Method for high-throughput screening of microbial strains by electrospray single emulsion and its application

Abstract

The present invention relates to microbial screening technology, and discloses a method for high-throughput screening of microbial strains by electrospraying a single emulsion and its application. The method comprises: establishing a microbial mutant library, obtaining a culture solution of the microbial mutant library; mixing the culture solution with a solution containing a gelling agent to obtain a dispersion, electrospraying the dispersion to form monodisperse droplets; contacting the monodisperse droplets with a collection solution containing a crosslinking agent to solidify to form microspheres, and subjecting the microspheres to microfluidic sorting after culturing; wherein the gelling agent and the crosslinking agent are in contact to generate a crosslinking and curing reaction. The method does not require the use of an oil phase to generate single-encapsulated droplets, and the fluorescent staining and screening process are simpler, effectively improving the effectiveness and accuracy of strain screening.

Description

Technical Field

[0001] The invention relates to a microorganism screening technology, and in particular to a method for high-throughput screening of microorganism strains by electrospraying a single emulsion and an application thereof. Background Art

[0002] Schizochytrium is a typical cell factory for biological manufacturing. Its oil content accounts for up to 55% of the cell dry weight. The oil it produces can be used as biodiesel, food or nutritional supplements. Among them, DHA is a polyunsaturated fatty acid necessary for the human body. It was extracted from deep-sea fish oil in the early days. The DHA content of Schizochytrium accounts for about 20% of the cell dry weight. It is considered to be a microorganism with great potential for industrial production of DHA.

[0003] In order to quickly obtain Schizochytrium with higher lipid production, high-throughput screening of the Schizochytrium mutant library is essential. The most advanced method at present is the microfluidic high-throughput screening strategy, but because the microfluidic method requires the use of an oil phase, it is cumbersome to clean the droplets when staining with Nile red, and the droplets are lost more during the cleaning process, which has an adverse effect on the effectiveness and accuracy of strain screening. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and provide a method for high-throughput screening of microbial strains by electrospraying single emulsion and its application. The method does not require the use of an oil phase to generate single-encapsulated droplets, and the fluorescent staining and screening processes are simpler, effectively improving the effectiveness and accuracy of strain screening.

[0005] In order to achieve the above object, the present invention provides a method for high-throughput screening of microbial strains by electrospraying single emulsion in a first aspect, the method comprising the following steps:

[0006] S1. Establishing a microbial mutant library and obtaining a culture solution of the microbial mutant library;

[0007] S2, mixing the culture solution with a solution containing a gelling agent to obtain a dispersion, and electrospraying the dispersion to form monodisperse droplets;

[0008] S3, contacting the monodisperse droplets with a collection solution containing a crosslinking agent to solidify them to form microspheres, and culturing the microspheres and performing microfluidic sorting;

[0009] Wherein, the gelling agent and the cross-linking agent can undergo a cross-linking and curing reaction when in contact.

[0010] Preferably, the establishment of the microbial mutant library in step S1 adopts at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis.

[0011] Preferably, the OD of the culture medium in step S1 is600 The volume ratio of the culture solution to the solution containing the gelling agent in step S2 is 1:800-1200.

[0012] Preferably, the concentration of the gelling agent in the solution containing the gelling agent is 1-3% by weight, and the concentration of the cross-linking agent in the collected solution is 2-5% by weight.

[0013] Preferably, the gelling agent is sodium alginate and / or chitosan, and the cross-linking agent is calcium chloride and / or ferric chloride.

[0014] Preferably, the channel diameter of the dispersion liquid in step S2 is 40-100 μm.

[0015] Preferably, the electrospraying conditions include: a voltage of 4-20 kV, a flow rate of the dispersion liquid less than or equal to 5 mL / h, and a distance between the droplet outlet of the channel and the collection liquid of 0-5 cm.

[0016] Preferably, the culture conditions in step S3 include: a temperature of 25-35° C. and a culture time of 3-100 days.

[0017] Preferably, the screening signal used in the microfluidic sorting in step S3 is selected from at least one of fluorescence, absorbance, Raman spectroscopy and mass spectrometry.

