CN110734944B

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

Info

Publication number: CN110734944B
Application number: CN201911097273.1A
Authority: CN
Inventors: 马媛媛; 汪振洋; 宋浩; 洪解放; 来庆英; 刘文斌; 张敏华; 刘伟
Original assignee: Tianjin University; Sinochem Health Co Ltd
Current assignee: Tianjin University; Sinochem Health Co Ltd
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2023-02-21
Anticipated expiration: 2039-11-11
Also published as: CN110734944A

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

Method for synthesizing rebaudioside M by one-step method

Technical Field

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

Background

Stevioside is a novel natural low-calorie sweetener stevioside compound extracted from stevia leaves of Compositae herbaceous plants, and has high sweetness, low calorie, no toxicity, high temperature resistance and high temperature resistanceGood acid-base and water solubility, etc., is certified as safe by the American food and drug administration, and can be applied to food industry ^[1-5] . Rebaudioside M (RebM) has better taste properties, but its content of dry weight of the leaves is less than 0.1%, resulting in high separation cost and high price. The biocatalytic method for obtaining RebM in high concentration has attracted attention of scholars. It is reported that recombinant enzyme derived from stevia rebaudiana can catalyze rebD to produce rebM, but the yield is low ^[3-4] 。

RebD is taken as a substrate, and RebM can be obtained through a biocatalysis method, but the following problems mainly exist in the biocatalysis process at present: (1) The biological catalysis needs high-efficiency glycosyltransferase, the content of the glycosyltransferase in plants is very low and can not reach the level of practical application, and the glycosyltransferase is difficult to purify; (2) The recombinant glycosyltransferase expressed in the cells can catalyze the generation of a RebD product RebM, but the content of RebD in stevia is lower than 0.1 percent, and the price is higher; (3) Recombinant enzyme produced by intracellular expression can be catalyzed only by processes of centrifugally collecting thalli, breaking cells and the like, and the steps are complicated; (4) During the reaction, the glycosyltransferase is mostly deactivated by heat inhibition. (5) Recombinant bacteria to obtain recombinant enzyme, preparation of enzyme and catalysis are time-consuming, labor-consuming and financial-consuming ^[3-9] . Based on the above current research situation, there is a need for: the rebaudioside M is obtained by a method which is low in price, large in yield, few in steps, quick and effective and low in cost.

This technology would also be a viable route to the industrial biocatalytic preparation of RebM.

Reference documents:

[1] general description of the use of herquan non-synthetic sweeteners in the food industry [ J ] biology teaching, 2017, 42 (10): 10-12.

[2] Extraction of inulin and its use in bread research progress [ J ] food safety guidance, 2018, 09:142.

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

[4] functional study progress of the steviol glycosides [ D ] food science 2015, 36 (17): 264-269.

[5] Tang Shi Qi, the rise of stevia sugar and development strategy [ J ]. Chinese food industry, 1999, (2): 52-52.

[6] Wailawa, compound food additive [ M ] Beijing, chemical industry Press, 2006.

[7] Application progress of genetic engineering and metabolic engineering to stevia sugar production [ J ] biotechnological report, 2015, 31 (9): 8-14

[8]Kumari N and Kumar S,Chemistry and analytical techniques for ent-kaurene-glycosides of Stevia rebaudianaBertoni-Areview.2017.JournalofAppliedandNatural Science9(4):2114-2126

[9] Plum brilliant, clear, and stale amount a method for preparing rebaudioside M by an enzymatic process [ P ]. China: 107666834, 2018-08-10.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a one-step method for synthesizing rebaudioside M.

The technical scheme of the invention is summarized as follows:

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 lecitin M;

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

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

Step (1) is preferably: 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 culturing for 2h-3 days, the ratio of the cell concentrations of the recombinant bacterium 1 and the recombinant bacterium 2 in inoculation is 1.

