Genetically engineered bacteria and application thereof to preparation of rebaudioside A

A technology of genetically engineered bacteria and engineered bacteria, applied in the field of bioengineering, can solve the problems of low adsorption selectivity of RA glycosides, low yield of rebaudioside A, complicated recrystallization process, etc., so as to improve cell stability and reduce Cell damage, potent effect

Active Publication Date: 2017-07-07
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Separation and purification process is used to separate RA glycosides and St glycosides to produce RA glycosides. Among them, the effect of separating RA glycosides and St glycosides by HPLC method is the best, but its processing capacity is too small, and it is difficult to scale up for industrial production; the recrystallization method is complicated. , high energy consumption, long time period, and problems such as organic solvent residues; although resin adsorption has large adsorption and exchange capacity, and is easy to scale production, but rebaudioside A (RA glycoside) and stevioside (St glycoside) with high content ) are very similar in chemical structure and polarity, resulting in relatively small adsorption selectivity to RA glycosides, which affects the separation effect and makes the yield of rebaudioside A low, and there are certain difficulties in industrial practical application
The current bioenzymatic method for synthesizing RA glycosides needs to add expensive UDP-glucose as a substrate, and through the action of UDP-glucosyltransferase, catalyzes St glycosides to generate RA glycosides, resulting in poor economic efficiency of enzymatically catalyzing St glycosides to synthesize RA glycosides , Lack of market competitiveness

Method used

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  • Genetically engineered bacteria and application thereof to preparation of rebaudioside A
  • Genetically engineered bacteria and application thereof to preparation of rebaudioside A
  • Genetically engineered bacteria and application thereof to preparation of rebaudioside A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of recombinant yeast

[0048] 1. Acquisition of glycosyltransferase UGT gene:

[0049] According to the AY345974.1 gene sequence, the codon optimization was carried out, and the optimized gene sequence was named UGT, and the gene synthesis was completed by Nanjing Jinsirui Company. The sequence is shown in SEQ.NO.2.

[0050] Design primers based on UGT gene sequence

[0051] Upstream primers (-sense containing Sal i) For P1:

[0052] 5'-cactatagggcccgggcgtcgacATGTCTGAAAATAAGACTGAAACT-3'

[0053] Downstream primers (-sense containing xho i) For P2:

[0054] 5'-TCTTAGCTAGCCGCGGTACCAAGCTTACTCGAGTTATAATGATGAAAT-3'

[0055] All primers were synthesized by Shanghai Shenergy Gaming Company.

[0056] Gene PCR conditions (50 uL system):

[0057] PCR system: 50μL

[0058] ddH2O: 19 μL

[0059] P1: 2 μL

[0060] P2: 2 μL

[0061] Template: 2 μL

[0062] 2×Phanta Master Mix: 25μL

[0063] Denaturation at 94°C for 5min, followed by 30 cycles...

Embodiment 2

[0085] Example 2: Recombinant Saccharomyces cerevisiae co-express UGT and UGPase

[0086] Activation of strains: In a sterile environment, spread the strains preserved in glycerol tubes on YPDA plates and grow at 30°C for 24-36 hours. Pick a single colony on the YPDA plate and spread it on the SC screening medium in three areas, and grow at 30°C for 24-36 hours.

[0087] Fermentation culture of recombinant bacteria: Pick a single colony on the SC screening plate and transfer it to liquid SC medium, grow at 30°C for 16-20 hours, centrifuge at 6000rpm for 5min, and wash the bacteria twice with potassium phosphate buffer.

[0088] Among them, the nutrient-rich medium YPDA formula is: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, 0.75g / L adenine. Screening medium SC formula is: 6.7 g / L LYNB, 20 g / L glucose, 0.1 g / L adenine, 0.1 g / L cysteine, 0.1 g / L arginine, 0.1 g / L uracil, 0.1g / L Threonine, 0.1g / L Lysine, 0.1g / L Tryptophan, 0.05g / L Histidine, 0.05g / L Aspartic Acid, 0.05g / L ...

Embodiment 3

[0089] Embodiment 3: the establishment of enzyme activity assay method

[0090] The recombinant Saccharomyces cerevisiae cells collected by centrifugation were washed twice with 0.1 mM potassium phosphate buffer (pH 8.0), and the cells were broken by high pressure. Then 4°C, centrifuged at 6000r / min for 30 min to collect the crude enzyme extract for later use.

[0091] Determination of UGT enzyme activity reaction system:

[0092] In a 3 mL reaction system (1.2 mM St glycoside, 4 mM UDPG, 3 mM MgCl 2 , 0.89 mg crude enzyme, pH = 8.0), add the crude enzyme extract to react, after 3 h at room temperature at 30 °C, add water-saturated n-butanol and vortex to terminate the reaction. The supernatant obtained by extraction with n-butanol was analyzed by high performance liquid chromatography (HPLC). Control bacteria were treated in the same way.

[0093] Determination of UGP enzyme activity reaction system:

[0094] Using UGP reversible reaction, its catalyzed reaction generate...

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Abstract

The invention discloses genetically engineered bacteria and application thereof to preparation of rebaudioside A. The genetically engineered bacteria capable of producing stevia rebaudiana glycosyl transferase UGT76G1 and UDP-glucose pyrophosphorylase UGPase are obtained through construction, and perform overexpression on the UDP-glucose pyrophosphorylase UGPase on the basis of performing constitutive expression on recombinant bacteria of the stevia rebaudiana glycosyl transferase UGT to increase the glycosyl donor quantity of glycosylation reaction. A whole-cell catalytic reaction system is established, and stevioside is converted into the rebaudioside A. The dosage concentration of the bacteria is 1 to 10 g / L, the dosage of glucose is 20 to 120 g / L and the dosage of the substrate stevioside is 1 to 20 g / L. In the catalytic reaction process, a cell catalyst is subjected to surfactant treatment, the cell permeability is improved and the yield of the rebaudioside A is greatly increased. A synthetic route of intracellular UDPG is utilized, so the glycosyl donor quantity of the intracellular UDPG is increased, additional addition is avoided and regeneration cycle is realized; and cultivation of yeast and expression of recombinase are conducted simultaneously, so the cycle of the cultivation of the recombinant bacterial and the expression of the recombinase is shortened.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing stevia glycosyltransferase UGT76G1 and UDP-glucose pyrophosphorylase UGPase and its application in preparing rebaudioside A, belonging to the technical field of bioengineering. Background technique [0002] Stevioside is a natural sweetener extracted from stevia stems and leaves. Its main component is stevioside, which is a non-fermentable natural sweetener with high sweetness and low calorie value. The sweetness is about 200-300 times that of sucrose, and the sweetness of purified rebaudioside A sugar is about 450 times that of sucrose, and the taste is better. The caloric value of stevioside is only 1 / 300 of that of sucrose[1]. It has the characteristics of high strength, good taste, high temperature resistance, and good stability. It is not absorbed by the human body after ingestion, and does not generate heat [2]. It is a suitable sweetener for diabetics and obese patients. It ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/81C12N1/19C12P19/56C12R1/865
CPCC07K2319/00C12N9/1048C12N9/1241C12N15/81C12N2830/34C12P19/56C12Y207/07009
Inventor 李艳李阳阳周伯雅严明陈可泉郝宁张竹山于青海陈剑波
Owner NANJING UNIV OF TECH
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