阅读说明：本技术 一锅法生产莱鲍迪苷d的方法 (Method for producing rebaudioside D by one-pot method ) 是由 马媛媛 宋浩 洪解放 汪振洋 来庆英 张敏华 于 2019-11-11 设计创作，主要内容包括：本发明公开了一锅法生产莱鲍迪苷D的方法,包括如下步骤：构建能分泌表达糖基转移酶UGT1的重组菌1,所述糖基转移酶UGT1的氨基酸序列如SEQ ID NO.1所示；在pH3.5-7.8的培养基中培养重组菌1,得到培养液；在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,得到莱鲍迪苷D。本发明的一锅法生产莱鲍迪苷D在重组菌1发酵生成糖基转移酶UGT1的同时可催化莱鲍迪苷A生成莱鲍迪苷D,无需纯化UGT1和离心分离就能获得莱鲍迪苷D的。本发明的方法简单、高效、可以降低分离糖基转移酶UGT1的生产成本,可以商业化获得甜度高、口感好、高价值的莱鲍迪苷D。(The invention discloses a method for producing rebaudioside D by a one-pot method, which comprises the following steps: constructing a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1, wherein the amino acid sequence of the glycosyltransferase UGT1 is shown as SEQ ID NO. 1; culturing the recombinant bacterium 1 in a culture medium with the pH value of 3.5-7.8 to obtain a culture solution; adding a substrate rebaudioside A into the culture solution, adding uridine diphosphate glucose, magnesium sulfate or magnesium chloride and methanol, and reacting to obtain rebaudioside D. The rebaudioside D produced by the one-pot method can catalyze rebaudioside A to produce rebaudioside D while the recombinant bacterium 1 is fermented to produce the glycosyltransferase UGT1, and rebaudioside D can be obtained without purifying UGT1 and performing centrifugal separation. The method is simple and efficient, can reduce the production cost of separating glycosyltransferase UGT1, and can commercially obtain rebaudioside D with high sweetness, good taste and high value.)

1. The method for producing rebaudioside D by a one-pot method is characterized by comprising the following steps of:

(1) constructing a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1, wherein the amino acid sequence of the glycosyltransferase UGT1 is shown as SEQ ID NO. 1;

(2) culturing the recombinant bacterium 1 in a culture medium with the pH value of 3.5-7.8 to obtain a culture solution;

(3) and (3) adding a substrate rebaudioside A into the culture solution obtained in the step (2), adding uridine diphosphate glucose, magnesium sulfate or magnesium chloride and methanol, and reacting to obtain rebaudioside D.

2. The method according to claim 1, wherein the recombinant bacterium 1 is constructed by the steps of: the glycosyltransferase UGT1 gene is connected to an expression vector and then transferred into a host cell to obtain a first recombinant bacterium 1; or integrating a glycosyltransferase UGT1 gene into the genome of a host cell by a molecular biology technology to obtain a second recombinant bacterium 1; the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO. 2.

3. The method as claimed in claim 1, wherein the step (3) is: adding a substrate rebaudioside A with a final concentration of 0.3-20g/L into the culture solution obtained in the step (2), adding uridine diphosphate glucose with a final concentration of 0.2-1.5mM, adding magnesium sulfate or magnesium chloride with a final concentration of 0.5-5mM, and adding methanol with a final concentration of 0.5% -1.5% every 24 hours to react to obtain rebaudioside D.

4. The method according to claim 2, characterized in that the host cell is Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis.

5. The method according to claim 2, characterized in that the expression vector is a pPICZalphaA/B/C, pPIC9K, pPIC9, pPink α -HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vector.

6. The method as claimed in claim 1, wherein the signal peptide for secretion expression of glycosyltransferase UGT1 by recombinant strain 1 is signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha or SED1, and the corresponding nucleotide sequences of the signal peptide are sequentially shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No. 12.

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a method for producing rebaudioside D by a one-pot method, which comprises the steps of constructing a recombinant bacterium 1 capable of secreting UGT1 expression, culturing the recombinant bacterium 1 and directly adding a substrate RA to generate RD without separation and purification.

Background

Stevioside, a novel natural low-calorie sweetener extracted from stevia rebaudiana leaves of Compositae herbaceous plants, is certified as safe by the United states food and drug administration and can be applied to the food industry due to the advantages of high sweetness, low calorie, no toxicity, high temperature resistance, acid and alkali resistance, good water solubility and the like. Rebaudioside D (RD) has better mouthfeel characteristics, but stevia contains only about 0.5%, resulting in high separation cost and high price. Biocatalytic methods have attracted attention from scholars to obtain high concentrations of RD. At present, recombinant enzyme from stevia rebaudiana can catalyze RA to generate RD, but the yield is low and the cost is high.