Step (2) is preferably: adding substrate rebaudioside A with the final degree of 0.5-20g/L into the culture solution obtained in the step (1), adding uridine diphosphate glucose with the final concentration of 0.2-1.5mM, adding magnesium sulfate or magnesium chloride with the final concentration of 0.5-5mM, and adding methanol with the final concentration of 0.5% -1.5% every 24 hours to react to obtain the lecitin M.

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

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

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

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

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

the signal peptide used by the recombinant strain 2 for secreting UGT1 protein is signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha and SED1, and the nucleotide sequence corresponding to the signal peptide is shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 in sequence.

The invention has the advantages that:

1. overcomes the defect that the prior art can not purify a large amount of glycosyltransferase from stevia rebaudiana.

2. The one-step synthesis overcomes the complex steps of cell breaking, separation and purification or addition of a cell membrane penetrating agent and the like in catalysis in the prior art.

3. The rebaudioside M (RebM) can be produced only by using a substrate rebaudioside D (RebD) in the existing biocatalysis technology, the price of the RebD is high compared with that of the rebaudioside A (RebA) used as the substrate in the technology, and the RebA with low price is used as the substrate in the invention, so that the production cost is reduced.

4. The invention can produce the RebM in one step by mixing and fermenting and adding the RebA into the fermentation liquor. RebM is a sweetening agent with high sweetness and good taste, the method of the invention can obtain the glycosidase with secretion expression, can improve the yield and the thermal stability of recombinant bacteria glycosidase, and realizes the mixed bacteria catalysis one-step method for obtaining the RebM by directly using the low-value RebA as a substrate. Improve the catalytic efficiency, reduce the cost for enzyme purification and the cost for substrate, and obtain the RebM with high sweetness, good taste and high value.

Drawings

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

FIG. 2 is the PCR identification electropherogram of recombinant Pichia pastoris transformants of the glycosyltransferases UGT1 and UGT2 genes. Lane M is DNA Standard molecular weight Marker; wherein, each lane of a is the PCR product electrophoresis pattern of each transformant of UGT1 and the control strain X33. b are PCR product electrophoretograms of UGT2 transformants and a control strain X33. CK is the result of electrophoresis of PCR products using water as a template.

FIG. 3 electrophoretogram of total protein of supernatant of each transformant culture. Wherein a is an electrophoretogram of transformant secretion expression UGT 1; b is an electrophoresis picture of transformant secretion expression UGT2.

FIG. 4 recombinant Strain 1 and recombinant Strain 2 recombinant Pichia pastoris express UGT2 at various methanol concentrations. And (3) when the control bacterium X33, the recombinant strain 1 and the recombinant strain 2 are cultured on the 3 rd day, taking culture supernatant, and concentrating the culture supernatant into 5-fold electrophoresis images. a and b are electrophoretograms of UGT1 and UGT2 secreted by recombinant strain 1 and recombinant strain 2, respectively, when cultured at various methanol concentrations.

FIG. 5 SDS-PAGE patterns of purified UGT1 and UGT2. Purifying culture supernatants of the 3 rd day of the recombinant strain 1 and the recombinant strain 2, and then performing electrophoresis, wherein a is a purified UGT1 electrophoretogram; b is the purified UGT2 electrophoretogram.

FIG. 6 strain growth and protein concentration determination during one-step production of RebM using recombinant Strain 1 and recombinant Strain 2.

a is the growth curve of strains EX-3 and XS-35 under 4 experimental conditions, and b is the protein concentration of the culture supernatant at

day

3 and 4 of strain culture (i.e.at

day

2 and 3 after feeding). 1#: pH 6.0, EX-3 and XS-35 feed at ratio 1, day 2; 2#: pH 6.0, EX-3 and XS-35 feed at ratio 1, day 3; 3#: pH 7.3, EX-3 and XS-35 feed at ratio 1, day 3; 4#: pH 7.3, EX-3 and XS-35 feed at ratio 1, day 2.

Detailed Description

Coli TOP10F' (commercial).

The present invention will be further described with reference to the following examples.

