Method for producing rebaudioside D by two-step temperature control one-pot method

Abstract

The invention discloses a method for producing rebaudioside D by a two-step temperature-controlled one-pot method. The method uses an engineering bacterium capable of secreting and expressing glycosyltransferase to transform rebaudioside A into rebaudioside D. The method is characterized in that it comprises the following steps: an induction culture stage: at a temperature of 28°C-30°C, the engineering bacterium is induced to culture; and an enzymatic reaction stage: at a temperature of 32°C-42°C, rebaudioside A is added to carry out an enzymatic reaction, which far exceeds the upper limit of the conversion rate of preparing rebaudioside A by a one-step method.

Description

Technical Field

[0001] The invention relates to a production method of rebaudioside D, belonging to the field of biological fermentation engineering. Background Art

[0002] The Chinese invention patent specification with publication number CN 110846363 A discloses a method for producing rebaudioside D, which comprises the following steps: (1) constructing a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1, wherein the amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID NO.1; (2) culturing the recombinant bacterium 1 in a medium with a pH of 3.5-7.8 to obtain a culture solution; (3) adding substrate rebaudioside A, uridine diphosphate glucose, magnesium sulfate or magnesium chloride, and methanol to the culture solution obtained in step (2), and reacting to obtain rebaudioside D. The recombinant bacterium 1 is obtained by connecting the glycosyltransferase UGT1 gene to an expression vector and then transferring it into a host cell to obtain a first recombinant bacterium 1; or by integrating the glycosyltransferase UGT1 gene into the genome of a host cell by molecular biology technology to obtain a second recombinant bacterium 1; the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO.2.

[0003] The invention patent specification discloses a method for catalyzing rebaudioside A (RebA) to produce rebaudioside D (RebD) by one-pot method: 30 μL of glycerol tube culture of recombinant Pichia pastoris strain XE-3 (recombinant bacteria 1) was inoculated in 3 mL YPD and pre-cultured to OD600 of 6. 1% inoculum (0.25 mL) of the above culture was inoculated into 25 mL BMMY medium with pH = 3.5, at 28°C and 250 rpm, so that the initial OD600 was about 1.0. After culturing for 2 days at 28°C and 250 rpm, feeding was performed, adding RebA with a final concentration of 0.3 g / L, adding uridine diphosphate glucose (UDPG) with a final concentration of 0.2 mM, adding Mg2+ (magnesium sulfate) with a final concentration of 0.5 mM, culturing at 28°C and 250 rpm, adding methanol with a final volume concentration of 0.5% every 24 hours, and measuring the OD600 of the bacteria. The HPLC method was used to analyze the production level of the product RebD in the sample. The conversion rate can be calculated based on the concentration change of rebaudioside A, and the yield can be calculated based on the generated amount of rebaudioside D. When the recombinant bacteria 1 was cultured for 4 days, the conversion rate of substrate rebaudioside A was 39.59%, and the yield of product rebaudioside D was 38.85%.

[0004] The invention patent specification also discloses a second one-pot method for catalyzing rebaudioside A (RebA) to produce rebaudioside D (RebD): 30 μL of the glycerol tube culture of the recombinant Pichia pastoris strain XE-3 (recombinant bacteria 1) was inoculated in 3 mL YPD for pre-culture until the OD600 was 4. 1% inoculum (0.25 mL) of the above culture was inoculated into 25 mL BMMY medium with pH = 7.8, at 28°C and 250 rpm, so that the initial OD600 was about 1.0. 28°C and 250 rpm; feeding was performed at 0.2 days of culture, and RebA with a final concentration of 20 g / L, UDPG with a final concentration of 1.5 mM, and Mg2+ (magnesium chloride) with a final concentration of 5 mM were added, and cultured at 28°C and 250 rpm. Methanol with a final volume concentration of 1.5% was added every 24 hours, and the OD600 of the bacteria was measured. The HPLC method was used to analyze the production level of the product RebD in the sample. The conversion rate can be calculated based on the change in RebA concentration, and the yield can be calculated based on the amount of RebD produced. When the recombinant bacteria 1 was cultured for 4 days, the conversion rate of substrate RebA was 29.10%, and the yield of product RebD was 28.08%.

