CN110734944B - Method for synthesizing rebaudioside M by one-step method - Google Patents

Abstract

The invention discloses a method for synthesizing lecitin M by a one-step method, which comprises the following steps: (1) The recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacterium 2 capable of secreting and expressing glycosyltransferase UGT2 are mixed and inoculated in a culture medium containing methanol for culture; (2) Adding a substrate rebaudioside A into the culture solution obtained in the step (1), adding uridine diphosphate glucose, magnesium sulfate or magnesium chloride and methanol, and reacting to obtain lygodiiside M; the invention overcomes the complex steps of cell breaking, separation and purification or adding cell membrane penetrating agent and the like in the prior art; compared with the expensive rebaudioside D in the prior art, the raw material rebaudioside A has low price. The yield and the thermal stability of the recombinant bacterial glucoside transferase are improved, and the mixed bacteria catalysis one-step method for obtaining the RebM by directly using the low-value RebA as a substrate is realized. The catalytic efficiency is improved, and the cost for enzyme purification and the cost for substrate are reduced.

Description

One-step method for synthesizing rebaudioside M

technical field

The invention belongs to the field of bioengineering, and in particular relates to a method for synthesizing rebaudioside M in one step.

Background technique

Steviol glycoside is a new type of natural low-calorie sweetener steviol glycoside compound extracted from stevia leaves of Compositae herb, because of its high sweetness, low calorie, non-toxicity, high temperature resistance, acid and alkali resistance and good water solubility It has been certified as safe by the US Food and Drug Administration and can be used in the food industry ^[1-5] . Rebaudioside M (RebM) has better taste characteristics, but its content in dry weight of leaves is less than 0.1%, resulting in high isolation cost and high price. Obtaining high concentration of RebM by biocatalysis has attracted the attention of scholars. It is currently reported that the recombinase derived from Stevia rebaudiana can catalyze RebD to generate RebM, but the yield is low ^[3-4] .

Using RebD as a substrate, RebM can be obtained by biocatalysis, but there are currently the following problems in the biocatalysis process: (1) Biocatalysis requires efficient glycosyltransferases, and the content of glycosyltransferases in plants Very low, far below the level of practical application, and difficult to purify; (2) the recombinant glycosyltransferase expressed in the cell can catalyze the generation of RebD product RebM, but RebD content is also lower than 0.1% in Stevia and also expensive (3) The recombinant enzymes produced by intracellular expression need to be centrifuged to collect the cells and break down the cells to be catalyzed, and the steps are cumbersome; (4) During the reaction process, due to thermal inhibition, most of the activities of the glycosyltransferases will be reduced. lost. (5) Obtaining recombinant enzymes from recombinant bacteria, the preparation and catalysis of enzymes are time-consuming, labor-intensive and expensive ^[3-9] . Based on the above research status, there is an urgent need for a low-cost, large-yield substrate and a less-step, fast, effective, and low-cost method to obtain rebaudioside M.

This technology will also be a feasible way to realize the industrial biocatalytic preparation of RebM.

references:

[1] Hao Difei. Overview of the application of synthetic sweeteners in the food industry [J]. Biology Teaching, 2017, 42(10): 10-12.

[2] Yuan Guangmei, Zuo Meiwen, Tan Ying, etc. Research progress on the extraction of inulin and its application in bread [J]. Food Safety Guide, 2018, 09: 142.

[3] Prakash I, Markosyan A, Bunders C. Development of next generation stevia sweetener: rebaudioside M [J]. Foods, 2014, 3(1): 162-175.

[4] Wan Huida, Li Dan, Xia Yongmei. Progress in functional research of steviol glycosides [D]. Food Science, 2015, 36(17): 264-269.

[5] Tang Zhifa. The Rise and Development Strategy of Stevia [J]. China Food Industry, 1999, (2): 52-52.

[6] Hu Guohua. Compound food additives [M]. Beijing: Chemical Industry Press, 2006.

[7] Yu Boying, Wang Ninglin, Li Guojing, etc. Application progress of genetic engineering and metabolic engineering in stevioside production [J]. Biotechnology Bulletin, 2015, 31(9): 8-14

[8] Kumari N and Kumar S, Chemistry and analytical techniques for ent-kaurene-glycosides of Stevia rebaudiana Bertoni-Areview. 2017. Journal of Applied and Natural Science 9(4): 2114-2126

[9] Li Yan, Yan Ming, Chen Liangliang. A method for enzymatically preparing rebaudioside M [P]. China: 107666834, 2018-08-10.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for synthesizing rebaudioside M in one step.

Technical scheme of the present invention is summarized as follows:

The method for synthesizing recidiside M by one-step method comprises the steps:

(1) The recombinant bacteria 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacteria 2 capable of secreting and expressing glycosyltransferase UGT2 are mixed and inoculated in a medium containing methanol for cultivation;

(2) Add the substrate rebaudioside A to the culture medium obtained in step (1), add uridine diphosphate glucose, magnesium sulfate or magnesium chloride, methanol, and react to obtain rebaudioside M;

The amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID NO.1;

The amino acid sequence of the glycosyltransferase UGT2 is shown in SEQ ID NO.5.

Step (1) is preferably: the recombinant bacteria 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacteria 2 capable of secreting and expressing glycosyltransferase UGT2 are mixed and inoculated in a medium containing methanol for 2h-3 days, and the The ratio of the cell concentration when the recombinant bacterium 1 and the recombinant bacterium 2 were inoculated was 1:0.7-2, the total concentration of the recombinant bacterium 1 and the recombinant bacterium 2 was controlled at the light absorbing value OD600 of the bacterial liquid to be 1, and the pH of the medium containing methanol= 5.5-8, the volume concentration of methanol in the medium containing methanol is 0.5%-1.5%.

Step (2) is preferably: to the culture medium obtained in step (1), adding a final concentration of 0.5-20g/L substrate rebaudioside A, adding a final concentration of 0.2-1.5mM uridine diphosphate glucose, adding a final Magnesium sulfate or magnesium chloride with a concentration of 0.5-5mM, and methanol with a final concentration of 0.5%-1.5% were added every 24 hours for reaction to obtain recidiside M.

The recombinant bacterium 1 is constructed by the following steps: the glycosyltransferase UGT1 gene is connected to the expression vector and then transferred into the host cell 1 to obtain the first recombinant bacterium 1; or the glycosyltransferase UGT1 gene is integrated into the The genome of the host cell 1 is used to obtain the second recombinant bacterium 1; the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO.2.