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

Example 1 Synthesis of transferase UGT2 and UGT1 genes and construction of expression vector

The glycosyl transferase UGT1 gene is synthesized by the Kinsley company, the nucleotide sequence of the gene is shown as SEQ ID NO.2 in the sequence table (SEQ ID NO.1 is the amino acid sequence of the glycosyl transferase UGT 1), the gene is connected to pPICZalphaA through a molecular biological method, and the pP-UGT1 plasmid of 4954bp shown as SEQ ID NO.3 in the sequence table is obtained. The pP-UGT1 plasmid (FIG. 1) contains the nucleotide sequence of the signal peptide alpha factor (SEQ ID NO. 9). The plasmid was transformed into a transformant obtained from E.coli TOP10F', and 15. Mu.g (or 10. Mu.g) of pPIC-UGT1 plasmid (a medium kit from Tiangen corporation) was extracted from the transformant. Cutting the 4954bp fragment shown in the linear SEQ ID NO.3 by PmeI enzyme, extracting by phenol/chloroform, precipitating the linear DNA fragment shown in the linear SEQ ID NO.3 by ethanol, drying, and dissolving in sterile water to prepare a linear DNA solution with the DNA concentration of 1 mu g/mu L (or 0.4 mu g/mu L).

A nucleotide sequence shown by SEQ ID No.4 in a sequence table (SEQ ID No.5 is an amino acid sequence of glycosyltransferase UGT 2) is synthesized according to the same method, and is connected to pPICZalphaA by a molecular biological method to obtain a 4861 bp-long pP-UGT2 plasmid (SEQ ID No.6 in the sequence table) and a linear DNA solution containing the UGT1 gene.

Example 2 integration of glucosyltransferase UGT2 and UGT1 genes into pichia pastoris chromosomes:

the competent cells of Pichia pastoris host bacteria X-33 and Gs115 were prepared by referring to the method for preparing yeast competent cells by electroporation transformation in the protocol for molecular biology (fourth edition) (P512-513), 80. Mu.L of the competent cells of Pichia pastoris host bacteria were mixed with 10. Mu.L of a linear DNA solution of 1. Mu.g/. Mu.L of pP-UGT1 plasmid, and then transferred to an electrode cup precooled at 4 ℃ (optionally at 0 to 4 ℃), and ice-cooled for 5min at a voltage of 1500V, and then shocked with an electrotransfer of eppendorf, 5ms, and immediately after shocking, 1mL of a 1M sorbitol aqueous solution precooled at 4 ℃ (optionally at 1 to 4 ℃) was added to the electrode cup, and mixed, and the mixture was transferred to a 15mL centrifuge tube, incubated at 29 ℃ for 1h (optionally at 1 to 2 h), and then plated on a YPD plate containing 200. Mu.g/mL of Zeocin (sold by ITrogen) until the first recombinant strain appeared after culturing in an inverted manner of 100. Mu.L (optionally at 50 to 200. Mu.L).

The pP-UGT2 plasmid was extracted in the same manner as described above, and the UGT2 gene was transformed into competent cells of X-33 and Gs115 by electric transformation after linearization to obtain the first recombinant strain 2.

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

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

The transformants at the early stage of the laboratory were identified by colony PCR method. Centrifuging the bacteria stored in the glycerinum tube, discarding supernatant, taking concentrated bacteria as a PCR amplification template, and amplifying by adopting exogenous gene specific primers such as sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence table, wherein if the recombinant plasmid is transformed into host cells, the target fragment with the expected size can be obtained by amplification.

20 mu L of glycerol bacteria are respectively taken, centrifuged at 12000rpm/4 ℃/5min, the supernatant is discarded, and the precipitate is used as a colony PCR template. Preparing 15 mu L of reaction system 2 xRapid Taq Master Mix 7.5 mu L in a 200 mu L PCR tube, wherein 0.5 mu L of nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8 are upstream and downstream primers, 6.5 mu L of sterile water, and pP-UGT2 plasmid is used as a template as a positive control; using wild pichia pastoris genome DNA as a template as a negative control; sterile water was used as a template as a negative control. The PCR thermal cycle program was 95 ℃ denaturation 30s,54 ℃ annealing 30s, and 72 ℃ extension 3min. 1% agarose gel (1% agar, 1.5% EB, TAE buffer) was prepared. And (5) taking the PCR product for spotting, and carrying out 170V electrophoresis for 30min. The electropherogram is scanned.