[0005] The invention patent specification also discloses a third one-pot method for catalyzing rebaudioside A (RebA) to produce rebaudioside D (RebD). Take 30 μL of the glycerol tube culture of the recombinant Pichia pastoris strain XE-3 (recombinant bacteria 1) and inoculate it in 3 mL YPD for pre-culture until OD600 is 3. Inoculate 1% of the above culture (0.25 mL) into 25 mL BMMY medium with pH = 6.5, 28°C, 250 rpm, so that the initial OD600 is about 1.0. After culturing for 1 day at 28°C and 250 rpm, feed is added, RebA with a final concentration of 1 g / L, UDPG with a final concentration of 1 mM, Mg2+ (magnesium sulfate) with a final concentration of 3 mM, and culture at 28°C and 250 rpm. Methanol with a final volume concentration of 1% is added every 24 hours, and the OD600 of the bacteria is measured. The HPLC method is used to analyze the production level of the product RebD in the sample. The conversion rate can be calculated based on the change in RebA concentration, and the yield can be calculated based on the amount of RebD produced. When the recombinant bacteria 1 was cultured for 4 days, the conversion rate of substrate RebA was 68.97%, and the yield of product RebD was 57.75%.

[0006] It can be seen that the RebA conversion rate of the method disclosed in the invention patent specification does not exceed 70%. There are factors that limit the progress of the reaction.

[0007] References:

[0008] [1] Liwen Fei, Yong Wang. Efficient biocatalytic synthesis of sweetener rebaudioside D[J]. Food and Fermentation Industries, 2018, 44(4): 1-7.

[0009] [2] Yang Yufeng, Fei Liwen, Li Jianhua, et al. Synthesis of rebaudioside D by recombinant Escherichia coli whole cells[J]. Industrial Microbiology, 2017, 47(5): 1-7.

[0010] [3] Wang Zhenyu, Yu Hongshan, Jin Fengxie. Activity of endocellulase and glucosidase produced by thermophilic anaerobic bacteria. Journal of Dalian University of Light Industry, 2005, 24(2), 110-114.

[0011] [4] Guan Jin. Application status and development prospects of sweeteners [J]. Contemporary Chemical Industry, 2002, 31(2): 89-91. Zheng Jianxian. Functional food additives [M]. Beijing: China Light Industry Press, 1997.

[0012] [5] Tang Zhifa. The rise and development strategy of stevia[J]. China Food Industry, 1999, (2): 52-52.

[0013] [6] Hellfritsch, C.: Brockhoff, A.; St ahler, F.; Meyerhof, W.; Hofmann, T., Humanpsychometricandtastereceptorresponsestosteviolglycosides. J. Agric. Food Chem. 2012, 60: 6782-6793.

[0014] [7]Olsson, K.; Carlsen, S.; Semmler, A.; Simon, E.; Mikkelsen, MD; BL, Microbial production of next-generation stevia sweeteners. Microb. Cell Fact. 2016, 15, 207-222. Summary of the invention

[0015] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a method for producing rebaudioside D by a two-step temperature-controlled one-pot process that breaks through the upper limit of conversion rate.

[0016] The technical solution adopted by the present invention to solve the above problems is: a method for producing rebaudioside D by a two-step temperature-controlled one-pot method, wherein the method uses an engineered bacterium capable of secreting and expressing glycosyltransferase to transform rebaudioside A into rebaudioside D, and the method is characterized in that it comprises the following steps:

[0017] 1) Induction culture stage: the engineered bacteria are induced to culture at a temperature of 28° C. to 30° C., and this stage lasts for 2 days;

[0018] 2) Enzymatic reaction stage: Enzymatic reaction stage: at a temperature of 32° C.-42° C., rebaudioside A is added for enzymatic reaction, and methanol is added in batches, and the method of adding methanol is related to the temperature.

[0019] Preferably, in the step 2) of the present invention, the enzymatic reaction stage is carried out at a temperature of 35°C.

[0020] Preferably, in the enzymatic reaction stage of step 2) of the present invention, rebaudioside A, UDP-glucose and magnesium chloride are added at once, and methanol is added in batches to carry out the reaction.

[0021] Preferably, during the enzymatic reaction stage of the present invention, methanol is added every 24 hours.