The expression vector is pPICZalphaA/B/C, pPIC9K, pPIC9, pPinkα-HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vector.

The host cell 1 is Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis.

The recombinant bacterium 2 is constructed by the following steps: the glycosyltransferase UGT2 gene is connected to the expression vector and then transferred into the host cell 1 to obtain the first recombinant bacterium 2; or the glycosyltransferase UGT2 gene is integrated into the The genome of the host cell 1 is used to obtain the second recombinant bacterium 2; the nucleotide sequence of the glycosyltransferase UGT2 gene is shown in SEQ ID NO.4.

The expression vector is pPICZalphaA/B/C, pPIC9K, pPIC9, pPinkα-HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vector.

The host cell 1 is Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis.

The signal peptide used by recombinant strain 1 to secrete UGT1 protein is the signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha, and SED1, and the nucleotide sequence corresponding to the signal peptide is SEQ ID NO.9, SEQ ID NO .10, shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15;

The signal peptides used by recombinant strain 2 to secrete UGT1 protein are the signal peptides of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha, and SED1, and the nucleotide sequences corresponding to the signal peptides are SEQ ID NO.9, SEQ ID NO .10, shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15.

Advantages of the present invention:

1. Overcome the deficiency that the prior art cannot purify glycosyltransferase in large quantities from stevia rebaudiana.

2. One-step synthesis overcomes the cumbersome steps of cell destruction, separation and purification, or addition of cell membrane permeabilizers required for catalysis in the prior art.

3. The existing biocatalysis technology uses the substrate rebaudioside D (RebD) to produce rebaudioside M (RebM). Compared with the substrate rebaudioside A (RebA) used in this technology, RebD is more The price is expensive, and the present invention uses relatively cheap RebA as a substrate to reduce production costs.

4. The mixed bacteria fermentation of the present invention, adding RebA to the fermentation liquid, can produce RebM in one step. RebM is a sweetener with high sweetness and good taste. The method of the present invention can obtain secreted and expressed glycosyltransferase, which can improve the yield and thermal stability of recombinant bacterial glycosyltransferase, and realize the direct use of RebA with low value as RebM was obtained by a one-step method catalyzed by mixed bacteria with substrates. Improve catalytic efficiency, reduce the cost of enzyme purification and substrate costs, and obtain RebM with high sweetness, good taste and high value.

Description of drawings

Fig. 1 is a construction diagram of a recombinant vector containing glycosyltransferase UGT1 and UGT2 genes. Wherein a is the map of plasmid pP-UGT1, including UGT1 gene; b is the map of pP-UGT2 plasmid, including UGT2 gene.

Fig. 2 is the electrophoretic pattern of PCR identification of recombinant Pichia pastoris transformants of glycosyltransferase UGT1 and UGT2 genes. Lane M is DNA standard molecular weight marker; the lanes in a are the electrophoresis patterns of PCR products of UGT1 transformants and control strain X33 respectively. b Each lane is the electrophoresis pattern of PCR products of UGT2 transformants and control strain X33, respectively. CK is the result of electrophoresis of PCR products with water as a template.

Fig. 3 Electrophoresis diagram of total protein in culture supernatant of each transformant. Wherein a is the electrophoresis pattern of UGT1 secreted and expressed by the transformant; b is the electrophoresis pattern of UGT2 secreted and expressed by the transformant.

Figure 4 Recombinant strain 1 and recombinant strain 2 expressed UGT2 in recombinant Pichia pastoris under various methanol concentrations. On the 3rd day of culture for the control bacteria X33, the recombinant strain 1 and the recombinant strain 2, the culture supernatant was taken and concentrated 5 times for electrophoresis. a and b are the electropherograms of UGT1 and UGT2 secreted by recombinant strain 1 and recombinant strain 2, respectively, when cultured at various methanol concentrations.

Figure 5 SDS-PAGE of purified UGT1 and UGT2. Electrophoresis of purified culture supernatants of recombinant strain 1 and recombinant strain 2 on day 3, a is the electrophoresis of purified UGT1; b is the electrophoresis of purified UGT2.

Fig. 6 Determination of strain growth and protein concentration during the one-step production of RebM by recombinant strain 1 and recombinant strain 2.

a is the growth curve of bacterial strain EX-3 and XS-35 under 4 kinds of experimental conditions, and b is the growth curve of the culture supernatant of the 3rd day and the 4th day (that is, the 2nd day and the 3rd day after feeding) of the bacterial strain protein concentration. 1#: pH 6.0, ratio of EX-3 and XS-35 1:1, feeding on the second day; 2#: pH 6.0, ratio of EX-3 and XS-35 1:2, feeding on the third day; 3# : pH 7.3, ratio of EX-3 and XS-35 1:1, feeding on the 3rd day; 4#: pH 7.3, ratio of EX-3 and XS-35 1:2, feeding on the 2nd day.

Detailed ways

Escherichia coli TOP10F' (commercial).

The present invention will be further described below in conjunction with specific examples.

The expression vectors are known as pPICZalphaA/B/C, pPIC9K, pPIC9, pPinkα-HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vectors.

Embodiment 1 The synthesis of transfer glycosidase UGT2 and UGT1 gene and the construction of expression vector

GenScript's synthetic glycosyltransferase UGT1 gene, the nucleotide sequence of this gene is shown in SEQ ID NO.2 in the sequence table (SEQ ID NO.1 is the amino acid sequence of glycosyltransferase UGT1), the gene is passed The method of molecular biology was connected to pPICZalphaA to obtain the 4954bp pP-UGT1 plasmid shown in SEQ ID NO.3 in the sequence listing. The pP-UGT1 plasmid (FIG. 1) contains the nucleotide sequence (SEQ ID NO. 9) of the signal peptide alpha factor. Transform the transformant obtained by transforming Escherichia coli TOP10F' with this plasmid, and extract 15 μg (or 10 μg) of pPIC-UGT1 plasmid (Mid-quantity kit from Tiangen Company) from the transformant. Cut the 4954bp fragment shown in linear SEQ ID NO.3 with PmeI enzyme, after phenol/chloroform extraction, ethanol precipitation linear DNA fragment shown in linear SEQ ID NO.3, be dissolved in sterile water after drying, prepare Prepare a linear DNA solution with a DNA concentration of 1 μg/μL (or 0.4 μg/μL).