The PCR products were electrophoresed and detected as shown in FIG. 2.

The results of PCR identification of transformants containing the UGT1 gene are shown in FIG. 2a, and PCR can amplify a 1950bp target gene UGT1 and a 2.2kb AOX1 gene band, which indicates that the UGT1 gene of these transformants has been successfully integrated into the chromosome of the Pichia pastoris X-33 strain (FIG. 2 a). As shown in FIG. 2b, the PCR-identified transformants containing the UGT2 gene amplified a band of 1857bp of the target gene and 2.2kb of the AOX1 gene, indicating that the UGT2 gene of these transformants had been successfully integrated into the chromosome of P.pastoris X-33 strain (FIG. 2 b).

Example 4 methanol Induction of UGT1 Gene expression in Pichia pastoris transformants

(1) Inoculating the 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 identified in example 3 and the Pichia pastoris host strain X33 to 2ml of YPD medium with pH =5.5 for pre-culture at 220 rpm, and culturing at 29 ℃ to OD ₆₀₀ ＝4；

(2) The pre-cultured strain of step (1) was transferred to 25ml of BMGY medium at a volume ratio of 1 ₆₀₀ ＝4；

(3) Transferring the bacterial liquid cultured in the step (2) into 50ml of BMMY culture medium with pH =5.5 and methanol volume concentration of 0.75% respectively to ensure that the concentration OD of the bacterial liquid ₆₀₀ And =1, culturing in a shaking table at 29 ℃ at 220 r/min, supplementing methanol every 24h, wherein the adding volume of the methanol is 0.75% of the volume of the culture medium, and inducing the recombinant bacteria to secrete and express UGT1. In order to detect that the glycosyltransferase gene UGT1 can be effectively expressed and secrete the target protein to the outside of the cells of the recombinant Pichia pastoris, a culture medium for growing the recombinant bacteria is taken from 1 to 8 days after induction to carry out activity determination, protein electrophoresis and protein concentration determination on a crude enzyme solution. The sampling mode is as follows: taking 500 mul of culture medium, centrifuging, and taking supernatant to determine enzyme activity. Along with the increase of the induction time, 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 graph), UGT1 secreted and expressed by each strain is different, wherein the recombinant protein UGT1 secreted by the strains such as recombinant bacteria XE3 and XE4 is larger in amount. The XE-3 strain expresses the highest expression level of the target protein UGT1 and is named as a recombinant strain 1. The recombinant strain 1 can express UGT1 when being cultured for 1-7 days, and the expression level reaches a higher level already at the 3 rd day.

The rotating speed of the culture in the steps (1) and (2) can be any value of 200-250 r/min; the culture temperature may be any value of 28 ℃ to 30 ℃; concentration OD of the culture ₆₀₀ Or may be any value of 2 to 6. Methanol in BMMY culture medium in step (3)The volume concentration of (a) is any one of 0.5-1%; the concentration OD of the cells ₆₀₀ Or may be any value in the range of 0.8 to 1.5; the rotating speed can be any value of 200-250 revolutions per minute; the culture temperature can be any value of 28-30 ℃, methanol is supplemented every 24h, and the adding volume of the methanol is any value of 0.5-1% of the volume of the culture medium.

BMGY medium 10g/L yeast extract, 20g/L peptone, 13.4g/L yeast nitrogen base, 100mM potassium phosphate, 4X10 ^-4 Biotin in g/L, glycerol in 10g/L and water in the rest.

BMMY medium is 10g/L yeast extract, 20g/L peptone, 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.

The MMH culture medium is 13.4g/L yeast nitrogen base, 4x10 ^-4 g/L biotin, the volume concentration of methanol is 0.5-1%, and the balance is water.

