CN107513505A - A kind of engineered yeast biosystem of efficient detection sweetness intensities and its application - Google Patents

Abstract

The invention discloses one kind to have merged sweet receptor albumen T1R2/T1R3 and detection genetic circuits, the biosystem that can the sweetness intensities of determinand be carried out with efficient detection using yeast cells as host, including the sweet receptor albumen T1R2/T1R3 for carrying out modification and codon optimization, T1R2/T1R3 Gpa1 albumen is adapted to after transformation, signal interference, the G-protein coupled signal path for amplifying signal are eliminated, and induction type, there are the detection genetic circuits of fluorescin or enzyme as reporter gene.Determinand is added in culture environment, after sweet substance molecule is combined with sweet receptor, signal is amplified using host itself G-protein coupled signal path, activate detected downstream circuit, using the expression of reporter gene as signal output, the sweetness intensities of sweet substance are characterized by signal output intensity.Condition of culture of the invention is simple, cost is cheap, the cycle is short, detection range is wide, repeatability is high, stability is high, and the efficient detection to different sweet substance sweetness intensities can be achieved.

Description

A kind of engineered yeast biosystem of efficient detection sweetness intensities and its application

Technical field

The invention belongs to technical field of bioengineering, can be applicable to the industries such as varieties of food items, medicine, beverage, additive, can The sweetness intensities of various different sweet substances are detected.

Background technology

Sweet taste is the sense of taste most welcome by the mankind, and it can be used in improving the palatability of food and some edible properties, together When give people to bring the impression of pleasure, so sweetener is widely used in the industries such as food, beverage, medicine, additive.With life The flat raising of running water, the mankind are for the degree of dependence also more and more higher of sweet food, and excessive edible sweet food may result in obesity The problems such as, the World Health Organization analyzes point out by inquiry, and long-term consumption sugar can make the lost of life about 20 years of people.So product It is closely bound up with human lives that this work of sweetener in good taste, harmless, low in calories is found in pole research, to raising people Class living standard is significant.The power of sweet taste can represent that it is the important indicator of sweetener with sweetness intensities, current For the detection method of sweetness intensities, mainly there are organoleptic analysis, electronic tongues detection and analysis, cell line method.Wherein, Organoleptic method Using people as the main body of sensory evaluation, there is unstability and be easily disturbed；Electronic tongues detection method equipment cost is higher, It is ripe not enough to sample amounts analysis and sensor has selectivity and restricted, different samples need to carry out it is different grope, It is difficult to popularize；Cell line method adds the costs such as fund, time because cell culture condition is harsh, excessive cycle, it is difficult to using Among actual production.

The content of the invention

In order to overcome the middle cost of the sweetness intensities detection method of existing sweet substance is higher, stability is poor, be easily disturbed, The shortcomings of universality is inadequate, more unknown sweet substances are excavated, and formulate new sweetness standards, the present invention is clear using genetic background Clear, the Absolute oral safety and short saccharomyces cerevisiae of growth cycle is host, expression G-protein coupling sweet receptor T1R2/T1R3, Different sweet substances are added in culture environment respectively, after sweet substance molecule is combined with sweet receptor, utilize host itself G Protein signal path amplifies signal, activates detected downstream circuit, expresses fluorescin or enzyme, by than fluorescence intensity or corresponding Enzymatic situation characterize the sweetness intensities of sweet substance.Because condition of culture is simple, cost is cheap, the cycle is short, detection model The advantages such as wide, repeatable height, stability height are enclosed, the efficient detection to different sweet substance sweetness intensities can be achieved.

The patent technical solution adopted for solving the technical problem of the present invention is：One kind has merged T1R2/T1R3 sweet receptors With detection genetic circuits, the biosystem using yeast cells as host, including carried out the sweet receptor albumen of codon optimization T1R2/T1R3, is applied to sweet receptor albumen T1R2/T1R3 G-protein α subunit Gpa1 albumen after transformation, and eliminates letter Number interference, the G-protein coupled signal path of enhancing sweetness intensities signal, induction type, have fluorescin or enzyme as report base The detection genetic circuits of cause.