Synthesize the nucleotide sequence shown in SEQ ID NO.4 (SEQ ID NO.5 is the amino acid sequence of glycosyltransferase UGT2) in the sequence listing in the same way, and connect to pPICZalphaA with the method of molecular biology to obtain pP with a length of 4861bp -UGT2 plasmid (SEQ ID NO.6 in the sequence listing) and its linearized DNA solution containing the UGT1 gene.

Example 2 The glucosyltransferase UGT2 and UGT1 genes were respectively integrated into the Pichia pastoris chromosome:

Prepare competent cells of Pichia pastoris host strains X-33 and Gs115 by referring to the method for preparing yeast competent cells by electroporation transformation in the refined Molecular Biology Experiment Guide (Fourth Edition) (P512-513), and 80 μL Mix the competent cells of Pichia pastoris host bacteria with 10 μL of 1 μg/μL linear DNA solution of pP-UGT1 plasmid, and then transfer to 4°C (or any value between 0-4°C) for pre-treatment. In a cold electrode cup, ice bath for 5 minutes, voltage 1500V, electric shock with eppendorf company electroporator, electric shock 5ms, immediately add 1mL of 4 ℃ pre-cooled 1M sorbitol aqueous solution to the electrode cup, mix well, and mix Transfer the solution to a 15mL centrifuge tube, and culture it statically at 29°C for 1h (it can be any value in 1-2h), and smear 100μL on a YPD plate containing 200μg/mL Zeocin (sold by Invitrogen Company) (it can also be in 50 μL-200 μL) of the cultured mixture, and culture it upside down at 29°C until the first recombinant strain 1 appears.

The pP-UGT2 plasmid was extracted by the same method as above, and after linearization, the UGT2 gene was transformed into X-33 and Gs115 competent cells by electroporation to obtain the first recombinant strain 2.

YPD solid medium is yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 18g/L.

Example 3 PCR identification of Pichia pastoris transformants containing glucosyltransferase UGT1 and UGT2 genes

Transformants in the previous laboratory were identified by colony PCR. After centrifuging the bacteria preserved in glycerol tubes, the supernatant was discarded, and the concentrated bacteria were used as PCR amplification templates, using exogenous gene-specific primers as shown in SEQ ID NO.7 in the sequence listing and SEQ ID NO.8 in the sequence listing The sequence is amplified, and if the recombinant plasmid is transformed into the host cell, the target fragment of the expected size can be amplified.

Take 20 μL of glycerol bacteria, centrifuge at 12000 rpm/4°C/5 min, discard the supernatant, and precipitate as a colony PCR template. Prepare 15 μL reaction system 2×Rapid Taq Master Mix 7.5 μL in a 200 μL PCR tube, 0.5 μL of the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 are upstream and downstream primers, 6.5 μL of sterile water, use The pP-UGT2 plasmid was used as a template as a positive control; wild-type Pichia pastoris genomic DNA was used as a template as a negative control; sterile water was used as a template as a negative control. The thermal cycle program of the PCR reaction was denaturation at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 3 min. Prepare 1% agarose gel (1% agar, 1.5‰EB, TAE buffer). Take the PCR product spotting, 170V electrophoresis for 30min. Scan the electropherogram.

The electrophoresis of the PCR product was detected as shown in Figure 2.

The PCR identification results of the transformants containing the UGT1 gene are shown in Figure 2a. PCR can amplify the bands of the 1950bp target gene UGT1 and the 2.2kb AOX1 gene, indicating that the UGT1 genes of these transformants have been successfully integrated into Pasteurby Chromosome of Saccharomyces erythrocerevisiae X-33 strain (Fig. 2a). The results of PCR identification of transformants containing UGT2 gene are shown in Figure 2b. PCR can amplify the band of 1857bp target gene and 2.2kb AOX1 gene, indicating that the UGT2 gene of these transformants has been successfully integrated into Pasteur Pichia strain X-33 on the chromosome (Fig. 2b).

Example 4 Methanol induces UGT1 gene expression of Pichia pastoris transformants

① Inoculate recombinant Pichia pastoris XE-3, XE-4, XE-9, XE-12, XE-13, XE-14, XE- 15. XE-16, XE-17, XE-18, XE-19, XE-20, and Pichia pastoris host strain X33 were respectively transferred to 2ml of YPD medium with pH=5.5 for preculture, 220 rpm /min, cultured at 29°C until OD ₆₀₀ =4;

②Transfer the pre-cultured bacterial solution in step ① into 25ml of BMGY medium at a volume ratio of 1:100, culture at 220 rpm at 29°C until OD ₆₀₀ =4;

③Transfer the bacterial solution cultivated in step ② to 50ml BMMY medium with pH=5.5 and methanol volume concentration of 0.75% respectively, so that the concentration of the bacterial solution OD ₆₀₀ =1, shake at 220 rpm at 29°C Methanol was supplemented every 24 hours, and the volume of methanol added was 0.75% of the volume of the medium to induce the secretion and expression of UGT1 by the recombinant bacteria. In order to detect that the glycosyltransferase gene UGT1 can be effectively expressed and the target protein is secreted outside the cells of the recombinant Pichia pastoris, the growth medium of the recombinant bacteria was taken from the first day to the eighth day after induction for crude enzyme solution Activity determination, protein electrophoresis and protein concentration determination. The sampling method is as follows: take 500 μl of medium, after centrifugation, take the supernatant to measure the enzyme activity. As the induction time increases, the more the target protein UGT1 is secreted, while the control strain Pichia pastoris host strain X33 does not secrete the target protein UGT1 (Figure 3 upper figure), and the secreted and expressed UGT1 of each strain is also different , wherein recombinant strains such as XE3 and XE4 secrete a large amount of recombinant protein UGT1. The XE-3 strain expressed the highest expression level of the target protein UGT1 and was named recombinant strain 1. Recombinant strain 1 can express UGT1 when cultured for 1-7 days, and the expression level has reached a higher level on the 3rd day.

The speed of cultivation in steps ① and ② can also be any value in 200-250 rpm; the culture temperature can also be any value in 28°C-30°C; the concentration OD ₆₀₀ of the culture bacteria can also be 2- Any one of 6 values. In step ③, the volume concentration of methanol in the BMMY medium is any value in 0.5%-1%; the concentration _OD600 of the bacteria can also be any value in the range of 0.8-1.5; the rotating speed can also be 200-250 rpm Any value in the medium; the culture temperature can also be any value in the range of 28°C-30°C, methanol is supplemented every 24 hours, and the volume of methanol added is any value in the range of 0.5%-1% of the medium volume.