BMMH medium was 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 is water.

The recombinant strain 1 contains engineering strains of UGT1 genes, and the host cells can also be saccharomyces cerevisiae or bacillus subtilis.

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

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

When the host cell is bacillus subtilis, the expression vector is pHT01, pHT08 or pHT43.

In order to effectively secrete the target protein outside cells of the host strain, the selected signal peptides 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 Induction of UGT2 Gene expression in Pichia pastoris transformants

(1) Inoculating 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 inoculating to YPD culture medium of 2ml and pH =5.5 for pre-culture at 220 rpm, and culturing at 29 ℃ to OD ₆₀₀ ＝4；

(3) Respectively transferring the bacterial liquid cultured in the step (2) into 50ml of BMMY culture medium with pH =5.5 and methanol volume concentration of 0.75%, and enabling the concentration OD of the bacterial liquid ₆₀₀ And =1, culturing in a shaking table at 29 ℃ at 220 r/min, supplementing methanol every 24h, wherein the adding volume of the methanol is 0.75% of the volume of the culture medium, and inducing the recombinant bacteria to secrete and express UGT2. In order to detect that the glycosyltransferase gene UGT2 can be effectively expressed and secrete the target protein to the outside of the cells of the recombinant Pichia pastoris, a culture medium for growing the recombinant bacteria is taken from 1 to 8 days after induction to carry out activity determination, protein electrophoresis and protein concentration determination on a crude enzyme solution. The sampling mode is as follows: taking 500 mu L of culture medium, centrifuging, and taking supernatant to determine enzyme activity. The UGT2 is secreted more along with the increase of the induction time, while the Pichia pastoris host strain X33 serving as a control strain has no secretion of the UGT2 serving as the target protein (figure 3 lower graph), the UGT2 secreted and expressed by each strain is different, wherein the recombinant strain XS-35 secretes the largest amount of the recombinant protein UGT2 and is named as recombinant strain 2.

The rotating speed of the culture in the steps (1) and (2) can be any value of 200-250 r/min; the culture temperature may be any one of 28 ℃ to 30 ℃; concentration OD of the cultured bacteria ₆₀₀ Or may be any value of 2 to 6. In the step (3), the volume concentration of methanol in the BMMY culture medium is any one value of 0.5% -1%; OD concentration of cells ₆₀₀ Or may be any value in the range of 0.8 to 1.5; the rotating speed can be any value of 200-250 revolutions per minute; the culture temperature can be any value of 28-30 deg.C, methanol is supplemented every 24h, and the volume of methanol added is any of 0.5% -1% of the culture medium volumeMeaning a value.

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

The host cell is Saccharomyces cerevisiae expression vector pYES2, YCplac33, YEplac195,

In order to effectively secrete the target protein outside cells of the host strain, selected signal peptides 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 6 Effect of methanol concentration on the production of UGT1 and UGT2 by recombinant bacteria

Recombinant strain 1 was cultured in BMMY with methanol concentrations of 0.5%, 0.75%, 1.0%, 1.25%,1.5%, respectively, according to the method described in example 3, and growth and expression trends of glycosyltransferase in BMMY medium were examined, and the optimum methanol concentration for inducing recombinant strain 1 to secrete UGT1 was examined. The results of SDS-PAGE and protein concentration determination both showed that recombinant strain 1 expressed the protein of interest with an optimum methanol concentration of 0.75%, followed by 1.0% and 1.25%, with higher protein secretion expression at 1.5% or 0.5% (FIG. 4 top panel). Recombinant strain 2 was cultured in BMMY with methanol concentrations of 0.25%, 0.5%, 0.75%, 1.0%, 1.5% and 2.0%, respectively, according to the method described in example 4, and the optimum methanol concentration for inducing recombinant strain 2 to secrete UGT2 was examined. The results of SDS-PAGE and protein concentration determination both show that the methanol concentration has influence on the growth and expression of the recombinant strain XS-35, and the excessive methanol concentration has toxic action on the growth of pichia pastoris. The low concentration can lead to insufficient induced carbon source, the biomass and the expression quantity are reduced, and the maintenance of proper methanol concentration is the key for ensuring high expression quantity. This experiment shows that recombinant strain 2 secretes and expresses protein UGT2 with optimal methanol concentration of 0.75% (lower graph in FIG. 4), and 0.25%, 0.5%, 1.0%, 1.5%, and 2.0% all have higher UGT2 expression (lower graph in FIG. 4).

