阅读说明：本技术 一步法合成莱鲍迪苷m的方法 (method for synthesizing rebaudioside M ) 是由 马媛媛 汪振洋 宋浩 洪解放 来庆英 刘文斌 张敏华 刘伟 于 2019-11-11 设计创作，主要内容包括：本发明公开了一步法合成莱胞迪苷M的方法,包括如下步骤：(1)将能够分泌表达糖基转移酶UGT1的重组菌1和能够分泌表达糖基转移酶UGT2的重组菌2混合接种在含有甲醇的培养基中培养；(2)向步骤(1)获得的培养液中加入底物莱鲍迪苷A,加入尿苷二磷酸葡萄糖,硫酸镁或氯化镁,甲醇,反应,得到莱胞迪苷M；本发明克服了现有技术催化需要破胞、分离纯化或者加入细胞膜通透剂等繁琐步骤；本发明的原料莱鲍迪苷A相对于现有技术的价格昂贵莱鲍迪苷D做原料,价格低廉。提高重组菌糖苷转移酶的产量及热稳定性,实现直接用价值低的RebA为底物混菌催化一步法获得RebM。提高催化效率,降低酶纯化所需费用及底物的费用。(The invention discloses a step method for synthesizing rebaudioside M, which comprises the following steps of (1) mixing and inoculating a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1 and a recombinant bacterium 2 capable of secreting and expressing glycosyltransferase UGT2 in a culture medium containing methanol for culture, (2) adding a substrate rebaudioside A into a culture solution obtained in the step (1), and adding uridine diphosphate glucose, magnesium sulfate or magnesium chloride and methanol for reaction to obtain the rebaudioside M.)

1, A method for synthesizing lecytosine M, 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 in SEQ ID NO. 1;

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

2. The method according to claim 1, wherein step (1) is: 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 1:0.7-2, the total concentration of the recombinant bacterium 1 and the recombinant bacterium 2 is controlled to be 1 of the light absorption value OD600 of a bacterium solution, the pH value of the culture medium containing methanol is 5.5-8, and the volume concentration of methanol in the culture medium containing methanol is 0.5-1.5%.

3. The method according to claim 1, wherein the step (2) is: 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.

4. The method according to claim 1 or 2, wherein the recombinant bacterium 1 is constructed by the steps of connecting a glycosyltransferase UGT1 gene to an expression vector and then transferring the gene into a host cell 1 to obtain a th recombinant bacterium 1, or integrating a glycosyltransferase UGT1 gene into the genome of the host cell 1 through a molecular biology technology to obtain a second recombinant bacterium 1, wherein the nucleotide sequence of the glycosyltransferase UGT1 gene is shown as SEQ ID NO. 2.

5. The method according to claim 4, wherein the expression vector is pPICZalphaA/B/C, pPIC9K, pPIC9, pPink α -HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT 43.

6. The method according to claim 4, characterized in that the host cell 1 is Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis.

7. The method according to claim 1 or 2, wherein the recombinant bacterium 2 is constructed by connecting a glycosyltransferase UGT2 gene to an expression vector and then transferring the gene into a host cell 1 to obtain a th recombinant bacterium 2, or integrating a glycosyltransferase UGT2 gene into the genome of the host cell 1 through a molecular biology technology to obtain a second recombinant bacterium 2, wherein the nucleotide sequence of the glycosyltransferase UGT2 gene is shown as SEQ ID NO. 4.

8. The method according to claim 7, wherein the expression vector is pPICZalphaA/B/C, pPIC9K, pPIC9, pPink α -HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vector.

9. The method according to claim 7, characterized in that the host cell 1 is Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis.

10. The method as claimed in claim 1, wherein the signal peptide for the UGT1 protein secretion of recombinant strain 1 is the 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;

the signal peptide used by the recombinant strain 2 for secreting UGT1 protein is the signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha and SED1, and the corresponding nucleotide sequences of the signal peptide are sequentially 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.

Technical Field

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

Background

The stevioside is novel natural low calorie sweetener stevioside compounds extracted from stevia leaf of Compositae herb, and has high sweetness, low calorie, no toxicity, high temperature resistance, acid and alkali resistance, good water solubility, etc. and is certified as safe by the American food and drug administration, and can be applied in 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. Biocatalytic methods to obtain high concentrations of RebM have attracted attention from 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 bacteriumObtaining recombinase, preparation of enzyme and catalysis are time-consuming, labor-consuming and financial-consuming^[3-9]Based on the current research situation, methods with low price, large yield, few steps, rapidness, effectiveness and low cost are urgently needed to obtain rebaudioside M.