BMGY medium is 10g/L yeast extract, 20g/L peptone, 13.4g/L yeast nitrogen base, 100mM potassium phosphate, 4x10 ^-4 g/L biotin, 10g/L glycerol, and the balance is water.

BMMY medium is 10g/L yeast extract, 20g/L peptone, 13.4g/L yeast nitrogen base, 100mM potassium phosphate, 4x10 ^-4 g/L biotin, the volume concentration of methanol is 0.5%-1%, and the balance for water.

The MMH medium is 13.4g/L yeast nitrogen base, 4×10 ^-4 g/L biotin, the volume concentration of methanol is 0.5%-1%, and the balance is water.

The BMMH medium is 13.4g/L yeast nitrogen base, 100mM potassium phosphate, 4× ^{10 -4} g/L biotin, the volume concentration of methanol is 0.5%-1%, and the balance is water.

Recombinant strain 1 is an engineering strain containing UGT1 gene, and the host cell can also be Saccharomyces cerevisiae or Bacillus subtilis.

When the host cell is Pichia pastoris, the expression vector is pPIC9K, pPIC9 or pPinkα-HC.

The host cell is Saccharomyces cerevisiae, and the expression vectors selected are pYES2, YCplac33, YEplac195,

When the host cell is Bacillus subtilis, the expression vectors selected are pHT01, pHT08 or pHT43.

In order to effectively secrete the target protein outside the host strain cells, the signal peptides selected are alpha factor (the nucleic acid sequence is SEQ ID NO.9), xyn2s (the nucleic acid sequence is SEQ ID NO.10), phoA (the nucleic acid sequence is SEQ ID NO. 11), phoD (the nucleic acid sequence is SEQ ID NO.12), amyE (the nucleic acid sequence is SEQ ID NO.13), MFalpha (the nucleic acid sequence is SEQ ID NO.14) and SED1 (the nucleic acid sequence is SEQ ID NO.15).

Example 5 Methanol induces UGT2 gene expression of Pichia pastoris transformants

① Inoculation of Pichia pastoris host strain X33 and recombinant Pichia pastoris XS-21, XS-22, XS-28, XE-35 containing glucosyltransferase gene UGT2 identified in Example 3 , respectively transferred to 2ml of YPD medium with pH=5.5 for pre-cultivation, 220 rpm, 29°C to OD ₆₀₀ =4;

②Transfer the pre-cultured bacterial solution in step ① into 25ml of BMGY medium at a volume ratio of 1:100, culture at 220 rpm at 29°C until OD ₆₀₀ =4;

③Transfer the bacterial solution cultivated in step ② to 50ml BMMY medium with pH=5.5 and methanol volume concentration of 0.75% respectively, so that the concentration of the bacterial solution OD ₆₀₀ =1, shake at 220 rpm at 29°C In medium culture, methanol was supplemented every 24 hours, and the volume of methanol added was 0.75% of the medium volume to induce the recombinant bacteria to secrete and express UGT2. In order to detect that the glycosyltransferase gene UGT2 can be effectively expressed and the target protein is secreted outside the cells of the recombinant Pichia pastoris, the growth medium of the recombinant bacteria was taken from the first day to the eighth day after induction for crude enzyme solution Activity determination, protein electrophoresis and protein concentration determination. The sampling method is as follows: take 500 μL of medium, after centrifugation, take the supernatant to measure the enzyme activity. As the induction time increases, the more UGT2 is secreted, while the control strain Pichia pastoris host strain X33 has no secretion of the target protein UGT2 (Figure 3, lower figure), and the UGT2 secreted and expressed by each strain is also different. Recombinant strain XS-35 secreted the largest amount of recombinant protein UGT2, named recombinant strain 2.

The speed of cultivation in steps ① and ② can also be any value in 200-250 rpm; the culture temperature can also be any value in 28°C-30°C; the concentration OD ₆₀₀ of the culture bacteria can also be 2- Any one of 6 values. In step ③, the volume concentration of methanol in the BMMY medium is any value in 0.5%-1%; the concentration _OD600 of the bacteria can also be any value in the range of 0.8-1.5; the rotating speed can also be 200-250 rpm Any value in the medium; the culture temperature can also be any value in the range of 28°C-30°C, methanol is supplemented every 24 hours, and the volume of methanol added is any value in the range of 0.5%-1% of the medium volume.

The recombinant strain 2 is an engineering strain containing the UGT2 gene, and the host cell can also be Saccharomyces cerevisiae or Bacillus subtilis.

When the host cell is Pichia pastoris, the expression vector is pPIC9K, pPIC9 or pPinkα-HC.

The host cell is Saccharomyces cerevisiae, and the expression vectors selected are pYES2, YCplac33, YEplac195,

When the host cell is Bacillus subtilis, the expression vectors selected are pHT01, pHT08 or pHT43.

In order to effectively secrete the target protein outside the host strain cells, the signal peptides selected are alpha factor (the nucleic acid sequence is SEQ ID NO.9), xyn2s (the nucleic acid sequence is SEQ ID NO.10), phoA (the nucleic acid sequence is SEQ ID NO. 11), phoD (the nucleic acid sequence is SEQ ID NO.12), amyE (the nucleic acid sequence is SEQ ID NO.13), MFalpha (the nucleic acid sequence is SEQ ID NO.14) and SED1 (the nucleic acid sequence is SEQ ID NO.15).