Example 7 purification and detection of biological Activity of UGT1 and UGT2

Day 3 fermentation supernatants of XE-3 and XS-35 strains were purified using Ni-NTA His resin from Merk corporation. 13000g/4 ℃/30min centrifugation of 20mL of the culture was performed, the supernatant was filtered with 0.45 μm filter, the filtered sample was added to a 10kD Millipore ultrafiltration tube, centrifuged at 5000g/4 ℃/20min, 1 XNi-NTA binding buffer (50 mmol/L NaH2PO4, 300mmol/L NaCl) was added to the tube to make up the volume to 13mL, centrifuged under the above conditions, and concentrated to 2mL. 2mL of Ni-NTA His Bind resin (Novagen, 139311725) 50% was put into a gravity column (Biorad, 732-1010), 2mL of the ultrafiltrated and concentrated protein solution was mixed with 6mL of 1 XNi-NTA binding buffer, and the mixture was gently shaken and mixed (200 rpm/4 ℃/1 hour). Collecting effluent, adding 1mL rinsing buffer solution (50 mmol/L NaH2PO4, 300mmol/L NaCl,2mmol/L imidazole) and rinsing for 1 time; 4mL of elution buffer (50 mmol/L NaH2PO4, 300mmol/L NaCl,150mmol/L imidazole) eluted the target protein. Washing an equilibrium desalting chromatographic column (GE, PD-10, sephadex G-25 filler) by using 3.5mL of 10mmol/L phosphate buffer solution, supplementing the volume of the eluted 4mL protein sample to 5mL by using 10mmol/L phosphate buffer solution, respectively adding the protein sample to the well-balanced desalting chromatographic column for 2.5mL, discarding the effluent, and eluting the target protein by using 3.5mL of 10mmol/L phosphate buffer solution to obtain the purified protein with the imidazole removed. The purified and desalted pure enzyme has obvious target bands, which indicates that UGT1 and UGT2 are successfully separated and purified (figure 5).

Purified UGT1 and UGT2 were used for catalytic experiments: to 500. Mu.L of the reaction system were added 50mmol/L phosphoric acid buffer solution (pH 7.2), 0.5g/L RebA,3mmol/L MgCl ₂ 1mmol/L UDPG, 70. Mu.L pure enzyme, reacted at 30 ℃ for 2 days. Purified UGT2 was used for catalytic experiments: to 500. Mu.L of the reaction system were added 50mmol/L phosphoric acid buffer solution (pH 7.2), 0.5g/L RebA or RebD,3mmol/L MgCl2,1mmol/L UDPG, and 70. Mu.L of pure enzyme, and the reaction was carried out at 30 ℃ for 2 days. After completion of the reaction, 500. Mu.L of 60% (v/v) acetonitrile was added thereto, and after shaking and mixing, the mixture was centrifuged at 12000rpm at room temperature for 10 minutesmin, the supernatant passes through a 0.2 μm organic membrane. HPLC using a Luna C18 reverse phase bonded silica gel column (4.6 mm. Times.250mm, 5 μm), mobile phase using acetonitrile: sodium phosphate buffer solution (pH 2.6) =32, flow rate 1mL/min, column temperature 40 ℃. An ultraviolet detector VWD and a differential refraction detector RID are adopted, the wavelength of the VWD detector is 210nm, the temperature of an optical unit of the RID detector is 40 ℃, and the sample injection amount is 20 mu L. The amount of enzyme required to catalyze the production of 1. Mu. Mol of product from a substrate molecule per minute is 1 unit U. Specific activity means the enzyme activity per mg of enzyme protein, unit U/mg. The concentration of the purified protein UGT1 is measured to be 0.232mg/mL, and the specific activity of UGT1 recombinase is 0.011U/mg, which indicates that the active recombinase UGT1 can be successfully purified from the culture of the 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, and MgCl ₂ The concentration of (b) is 0 or any one of 0.5 to 20mmol/L, 0.3 to 3mmol/L UDPG,1 to 70. Mu.L of pure enzyme, and the reaction is carried out at 30 ℃ for 0.5 to 72 hours.

