully solved the key technical bottlenecks of difficulty in real-time matching of enzyme activity ratio, large mass transfer resistance, and high enzyme activity loss in multi-enzyme in vitro catalytic systems; Achieving synergistic catalysis: By using a co-expression strategy, the three enzymes coexist in the cell in the optimal ratio, achieving highly efficient synergistic catalysis, an effect that cannot be achieved by simple mixing. Attached Figure Description

[0018] Figure 1 This is a standard curve showing the relationship between protein content and absorbance in Example 2; Figure 2 In Example 5 E . coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacting in a 1 mL catalytic system for 20 min; Figure 3 In Example 5 E . coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacting in a 1 mL catalytic system for 1 h. Figure 4 In Example 6 E . coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacting in a 100 mL catalytic system for 1 h. Figure 5 In Example 6 E . coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacted in a 100 mL catalytic system for 10 h. Detailed Implementation

[0019] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods. For example, molecular biology experimental methods involved in the embodiments can be found in the 3rd edition of *Molecular Cloning: A Laboratory Manual*. Where specific techniques or conditions are not specified in the experiments, they should be performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the reagents and materials mentioned are commercially available.

[0020] Example 1 This embodiment prepared a multi-enzyme co-expression genetically engineered bacterium, the preparation method of which includes the following steps: (1) Construct a first expression vector containing the gene encoding one of the enzymes: sucrose synthase (SUS), β-1,2-glycosyltransferase (β-1,2-GT), or β-1,3-glycosyltransferase (β-1,3-GT); (2) Construct a second expression vector containing the genes encoding two other enzymes; (3) The vectors constructed in steps (1) and (2) are combined and converted to E. coli The multi-enzyme co-expressing genetically engineered bacteria were obtained by colony PCR and sequencing in DH5α competent cells and stored at -80℃.

[0021] The expression vectors pETDuet-1, pACYCDuet-1, and pRSFDuet-1 used in this invention are commercially available vectors from Merck. The complete nucleotide sequences of these vectors can be obtained from the public database Addgene, with plasmid numbers pETDuet-1 (Addgene#71147), pACYCDuet-1 (Addgene#71146), and pRSFDuet-1 (Addgene#71341), respectively.

[0022] by E . coli Taking BL21(DE3) / pACYCDuet-1-β-1,2-GT+pETDuet-1-β-1,3-GT-SUS as an example, it represents the β-1,2-GT enzyme encoding gene being ligated to the vector pACYCDuet-1, and β-1,3-GT and SUS being ligated to the vector pETDuet-1. Both are then co-transformed into... E. coli In DH5α competent cells, co-expressing genetically engineered bacteria were obtained; the same method was used for other engineered bacteria.

[0023] The multi-enzyme co-expression genetically engineered bacteria constructed in this embodiment are as follows: E . coliBL21(DE3) / pACYCDuet-1-β-1,2-GT+pETDuet-1-β-1,3-GT-SUS; E . coli BL21(DE3) / pACYCDuet-1-SUS+pETDuet-1-β-1,3-GT-β-1,2-GT; E . coli BL21(DE3) / pETDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS; E . coli BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS; E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pETDuet-1-β-1,2-GT-SUS; E . coli BL21(DE3) / pETDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS; E . coli BL21(DE3) / pACYCDuet-1-β-1,3-GT+pETDuet-1-β-1,2-GT-SUS; E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS; In addition, genetically engineered bacteria expressing single enzymes were constructed. E . coli BL21(DE3) / pET28a(+)-SUS; E . coli BL21(DE3) / pET28a(+)-β-1,2-GT; E . coli BL21(DE3) / pET28a(+)-β-1,3-GT.

