A kind of genetically engineered bacteria and its application in the preparation of rebaudioside A

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 ...

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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

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