The influence of embodiment 6 methanol concentration on recombinant bacteria producing UGT1 and UGT2

According to the method described in Example 3, the recombinant bacterial strain 1 was cultivated in BMMY containing methanol concentrations of 0.5%, 0.75%, 1.0%, 1.25%, and 1.5%, and the growth and development of glycosyltransferase in the BMMY medium were investigated. The expression trend was investigated to investigate the optimum concentration of methanol for inducing the secretion of recombinant strain 1 to produce UGT1. The results of SDS-PAGE and protein concentration determination all show that the most suitable methanol concentration for recombinant strain 1 to express the target protein is 0.75%, followed by 1.0% and 1.25%, and there is also a higher secretory expression of protein at 1.5% or 0.5% (Fig. 4 above). According to the method described in Example 4, the recombinant bacterial strain 2 was cultivated in BMMY containing methanol concentrations of 0.25%, 0.5%, 0.75%, 1.0%, 1.5% and 2.0%, respectively, and the most suitable method for inducing the secretion of recombinant bacterial strain 2 to produce UGT2 was investigated. methanol concentration. The results of SDS-PAGE and protein concentration test showed that the concentration of methanol had an effect on the growth and expression of the recombinant strain XS-35. Too high concentration of methanol may have a toxic effect on the growth of Pichia pastoris. If the concentration is too low, the induced carbon source will be insufficient, and the biomass and expression will decrease. Maintaining an appropriate methanol concentration is the key to ensuring high expression. This experiment shows that the optimal concentration of methanol for secreting and expressing protein UGT2 of recombinant strain 2 is 0.75% (Fig. The following figure).

Example 7 Purification and biological activity detection of UGT1 and UGT2

The 3rd day fermentation supernatants of XE-3 and XS-35 strains were purified by Ni-NTA His.Bind resin from Merk Company. Take 20mL of the culture and centrifuge at 13000g/4℃/30min, the supernatant is suction-filtered with a 0.45μm filter membrane, and the filtered sample is added to a 10kD ultrafiltration tube of Milipore Company, centrifuged at 5000g/4℃/20min, Add 1× Ni-NTA binding buffer (50mmol/L NaH2PO4, 300mmol/L NaCl) to make up the volume to 13mL, centrifuge according to the above conditions, and concentrate to 2mL. Take 2 mL of 50% Ni-NTA His Bind resin (Novagen, 139311725) and add it to a gravity column (Biorad, 732-1010), add 2 mL of ultrafiltered and concentrated protein solution and 6 mL of 1×Ni-NTA binding buffer, gently Shake to mix, combine (200rpm/4°C/1h). Collect the effluent, add 1mL washing buffer (50mmol/L NaH2PO4, 300mmol/LNaCl, 2mmol/L imidazole) to rinse once; 4mL elution buffer (50mmol/L NaH2PO4, 300mmol/L NaCl, 150mmol/L imidazole) elute the protein of interest. Wash the equilibrated desalting chromatography column (GE, PD-10, Sephadex G-25 filler) with 3.5mL 10mmol/L phosphate buffer, and make up the volume of 4mL protein sample after the above elution to 5mL with 10mmol/L phosphate buffer, respectively Add 2.5mL to a well-balanced desalting chromatography column, discard the effluent, and elute the target protein with 3.5mL 10mmol/L phosphate buffer to obtain the purified protein of deimidazole. Purified and desalted pure enzymes had obvious target bands, indicating that UGT1 and UGT2 were successfully separated and purified ( FIG. 5 ).

Purified UGT1 and UGT2 were used for catalytic experiments: 50mmol/L phosphate buffer solution (pH 7.2), 0.5g/L RebA, 3mmol/L MgCl ₂ , 1mmol/L UDPG, 70μL pure enzyme were added to 500μL reaction system, 30℃ Response 2 days. Purified UGT2 was used for catalytic experiments: 50mmol/L phosphate buffer solution (pH 7.2), 0.5g/L RebA or RebD, 3mmol/L MgCl2, 1mmol/L UDPG, 70μL pure enzyme were added to the 500μL reaction system, and the reaction was performed at 30℃ for 2 sky. After the reaction was completed, 500 μL of 60% (v/v) acetonitrile was added, shaken and mixed, centrifuged at 12000 rpm for 10 min at room temperature, and the supernatant was passed through a 0.2 μm organic membrane. HPLC adopts Luna C18 reverse-phase bonded silica gel separation column (4.6mm×250mm, 5μm), the mobile phase adopts acetonitrile:sodium phosphate buffer solution (pH 2.6)=32:68, the flow rate is 1mL/min, and the column temperature is 40°C. The ultraviolet detector VWD and the differential refractive index detector RID are adopted, the wavelength of the VWD detector is 210nm, the temperature of the optical unit of the RID detector is 40°C, and the injection volume is 20 μL. The amount of enzyme required to catalyze 1 μmol of product per minute of enzyme-catalyzed substrate molecule is 1 unit U. Specific activity refers to the enzyme activity per mg of enzyme protein, unit U/mg. The concentration of the purified protein UGT1 was measured to be 0.232 mg/mL, and the specific activity of the UGT1 recombinase was 0.011 U/mg, indicating that the active recombinase UGT1 could be successfully purified from the culture of recombinant strain 1. The concentration of the purified protein UGT2 is 0.232mg/mL, and the specific activity of UGT2 recombinase is 0.016U/g. The concentration of the phosphate buffer solution is 1-100mmol/L, the concentration of the substrate RebA can be 0.5-20g/L, the concentration of _MgCl2 can be 0 or any value in 0.5-20mmol/L, 0.3-3mmol/LUDPG , 1-70μL pure enzyme, react at 30℃ for 0.5-72 hours.

Mixed fermentation of embodiment 8 recombinant strain 1 and recombinant strain 2

According to the method described in Examples 3 and 4, two strains were cultivated in BMMY medium, the pH was controlled at 5 to 6, the concentration of methanol was 0.75%-1.0%, and the mixed bacteria ratio was 1:1 or 2:1. After 4 days of fermentation, 120 μL of the supernatant was put into the enzymatic system described in Example 6 to react for 48 hours, and the efficiency of converting 0.4-0.6 g/L of RebA into RebM was investigated. The conversion rates of RebA were 44.89%, 50.35%, 69.24% and 52.46%, respectively. The production amounts of RebM were 0.15, 0.17, 0.23 and 0.18g/L, and the yields were 37.41%, 41.96%, 57.70% and 43.71%, respectively. Through the analysis of orthogonal experiments, theoretically, under the conditions of 0.75% methanol and pH 6.0, the recombinant strain 1 and recombinant strain 2 were inoculated into BMMY at a ratio of 2:1, and the crude enzyme solution produced had the highest conversion yield of RebA to RebM. . These results indicated that mixed cultures containing UGT1 and UGT2 could catalyze RebA to RebM in one step. In the above experiment, the time for the mixed culture of the recombinant strain 1 and the recombinant strain 2 can be 0-10 days, the pH can be 3-7, and the temperature can be 26-30 degrees. 1-120ul of the culture can be taken and added to the enzymatic reaction system described in Example 6, the final concentration of the substrate rebaudioside A can be 0.5-120g/L, and the reaction is 1-240 hours.