[0024] Example 2 This example demonstrates the preparation of whole-cell crude enzyme solution, using the following method: (1) Pick a single colony of the genetically engineered bacteria and inoculate it into 20 mL of liquid LB medium containing the corresponding antibiotic. Incubate at 37°C and 200 rpm for 10 h with shaking. Inoculate 1% of the bacteria into 50 mL of liquid TB medium containing the corresponding antibiotic. Incubate at 37°C and 200 rpm for 1.5 h-2 h with shaking. When the OD600 reaches 0.6, add IPTG to a final concentration of 0.5 mmol / L and incubate at 25°C and 200 rpm for 20 h with shaking. (2) Centrifuge the bacterial culture at 4℃ and 4,000 rpm for 10 min, discard the supernatant, and collect the bacteria; resuspend the precipitate in 1×PBS, the amount of 1×PBS being 5 times the mass of the bacterial culture used; then use an ultrasonic disruptor to disrupt the culture at 40% power (total power 560W), disrupt for 2 s, stop for 4 s, and disrupt for 15 min to obtain the whole cell crude enzyme solution. Detect its protein concentration. The protein concentration measurement method is as follows: S1. Create the standard track: Take 7 test tubes, number them (each numbered tube should be repeated 3 times), add reagents according to Table 1, stopper them, shake well, let stand for 2 minutes, and then measure the colorimetric value at a wavelength of 595 nm (the colorimetric measurement should be completed within 1 hour). Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis. Figure 1 ).

[0025] Table 1. Reagent Addition Amount

[0026] S2. Take another test tube, accurately add 100 μL of sample, then add 0.9 mL of distilled water and 5 mL of Coomassie Brilliant Blue G-250 reagent, mix thoroughly, let stand for 2 min, use the standard curve tube No. 1 as a reference, measure the absorbance at a wavelength of 595 nm, record the absorbance value, calculate the protein concentration, and the results are shown in Table 2.

[0027] Table 2 Protein concentration of crude enzyme solution

[0028] Example 3 In this embodiment, Reb A was synthesized into Reb M using crude enzyme solution as the catalysis. The total system volume was 1 mL. Using phosphate buffer as the solvent and reaction environment, 60 g / L sucrose, 2 g / L Reb A, and 0.1 g / L ADP were added, followed by 0.5 g / L crude enzyme solution (based on protein concentration). The pH of the reaction system was adjusted to 6.5. The reaction was carried out at 65°C and 800 rpm for 1 hour, then terminated by water bath treatment at 95°C for 10 minutes. After the reaction, the mixture was centrifuged at 12,000 rpm for 2 minutes, filtered through a 0.22 μm filter, and the Reb A and Reb M contents were determined by HPLC.

[0029] The control group was E. coli BL21(DE3) / pET28a(+)-SUS, E. coli Crude enzyme solutions were prepared from BL21(DE3) / pET28a(+)-β-1,2-GT and E. coli BL21(DE3) / pET28a(+)-β-1,3-GT. The final concentrations of the three enzymes were added to the reaction system at protein concentrations of 0.04 g / L, 0.06 g / L, and 0.1 g / L, respectively, while the concentrations of other substances remained unchanged.

[0030] The results are shown in Table 3. After 1 hour of reaction... E. coli The Reb M formation of BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS was 3.25 g / L. E. coli The Reb M production of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS was 3.07 g / L, which was significantly higher than that of the control group (Reb M production was 0.46 g / L).

[0031] Table 3 Catalytic Results Data Table

[0032] Example 4 In this embodiment, Reb A is synthesized into Reb M using crude enzyme solution as a catalyst. The method is the same as in Example 3, except that the amount of Reb A added to the substrate is 6 g / L.

[0033] The results are shown in Table 4. After 1 hour of reaction, E . coli The Reb M formation of BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS was 8.89 g / L. E . coli The Reb M production of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS was 9.56 g / L, which was significantly higher than that of the control group.

[0034] Table 4 Catalytic Results Data Table

[0035] Example 5 This embodiment utilizes crude enzyme solution to catalyze the synthesis of Reb M from Reb A, following the method described in Example 3, except that the amount of Reb A added to the substrate is 10 g / L. This embodiment also showed that the Reb M production in Example 4 was significantly higher than in other groups. E . coli BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS and E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS.

[0036] The results are shown in Table 5. After 1 hour of reaction, the Reb M production of both co-expressing genetically engineered bacteria was significantly higher than that of the control group, especially... E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS showed the best performance, with a Reb M production of 10.56 g / L and a Reb A conversion rate of 80.40%. Figure 2 for E. coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacted in a 1 mL catalytic system for 20 min. Figure 3 The HPLC chromatogram shows the reaction of E. coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS in a 1 mL catalytic system for 1 h.