[0022] Preferably, the step 1) induction culture stage and the step 2) enzymatic reaction stage of the present invention are carried out in the same BMMY culture medium.

[0023] Preferably, in the step 1) of the present invention, the induction culture stage is carried out in BMMY medium, the pH of the BMMY medium is 5.5-7.0, and methanol is added in the induction culture stage of step 1), and the concentration of methanol is 0.5%-1.5%.

[0024] Preferably, the relationship between the pH of the BMMY medium of the present invention and the temperature T selected in step 2) is: the conversion rate of rebaudioside A = the conversion rate of rebaudioside A = -0.1537PH 3 +2.875PH 2 -17.311PH+0.000264T 3 -0.02793T 2 +1.02032T+22.68791.

[0025] Preferably, in the step 2) of the present invention, rebaudioside A is added to a final concentration of 1 g / L, uridine diphosphate glucose is added to a final concentration of 1 mM, magnesium chloride is added to a final concentration of 3 mM, and methanol is added to a final concentration of 0.75% every 24 hours.

[0026] Preferably, the temperature needs to be increased when switching from step 1) to step 2) of the present invention, and the temperature increase rate is 0.5-1°C / h.

[0027] Preferably, the temperature of step 2) of the present invention is 42°C.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] (1) Compared with the method described in the specification of publication number CN 110846363 A, the present invention divides one step into two steps, and only adds the substrate rebaudioside A in the second step, thereby reducing the influence of the substrates rebaudioside A and rebaudioside D on the gene expression of the engineered bacteria, greatly improving the conversion rate of rebaudioside A, and breaking through the upper limit of the conversion rate of rebaudioside A in the method described in the specification of publication number CN 110846363 A. In particular, when the temperature in step 2) is selected to be 35°C, the conversion rate can reach 96.92%, which far exceeds the upper limit of the method described in the specification of publication number CN 110846363 A.

[0030] (2) Compared with the method described in the specification of publication number CN 110846363 A, the inventors split the method into two steps. The temperature is set at about 28°C during the induction culture stage, which can effectively express the gene of glycosyltransferase UGT1, so that there is sufficient glycosyltransferase UGT1. If the temperature is increased during the induction culture stage, the expression of the gene of glycosyltransferase UGT1 will be inhibited, and the concentration of glycosyltransferase UGT1 will be reduced. When the concentration of glycosyltransferase UGT1 accumulates to a certain degree (enzymatic reaction stage), the temperature is increased, which can greatly improve the activity of glycosyltransferase UGT1. However, the side effect is that the glycosylation rate of glycosyltransferase UGT1 is also increased, and the expression of glycosyltransferase UGT1 gene is inhibited. Therefore, the temperature and timing need to be considered to improve the conversion rate of rebaudioside A in the one-pot method. The present invention can add methanol in batches, and adjust the timing of methanol addition according to the temperature of the increase, which can break through the ceiling of the conversion rate of rebaudioside A in the one-pot method.

[0031] (3) Extracellular free enzymes have higher substrate accessibility and higher specific activity than immobilized enzymes, so this method is more efficient.

[0032] (4) Rebaudioside D has low solubility (<0.2 g / L) and is easily precipitated. Rebaudioside D can be produced by continuous addition and product removal. This allows the concentration of Rebaudioside D to be reduced, the concentration of Rebaudioside A to be increased, and the production efficiency to be increased.

[0033] (5) During the enzymatic reaction stage, the temperature was 35°C, which is a relatively balanced temperature for various reactions and a relatively balanced temperature for the catalytic reaction activity rate of glycosyltransferase UGT1 and the denaturation and inactivation rate of glycosyltransferase UGT1.

[0034] (6) In step 2) of the present invention, the conversion rate of rebaudioside A is increased again when the temperature is 42°C. Although its final conversion rate of rebaudioside A is not as high as that at 35°C, it is not much different from the final conversion rate at 35°C. The conversion rate of rebaudioside A at 42°C is much faster than that at 35°C. If intermittent production is used during production, the production time can be shortened and the production efficiency can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a curve chart of pH and rebaudioside A conversion rate of Examples 2 to 6.

[0036] Figure 2 It is a curve diagram of temperature T and rebaudioside A conversion rate in Example 7-Example 10. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and by way of examples. The following examples are intended to explain the present invention but the present invention is not limited to the following examples.