Example 9

The method for synthesizing recidiside M by one-step method comprises the steps:

(1) The recombinant bacteria 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacteria 2 capable of secreting and expressing glycosyltransferase UGT2 were mixed for 2 hours; The ratio is 1:0.7, the total concentration of recombinant bacteria 1 and recombinant bacteria 2 is controlled so that the absorbance value OD600 of the bacterial solution is 1, the pH of the medium is 5.5, and the volume concentration of methanol in the medium containing methanol is 0.5%;

(2) In the culture medium that step (1) obtains, add final concentration and be 0.5g/L substrate rebaudioside A, add final concentration and be 0.2mM uridine diphosphate glucose, add the magnesium sulfate that final concentration is 0.5mM , adding methanol with a final concentration of 0.5% every 24 hours for reaction to obtain recidiside M. The yield of recidiside M was 36.50%.

Example 10

The method for synthesizing recidiside M by one-step method comprises the steps:

(1) The recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacterium 2 capable of secreting and expressing glycosyltransferase UGT2 were mixed and cultured for 2 days; The ratio is 1:1, the total concentration of recombinant bacteria 1 and recombinant bacteria 2 is controlled at the absorbance value OD600 of the bacteria solution is 1, the pH of the medium containing methanol is 6.0, and the volume concentration of methanol in the medium containing methanol is 0.75 %;

(2) In the culture solution that step (1) obtains, add final concentration and be 2g/L substrate rebaudioside A, add final concentration and be 1.5mM uridine diphosphate glucose, add the magnesium chloride that final concentration is 5mM, every 24 Methanol with a final concentration of 0.75% was added after 1 hour for reaction to obtain recidiside M. The yield of recidiside M was 56.50%.

Example 11

The method for synthesizing recidiside M by one-step method comprises the steps:

(1) The recombinant bacteria 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacteria 2 capable of secreting and expressing glycosyltransferase UGT2 were mixed and inoculated in a medium containing methanol for 3 days; the recombinant bacteria 1 and recombinant bacteria The ratio of cell concentration during inoculation of 2 is 1:2, the total concentration of recombinant bacteria 1 and recombinant bacteria 2 is controlled at the absorbance value OD600 of the bacterial solution is 1, the pH of the medium containing methanol is 8.0, and the medium containing methanol The volume concentration of methanol in the medium is 1.5%;

(2) In the culture solution that step (1) obtains, add final concentration and be 20g/L substrate rebaudioside A, add final concentration and be 1.5mM uridine diphosphate glucose, add the magnesium chloride that final concentration is 5mM, every 24 After 1 hour, add methanol with a final concentration of 1.5%, and react to obtain recidiside M. The yield of recidiside M was 28.10%.

sequence listing

<110> Sinochem Health Industry Development Co., Ltd.

Tianjin University

<120> One-step method for synthesizing rebaudioside M

<140> 2019110972731

<141> 2019-11-11

<160> 15

<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 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 His

485 490 495

His

<210> 2

<211> 1485

<212>DNA

<213> Artificial Sequence

<400> 2

atggactctg gttactcttc ttcttacgct gctgctgctg gtatgcacgt tgttatctgt 60

ccatggttgg ctttcggtca cttgttgcca tgtttggact tggctcaaag attggcttct 120

agaggtcaca gagtttcttt cgtttctact ccaagaaaca tctctagatt gccaccagtt 180

agaccagctt tggctccatt ggttgctttc gttgctttgc cattgccaag agttgaaggt 240

ttgccagacg gtgctgaatc tactaacgac gttccaacacg acagaccaga catggttgaa 300

ttgcacagaa gagctttcga cggtttggct gctccattct ctgaattttt gggtactgct 360

tgtgctgact gggttatcgt tgacgttttc caccactggg ctgctgctgc tgctttggaa 420

cacaaggttc catgtgctat gatgttgttg ggttctgctc acatgatcgc ttctatcgct 480

gacagaagat tggaaagagc tgaaactgaa tctccagctg ctgctggtca aggtagacca 540

gctgctgctc caactttcga agttgctaga atgaagttga tcagaactaa gggttcttct 600

ggtatgtctt tggctgaaag attctctttg actttgtcta gatcgtcttt ggttgttggt 660

agatcgtgtg ttgaatttga accagaaact gttccattgt tgtctacttt gagaggtaag 720

ccaatcactt tcttgggttt gatgccacca ttgcacgaag gtagaagaga agacggtgaa 780

gacgctactg ttagatggtt ggacgctcaa ccagctaagt ctgttgttta cgttgctttg 840

ggttctgaag ttccattggg tgttgaaaag gttcacgaat tggctttggg tttggaattg 900

gctggtacta gattcttgtg ggctttgaga aagccaactg gtgtttctga cgctgacttg 960

ttgccagctg gtttcgaaga aagaactaga ggtagagaggtg ttgttgctac tagatgggtt 1020

ccacaaatgt ctatcttggc tcacgctgct gttggtgctt tcttgactca ctgtggttgg 1080

aactctacta tcgaaggttt gatgttcggt cacccattga tcatgttgcc aatcttcggt 1140

gaccaaggtc caaacgctag attgatcgaa gctaagaacg ctggtttgca agttgctaga 1200

aacgacggtg acggttcttt cgacagagaa ggtgttgctg ctgctatcag agctgttgct 1260

gttgaagaag aatcttctaa ggttttccaa gctaaggcta agaagttgca agaaatcgtt 1320

gctgacatgg cttgtcacga aagatacatc gacggtttca tccaacaatt gagatcgtac 1380

aaggacttgg tacctcgagc cgcggcggcc gccagctttc tagaacaaaa actcatctca 1440

gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcat 1485

<210> 3

<211> 4954

<212>DNA

<213> Artificial Sequence

<400> 3

agatctaaca tccaaagacg aaaggttgaa tgaaacctttttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

aacaccatgac 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 caaatggggga 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 ggattttttttgtcattttg 2880

tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg 2940

gtagggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt 3000

cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt 3060

caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt 3120

accacttcaa aacacccaag cacagcatac taaattttcc ctctttcttc ctctagggtg 3180

tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt 3240

cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa attttttttt 3300

ttagtttttt tctctttcag tgacctccat tgatatttaa gttaataaac ggtcttcaat 3360

ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact 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 cggccacgg gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg 3960

atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa 4020

ccgaaaagga aggagttaga caacctgaag tctaggtccc tattatttt 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 aggtggcgaa acccgacagg actataaaga taccaggcgt 4380

