High-throughput screening method of E-beta-farnesene sample

Abstract

The invention discloses a high-throughput screening method for an E-beta-farnesene sample. The high-throughput screening method comprises the following steps: preparing a 4-NP solution and preparing an E-beta-farnesene standard sample; adding 100 [mu] l of an E-beta-farnesene standard sample into each pore of the pore plate; respectively adding 100 [mu] l of a 4-NP solution into each pore of the pore plate so as to mix the E-beta-farnesene standard sample and the 4-NP solution; detecting the fluorescence intensity in each pore of the pore plate through a detector; substituting the detected fluorescence intensity data into the standard curve, and calculating the concentration of the E-beta-farnesene sample; according to the method, 96 samples can be treated in a short time, the detection efficiency is greatly improved, errors are small, and the method is suitable for high-throughput screening of E-beta-farnesene samples.

Description

Technical Field

[0001] The invention belongs to the technical field of active compound screening, and in particular relates to a high-throughput screening method for E-β-farnesene samples. Background Art

[0002] E-β-farnesene is a widely used acyclic sesquiterpene. Due to its unique structure and physicochemical properties, it has significant hydrophobicity and high energy density. E-β-farnesene has extremely important application value in the fields of bioenergy, medicine, agriculture and industry. In nature, E-β-farnesene is an important signal molecule that can serve as an aphid alarm pheromone to prevent aphids from approaching crops and prevent potential damage. E-β-farnesene can also be used as a precursor for specialty chemical products (including solvents, emollients and vitamin E). In addition, in the energy field, due to its advantages of high cetane number, low temperature function and high energy density, E-β-farnesene can also be used as a lubricant and high-performance fuel.

[0003] The global demand for E-β-farnesene is growing, but existing production methods are unable to meet the growing demand. Environmental factors such as seasonality and regional climate have a great impact on plant growth, and the concentration of E-β-farnesene in plant matter is usually low. Therefore, the extraction of E-β-farnesene from natural sources is inherently challenging. Currently, more and more studies are exploring chemical and biosynthetic methods for E-β-farnesene. The chemical synthesis of E-β-farnesene mainly relies on precursors such as nerol, farnesol, myrcene, etc. However, due to low overall yield, harsh reaction conditions and complex processes, the large-scale production of high-purity E-β-farnesene remains difficult. The biosynthesis of E-β-farnesene mainly passes through the mevalonate (MVA) pathway to produce isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMPP). These two compounds are then condensed to form the precursor farnesyl diphosphate (FPP). E-β-farnesene can be produced from FPP by heterologous expression of E-β-farnesene synthase. The design of metabolic pathways, regulation of intermediate fluxes and optimization of the culture process have greatly improved the yield of E-β-farnesene.

[0004] Therefore, a robust and scalable high-throughput culture system as well as a simple and rapid high-throughput screening method are essential for screening high-producing strains.

[0005] Existing screening methods involve growing strains in shake flasks and then measuring the target product in each culture using high-performance liquid chromatography, gas chromatography, and ultraviolet spectrophotometry. However, the high cost and low efficiency of chromatographic methods limit the implementation of high-throughput screening methods. Although absorbance-based detection methods can detect a variety of compounds, their sensitivity is often low.

[0006] The current E-β-farnesene screening method has the disadvantages of small number of samples detected simultaneously and long detection time, which is not suitable for high-throughput screening. Therefore, it is necessary to establish a simple and rapid high-throughput screening method to screen high-yield strains and increase the yield of E-β-farnesene. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a high-throughput screening method for E-β-farnesene samples, which can process 96 samples in a short time, greatly improve the detection efficiency, have a small error, and is suitable for high-throughput screening of E-β-farnesene samples.

[0008] To achieve the above object, the specific scheme of the present invention is as follows: A high-throughput screening method for E-β-farnesene samples, comprising:

[0009] Prepare 4-NP solution and E-β-farnesene standard;

[0010] Add 100 μl of E-β-farnesene standard to each well of the well plate;

[0011] Add 100 μl of 4-NP solution to each well of the well plate to mix the E-β-farnesene standard and 4-NP solution;

[0012] The fluorescence intensity in each well of the well plate is detected by a detector;

[0013] The fluorescence intensity data obtained by the detection was substituted into the standard curve to calculate the concentration of the E-β-farnesene sample.