[0038] In the embodiment of the present invention:

[0039] The engineered bacteria refers to the recombinant bacteria 1 described in the Chinese invention patent specification with publication number 107446874B.

[0040] Embodiment 1:

[0041] The recombinant bacteria 1 described in the specification with the publication number CN110846363A was used as the engineering bacteria in this embodiment. The construction process of the recombinant bacteria 1 described in the specification of CN110846363A is as follows: GenScript synthesized the glycosyltransferase UGT1 gene, the nucleotide sequence of which is shown in SEQ ID NO.2 in the sequence list (SEQ ID NO.1 is the amino acid sequence of glycosyltransferase UGT1), and the gene was connected to pPICZalphaA by molecular biological methods to obtain the 4954bp pP-UGT1 plasmid shown in SEQ ID NO.3 in the sequence list. The pP-UGT1 plasmid contains the nucleotide sequence of the signal peptide alpha factor (SEQ ID NO.6); the plasmid was transformed into Escherichia coli TOP10F' to obtain the transformant, and 15 μg (or 10 μg) of the pPIC-UGT1 plasmid was extracted from the transformant (Tiangen Company medium-quantity kit). The 4954bp fragment shown in SEQ ID NO.3 was cut into linear fragments with PmeI, extracted with phenol / chloroform, and the linear DNA fragments shown in SEQ ID NO.3 were precipitated with ethanol, dried and dissolved in sterile water to prepare a linear DNA solution with a DNA concentration of 1 μg / μL (or 0.4 μg / μL). The competent cells of Pichia pastoris host bacteria X-33 and Gs115 were prepared by referring to the electroporation transformation method in the Concise Molecular Biology Experiment Guide (Fourth Edition) (P512-513). 80 μL of the competent cells of the Pichia pastoris host bacteria were mixed with 10 μL of 1 μg / μL of the linear DNA solution of the pP-UGT1 plasmid, and then transferred to an electrode cup precooled at 4°C (or any value between 0-4°C), ice bathed for 5 minutes, voltage 1500V, and Eppendorf public The electroporator of the company was used for electric shock for 5ms. Immediately after the electric shock, 1mL of 1M sorbitol aqueous solution precooled at 4°C was added to the electrode cup, mixed, and the mixture was transferred to a 15mL centrifuge tube, and cultured at 29°C for 1h (can be any value between 1-2h), and 100μL (can also be any value between 50μL-200μL) of the cultured mixture was applied to a YPD plate containing 200μg / mL Zeocin (sold by Invitrogen), and cultured inverted at 29°C until the first recombinant bacteria 1 appeared. The first recombinant bacteria 1 was used as the engineering bacteria of this embodiment.

[0042] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0043] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600=4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0044] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0045] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to 0D 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0046] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 5.5 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0047] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 35°C for 4 days;

[0048] ⑥ Detection of conversion rate: The conversion rate of the substrate rebaudioside A of the sample was analyzed by HPLC.

[0049] In the present invention, UGT1 is a glycosyltransferase that can catalyze rebaudioside A to produce rebaudioside D. In this embodiment, the gene of the glycosyltransferase UGT1 is OsEUGTl1, but the glycosyltransferase gene of the present invention is not limited to OsEUGT11.

[0050] Embodiment 2:

[0051] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0052] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0053] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0054] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to 0D 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0055] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution OD600 = 1, and cultured at 220 rpm and 28°C. 1100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0056] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 28°C for 4 days;

[0057] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC method, and finally the conversion rate of rebaudioside A reached 70.07%.

[0058] Embodiment 3:

[0059] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0060] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0061] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0062] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to 0D 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0063] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0064] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 32°C for 4 days;

[0065] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 86.44%.

[0066] Embodiment 4:

[0067] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0068] ① Pre-culture stage: transfer the engineered bacteria to 5 ml of YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until OD 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0069] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0070] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0071] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0072] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 35°C for 4 days;

[0073] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached the highest 96.92%. This conversion rate far exceeds the method described in the specification of publication number CN110846363A.

[0074] Embodiment 5:

[0075] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0076] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0077] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0078] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0079] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0080] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 38°C for 4 days;

[0081] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 82.13%.