[0018] Preferably, the microfluidic sorting process includes: after fluorescent staining the cultured microspheres, detecting the fluorescence intensity thereof and setting a fluorescence screening threshold, and using a sorting chip to sort out microparticles with a fluorescence intensity higher than the fluorescence screening threshold.

[0019] Preferably, the fluorescent dye is a dimethyl sulfoxide solution containing Nile red; and the sorting chip is designed as a carrier oil physical sorting chip or an electric response valve sorting chip.

[0020] Preferably, the carrier oil physical sorting chip comprises a physical sorting chip body, a physical sorting channel located on the physical sorting chip body, a carrier oil injection channel connected to the side wall of the physical sorting channel, and a physical collection channel connected to the side wall of the physical sorting channel. The carrier oil injection channel and the physical collection channel are arranged on both sides of the physical sorting channel and are located on the same straight line, so that when the physical sorting chip body detects that the fluorescence intensity of the microspheres is higher than the fluorescence screening threshold, additional carrier oil can be injected from the carrier oil injection channel into the physical sorting channel to push the microspheres into the physical collection channel.

[0021] Preferably, the electric response valve sorting chip includes an electric sorting chip body, an electric sorting channel located on the electric sorting chip body, an electric collection channel connected to the side wall of the electric sorting channel, and an electric response valve located in the electric sorting channel. The electric response valve is located at the connection between the electric collection channel and the electric sorting channel, so that when the electric sorting chip body detects that the fluorescence intensity of the microsphere is higher than the fluorescence screening threshold, the electric response valve can be opened to push the microsphere into the electric collection channel.

[0022] The second aspect of the present invention provides the use of the above method in screening Schizochytrium with high lipid production.

[0023] Through the above technical solution, the beneficial effects of the present invention are:

[0024] The present invention relies on electrospray technology and utilizes electric field force to pull and generate monodisperse droplets, and the preparation method is simple and easy. Compared with the traditional strain screening method, the screening method provided by the present invention encapsulates the strain in gel microspheres, utilizes the water retention and supporting force of the gelling agent, provides a stable space for strain growth, ensures the independent growth environment of a single strain, and further increases the accuracy of screening. When the present invention utilizes electrospray technology to generate single-encapsulated droplets, there is no need to use an oil phase, so that the step of fluorescent staining the droplets in the screening process is simpler to operate, effectively improving the effectiveness and accuracy of strain screening.

[0025] Furthermore, the present invention innovatively proposes a carrier oil physical sorting chip or an electric response valve sorting chip, which can be adapted to a variety of detection signals such as fluorescence, Raman, mass spectrometry, etc., and can cover the sorting of almost all types of products, with a wider range of applications and higher screening accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart of the electrospray single emulsion high-throughput screening of Schizochytrium in Example 1;

[0027] Figure 2 It is a structural schematic diagram of a specific embodiment of the carrier oil physical sorting chip of the present invention;

[0028] Figure 3 It is a structural schematic diagram of a specific embodiment of the electric response valve sorting chip in the present invention;

[0029] Figure 4 Characterization diagram of Schizochytrium in Example 1 after 0 day and 72 days of growth in monodisperse droplets;

[0030] Figure 5The average particle size diagram of monodisperse droplets formed by electrospraying under different conditions, where (a) is the different flow rate of the dispersion, (b) is the different voltage, (c) is the different channel diameters of the dispersion, and (d) is the different distances between the droplet outlet of the dispersion channel and the collecting liquid.

[0031] Description of Reference Numerals

[0032] 1. Carrier oil physical sorting chip; 11. Physical sorting chip body;

[0033] 12 physical sorting channel; 13 carrier oil injection channel;

[0034] 14 physical collection channels; 2 electric response valve sorting chips;

[0035] 21 electric sorting chip body; 22 electric sorting channel;

[0036] 23 electric collection channel; 24 electric response valve. DETAILED DESCRIPTION

[0037] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0038] The first aspect of the present invention provides a method for high-throughput screening of microbial strains by electrospraying single emulsion, the method comprising the following steps:

[0039] S1. Establishing a microbial mutant library and obtaining a culture solution of the microbial mutant library;

[0040] S2, mixing the culture solution with a solution containing a gelling agent to obtain a dispersion, and electrospraying the dispersion to form monodisperse droplets;

[0041] S3, contacting the monodisperse droplets with a collection solution containing a crosslinking agent to solidify them to form microspheres, and culturing the microspheres and performing microfluidic sorting;

[0042] Wherein, the gelling agent and the cross-linking agent can undergo a cross-linking and curing reaction when in contact.