[0014] The present invention is further configured such that the concentration of the 4-NP solution is greater than or equal to 0.05 mM and less than or equal to 2 mM.

[0015] The present invention is further configured such that the concentration of the 4-NP solution is 2 mM.

[0016] The present invention is further configured such that the solvent of the 4-NP solution is methanol, ethyl acetate, acetonitrile or ethanol.

[0017] The present invention is further configured such that the solvent of the 4-NP solution is ethanol.

[0018] The present invention is further configured that the preparation of the E-β-farnesene standard sample comprises the following steps:

[0019] Select the E-β-farnesene engineered bacteria that have been subjected to mutagenesis treatment, and culture the mutagenized yeast strain on YPD solid medium;

[0020] YPD liquid culture medium was prepared, the strain was expanded and cultured in a deep-well plate, and n-nonane was added for extraction to obtain an E-β-farnesene standard sample.

[0021] The present invention is further configured to perform fluorescence intensity detection when the E-β-farnesene standard sample and the 4-NP solution are mixed and reacted for ten minutes.

[0022] The present invention is further configured such that the detector is an ELISA reader or a fluorescence spectrophotometer.

[0023] The beneficial effects of the present invention are as follows: the present method can process 96 samples in a short time, and the detection time only takes 1 minute, which greatly improves the detection efficiency. In addition, the present method has a small error and is suitable for high-throughput screening of E-β-farnesene samples, providing a fast and efficient high-throughput screening method for E-β-farnesene samples.

[0024] This method is based on the fluorescence mechanism of intramolecular charge transfer promoted by the Diels-Alder cycloaddition reaction of 4-NP fluorescent probe and E-β-farnesene. The concentration of E-β-farnesene is calculated by measuring the fluorescence intensity of the sample. Compared with traditional detection methods, this method has higher detection efficiency and smaller error, and is particularly suitable for large-scale screening of E-β-farnesene samples and its application development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the mechanism by which the 4-NP fluorescent probe releases fluorescence due to a Diels-Alder cycloaddition reaction with E-β-farnesene.

[0026] Figure 2 It is a schematic diagram of the structure of a 96-well plate.

[0027] Figure 3 This is a schematic diagram of the 96-well plate operation steps. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the implementation scope of the present invention is not limited thereto.

[0029] The term "high-throughput screening" used in the present invention means that the number of tests in a single experiment is greater than 96, and the screening method can simultaneously detect the concentrations of 96 samples each time it is applied.

[0030] like Figures 1 to 3 As shown, the high-throughput screening method of an E-β-farnesene sample described in this embodiment specifically includes:

[0031] Prepare 4-NP (N-methyl-4-nitroso-phthalimide, CAS: 2458145-59-8) solution: Use 4-NP as E-β-farnesene fluorescent probe. The fluorescence mechanism of 4-NP fluorescent probe is as follows: Figure 1 As shown, from Figure 1It can be seen that the reason for the fluorescence release of 4-NP is that the nitroso group in the 4-NP fluorescent probe molecule undergoes a Diels-Alder cycloaddition reaction with E-β-farnesene, resulting in intramolecular charge transfer, thereby releasing fluorescence.

[0032] The concentration of the 4-NP solution is greater than or equal to 0.05 mM and less than or equal to 2 mM, preferably, the concentration of the 4-NP solution is 2 mM. The solvent of the 4-NP solution is methanol, ethyl acetate, acetonitrile or ethanol; preferably, the solvent of the 4-NP solution is ethanol.

[0033] The preparation method of 4-NP solution is as follows:

[0034] To a solution of N-methyl-4-nitrophthalimide (2.1 g, 10.0 mmol) in ethyl acetate (100.0 mL) (i.e., N-methyl-4-nitrophthalimide dissolved in ethyl acetate) was added a few drops of concentrated acetic acid, and the mixture was hydrogenated with palladium carbide (10%, 300 mg) at room temperature and normal pressure for 8 h, and the reaction progress was monitored by thin layer chromatography.

[0035] After the reaction was completed, palladium carbon was removed by filtration and then concentrated by rotary evaporation to obtain crude N-methyl-4-aminophthalimide.