Example 8 Mixed fermentation of recombinant Strain 1 and recombinant Strain 2

Two strains were cultured in BMMY medium according to the methods described in examples 3 and 4, with pH controlled between 5 and 6, methanol concentration 0.75% -1.0%,

mixed ratio

1 or 2. After 4 days of fermentation, 120. Mu.L of supernatant was taken to react in the enzymatic system described in example 6 for 48 hours, and the efficiency of converting RebA into RebM was examined at 0.4-0.6 g/L. The conversion of RebA was 44.89%, 50.35%, 69.24%, and 52.46%, respectively. The amounts of production of RebM were 0.15, 0.17, 0.23 and 0.18g/L, respectively, and the yields were 37.41%, 41.96%, 57.70% and 43.71%, respectively. Analysis by orthogonal experiments showed that theoretically, the yield of conversion of crude enzyme solution catalyzing RebA to RebM was the highest when recombinant strain 1 and recombinant strain 2 were inoculated into BMMY under conditions of 0.75% methanol, pH 6.0, 2. These results indicate that the mixed culture containing UGT1 and UGT2 can catalyze the one-step method for the generation of RebM by RebA. The above experiment may be carried out by culturing recombinant strain 1 and recombinant strain 2 in a mixed state at a pH of 3-7 and a temperature of 26-30 ℃ for 0-10 days. 1-120ul of the culture can be added into the enzymatic reaction system described in example 6, the final degree of the substrate rebaudioside A can be 0.5-120g/L, and the reaction lasts for 1-240 hours.

Example 9

(1) Carrying out mixed culture on a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1 and a recombinant bacterium 2 capable of secreting and expressing glycosyltransferase UGT2 for 2h; the ratio of the cell concentration of the recombinant bacteria 1 to the cell concentration of the recombinant bacteria 2 during inoculation is 1.7, the total concentration of the recombinant bacteria 1 and the recombinant bacteria 2 is controlled to be 1 at the light absorption value OD600 of a bacteria liquid, the pH =5.5 of a culture medium, and the volume concentration of methanol in the culture medium containing methanol is 0.5%;

(2) Adding substrate rebaudioside A with the final degree of 0.5g/L into the culture solution obtained in the step (1), adding uridine diphosphate glucose with the final concentration of 0.2mM, adding magnesium sulfate with the final concentration of 0.5mM, adding methanol with the final concentration of 0.5% every 24 hours, and reacting to obtain the lecitin M. The yield of rebaudioside M was 36.50%.

Example 10

(1) Carrying out mixed culture on a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1 and a recombinant bacterium 2 capable of secreting and expressing glycosyltransferase UGT2 for 2 days; the ratio of the cell concentration of the recombinant bacteria 1 to the cell concentration of the recombinant bacteria 2 during inoculation is 1, the total concentration of the recombinant bacteria 1 and the recombinant bacteria 2 is controlled to be 1 at the light absorption value OD600 of a bacterial liquid, the pH =6.0 of a culture medium containing methanol, and the volume concentration of the methanol in the culture medium containing methanol is 0.75%;

(2) Adding substrate rebaudioside A with the final degree of 2g/L into the culture solution obtained in the step (1), adding uridine diphosphate glucose with the final concentration of 1.5mM, adding magnesium chloride with the final concentration of 5mM, and adding methanol with the final concentration of 0.75% every 24 hours to react to obtain the rebaudioside M. The yield of rebaudioside M was 56.50%.