RD can be obtained by a biocatalysis method by taking RA as a substrate, but the biocatalysis process mainly has the following problems: (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 separate and purify; (2) the recombinant glycosyltransferase can catalyze the generation of a product RD, but the recombinant enzyme produced by intracellular expression can be catalyzed only by the processes of centrifugally collecting thalli, breaking cells and the like, and the steps are complicated; (3) during the reaction, most of the activity of the UGT enzyme is lost due to thermal inhibition. Due to the fact thatThus, efficient biocatalytic acquisition of RD requires highly efficient, simple, and low cost techniques^[3]。

Since the yield of the natural glycosyltransferase is low, it is difficult to isolate and purify the glycosyltransferase. The intracellular expression for producing the glycosyltransferase requires complicated processes such as collection of thalli, cell breaking and the like. If the generation of RD is realized by constructing the recombinant strain and secreting and producing glycosyltransferase and directly feeding the substrate RA in the culture process of culturing the recombinant strain, the process steps for generating RD can be reduced, the production cost is reduced, and the method is a feasible way for the industrial scale generation of RD.

Reference documents:

[1] ferrieron, Wangyong, high-efficiency biocatalytic synthesis of sweetener rebaudioside D [ J ]. food and fermentation industry, 2018,44(4):1-7.

[2] The recombinant colibacillus is used to catalytically synthesize rebaudiodside D [ J ] in whole cell, 2017,47(5):1-7.

[3] Wang Zhengyu, Fish Red flashing, Jinfeng harmonize, high temperature anaerobe for producing cellulose endoenzyme and activity of glucosidase, proceedings of Dalian institute of Industrial science 2005, 24(2), 110-l 14.

[4] A gem, the current application and development prospect of the sweetener [ J ]. the contemporary chemical industry, 2002,31(2):89-91.

Zhengjian Xian, functional food additive [ M ]. Beijing, published by the light industry of China, 1997.

[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.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for producing rebaudioside D by a one-pot method.

The technical scheme of the invention is summarized as follows:

a one-pot method for producing rebaudioside D comprising the steps of:

(1) constructing a recombinant bacterium 1 capable of secreting and expressing glycosyltransferase UGT1, wherein the amino acid sequence of the glycosyltransferase UGT1 is shown as SEQ ID NO. 1;

(2) culturing the recombinant bacterium 1 in a culture medium with the pH value of 3.5-7.8 for 0.2-2 days to obtain a culture solution;

(3) and (3) adding a substrate rebaudioside A into the culture solution obtained in the step (2), adding uridine diphosphate glucose, magnesium sulfate or magnesium chloride and methanol, and reacting to obtain rebaudioside D.

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 to obtain a first recombinant bacterium 1; or integrating a glycosyltransferase UGT1 gene into the genome of a host cell by a molecular biology technology to obtain a second recombinant bacterium 1; the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO. 2.

Step (3) is preferably: adding a substrate rebaudioside A with a final concentration of 0.3-20g/L into the culture solution obtained in the step (2), adding uridine diphosphate glucose with a final concentration of 0.2-1.5mM, adding magnesium sulfate or magnesium chloride with a final concentration of 0.5-5mM, and adding methanol with a final concentration of 0.5% -1.5% every 24 hours to react to obtain rebaudioside D.

The host cell is preferably: saccharomyces cerevisiae, Pichia pastoris, or Bacillus subtilis.

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

The recombinant bacterium 1 secretes and expresses a signal peptide used by glycosyltransferase UGT1, wherein the signal peptide is a signal peptide of alpha factor, xyn2s, phoA, phoD, amyE, MFalpha or SED1, and the corresponding nucleotide sequences of the signal peptide are sequentially shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.

THE ADVANTAGES OF THE PRESENT INVENTION

The rebaudioside D produced by the one-pot method can catalyze rebaudioside A to produce rebaudioside D while the recombinant bacterium 1 is fermented to produce the glycosyltransferase UGT1, and rebaudioside D can be obtained without purifying UGT1 and performing centrifugal separation. The method is simple and efficient, can reduce the production cost of separating glycosyltransferase UGT1, and can commercially obtain rebaudioside D with high sweetness, good taste and high value.

Drawings

FIG. 1 is a schematic diagram of a recombinant vector construction scheme of UGT1 gene containing glycosyltransferase.

FIG. 2 is a PCR identification electropherogram of recombinant Pichia pastoris transformants of UGT1 gene for glycosyltransferase. Lane M is DNA Standard molecular weight Marker; each lane shows the electrophoretogram of PCR products of each transformant and a control strain X33, CK (water template).

FIG. 3 Total protein concentration and electrophoretogram of the culture supernatant of each transformant.

FIG. 3a is the total protein concentration of the supernatant of each transformant culture, and FIG. 3b is an electrophoretogram of the total protein of the supernatant of each transformant culture.

FIG. 4 SDS-PAGE patterns of UGT1 in culture supernatants at 0-7 days of fermentation. The supernatant of the fermentation broth was concentrated 5 times and then 10ul of the supernatant was applied.

FIG. 5 recombinant Pichia pastoris expression of UGT1 at various methanol concentrations. a is the total protein concentration secreted by the recombinant bacterium 1 when cultured in various methanol concentrations, and fig. 5b is an electrophoresis chart of UGT1 secreted by the recombinant bacterium 1 when cultured in various methanol concentrations.

FIG. 6 is an SDS-PAGE pattern of purified UGT 1.

Detailed Description

Coli TOP 10F' (commercial product).

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 α -HC, pYES2, YCplac33, YEplac195, pHT01, pHT08 or pHT43 vectors.