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 journal, 2018, 09: 142.

[3]Prakash I,Markosyan A,Bunders C.Development ofnext generationstevia 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] Yubo glu, Wangning Lin, Lizhou Jing, etc. the application of genetic engineering and metabolic engineering in the production of stevioside is advanced [ J ]. report on biotechnology 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] Plums are bright, strict and aging, method for preparing rebaudioside M by enzyme method [ P ]. China, 107666834, 2018-08-10.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides methods for synthesizing rebaudioside M by a step method.

The technical scheme of the invention is summarized as follows:

Process for the synthesis of lecytosine M 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 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 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 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 1:0.7-2, the total concentration of the recombinant bacterium 1 and the recombinant bacterium 2 is controlled to be 1 of the light absorption value OD600 of a bacterium solution, the pH value of the culture medium containing methanol is 5.5-8, and the volume concentration of methanol in the culture medium containing methanol is 0.5-1.5%.

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 of connecting a glycosyltransferase UGT1 gene to an expression vector and then transferring the gene into a host cell 1 to obtain a th recombinant bacterium 1, or integrating a glycosyltransferase UGT1 gene into a genome of the host cell 1 through a molecular biology technology to obtain a second recombinant bacterium 1, wherein 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 α -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 of connecting a glycosyltransferase UGT2 gene to an expression vector and then transferring the gene into a host cell 1 to obtain a th recombinant bacterium 2, or integrating a glycosyltransferase UGT2 gene into a genome of the host cell 1 through a molecular biology technology to obtain a second recombinant bacterium 2, wherein the nucleotide sequence of the glycosyltransferase UGT2 gene is shown as SEQ ID NO. 4.

The expression vector is pPICZalphaA/B/C, pPIC9K, pPIC9, pPink α -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 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 sequentially 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;

the signal peptide used by the recombinant strain 2 for secreting UGT1 protein is the signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha and SED1, and the corresponding nucleotide sequences of the signal peptide are sequentially 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.

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, steps, and overcomes the complex steps of cell breaking, separation and purification or adding cell membrane penetrating agent in the prior art.

3. The existing biocatalysis technology uses a substrate rebaudioside D (RebD) to produce rebaudioside M (RebM), the RebD is expensive compared with the substrate rebaudioside A (RebA) used in the technology, and the invention uses the RebA with relatively low price as the substrate to reduce the production cost.

4. The method can be used for producing the RebM which is sweetening agents with high sweetness and good mouthfeel by adding the steps of RebA, &lTtTtransfer = one & ' gTt-one & ' lTt/T & ' gTt into fermentation liquor through mixed fermentation, can obtain the glycosidase with secretory expression, can improve the yield and the thermal stability of recombinant bacteria glycosidase, realizes that the RebM is obtained by directly using the RebA with low value as a substrate mixed bacteria catalysis step method, improves the catalysis efficiency, reduces the cost required by enzyme purification and the cost of the substrate, and obtains the RebM with high sweetness, good mouthfeel and high value.

Drawings

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

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

FIG. 3 is an electrophoretogram of total protein of the culture supernatant of each transformant. Wherein a is an electrophoretogram of transformant secretion expression UGT 1; b is an electrophoresis picture of transformant secreting and expressing UGT 2.

FIG. 4 recombinant Strain 1 and recombinant Strain 2 recombinant Pichia pastoris expression of UGT2 at various methanol concentrations. The control bacterium X33, the recombinant strain 1 and the recombinant strain 2 were cultured on day 3, and the culture supernatant was collected and concentrated 5-fold in an electrophoretogram. 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 UGT 2. 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 electrophoretogram of purified UGT 2.

FIG. 6 strain growth and protein concentration determination during the production of RebM by recombinant Strain 1 and recombinant Strain 2 steps.

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 day 4 of strain culture (i.e., day 2 and day 3 after feeding). 1 #: feeding materials at the pH of 6.0, the ratio of EX-3 to XS-35 of 1:1 and on the 2 nd day; 2 #: feeding materials at 3 days with the pH of 6.0 and the ratio of EX-3 to XS-35 of 1: 2; 3 #: feeding materials at the 3 rd day with the pH of 7.3 and the ratio of EX-3 to XS-35 of 1: 1; 4 #: pH 7.3, EX-3 and XS-35 at a ratio of 1:2, fed on day 2.

Detailed Description

Coli TOP 10F' (commercial product).

The invention is further illustrated in with reference to specific examples.

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