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Genetically engineered bacteria used for producing stevia glycosyltransferase UGT76G1 and application thereof

A technology of UGT76G1, genetically engineered bacteria, applied in genetic engineering, transferase, application, etc., can solve the problems of not being a solution, high price, difficult purification process, etc.

Inactive Publication Date: 2012-07-11
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the strategies for increasing the relative content of rebaudioside A glycosides reported in the literature include: (1) Cultivate stevia varieties with high content of rebaudioside A glycosides. Domestic attempts have been made to obtain varieties with rebaudioside A glycosides content exceeding 40% through grafting, but this variety does not have the common Adaptability, and the variety is easy to degenerate, so it is not suitable for large-scale planting [11] (2) improve the ratio of rebaudioside A glucoside and stevioside through purification, this method is because rebaudioside A glycoside content is lower than stevioside in the natural plant, and the two physical properties are very similar, making the purification process very difficult, and the yield is low
The high cost of these two methods has caused the high price of stevia with high rebaudioside A content in the market, and the market price of stevioside containing 80% rebaudioside A is 4 to 5 times higher than that of general mixed stevia
It is also reported that the taste and taste of stevioside can be improved by using cyclodextrin glycosyltransferase modification method, but this method seriously reduces the sweetness, so it is not the best solution
In short, the existing methods are not suitable for promotion

Method used

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  • Genetically engineered bacteria used for producing stevia glycosyltransferase UGT76G1 and application thereof
  • Genetically engineered bacteria used for producing stevia glycosyltransferase UGT76G1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of recombinant yeast.

[0051] 1. Acquisition of glycosyltransferase UGT gene:

[0052] According to the AY345974.1 gene sequence, codon optimization was carried out, and the optimized gene sequence was named UGT, and the gene synthesis was completed by Nanjing Jinsirui Company.

[0053] Design primers based on UGT gene sequence

[0054] The upstream primer (-sense contains EcoRI) is:

[0055] 5′-CGGAATTCAAACAATGTCTGAAAATAAGACTGAAACTACTG-3′

[0056] The downstream primer (-sense contains XhoI) is:

[0057] 5′-CCGCTCGAGTTATAATGATGAAATATAAGAAACCAA-3′

[0058] All primers were synthesized by Shanghai Shenergy Gaming Company.

[0059] Gene PCR conditions (50μL system):

[0060] Denaturation at 94°C for 5 minutes;

[0061] Cycle 30 times according to the following parameters: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min;

[0062] Finally, extend at 72°C for 10 min.

[0063] 2. The recombinant engineered...

Embodiment 2

[0069] Example 2: Induced expression of recombinant yeast.

[0070] Pick a single colony of the recombinant engineered bacteria into SC-U medium, and cultivate overnight at 30°C with shaking. Then inoculate on the fresh medium whose carbon source is glucose (final concentration is 20g / L) according to 2% inoculation amount, cultivate 8h, this part is the accumulation of biomass. Then in a sterile environment, collect the bacteria and discard the supernatant, wash the bacteria and transfer the bacteria to a fresh selection medium whose carbon source is galactose (final concentration is 20g / L) for induction. The induction time is 48h. The bacterial solution was centrifuged at 6000 rpm at 4°C for 10 min, and the supernatant was discarded.

[0071] Among them, the medium formula with glucose as the carbon source is: 6.7g / L YNB, 20g / L glucose, 0.1g / L adenine, 0.1g / L arginine, 0.1g / L cysteine, 0.1g / L L Leucine, 0.1g / L Lysine, 0.1g / L Threonine, 0.1g / L Tryptophan, 0.05g / L Aspartic A...

Embodiment 3

[0073] Example 3: Establishment of enzyme activity assay method.

[0074] Get the thalline precipitation in Example 2, thalline 20mg, wash twice with potassium phosphate buffer (pH7.0), the precipitation after washing is ground and broken in liquid nitrogen, and wash with potassium phosphate buffer (pH7.0) The broken bacterial solution was centrifuged at 12000rpm and 4°C for 15min. The supernatant is the crude enzyme solution. Precisely weigh the sample and prepare a 1.4ml system, in which the final concentration of stevioside is 1g / L, UDP-glucose is 1g / L, and 2g / L Mgcl is added 2 ; Mix 10mg / L BSA, and finally add 400μl of crude enzyme solution and potassium phosphate buffer (pH7.0) until the system is 1.4ml, and start the reaction. After heat preservation at 30°C for 12h, the reaction was terminated by boiling at high temperature. Centrifuge and take the supernatant as a sample. The content of rebaudiosideA glycoside in the reaction system was detected by HPLC. The exper...

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Abstract

The invention discloses a genetically engineered bacteria used for producing stevia glycosyltransferase UGT76G1; a UGT76G1 coding gene is inserted between the restriction enzyme cutting sites EcoRI and XhoI of a PYes2 carrier, so as to construct a recombinant plasmid, and then the recombinant plasmid is introduced to expression host Saccharomyces cerevisiae YPH499 to obtain the engineered bacteria; and the coding gene of the UGT76G1 is GenBank, No. GenBank: AY345974.1, and the gene sequence of the coding gene is named as UGT (Udp Glucuronyl Transferase). The invention also discloses a construction method of the genetically engineered bacteria and the application of the bacteria to the production of rebaudioside A. According to the invention, under the condition that expensive UDPG (Uridine Diphosphate Glucose) is not added, cheap carbon source glucose is used as a substrate, the metabolic pathway of UDPG in the yeast is regulated, and then the rebaudioside A is produced from St glycosides through whole cell catalysis.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing stevia glycosyltransferase UGT76G1 and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Sweeteners are the most widely used additives in the food industry, and can be divided into natural sweeteners and artificial sweeteners according to their sources. Among natural sweeteners, sucrose is the most widely used sweetener, but sucrose is a high-calorie sweetener, and excessive intake can lead to obesity, diabetes, dental caries and other diseases. [1，2，3，4] . Artificially synthesized sweeteners, such as dulin (p-ethoxyphenylurea) and cyclamata, although they can satisfy people's sweetness, have been found to have toxic side effects and have been banned. Prohibited; saccharin (saccharin, o-sulfonyl benzimide), may also be restricted due to reported carcinogenic effects. Finding non-toxic, safe, low-calorie, high-sweet natural sw...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/54C12N15/63C12N9/10C12P19/56C12R1/865
Inventor 严明李艳刘欢许琳郝宁魏淼许昇安明东王珊珊安芳芳郝思清顾金海
Owner NANJING UNIV OF TECH
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