[0082] Embodiment 6:

[0083] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0084] ① Pre-culture stage: transfer the engineered bacteria to 5 ml of YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until OD 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0085] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0086] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 600 25, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0087] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0088] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 42°C for 4 days;

[0089] ⑥ Detection of conversion rate: The conversion rate of the substrate rebaudioside A of the sample was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 86.79%.

[0090] The conditions of Example 2-Example 6 are respectively:

[0091] Table 1: Example 2-Example 6 Rebaudioside A conversion rate table

[0092]

[0093] like Figure 1 As shown in Table 1 above, it can be seen that the temperature is raised from 28°C. Because it is a one-pot method, the induction culture stage ④ and the enzymatic reaction stage ⑤ are in the same BMMY medium. The temperature of the enzymatic reaction stage ⑤ is 28°C, 32°C, and 35°C. As the temperature increases, the conversion rate of rebaudioside A conversion rate also increases. When the temperature of the enzymatic reaction stage ⑤ is 35°C-38°C, the conversion rate of rebaudioside A conversion rate decreases. At the temperature of the enzymatic reaction stage ⑤, 35°C, the highest rebaudioside A conversion rate of 96.92% is obtained. This conversion rate is much higher than the rebaudioside A conversion rate that can be obtained by the method recorded in the specification of the publication number CN 110846363 A. When the temperature of ⑤ the enzymatic reaction stage is 42℃, the conversion rate of rebaudioside A begins to increase to 86.79%. Although the conversion rate of rebaudioside A at this temperature is lower than that at 35℃, due to its higher temperature and faster reaction rate, equilibrium can be reached in a shorter time, and the loss of rebaudioside A conversion rate is not much compared with that at 35℃. This phenomenon can be used to use 42℃ in production to increase production speed and achieve a higher rebaudioside A conversion rate.

[0094] Embodiment 7:

[0095] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0096] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0097] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0098] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 60025, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0099] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 5.5 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0100] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 28°C for 4 days;

[0101] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 27.87%.

[0102] Embodiment 8:

[0103] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0104] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0105] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0106] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 60025, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0107] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 6.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0108] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 28°C for 4 days;

[0109] ⑥ Detection of conversion rate: The conversion rate of the substrate rebaudioside A of the sample was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 43.05%.

[0110] Embodiment 9:

[0111] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0112] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0113] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0114] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 60025, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0115] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 6.5 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0116] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 28°C for 4 days;

[0117] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC method, and finally the conversion rate of rebaudioside A reached 62.91%.

[0118] Embodiment 10:

[0119] The method for producing rebaudioside D by the two-step temperature-controlled one-pot method of this embodiment comprises the following steps:

[0120] ① Pre-culture stage: transfer the engineered bacteria to 5 ml YPD medium at a 1% inoculum volume for pre-culture at 220 rpm and 28°C until 0D 600 =4, YPD medium is 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the balance is water;

[0121] ② The second culture stage: The pre-cultured bacterial solution of step ① was transferred to 25 ml of BMGY medium at an inoculum size of 1%, and cultured at 220 rpm and 28°C until OD600 = 4. The BMGY medium contained 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0), 4 x 10 -5 % biotin, 10 g / L glycerol, the balance is water;

[0122] ③ Centrifugation stage: Centrifuge the cultured bacterial solution in step ② at 5000×g and 4°C for 10 min to harvest the bacterial cells, discard the supernatant, and resuspend the bacterial cells in BMMY medium (pH 5.5) to an OD of 0. 60025, BMMY medium is 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB) without amino acids, 100 mM potassium phosphate (pH = 5.0, 5.5, 6.0, 6.5, 7.0), 4x10 -5 % biotin, the volume concentration of methanol is 0.75%, and the balance is water;

[0123] ④ Induction culture stage: 1 ml of the resuspended bacterial solution was transferred to 24 ml of BMMY medium with pH = 7.0 and a final methanol concentration of 0.75%, so that the concentration of the bacterial solution was OD600 = 1, and cultured at 220 rpm and 28°C. 100% methanol was added every 24 hours to a final concentration of 0.75% to induce the engineered bacteria to secrete and express UGT1 for two days;

[0124] ⑤ Enzymatic reaction stage: on the third day, rebaudioside A, UDP-glucose and magnesium chloride were added to start the enzymatic catalytic reaction stage, and the temperature was raised to 28°C for 4 days;

[0125] ⑥ Detection of conversion rate: The conversion rate of the sample substrate rebaudioside A was analyzed by HPLC, and finally the conversion rate of rebaudioside A reached 62.41%.