[0043] The inventors of the present invention creatively discovered during the research process that, in high-throughput screening of microorganisms, after mixing the microbial strain with a gelling agent, electrospray technology is used to achieve single encapsulation of the strain to form monodisperse droplets. The size of the droplets can be precisely controlled by adjusting the channel diameter, flow rate and electric field parameters of the dispersion liquid to generate spherical particles of uniform size, so that the monodisperse droplets have the advantages of controllable size and adjustable morphology. The process of preparing the droplets is simple and does not require the use of an oil phase, making the step of fluorescent staining the droplets in the screening process simpler, effectively improving the effectiveness and accuracy of strain screening; the monodisperse droplets contain a gelling agent and the collecting liquid contains a cross-linking agent, so that the charged droplets enter the collecting liquid containing the cross-linking agent and solidify into gel particles, and the strain is single-encapsulated in the gel microspheres. The water retention and supporting strength of the gelling agent are used to provide a stable space for strain growth, ensure the independent growth environment of a single strain, and further increase the accuracy of screening.

[0044] The method for high-throughput screening of microbial strains provided by the present invention can be applied to the screening of any microbial species, and the detection signal and parameters of the microfluidic sorting in step S3 can be set according to the purpose of screening. For example, if the purpose of screening is to screen strains with high yield of a certain product, the detection signal of the microfluidic sorting can be determined according to the detection method of the product. For example, if it is necessary to screen strains with high oil yield, the fluorescence, absorbance, Raman spectrum, mass spectrum and other signals for detecting oil can be used as the signal detection strategy for microfluidic sorting, and the screening of strains with high oil yield can be achieved through signal comparison.

[0045] In the present invention, when screening microbial strains with high oil production, any microorganism that can synthesize oil during growth and metabolism can be used for screening, for example, Schizochytrium, Thraustochytrium, Chlorella, etc. Preferably, the microorganism is Schizochytrium, and the microfluidic sorting obtains a Schizochytrium strain with high oil production by screening signals.

[0046] In the present invention, the microbial mutant library established in step S1 can be mutated by a conventional microbial mutagenesis method to a certain microbial strain to obtain a mutant library containing multiple microbial strains. Preferably, the microbial mutant library established in step S1 adopts at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis. Among them, ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis can be carried out using a commercially available multifunctional plasma mutagenesis system.

[0047] In the present invention, the culture medium for obtaining the microbial mutant library can select a suitable culture medium and culture conditions according to the strain type of the microorganism. For example, when the microorganism is Schizochytrium, it is necessary to first subculture 2-4 times in the seed culture medium, culture for 20-30 hours each time, and then inoculate into the fermentation medium and culture for 40-60 hours, supplement sugar every 20-30 hours, and culture in a constant temperature shaker at 25-30°C.

[0048] The seed culture medium preferably comprises: sodium sulfate 15-25 g / L, magnesium sulfate heptahydrate 3-6 g / L, ammonium sulfate 4-6 g / L, sodium chloride 1-1.5 g / L, potassium chloride 0.5-1.5 g / L, monosodium glutamate 15-25 g / L, glucose 40-60 g / L, dipotassium hydrogen phosphate 3-5 g / L, yeast powder 3-5 g / L, vitamin B1 0.5-1.5 mL, vitamin B6 0.5-1.5 mL, vitamin B 12 0.5-1.5mL.

[0049] The formula of the fermentation medium is preferably sodium sulfate 10-15g / L, magnesium sulfate heptahydrate 1-3g / L, ammonium sulfate 3-5g / L, potassium sulfate 0.5-1g / L, potassium chloride 0.3-0.8g / L, calcium chloride 0.1-0.2g / L, sodium glutamate 15-25g / L, glucose 80-120g / L, potassium dihydrogen phosphate 3-5g / L, yeast powder 3-5g / L, vitamin B1 0.5-1.5mL, vitamin B6 0.5-1.5mL, vitamin B 12 0.5-1.5mL.