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4440

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 4500

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4560

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4620

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4680

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 4740

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4800

aacaaaccac cgctggtagc ggtggtttttttgtttgcaa gcagcagatt acgcgcagaa 4860

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4920

aaaactcacg ttaagggatt ttggtcatga gatc 4954

<210> 4

<211> 1395

<212>DNA

<213> Artificial Sequence

<400> 4

atggaaaaca agactgagac tactgttaga agaagaagaa gaatcatctt gttcccagtt 60

ccttttcaag gtcatattaa cccaattttg caattggcta acgttttgta ctctaagggt 120

ttctctatca ctatcttcca tactaacttc aataagccaa agacttctaa ctaccctcac 180

ttcactttta gattcatttt ggataacgat ccacaagatg aaagaatttc taatttgcca 240

actcatggtc ctttggctgg tatgagaatc ccaattatta acgaacacgg tgctgatgag 300

ttgagaagag aattggagtt gttgatgttg gcttctgaag aggatgaaga ggtttcttgt 360

ttgattactg atgctttgtg gtactttgct caatctgttg ctgattcttt gaacttgaga 420

agattggttt tgatgacttc ttctttgttc aatttccatg ctcacgtttc tttgccacaa 480

ttcgatgaat tgggttactt ggatcctgat gataagacta gattggaaga gcaggcttct 540

ggttttccaa tgttgaaggt taaggatatc aagtctgctt actctaactg gcaaatcttg 600

aaggagatct tgggtaaaat gatcaagcaa actaaggctt cttctggtgttatttggaac 660

tcttttaagg agttggaaga gtctgaattg gagactgtta ttagagagat tccagctcca 720

tctttcttga ttccattgcc taaacatttg actgcttctt cttcttcttt gttggatcac 780

gatagaactg ttttccaatg gttggatcaa caaccacctt cttctgtttt gtacgtttct 840

ttcggttcta cttctgaagt tgatgagaaa gatttcttgg aaattgctag aggtttggtt 900

gattctaagc aatctttctt gtgggttgtt agaccaggtt ttgtcaaagg ttctacttgg 960

gttgaaccat tgcctgatgg tttcttggga gagagaggta gaattgttaa atgggttcct 1020

caacaagaag ttttggctca tggtgctatt ggtgcttttt ggactcactc tggttggaac 1080

tctactttgg aatctgtttg tgagggtgtt ccaatgattt tctctgattt tggtttggat 1140

caacctttga atgctagata catgtctgat gttttgaagg ttggtgttta tttggaaaac 1200

ggttgggaaa gaggtgagat tgctaatgct attagaagag ttatggttga tgaagaggga 1260

gagtacatca gacaaaacgc tagagttttg aagcaaaaag ctgatgtttc tttgatgaaa 1320

ggtggttctt cttacgaatc tttggagtct ttggtttctt aatttcttc tttgcatcat 1380

catcatcatc attga 1395

<210> 5

<211> 466

<212> PRT

<213> Artificial Sequence

<400> 5

Glu Phe Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Arg

1 5 10 15

Ile Ile Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu

20 25 30

Gln Leu Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe

35 40 45

His Thr Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr

50 55 60

Phe Arg Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn

65 70 75 80

Leu Pro Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn

85 90 95

Glu His Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu

100 105 110

Ala Ser Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu

115 120 125

Trp Tyr Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu

130 135 140

Val Leu Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu

145 150 155 160

Pro Gln Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg

165 170 175

Leu Glu Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile

180 185 190

Lys Ser Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys

195 200 205

Met Ile Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe

210 215 220

Lys Glu Leu Glu Glu Ser Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro

225 230 235 240

Ala Pro Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser

245 250 255

Ser Ser Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln

260 265 270

Gln Pro Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu

275 280 285

Val Asp Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser

290 295 300

Lys Gln Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser

305 310 315 320

Thr Trp Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg

325 330 335

Ile Val Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile

340 345 350

Gly Ala Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val

355 360 365

Cys Glu Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro

370 375 380

Leu Asn Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu

385 390 395 400

Glu Asn Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val

405 410 415

Met Val Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu

420 425 430

Lys Gln Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu

435 440 445

Ser Leu Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu His His His His His

450 455 460

His His

465

<210> 6

<211> 4861

<212>DNA

<213> Artificial Sequence

<400> 6

agatctaaca tccaaagacg aaaggttgaa tgaaacctttttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

aacaccatgac 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 caaatggggga 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 ttcatggaaa ataaaaccga aaccaccgtc cgccgtcgtc gccgtatcat 1260