[0037] Table 5 Catalytic Results Data Table

[0038] Example 6 This embodiment utilizes crude enzyme solution to catalyze the synthesis of Reb M from Reb A, and expands the total catalytic system to 100 mL. E . coli BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS and E. coliThe application efficacy of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS was verified. Using phosphate buffer as the solvent and reaction environment, 100 g / L sucrose, 100 g / L Reb A, and 0.4 g / L ADP were added, followed by 0.5 g / L crude enzyme solution (based on protein concentration). The pH of the reaction system was adjusted to 6.5. The reaction was carried out at 65℃ and 800 rpm for 10 h, then terminated by water bath at 95℃ for 10 min. After the reaction, the mixture was centrifuged at 12,000 rpm for 2 min, filtered through a 0.22 μm filter, and the contents of Reb A, Reb D, and Reb M were determined by HPLC.

[0039] The control group was E. coli BL21(DE3) / pET28a(+)-SUS, E. coli BL21(DE3) / pET28a(+)-β-1,2-GT and E. coli The crude enzyme solution prepared by BL21(DE3) / pET28a(+)-β-1,3-GT was added to the reaction system at final concentrations of 0.1 g / L, 0.3 g / L, and 0.6 g / L protein, while the concentrations of other substances remained unchanged.

[0040] The results are shown in Table 6. After reacting for 10 hours in a 100 mL catalytic system, the Reb M production of both co-expressing genetically engineered bacteria was higher than that of the control group. E . coli The Reb M production of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS was 74.98 g / L, and the Reb A conversion rate was 99.75%. Figure 4 for E. coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacted in a 100 mL catalytic system for 1 h. Figure 5 for E. coli HPLC chromatogram of BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS reacted in a 100 mL catalytic system for 10 h.

[0041] Table 6 Catalytic Results Data Table

[0042] Example 7 Referring to Example 1, this example prepared a genetically engineered bacterium capable of co-expressing wild-type sucrose synthase, wild-type β-1,2-glycosyltransferase, and wild-type β-1,3-glycosyltransferase: wild-type E . coli BL21(DE3) / pACYCDuet-1-β-1,3-GT+pRSFDuet-1-β-1,2-GT-SUS and wild type E . coli BL21(DE3) / pRSFDuet-1-β-1,3-GT+pACYCDuet-1-β-1,2-GT-SUS, wherein the amino acid sequence of the wild-type sucrose synthase is shown in SEQ ID NO.7, the amino acid sequence of the wild-type β-1,2-glycosyltransferase is shown in SEQ ID NO.8, and the amino acid sequence of the wild-type β-1,3-glycosyltransferase is shown in SEQ ID NO.9.

[0043] Whole-cell crude enzyme solution was prepared using the above-mentioned genetically engineered bacteria and used to catalyze the synthesis of Reb M from Reb A. The catalytic method was the same as in Example 6, and the catalytic system was 100 mL. After 10 h of catalytic reaction, the contents of Reb A and Reb M were detected by HPLC. The results are shown in Table 7. It can be seen that the amount of Reb M produced by the genetically engineered bacteria in Example 6 of this invention is significantly higher than that of the wild-type co-expressed genetically engineered bacteria in Example 7.