Example 11

A method for synthesizing lecitinib M in a one-step process, comprising the steps of:

(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 3 days; the cell concentration ratio of the recombinant bacteria 1 to the recombinant bacteria 2 during inoculation is 1;

(2) Adding substrate rebaudioside A with the final degree of 20g/L into the culture solution obtained in the step (1), adding uridine diphosphate glucose with the final concentration of 1.5mM, adding magnesium chloride with the final concentration of 5mM, and adding methanol with the final concentration of 1.5% every 24 hours to react to obtain the rebaudioside M. The yield of rebaudioside M was 28.10%.

Sequence listing

<110> Zhonghua 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 (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> 1485

<212> DNA

<213> Artificial Sequence (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 gttccacacg 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 ggtagaggtg 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 (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 atatcttgtg 2940

gtaggggttt 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 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 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 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4860

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4920

aaaactcacg ttaagggatt ttggtcatga gatc 4954

<210> 4

<211> 1395

<212> DNA

<213> Artificial Sequence (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 cttctggtgt tatttggaac 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 acatttcttc tttgcatcat 1380

catcatcatc attga 1395

<210> 5

<211> 466

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Phe Met Glu Asn Lys Thr Glu Thr Thr Val 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 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

450 455 460

His His

465

<210> 6

<211> 4861

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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 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

tatttaagtt 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 gtggtccggg 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 cgaacccccc 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 agggattttg gtcatgagat 4860

c 4861

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gactggttcc aattgacaag c 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcaaatggca ttctgacatc c 21

<210> 9

<211> 267

<212> DNA

<213> Artificial Sequence (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 (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 (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 (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 tttattgctg 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

1. The one-step method for synthesizing rebaudioside M is characterized by comprising the following steps of:

(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 culturing for 2h-3 days, the ratio of the cell concentrations of the recombinant bacterium 1 and the recombinant bacterium 2 during inoculation is 1;

(2) Adding substrate rebaudioside A with the final degree of 0.5-20g/L into the culture solution obtained in the step (1), adding uridine diphosphate glucose with the final concentration of 0.2-1.5mM, adding magnesium sulfate or magnesium chloride with the final concentration of 0.5-5mM, and adding methanol with the final concentration of 0.5% -1.5% every 24 hours for reaction to obtain rebaudioside M;

the amino acid sequence of the glycosyltransferase UGT2 is shown as SEQ ID NO. 5;

the recombinant bacterium 1 is constructed by the following steps: connecting a glycosyltransferase UGT1 gene with a nucleotide sequence shown as SEQ ID NO.2 to an expression vector pPICZalphaA to obtain a pP-UGT1 plasmid with 4954bp shown as SEQ ID NO.3, wherein the pP-UGT1 plasmid contains a signal peptide alpha factor, and the nucleotide sequence of the signal peptide alpha factor is shown as SEQ ID NO. 9; extracting the plasmid from recombinant escherichia coli containing pP-UGT1, cutting a linear 4954bp fragment shown in SEQ ID NO.3 by PmeI enzyme, and transferring the linear 4954bp fragment into a host cell 1 to obtain a first recombinant bacterium 1;

the recombinant bacterium 2 is constructed by the following steps: connecting a glycosyltransferase UGT2 gene with a nucleotide sequence shown as SEQ ID NO.4 to an expression vector pPICZalphaA to obtain a 4861bp pP-UGT2 plasmid shown as SEQ ID NO.6, wherein the pP-UGT2 plasmid contains a signal peptide alpha factor, and the nucleotide sequence of the signal peptide alpha factor is shown as SEQ ID NO. 9; the plasmid is extracted from recombinant Escherichia coli containing pP-UGT2, a linear 4861bp fragment shown in SEQ ID NO.6 is cut by PmeI, and the linear 4861bp fragment is transferred into a host cell 1 to obtain a first recombinant bacterium 2.

2. The method according to claim 1, wherein the host cell 1 is Pichia pastoris.