[0036] The crude product was purified by recrystallization from ethanol to obtain yellow needle-shaped crystals with a yield of 66.7%. N-methyl-4-aminophthalimide was dissolved in H2O / CH2Cl2 (4:1 v / v) and potassium persulfate was added. The solution was stirred at room temperature until TLC showed that the raw material was completely consumed. The aqueous layer was extracted with CH2Cl2 (3×30mL), washed with 1.0% hydrochloric acid, 1.0% NaHCO3 and brine, and the combined organic matter was dried over anhydrous magnesium sulfate, filtered and concentrated. The crude product was purified by column chromatography to obtain N-methyl-4-nitrosophthalimide, and 4-NP solution was obtained.

[0037] Preparation of E-β-farnesene standard sample: The E-β-farnesene sample is derived from the fermentation broth and obtained by extraction. Preferably, since the E-β-farnesene sample to be tested is extracted from the fermentation broth, n-nonane is a preferred solvent for extracting hydrophobic compounds in living cultures due to its low toxicity and good phase separation. Therefore, the E-β-farnesene standard sample is obtained by extraction with n-nonane.

[0038] Preferably, a target gene fragment with different base sequences is obtained by random mutation, and a recombinant plasmid is constructed to express E-β-farnesene synthase in Saccharomyces cerevisiae; then, an E-β-farnesene engineered bacterium that has been subjected to mutagenesis treatment is selected, and the mutagenized yeast strain is cultured on a YPD solid medium;

[0039] Prepare YPD liquid medium, expand the strain culture in a deep-well plate, add n-nonane to extract and obtain E-β-farnesene standard sample. The deep-well plate is a 96-deep-well plate.

[0040] In this embodiment, E-β-farnesene extracted from yeast fermentation broth is used as a sample for screening, which is closer to the actual application scenario, and the screening result has a higher credibility.

[0041] 100 μl of E-β-farnesene standard is added to each well of the well plate; 100 μl of 4-NP solution is added to each well of the well plate to mix the E-β-farnesene standard and the 4-NP solution; specifically, the well plate is a 96-well plate, so that 96 samples can be processed in a short time, and the detection time only takes 1 minute, which improves the detection effect. Specifically, the 4-NP solution is transferred to the 96-well plate in batches through an automatic pipetting station to ensure that the initial fluorescence intensity of the 4-NP solution in each well is consistent.

[0042] The fluorescence intensity in each well of the well plate is detected by a detector; preferably, the detector is an enzyme reader or a fluorescence spectrophotometer, which can achieve the effect of simultaneous detection of multiple samples based on conventional detection means such as an enzyme reader or a fluorescence spectrophotometer, and is a low-cost high-throughput screening method. Preferably, the fluorescence intensity is detected when the E-β-farnesene standard sample and the 4-NP solution are mixed and reacted for ten minutes.

[0043] Specifically, the fluorescence intensity in each well is detected by an ELISA instrument, and the screening principle is explained by taking the specific identification of E-β-farnesene by the 4-NP fluorescent probe as an example: the screening tool 4-NP designed in this application is a conjugated diene fluorescent probe. When a conjugated diene is present, such as E-β-farnesene, the fluorescence intensity of 4-NP is released due to the Diels-Alder cycloaddition reaction between the nitroso group of 4-NP and the conjugated diene, and its fluorescence intensity is quantitatively related to the amount of conjugated diene it reacts with, so a rapid quantitative analysis can be performed using an ELISA instrument.

[0044] The specific detection steps for detection by microplate reader are as follows: first set the microplate reader to run the program, oscillate the well plate for ten seconds before the formal measurement to ensure sufficient mixing, the excitation wavelength for fluorescence intensity detection is 360nm, and the emission wavelength is 535nm. After the reaction has been carried out for ten minutes, the reaction can be considered to be completed, the program is closed, and the data is stored.