[0126] The parameters of Examples 7-10 are shown in the following table:

[0127] Table 2: Rebaudioside A conversion rate corresponding to the initial pH value of BMMY culture medium at different induction culture stages.

[0128]

[0129] In Examples 7 to 10, as the pH value increases, when pH=5.5 to 6.5, the conversion rate of rebaudioside A increases, and when pH=6.5 to 7.0, the conversion rate of rebaudioside A decreases. However, in Example 9 at pH=6.5 and Example 10 at pH=7.0, the conversion rates of rebaudioside A are not much different. Sequence Listing <110> Tianjin University Sinochem Health Industry Development Co., Ltd. <120> Method for producing rebaudioside D by two-step temperature control one-pot method <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 497 <212> PRT <213> Artificial Sequence <400> 1 Glu Phe Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly 1 5 10 15 Met His Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro 20 25 30 Cys Leu Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser 35 40 45 Phe Val Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro 50 55 60 Ala Leu Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val 65 70 75 80 Glu Gly Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp 85 90 95 Arg Pro Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala 100 105 110 Ala Pro Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile 115 120 125 Val Asp Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys 130 135 140 Val Pro Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser 145 150 155 160 Ile Ala Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala 165 170 175 Ala Gly Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg 180 185 190 Met Lys Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu 195 200 205 Arg Phe Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser 210 215 220 Cys Val Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg 225 230 235 240 Gly Lys Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly 245 250 255 Arg Arg Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln 260 265 270 Pro Ala Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu 275 280 285 Gly Val Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly 290 295 300 Thr Arg Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala 305 310 315 320 Asp Leu Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val 325 330 335 Val Ala Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala 340 345 350 Val Gly Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly 355 360 365 Leu Met Phe Gly His Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln 370 375 380 Gly Pro Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val 385 390 395 400 Ala Arg Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala 405 410 415 Ala Ile Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln 420 425 430 Ala Lys Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His 435 440 445 Glu Arg Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp 450 455 460 Leu Val Pro Arg Ala Ala Ala Ala Ala Ser Phe Leu Glu Gln Lys Leu 465 470 475 480 Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His 485 490 495 His <210> 2 <211> 1758 <212> DNA <213> Artificial Sequence <400> 2 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240 tctctcgaga aaagagaggc tgaagctgaa ttcatggact ctggttactc ttcttcttac 300 gctgctgctg ctggtatgca cgttgttatc tgtccatggt tggctttcgg tcacttgttg 360 ccatgtttgg acttggctca aagattggct tctagaggtc acagagtttc tttcgtttct 420 actccaagaa acatctctag attgccacca gttagaccag ctttggctcc attggttgct 480 ttcgttgctt tgccattgcc aagagttgaa ggtttgccag acggtgctga atctactaac 540 gacgttccac acgacagacc agacatggtt gaattgcaca gaagagcttt cgacggtttg 600 gctgctccat tctctgaatt tttgggtact gcttgtgctg actgggttat cgttgacgtt 660 ttccaccact gggctgctgc tgctgctttg gaacacaagg ttccatgtgc tatgatgttg 720 ttgggttctg ctcacatgat cgcttctatc gctgacagaa gattggaaag agctgaaact 780 gaatctccag ctgctgctgg tcaaggtaga ccagctgctg ctccaacttt cgaagttgct 840 agaatgaagt tgatcagaac taagggttct tctggtatgt ctttggctga aagattctct 900 ttgactttgt ctagatcgtc tttggttgtt ggtagatcgt gtgttgaatt tgaaccagaa 960 actgttccat tgttgtctac tttgagaggt aagccaatca ctttcttggg tttgatgcca 1020 ccattgcacg aaggtagaag agaagacggt gaagacgcta ctgttagatg gttggacgct 1080 caaccagcta agtctgttgt ttacgttgct ttgggttctg aagttccatt gggtgttgaa 1140 aaggttcacg aattggcttt gggtttggaa ttggctggta ctagattctt gtgggctttg 1200 agaaagccaa ctggtgtttc tgacgctgac ttgttgccag ctggtttcga agaaagaact 1260 agaggtagag gtgttgttgc tactagatgg gttccacaaa tgtctatctt ggctcacgct 1320 gctgttggtg ctttcttgac tcactgtggt tggaactcta ctatcgaagg tttgatgttc 1380 ggtcacccat tgatcatgtt gccaatcttc ggtgaccaag gtccaaacgc tagattgatc 1440 gaagctaaga acgctggttt gcaagttgct agaaacgacg gtgacggttc tttcgacaga 1500 gaaggtgttg ctgctgctat cagagctgtt gctgttgaag aagaatcttc taaggttttc 1560 caagctaagg ctaagaagtt gcaagaaatc gttgctgaca tggcttgtca cgaaagatac 1620 atcgacggtt tcatccaaca attgagatcg tacaaggact tggtacctcg agccgcggcg 1680 gccgccagct ttctagaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac 1740 catcatcatc atcatcat 1758 <210> 3 <211> 4954 <212> DNA <213> Artificial Sequence <400> 3 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900 caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960 tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140 tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1200 tgaagctgaa ttcatggact ctggttactc ttcttcttac gctgctgctg ctggtatgca 1260 cgttgttatc tgtccatggt tggctttcgg tcacttgttg ccatgtttgg acttggctca 1320 aagattggct tctagaggtc acagagtttc tttcgtttct actccaagaa acatctctag 1380 attgccacca gttagaccag ctttggctcc attggttgct ttcgttgctt tgccattgcc 1440 aagagttgaa ggtttgccag acggtgctga atctactaac gacgttccac acgacagacc 1500 agacatggtt gaattgcaca gaagagcttt cgacggtttg gctgctccat tctctgaatt 1560 tttgggtact gcttgtgctg actgggttat cgttgacgtt ttccaccact gggctgctgc 1620 tgctgctttg gaacacaagg ttccatgtgc tatgatgttg ttgggttctg ctcacatgat 1680 cgcttctatc gctgacagaa gattggaaag agctgaaact gaatctccag ctgctgctgg 1740 tcaaggtaga ccagctgctg ctccaacttt cgaagttgct agaatgaagt tgatcagaac 1800 taagggttct tctggtatgt ctttggctga aagattctct ttgactttgt ctagatcgtc 1860 tttggttgtt ggtagatcgt gtgttgaatt tgaaccagaa actgttccat tgttgtctac 1920 tttgagaggt aagccaatca ctttcttggg tttgatgcca ccattgcacg aaggtagaag 1980 agaagacggt gaagacgcta ctgttagatg gttggacgct caaccagcta agtctgttgt 2040 ttacgttgct ttgggttctg aagttccatt gggtgttgaa aaggttcacg aattggcttt 2100 gggtttggaa ttggctggta ctagattctt gtgggctttg agaaagccaa ctggtgtttc 2160 tgacgctgac ttgttgccag ctggtttcga agaaagaact agaggtagag gtgttgttgc 2220 tactagatgg gttccacaaa tgtctatctt ggctcacgct gctgttggtg ctttcttgac 2280 tcactgtggt tggaactcta ctatcgaagg tttgatgttc ggtcacccat tgatcatgtt 2340 gccaatcttc ggtgaccaag gtccaaacgc tagattgatc gaagctaaga acgctggttt 2400 gcaagttgct agaaacgacg gtgacggttc tttcgacaga gaaggtgttg ctgctgctat 2460 cagagctgtt gctgttgaag aagaatcttc taaggttttc caagctaagg ctaagaagtt 2520 gcaagaaatc gttgctgaca tggcttgtca cgaaagatac atcgacggtt tcatccaaca 2580 attgagatcg tacaaggact tggtacctcg agccgcggcg gccgccagct ttctagaaca 2640 aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg 2700 agtttgtagc cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg 2760 gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct 2820 tcatttttga tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg 2880 tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga atactcttg 2940 gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt 3000 cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt 3060 caaaatgttt ctactcctttt tttactctttc cagatttct cggactccgc gcatcgccgt 3120 accacttcaa aacccaag cacagcatac taattttcc ctctttcttc ctctagggtg 3180 tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt 3240 cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa attttttttt 3300 ttagttttt tctctttcag tgacctccat tgatatttaa gttaataaac ggtcttcaat 3360 ttctcaagtt tcagtttcat ttttcttgtt ctattacaac ttttttact tcttgttcat 3420 tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt gacaattaat catcggcata 3480 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag 3540 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 3600 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 3660 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt 3720 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 3780 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt 3840 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca 3900 cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg 3960 atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa 4020 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta 4080 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg 4140 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg 4200 ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4260 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4320 aaaatcgacg ctcaagtcag aggtggcga acccgacagg actataaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4440 tgtccgcctt tctcccttcg ggagcgtgg cgctttctca atgctcacgc tgtaggtatc 4500. tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4560. ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatc <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 gactggttcc aattgacaag c <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 gcaaatggca ttctgacatc c <210> 6 <211> 267 <212> DNA <213> Artificial Sequence <400> 6 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 120. ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc tgaagct <210> 7 <211> 99 <212> DNA <213> Artificial Sequence <400> 7 atggtctcct tcacctccct cctcgccggc gtcgccgcca tctcggggcgt cttggccgct cccgccgccg aggtcgatc cgtggctgtg gagagcgc <210> 8 <211> 123 <212> DNA <213> Artificial Sequence <400> 8 atgaaaaaaa tgagtttgtt tcaaaatatg aaatcaaaac ttctgccaat cgccgctgtt 60 tctgtcctta cagctggaat ctttgccgga gctgagcttc agcaaacaga aaaggccagc 120 gcc 123 <210> 9 <211> 168 <212> DNA <213> Artificial Sequence <400> 9 atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 60 aacaatacgt ttgaccgccg caaatttatt caaggagcgg ggaagattgc aggactttct 120 cttggattaa cgattgccca gtcggttggg gcctttgaag taaatgct 168 <210> 10 <211> 99 <212> DNA <213> Artificial Sequence <400> 10 atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60 tttcatttgg ttctggcagg accggcggct gcgagtgct 99 <210> 11 <211> 57 <212> DNA <213> Artificial Sequence <400> 11 atgagatttc catctatttt tactgctgtt ttgtttgctg cttcttctgc tttggct 57 <210> 12 <211> 57 <212> DNA <213> Artificial Sequence <400> 12 atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcccaa 57

