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Engineered yeast biological system for efficiently detecting sweetness strength and application thereof

A sweet intensity, biological system technology, applied in the field of bioengineering, can solve the problems of insufficient universality, easy interference, poor stability, etc., and achieve the effects of stable host, low culture cost, and mild culture conditions.

Inactive Publication Date: 2017-12-26
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to overcome the shortcomings of the existing sweetness intensity detection methods, such as high cost, poor stability, susceptibility to interference, and insufficient universality, to discover more unknown sweetness substances, and to formulate new sweetness standards, this paper The invention uses Saccharomyces cerevisiae with clear genetic background, absolute biosafety and short growth cycle as the host to express G protein-coupled sweet receptors T1R2 / T1R3, and add different sweet substances into the culture environment respectively. After the taste receptor binds, the host's own G protein signaling pathway is used to amplify the signal, activate the downstream detection circuit, express the fluorescent protein or enzyme, and characterize the sweetness intensity of the sweet substance by the specific fluorescence intensity or the corresponding enzyme catalysis

Method used

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  • Engineered yeast biological system for efficiently detecting sweetness strength and application thereof
  • Engineered yeast biological system for efficiently detecting sweetness strength and application thereof
  • Engineered yeast biological system for efficiently detecting sweetness strength and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The scheme is to set the concentration of α factor as 0 μmol / L, 1 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, and 15 μmol / L for gradient shaking culture, and the 0, 4, 8, 10, 12, 14 , 16, 18, 20, 22, 24, 26, 28, 30h, take about 5ml of bacterial culture suspension, and measure the growth curve. The absorbance value (OD) of bacterial solution at each time point 600 ) as the ordinate, and the culture time as the abscissa, draw the growth curve of yeast engineered bacteria.

[0027] Pick a single colony of blank host cells on a solid plate, inoculate it into a shaker flask with 100ml of YPD liquid medium, place it on a shaker, 28-32°C, 100-250rpm, 8-12 hours, to obtain a seed solution, and use 10% Ratio Inoculate the seed solution into a 96-well plate with YPD liquid medium to 150 μL of bacterial solution per well, add the above-mentioned different concentrations of α factors to each well, and the growth curve is as follows: figure 2 , as the concentration of α-pheromone in...

Embodiment 2

[0029] According to the scheme of Example 1, a single yeast cell colony of △sst2 was picked, and its growth curve was determined using a histidine-deficient medium. The growth curve was as follows: image 3 ,Depend on image 3 It can be seen that after the sst2 gene is knocked out, the signal pathway can amplify the signal.

Embodiment 3

[0031] According to the protocol of Example 1, single yeast cell colonies of △far1+△sst2 were picked, and their growth curves were measured using histidine and uracil-deficient medium, and the results were as follows Figure 4 As shown, compared with Example 2, it can be seen that after the knockout of the far1 gene, the growth inhibitory effect of the signaling pathway itself on the bacteria was released.

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Abstract

The invention discloses a biological system for efficiently detecting sweetness strength of a to-be-detected matter by taking yeast cell as host on the basis of combining sweetness receptor protein T1R2 / T1R3 with a detection gene path. The biological system comprises the sweetness receptor protein T1R2 / T1R3 subjected to modification and codon optimization, a modified Gpa1 protein adaptive to T1R2 / T1R3, a G protein coupling signal path for signal amplification without signal interference and an inducible detection gene path with fluorescent protein or enzyme as a reporter gene. After the to-be-detected matter is added into a culture environment and the sweet matter molecule is combined with the sweetness receptor, the host G protein coupling signal path is utilized to amplify the signal and activate a downstream detection path, the expression condition of the report gene is taken as signal output, and the sweetness strength of the sweet matter is represented by the signal output strength. The biological system disclosed by the invention has the advantages of simple culture condition, low cost, short period, wide detection scope, high repeatability, high stability and capability of realizing the efficient detection for the sweetness strength of different sweet matters.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, can be applied in various industries such as food, medicine, beverage, additives, etc., and can detect the sweetness intensity of various sweet substances. Background technique [0002] Sweetness is the most popular taste of human beings. It can be used to improve the palatability and certain eating properties of food, and at the same time bring people a pleasant feeling, so sweeteners are widely used in food, beverages, medicines, additives and other industries. With the improvement of living standards, human beings are more and more dependent on sweets, and excessive consumption of sweets may lead to obesity and other problems. The World Health Organization pointed out through investigation and analysis that long-term consumption of sugar can shorten human life by about 20 years. Therefore, active research to find sweeteners with good taste, harmless to human body and low calorie is cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19G01N21/64C12R1/865
CPCC07K14/705G01N21/6486
Inventor 李春胡鹏晶贾锦彤任师超阳洪宇白嘉琪倪江萍姜恬吕波
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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