[0045] The fluorescence intensity was measured by fluorescence spectrophotometer, and the standard curve was y = 0.0186x + 1.0679, where x is the concentration of the substance to be tested and y is the ratio of the measured fluorescence intensity to the blank group. The regression line R of the standard curve 2is 0.9969. This method has an upper detection limit and a lower detection limit, that is, the range in which the concentration of the analyte can be detected. First, the lower detection limit is calculated to be 2.72 mg / L through the standard curve. The upper detection limit of this method is related to the concentration of the 4-NP fluorescent probe. The most suitable concentration of 4-NP in ethanol is 2mM, and its upper detection limit is 200 mg / L. Therefore, the lowest E-β-farnesene concentration that can be detected by this method is 2.72 mg / L, and the highest E-β-farnesene concentration that can be detected is 200 mg / L.

[0046] The fluorescence intensity data obtained by the detection was substituted into the standard curve to calculate the concentration of the E-β-farnesene sample.

[0047] Embodiment 1:

[0048] The high-throughput screening method of E-β-farnesene samples in this embodiment specifically includes the following steps:

[0049] Step A1: prepare a 4-NP (N-methyl-4-nitroso-phthalimide, CAS: 2458145-59-8) solution of appropriate concentration, the concentration of the 4-NP solution is 2 mM, and the solvent is ethanol;

[0050] Prepare 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 150 mg / L and 200 mg / L of E-β-farnesene standard samples respectively.

[0051] Step A2: Add various concentrations of E-β-farnesene standard samples to a 96-well plate, with 100 μL added to each well.

[0052] The E-β-farnesene standard was mixed with the 4-NP solution by simultaneously adding a 2 mM 4-NP solution via an automated pipetting station.

[0053] Step A3: When the E-β-farnesene standard sample and the 4-NP solution are mixed and reacted for ten minutes, the fluorescence intensity is measured by a fluorescence spectrophotometer.

[0054] Step A4: Substitute the standard curve to obtain the concentration of the analyte:

[0055] After the fluorescence intensity data of the tenth minute is obtained, it is substituted into the standard curve to obtain the standard curve of y = 0.0186x + 1.0679, where x is the concentration of the substance to be tested and y is the ratio of the measured fluorescence intensity to the blank group; the regression line of the standard curve is R 2 is 0.9969; the concentration value of the analyte can be obtained.

[0056] This embodiment achieves the experimental purpose of rapid, accurate and high-throughput. The currently commonly used detection method is gas chromatography, and the detection time of a single sample is 20-30 minutes. This embodiment can detect 96 samples at the same time and the detection time is 1 minute, which greatly improves the detection efficiency.

[0057] Embodiment 2:

[0058] The E-β-farnesene engineering bacteria used in this embodiment are obtained by random mutation of the original E-β-farnesene engineering bacteria, and the E-β-farnesene production is increased or decreased. The specific steps are as follows:

[0059] (1) The drug-mutated plasmid was introduced into the yeast substrate, spread on a ball plate, and cultured in a constant temperature incubator at 28-30°C on a YPD solid medium plate.

[0060] (2) Prepare 100 ml of YPD liquid medium with the following ingredients: 1 g yeast extract powder, 2 g peptone, and 2 g glucose. Sterilize at 116°C for 25 min and transfer to a 96-well plate. Add 1 mL of YPD medium to each well.

[0061] To obtain seed solution, 96 single colonies were selected from the solid culture medium and transferred to a 96-deep-well plate containing 1 mL of YPD liquid medium and fermented on a well plate shaker at 30°C and 800 rpm for 24 h. 100 μL of seed solution was transferred to three sets of 96-deep-well plates containing 900 μL of YPD liquid medium and fermented at 30°C and 800 rpm for 72 h.

[0062] (3) After 72 hours of incubation in a 96-well plate, 30% (v / v) n-nonane was added to extract E-β-farnesene. The fermentation was continued for 24 hours and then allowed to stand. 100 μL of the upper organic phase was added to the 96-well plate using an automatic pipetting station, for a total of 96 E-β-farnesene standards.

[0063] (4) Prepare a 2 mM 4-NP solution and add it to a 96-well plate that has been pre-filled with E-β-farnesene standard sample using an automatic pipetting station. After the well plate is prepared, place it in an ELISA reader. Figure 3 The ELISA reader program settings are as follows: oscillation for 10 seconds before testing, temperature is 25°C, detection time is 60 seconds per test, and detection stops automatically after ten minutes.