Claims

1. A method for producing rebaudioside D by a two-step temperature-controlled one-pot method, wherein the method uses an engineered bacterium capable of secreting and expressing glycosyltransferase to transform rebaudioside A into rebaudioside D, characterized in that The steps include: 1) Induction culture stage: the engineered bacteria are induced to culture at a temperature of 28° C. to 30° C.; 2) Enzymatic reaction stage: adding rebaudioside A to carry out enzymatic reaction at a temperature of 32°C-45°C; Wherein, the glycosyltransferase is as shown in Sequence 1; the engineering bacteria is a Pichia pastoris host bacteria; The step 1) induction culture stage and the step 2) enzymatic reaction stage are performed in the same BMMY culture medium; In the step 2) of the enzyme-contacted reaction, rebaudioside A with a final concentration of 1-60 g / L, UDP-glucose with a final concentration of 1 mM, and magnesium chloride with a final concentration of 3 mM are added at once, and methanol with a final concentration of 0.75% is added every 24 hours.

2. The method for producing rebaudioside D by a two-step temperature-controlled one-pot process according to claim 1, characterized in that: In the step 2) of enzymatic reaction, the temperature is 35° C. for reaction.

3. The method for producing rebaudioside D by a two-step temperature-controlled one-pot process according to claim 1, characterized in that: In the step 1), the induction culture stage is carried out in BMMY medium, the pH of the BMMY medium is 5.0-8.0, and methanol is added in the induction culture stage of step 1), and the concentration of methanol is 0.5%-1.5%.

4. The method for producing rebaudioside D by a two-step temperature-controlled one-pot process according to claim 1, characterized in that: The temperature needs to be increased when switching from step 1) to step 2), and the temperature increase rate is 0.5-1°C / h.

5. The method for producing rebaudioside D by a two-step temperature-controlled one-pot process according to claim 1, characterized in that: The temperature of step 2) is 42°C.

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