Above-mentioned biosystem, the T1R2/T1R3 sweet proteins acceptor, gene order is obtained by PDB databases, gone forward side by side Row is applied to the codon optimization of yeast cells, then excellent by the gene order progress of given data and experimental verification to Gpa1 Change, the gene order of Gpa1, T1R2/T1R3 albumen is synthesized, then be conducted into host cell, after modification Gpa1 albumen is expressed with sweet receptor albumen T1R2/T1R3, solves Heterologous signal path incompatibility problem.

Above-mentioned transformation bacterial strain, the G-protein coupled signal path, by saccharomyces cerevisiae genome with G-protein signal path 3 relevant genes far1, ste2, sst2 carry out gene knockout.Ste2 genes exclusion pheromones are wherein knocked out for signal to lead to The interference on road, the signal transduction of sst2 genes amplification gpa1 genes is knocked out, knock out yeast sweet taste after far1 genes avoid transformation and believe Interference number for yeast normal physiological function, solves the problems such as stability is poor, is easily disturbed.

Above-mentioned transformation bacterial strain, the detection genetic circuits are the inducible promoter P for including specific activation_fus, fluorescence egg White or enzyme gene, terminator cyc1t expression cassette.

The effect that the present invention reaches is, by yeast cells heterogenous expression sweet receptor T1R2/T1R3, to be coupled downstream signal Path, existing most of sweetener on market today is verified, changed, establish new sweetness standards, and detect newly Sweet substance.Because the host that the present invention uses is stable, growth cycle is short, and condition of culture is gentle, and toxigenic capacity is low, sweet receptor There is universality for sweet substance, realize and the detection of sweet substance rapidly and efficiently is demarcated.

Brief description of the drawings

Fig. 1 is that sweet taste signal receives, conducts, amplifies, exports schematic diagram.

Fig. 2 is the growth curve of host when various concentrations α pheromones are added in host's culture environment.

Fig. 3 is the growth curve that △ sst2 bacterial strains are added after various concentrations α pheromones.

Fig. 4 is the growth curve that △ far1+ △ sst2 bacterial strains are added after various concentrations α pheromones.

After Fig. 5 is imports detection genetic circuits, fluorescence table of the yeast cells in the case of various concentrations α pheromones are added Up to curve.

Fig. 6 is that the △ far1+ △ sst2 yeast cells for importing detection genetic circuits is adding various concentrations α pheromones situations Under luciferase expression curve.

Fig. 7 is that the △ far1+ △ sst2+ △ ste2 yeast cells for importing detection genetic circuits is adding various concentrations α letters Luciferase expression curve in the case of breath element.

Fig. 8 detects genetic circuits, the △ far1+ △ sst2+ △ ste2 yeast cells of T1R2/T1R3 sweet receptors to import Immunofluorescence figure, left figure be light field under the conditions of, right figure be fluorescence excitation conditions under.

Embodiment

Below by embodiment, the present invention is furture elucidated, but not limited to this.

YPD culture mediums：About 10g/L yeast extracts, 20g/L glucose, 20g/L peptones, prepare solid medium and add 20g/L agar powders.

Deficiency culture medium：About 6.7g/L YNB (yeast nitrogen base without amino acids), Other nonessential amino acid powder (excluding leucine, histidine, tryptophan, uracil) of 0.6g-1.3g/L, 20g/L glucose Or galactolipin etc., in addition, adding following raw material according to different needs：

Leucine powder：60-100mg/L

Tryptophan powder：20-40mg/L

Histidine powder：20-40mg/L

Uracil powder：20-40mg/L

15-25g/L agar powders need to be added by preparing solid medium, adjust pH to 6.2-6.5.

【Embodiment 1】

Scheme is that setting α factor concentrations are 0 μm of ol/L, 1 μm of ol/L, 2.5 μm of ol/L, 5 μm of ol/L, 10 μm of ol/L, 15 μ Mol/L gradients shaken cultivation and 0 after culture, 4,8,10,12,14,16,18,20,22,24,26,28,30h take thalline Suspension 5ml or so is cultivated, determines growth curve.With the absorption photometric value (OD of each time point bacterium solution₆₀₀) it is ordinate, with culture Time is abscissa, draws out the growth curve of Yeast engineering bacteria.