tctgttcccg gtcccgttcc agggccacat caacccgatt ctgcaactgg cgaacgtgct 1320

gtattcgaaa ggtttcagca tcaccatctt ccatacgaac ttcaacaagc cgaagaccag 1380

caattacccg cactttacgt tccgttttat tctggataac gacccgcagg atgaacgcat 1440

ctctaatctg ccgacccacg gcccgctggc gggtatgcgt attccgatta tcaacgaaca 1500

cggcgcagat gaactgcgtc gcgaactgga actgctgatg ctggccagcg aagaagatga 1560

agaagtttct tgcctgatca ccgacgcact gtggtatttt gcccagtctg ttgcagatag 1620

tctgaacctg cgtcgcctgg tcctgatgac cagcagcctg ttcaattttc atgcccacgt 1680

tagtctgccg cagttcgatg aactgggtta tctggacccg gatgacaaaa cccgcctgga 1740

agaacaggcg agcggctttc cgatgctgaa agtcaaggat attaagtcag cgtactcgaa 1800

ctggcagatt ctgaaagaaa tcctgggtaa aatgattaag caaaccaaag caagttccgg 1860

cgtcatctgg aatagtttca aagaactgga agaatccgaa ctggaaacgg tgattcgtga 1920

aatcccggct ccgagttttc tgattccgct gccgaagcat ctgaccgcga gcagcagcag 1980

cctgctggat cacgaccgca cggtgtttca gtggctggat cagcaaccgc cgagttccgt 2040

gctgtatgtt agcttcggta gtacctcgga agtggatgaa aaggactttc tggaaatcgc 2100

tcgtggcctg gttgatagca aacaatcttt cctgtgggtg gttcgcccgg gttttgtgaa 2160

gggctctacg tgggttgaac cgctgccgga cggcttcctg ggtgaacgtg gccgcattgt 2220

caaatgggtg ccgcagcaag aagtgctggc gcatggcgcg attggcgcgt tttggaccca 2280

ctccggttgg aactcaacgc tggaatcggt ttgtgaaggt gtcccgatga ttttctcaga 2340

ttttggcctg gaccagccgc tgaatgcacg ttatatgtcg gatgttctga aagtcggtgt 2400

gtacctggaa aacggttggg aacgcggcga aattgcgaat gccatccgtc gcgttatggt 2460

cgatgaagaa ggcgaataca ttcgtcagaa tgctcgcgtc ctgaaacaaa aggcggacgt 2520

gagcctgatg aaaggcggtt catcgtatga aagtctggaa tccctggttt catacatcag 2580

ctctctgcat catcatcatc atcattgagt ttgtagcctt agacatgact gttcctcagt 2640

tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga 2700

atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata 2760

tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc 2820

tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg 2880

tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag accttcgttt 2940

gtgcggatcc cccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag 3000

attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac agcatactaa 3060

attttccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag 3120

aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca 3180

cgtttctttt tcttgaaatt ttttttttta gtttttttct ctttcagtga cctccattga 3240

tattaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 3300

ttacaacttt ttttacttct tgttcattag aaagaaagca tagcaatcta atctaagggg 3360

cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag 3420

gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc 3480

cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga 3540

cttcgccggt gtggtccggt acgacgtgac cctgttcatc agcgcggtcc aggaccaggt 3600

ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga 3660

gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat 3720

cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca 3780

cttcgtggcc gaggagcagg actgacacgt ccgacggcgg cccacgggtc ccaggcctcg 3840

gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt 3900

cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct 3960

aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa 4020

tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc 4080

ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc aacatgtgag 4140

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 4200

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 4260

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 4320

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 4380

tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 4440

gctgtgtgca cgaaccccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 4500

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 4560

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 4620

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 4680

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4740

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4800

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggatttg gtcatgagat 4860

c 4861

<210> 7

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 7

gactggttcc aattgacaag c 21

<210> 8

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 8

gcaaatggca ttctgacatc c 21

<210> 9

<211> 267

<212>DNA

<213> Artificial Sequence

<400> 9

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 tgaagct 267

<210> 10

<211> 99

<212>DNA

<213> Artificial Sequence

<400> 10

atggtctcct tcacctccct cctcgccggc gtcgccgcca tctcgggcgt cttggccgct 60

cccgccgccg aggtcgaatc cgtggctgtg gagaagcgc 99

<210> 11

<211> 123

<212>DNA

<213> Artificial Sequence

<400> 11

atgaaaaaaa tgagtttgtt tcaaaatatg aaatcaaaac ttctgccaat cgccgctgtt 60

tctgtcctta cagctggaat ctttgccgga gctgagcttc agcaaacaga aaaggccagc 120

gcc 123

<210> 12

<211> 168

<212>DNA

<213> Artificial Sequence

<400> 12

atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 60

aacaatacgt ttgaccgccg caaatttatt caaggagcgg ggaagattgc aggactttct 120

cttggattaa cgattgccca gtcggttggg gcctttgaag taaatgct 168

<210> 13

<211> 99

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttatgctg 60

tttcatttgg ttctggcagg accggcggct gcgagtgct 99

<210> 14

<211> 57

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgagatttc catctatttt tactgctgtt ttgtttgctg cttcttctgc tttggct 57

<210> 15

<211> 57

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcccaa 57

Claims (2)

1. the method for synthesizing rebaudioside M in one-step method is characterized in that comprising the steps: (1) The recombinant bacteria 1 capable of secreting and expressing glycosyltransferase UGT1 and the recombinant bacteria 2 capable of secreting and expressing glycosyltransferase UGT2 were mixed and inoculated in medium containing methanol for 2h-3 days. The recombinant bacteria 1 and The ratio of cell concentration when recombinant bacterium 2 is inoculated is 1:0.7-2, and the total concentration of recombinant bacterium 1 and recombinant bacterium 2 is controlled at the absorbance value OD600 of bacterium liquid to be 1, and the pH=5.5-8 of the medium containing methanol, The volume concentration of methanol in the medium containing methanol is 0.5%-1.5%; (2) Add the substrate rebaudioside A at a final concentration of 0.5-20 g/L to the culture medium obtained in step (1), at a final concentration of 0.2-1.5 mM uridine diphosphate glucose, at a final concentration of 0.5- 5mM magnesium sulfate or magnesium chloride, add methanol with a final concentration of 0.5%-1.5% every 24 hours, and react to obtain rebaudioside M; The amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID NO.1; The amino acid sequence of the glycosyltransferase UGT2 is shown in SEQ ID NO.5; The recombinant bacterium 1 is constructed by the following steps: the glycosyltransferase UGT1 gene whose nucleotide sequence is shown in SEQ ID NO.2 is connected to the expression vector pPICZalphaA to obtain the 4954bp pP- UGT1 plasmid, pP-UGT1 plasmid comprises signal peptide alpha factor, the nucleotide sequence of described signal peptide alpha factor is as shown in SEQ ID NO.9; Extract this plasmid from the recombinant escherichia coli containing pP-UGT1, use PmeI The enzyme cuts the 4954bp fragment shown in the linear SEQ ID NO.3, and transfers it into the host cell 1 to obtain the first recombinant bacterium 1; The recombinant bacteria 2 is constructed by the following steps: the glycosyltransferase UGT2 gene whose nucleotide sequence is shown in SEQ ID NO.4 is connected to the expression vector pPICZalphaA to obtain the 4861bp pP- UGT2 plasmid, pP-UGT2 plasmid comprises signal peptide alpha factor, the nucleotide sequence of described signal peptide alpha factor is as shown in SEQ ID NO.9; Extract this plasmid from the recombinant escherichia coli containing pP-UGT2, use PmeI The enzyme cuts into a linear 4861bp fragment shown in SEQ ID NO.6, and transforms it into host cell 1 to obtain the first recombinant bacterium 2 . 2. The method according to claim 1, characterized in that the host cell 1 is Pichia pastoris.

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