[0044] Table 7 Catalytic Results Data Table

[0045] The amino acid sequence of sucrase synthase is SEQ ID NO.1: MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQEGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME Nucleotide sequence of sucrose synthase SEQ ID NO.2: Amino acid sequence of β-1,2-glycosyltransferase SEQ ID NO.3: MHHHHEGVSDQTLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTLINLESIIKKIPEKYSESIRFVELHLPELPELPPHYHTTNGLPPHLNHTLHKALKMSKPNFSKILQNLKPDLVIYDILQPWAEHVVNEQNIPAVKILTSGAALFSYFFNFLKNPGVEFPFPAIYLPKVEQVKMREMFEKEPNEEDRLAEGNMQIMLMCTSRTIEAKYLDYCTELSNWKVVPVGPPFQDPITNDVDDMELIDWLGTKDENSTVFVCFGSEYFLSREDMEEVAFGLELSNVNFIWVARFPKGEEQNLEDVLPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESLDFGVPIIAMPMHNDQPINAKLIVELGVAMEIVRDDDGNIHRGEITETLKDVITGETGEILRGKVRDISKNLKSIREEEMNAAAEELIQLCRNSNKYK Nucleotide sequence of β-1,2-glycosyltransferase SEQ ID NO.4: The amino acid sequence of β-1,3-glycosyltransferase is SEQ ID NO.5: MPNKTETTVRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEED EEVSCLITDALWYFAQSVADSLNLRRLRLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESE LETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQE VLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL The nucleotide sequence of β-1,3-glycosyltransferase is SEQ ID NO.6: Amino acid sequence of wild-type sucrose synthase SEQ ID NO.7: MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQQGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME Amino acid sequence of wild-type β-1,2-glycosyltransferase SEQ ID NO.8: MATLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTLINLESIIKKIPEKYSESIRFVELHLPELPELPPHYHTTNGLPPHLNHTLHKALKMSKPNFSKILQNLKPDLVIYDILQPWAEHVVNEQNIPAVKILTSGAALFSYFFNFLKNPGVEFPFPAIYLPKVEQVKMREMFEKEPNEEDRLAEGNMQIMLMCTSRTIEAKYLDYCTELSNWKVVPVGPPFQDPITNDVDDMELIDWLGTKDENSTVFVCFGSEYFLSREDMEEVAFGLELSNVNFIWVARFPKGEEQNLEDVLPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESLDFGVPIIAMPMHNDQPINAKLIVELGVAMEIVRDDDGNIHRGEITETLKDVITGETGEILRGKVRDISKNLKSIREEEMNAAAEELIQLCRNSNKYK Amino acid sequence of wild-type β-1,3-glycosyltransferase SEQ ID NO.9: MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL。

Claims

1. A multi-enzyme co-expression genetically engineered bacterium, characterized in that: The engineered bacteria can co-express sucrose synthase, β-1,2-glycosyltransferase and β-1,3-glycosyltransferase.

2. The multi-enzyme co-expression genetically engineered bacterium as described in claim 1, characterized in that: The amino acid sequence of the sucrose synthase is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the β-1,2-glycosyltransferase is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the β-1,3-glycosyltransferase is shown in SEQ ID NO.5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

6.

3. The multi-enzyme co-expression genetically engineered bacterium as described in claim 2, characterized in that, The three enzymes are co-expressed in the host bacteria through two or three expression vectors.

4. The multi-enzyme co-expression genetically engineered bacterium as described in claim 3, characterized in that, When the three enzymes are co-expressed in the host bacteria using two expression vectors, the encoding genes for sucrose synthase and β-1,2-glycosyltransferase are linked to the same expression vector, while the encoding gene for β-1,3-glycosyltransferase is linked to another expression vector.

5. The multi-enzyme co-expression genetically engineered bacterium as described in claim 4, characterized in that, The host bacterium is Escherichia coli.

6. A method for preparing the multi-enzyme co-expression genetically engineered bacteria according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Construct a first expression vector containing the gene encoding β-1,3-glycosyltransferase; (2) Construct a second expression vector containing genes encoding sucrose synthase and β-1,2-glycosyltransferase; (3) The vectors constructed in steps (1) and (2) are transformed into the host bacteria to obtain the multi-enzyme co-expression genetically engineered bacteria.

7. A method for synthesizing rebaudioside M using the multi-enzyme co-expression genetically engineered bacteria according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Culture the multi-enzyme co-expression genetically engineered bacteria to obtain whole-cell crude enzyme solution; (2) Using rebaudine A, sucrose, and ADP as substrates, the reaction is carried out under the catalysis of the whole cell crude enzyme solution to generate rebaudine M.

8. The method as described in claim 7, characterized in that, Reaction conditions: pH 5.5-7.5, temperature 55-70℃.

9. The multi-enzyme co-expression genetically engineered bacteria according to any one of claims 1-5, or the method according to any one of claims 7-8, in the catalytic production of rebaudioside M from rebaudioside A.

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