[0064] (5) After the test is completed, the fluorescence intensity value is derived and substituted into the standard curve to obtain the sample concentration. The fluorescence intensity obtained in this experiment is shown in Table 1 below, and the sample concentration value is obtained by substituting into the standard curve:

[0065] Table 1

[0066]

[0067]

[0068]

[0069] (6) The upper organic phase was taken from the shake flask fermentation and tested by this method and gas chromatography respectively to verify the accuracy of the invention. The concentration and error were shown in Table 2 below:

[0070] Table 2

[0071] Detection Methods Determination concentration (mg / L) Standard Deviation Fluorescence detection (this method) 25.3028 2.14917 Gas chromatography 24.84317 0.70657

[0072] (7) It can be seen from the final experimental results that this method is within an acceptable range in terms of high-throughput screening, so this method can be applied to the high-throughput screening of E-β-farnesene samples. It can be seen from the above embodiments that compared with the method of detecting the concentration of E-β-farnesene by gas chromatograph, the 4-NP fluorescent probe is more efficient in the screening method, and the error is acceptable in the experiment. Such results are based on the correct judgment of the upper and lower limits of detection, which improves the detection efficiency in practical applications. The selection of the 4-NP fluorescent probe achieves high-speed and accurate detection of the concentration of E-β-farnesene samples, avoids the slowdown of the experimental progress due to the long detection time of a single sample, and reduces the frequency of experimental detection. Detecting the concentration of E-β-farnesene samples by the 4-NP fluorescent probe is an efficient and accurate detection method.

[0073] In order to verify the accuracy of this method, gas chromatography was used to detect shake flask fermentation samples. The results showed that the error between the E-β-farnesene concentration detected by this method and the gas chromatography detection result was within an acceptable range, further verifying the effectiveness and reliability of this method.

[0074] The present invention provides a rapid and efficient high-throughput screening method for E-β-farnesene samples. The method is based on the intramolecular charge transfer mechanism of the 4-NP fluorescent probe, and the concentration of E-β-farnesene is calculated by measuring the fluorescence intensity of the sample with an enzyme marker. Compared with traditional detection methods, the method has higher detection efficiency and smaller error, and is particularly suitable for large-scale screening of E-β-farnesene samples and its application development.

[0075] The above is only a preferred embodiment of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the protection scope of the patent application of the present invention.

Claims

1. A high-throughput screening method for E-β-farnesene samples, characterized in that: include: Prepare 4-NP solution and E-β-farnesene standard; Add 100 μl of E-β-farnesene standard to each well of the well plate; Add 100 μl of 4-NP solution to each well of the well plate to mix the E-β-farnesene standard and 4-NP solution; The fluorescence intensity in each well of the well plate is detected by a detector; The fluorescence intensity data obtained by the detection was substituted into the standard curve to calculate the concentration of the E-β-farnesene sample.

2. The high-throughput screening method for E-β-farnesene samples according to claim 1, characterized in that: The concentration of the 4-NP solution is greater than or equal to 0.05 mM and less than or equal to 2 mM.

3. The high-throughput screening method for E-β-farnesene samples according to claim 2, characterized in that: The concentration of 4-NP solution was 2 mM.

4. The high-throughput screening method for E-β-farnesene samples according to claim 1, characterized in that: The solvent of 4-NP solution is methanol, ethyl acetate, acetonitrile or ethanol.

5. The high-throughput screening method for E-β-farnesene samples according to claim 4, characterized in that: The solvent of the 4-NP solution was ethanol.

6. The high-throughput screening method for E-β-farnesene samples according to claim 1, characterized in that: The preparation of E-β-farnesene standard sample includes the following steps: Select the E-β-farnesene engineered bacteria that have been subjected to mutagenesis treatment, and culture the mutagenized yeast strain on YPD solid medium; YPD liquid culture medium was prepared, the strain was expanded and cultured in a deep-well plate, and n-nonane was added for extraction to obtain an E-β-farnesene standard sample.

7. The high-throughput screening method for E-β-farnesene samples according to claim 1, characterized in that: When the E-β-farnesene standard sample and the 4-NP solution were mixed and reacted for ten minutes, the fluorescence intensity was detected.

8. The high-throughput screening method for E-β-farnesene samples according to claim 1, characterized in that: The detector is an enzyme-labeled instrument or a fluorescence spectrophotometer.

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