Blank host cell single bacterium colony on picking solid plate, is inoculated into the shaking flask of 100mlYPD fluid nutrient mediums Shaking table is put into, 28-32 DEG C, 100-250rpm, 8-12 hour, seed liquor is obtained, has been inoculated into seed liquor with 10% ratio To every μ L of pore fungi liquid 150 among 96 orifice plates of YPD fluid nutrient mediums, the α factors of above-mentioned various concentrations are added into each hole, it is raw Long curve such as Fig. 2, as α pheromone concentrations raise, thalli growth inhibition level increase, it can thus be appreciated that host's its own signal path It can use.

【Embodiment 2】

According to the scheme of embodiment 1, picking △ sst2 yeast cells single bacterium colony, using histidine deficient culture medium, survey Its fixed growth curve, growth curve such as Fig. 3, as shown in Figure 3 by after sst2 gene knockouts, signal path can amplify signal.

【Embodiment 3】

According to the scheme of embodiment 1, picking △ far1+ △ sst2 yeast cells single bacterium colony, lacked using histidine, uracil Swaged culture medium, its growth curve is determined, as a result as shown in figure 4, after understanding far1 gene knockouts with the contrast of embodiment 2, solution Except signal path itself is to the growth inhibition effect of bacterium.

【Embodiment 4】

Scheme is that setting α factor concentrations are 0 μm of ol/L, 1 μm of ol/L, 2.5 μm of ol/L, 5 μm of ol/L, 10 μm of ol/L, 15 μ Mol/L gradients shaken cultivation and 0 after culture, 4,8,10,12,14,16,18,20,22,24,26,28,30h take thalline Suspension 5mL or so is cultivated, fluorescent excitation intensity (584/607) place is measured in ELIASA, with the fluorescence excitation of each time point bacterium solution Intensity is than absorption photometric value (OD₆₀₀) it is ordinate, using incubation time as abscissa, draw out the luciferase expression of Yeast engineering bacteria Curve.

The host cell single bacterium colony of detection genetic circuits has been introduced only on picking solid plate, has been inoculated into 100mlYPD liquid Be put into shaking table in the shaking flask of body culture medium, 28-32 DEG C, 100-250rpm, 8-12 hour, obtain seed liquor, by seed liquor with 10% is inoculated among 96 orifice plates of YPD fluid nutrient mediums to every μ L of hole 150, and above-mentioned various concentrations are added into each hole The α factors, its luciferase expression curve is determined, as a result as shown in figure 5, luciferase expression amount is directly proportional to α pheromone concentrations, it is known that detection Circuit can use.

【Embodiment 5】

According to embodiment 4, picking has imported the △ far1+ △ sst2 of detection genetic circuits yeast cells single bacterium colony, makes With histidine, uracil-deficient type culture medium, its luciferase expression curve, as a result as shown in fig. 6, compared with Fig. 5, its fluorescence are determined Expression quantity has a certain degree of enhancing.

【Embodiment 6】

According to embodiment 4, picking has imported the △ far1+ △ sst2+ △ ste2 of detection genetic circuits yeast cells list Bacterium colony, using histidine, uracil, tryptophan deficiency culture medium, determine its luciferase expression curve, as a result as shown in fig. 7, The knockout of ste2 genes, essentially eliminate interference of the α pheromones to this biosystem.

【Embodiment 7】

1. the △ far1+ △ sst2+ △ ste2 for having imported T1R2/T1R3 acceptors and having detected circuit are chosen from solid plate Single bacterium colony, 5ml leucines, histidine, tryptophan, uracil-deficient type fluid nutrient medium are inoculated in, are put into shaking table, 28-32 DEG C, 100-250rpm, 12-36 hour.2. taking bacterium solution 1.5ml to be put into centrifuge 2500rpm centrifugation 2min, supernatant is removed, with 1ml pH= 7.4 0.1M KPO₄Wash thalline one time.1.2M sorbierites (are dissolved in 0.1M KPO₄PH=7.4) thalline is resuspended in 600ul.3. add 30 DEG C of water-bath 30mins of 5ul lysozymes, centrifuge 2500rpm centrifugation 2min are put into, remove supernatant.4. with PBS (pH=7.4) weights It is outstanding, OD values are adjusted to 0.5, take 180ul to add 20ul37% formaldehyde.5. it is added dropwise using adhering on poly-D-lysine slide Bacterium solution treated 200ul, 20min is stood, remove supernatant, retain thalline on slide, PBS is washed three times, and 250ul mouse are added dropwise His/Myc primary antibodies (press 1 with PBS:250 dilutions), it is incubated 2 hours.6. removing primary antibody, PBS is washed four times；250ul goat-antis are added dropwise Mouse secondary antibody (presses 1 with PBS:250 dilutions), lucifuge dilution, dark is incubated 2 hours.7. removing secondary antibody, PBS is washed four times, at the step Reason all operates in the dark.8. the anti-fluorescence quenchers of 5ul (containing DAPI) lid cover glass is added dropwise, with colourless nail sheet for oil seal.9. make With confocal microscopy fluorescence, as a result as shown in figure 8, this biological detection system engineering bacteria has fluorescence appearance, illustrate sweet taste Acceptor T1R2/T1R3 successes are expressed on cell membrane.

【Embodiment 8】

Selection has imported T1R2/T1R3 acceptors from solid plate and the △ far1+ △ sst2+ △ ste2 of detection circuit are mono- Bacterium colony, 5mL leucines, histidine, tryptophan, uracil-deficient type fluid nutrient medium are inoculated in, are put into shaking table, 28-32 DEG C, 100-250rpm, 12-36 hour.Seed liquor is inoculated into 3 groups with 10% ratio and contains 5mL leucines, histidine, color ammonia Among sour, uracil-deficient type fluid nutrient medium test tube, and the fructose of addition 2%, 2% Sucralose are used as and treated thereto respectively Material is surveyed, 2% α information is added as control, is put into shaking table, 28-32 DEG C, 100-250rpm, 24 hours.Nutrient solution is taken, is determined Thalli growth situation (OD₆₀₀), and fluorescence intensity, by the use of the sign than fluorescence intensity (fluorescence intensity/OD) value as sweetness intensities Value, measures fructose, sucrose sweetness intensities value is respectively 44,42, meet the ratio sugariness of sucrose and fructose at 30 DEG C close to this The known fact.

【Embodiment 9】

According to embodiment 8, detection circuit selects different fluorescins (red fluorescent protein, green fluorescent protein and cyan Fluorescin etc.) and enzyme as reporter gene, obtain similar conclusion.

Sequence table

<110>Beijing Institute of Technology

<120>A kind of engineered yeast biosystem of efficient detection sweetness intensities and its application

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Claims (7)

1. engineering saccharomyces cerevisiae biosystem and its application of a kind of efficient and sensible detection material sweetness intensities, are included in wine brewing The G-protein coupling sweet receptor T1R2/T1R3 of mammal is expressed in yeast, is passed with reference to host's G-protein coupled signal path Lead and regulate and control stream signal, to start the expression of downstream reporter gene as signal output, and according to signal output intensity come table Levy the sweetness intensities of measured matter.

2. biosystem according to claim 1, it is characterised in that by the sweet receptor T1R2/T1R3 of mammal Modification and codon optimization accordingly are carried out with G-protein α subunits GPA1, is allowed to adapt to above-mentioned biosystem, to solve upstream Signal transduction problem.

3. biosystem according to claim 1, it is characterised in that knock out host itself and interference is produced to signal path Related gene, including sst2, ste2 and far1.

4. biosystem according to claim 1, it is characterised in that imported reporter gene expression box using defeated as downstream Go out signal include with P_fusFor promoter, the induction of reporter gene (including the reporter gene such as fluorescin and enzyme) and terminator Type detects circuit.

5. a kind of sweetness intensities detection and standard formulation method based on biosystem described in claim 1.

6. according to the method for claim 5, it is characterised in that with the signal output situation of detected downstream genetic circuits and its Whether measured matter pleasantly sweet and its sweetness intensities to characterize for intensity.

7. according to the method for claim 5, it is characterised in that a kind of pleasantly sweet test substance of selection, determine treat respectively Sweetness intensities of the material in various concentrations are surveyed, it is standard detection concentration to select suitable concn, then is distinguished with standard detection concentration The sweetness intensities of a variety of test substances are determined, select appropriate units, make sweetness intensities standard, can be according to different test substances Property formulates respective standard.