[0050] According to the present invention, the culture solution can be examined under a microscope, and according to the number of cells, a suitable volume of bacterial solution is selected and mixed with a solution containing a gelling agent for electrospraying. Preferably, the OD of the culture solution in step S1 is 600 0.5-0.7, such as the OD of the culture medium 600 If the OD value is too high, dilute it with water to the appropriate OD value. 600 ; In step S2, the volume ratio of the culture medium to the solution containing the gel is 1:800-1200. For example, the cell concentration in the culture medium is generally selected as OD 600 =0.6, take 1 μL of culture solution and add it to 1 mL of solution system containing gelling agent for electrospraying.

[0051] In the present invention, the solution containing the gelling agent may be an aqueous solution of the gelling agent, and the collecting liquid may be an aqueous solution of the cross-linking agent. Preferably, the concentration of the gelling agent in the solution containing the gelling agent is 1-3% by weight, specifically 1% by weight, 1.5% by weight, 2% by weight, 2.5% by weight, 3% by weight, or any value between the above two values; the concentration of the cross-linking agent in the collecting liquid is 2-5% by weight, specifically 2% by weight, 3% by weight, 4% by weight, 5% by weight, or any value between the above two values. Under this preferred embodiment, it is beneficial for the droplets to be completely solidified into microspheres through ionic cross-linking, thereby improving the accuracy of screening of microbial strains.

[0052] According to the present invention, the gelling agent can be a hydrogel, and a corresponding cross-linking agent is selected according to the hydrogel, and the cross-linking agent can be a chloride salt. Preferably, the gelling agent is sodium alginate and / or chitosan, more preferably sodium alginate; the cross-linking agent is calcium chloride and / or ferric chloride, more preferably calcium chloride. Under this preferred embodiment, the gelling agent and the cross-linking agent are solidified by ionic cross-linking, which causes less damage to the encapsulated strain, provides a stable space for strain growth, ensures the independent growth environment of a single strain, and further increases the accuracy of screening.

[0053] In the present invention, the gelling agent and the cross-linking agent can be prepared by conventional methods in the art, or can be commercially obtained.

[0054] In the present invention, the dispersion liquid can be electrosprayed using an electrospray device consisting of a peristaltic pump, a syringe fixed on the peristaltic pump, and a capillary connected to the needle of the syringe, wherein the needle of the syringe can be connected to the capillary by a PE tube, the wire of the high-voltage power supply of the electrospray device is connected to the needle of the syringe, and the ground wire is placed at the collection point of the droplets (i.e., the liquid surface of the collected liquid).

[0055] In the present invention, the particle size and morphology of the monodisperse droplets are adjusted by changing at least one of the channel diameter of the dispersion liquid, the flow rate of the dispersion liquid, the voltage of the electrospray, and the distance between the droplet outlet of the dispersion liquid channel and the collecting liquid to generate spherical particles of uniform size.

[0056] According to the present invention, preferably, the channel diameter of the dispersion liquid in step S2 is 40-100 μm, and the channel diameter of the dispersion liquid is specifically the inner diameter of the capillary in the above-mentioned electrospray device. Exemplarily, the inner diameter of the capillary can be 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or any value between the above two values.

[0057] According to the present invention, preferably, the conditions of the electrospray include: the voltage is 4-20kV, specifically 4kV, 8kV, 12kV, 16kV, 20kV, or any value between the above two values; more preferably 4-9kV. The flow rate of the dispersion liquid is less than or equal to 3mL / h, specifically 1mL / h, 1.5mL / h, 2mL / h, 2.5mL / h, 3mL / h, or any value between the above two values; the distance between the droplet outlet of the dispersion liquid channel (specifically the outlet of the capillary in the above electrospray device) and the collection liquid (specifically the liquid surface of the collection liquid) is 0-5cm, specifically 0cm, 1cm, 2cm, 3cm, 4cm, 5cm, or any value between the above two values.

[0058] In the present invention, the flow rate of the dispersion is controlled by the peristaltic pump in the above-mentioned electrospray device; generally, the outlet of the capillary is arranged to be located above the open glass bottle containing the collection liquid, so that the distance between the droplet outlet of the capillary and the collection liquid is convenient to adjust and meets the requirements.

[0059] According to the present invention, the culture conditions in step S3 can select appropriate culture conditions according to the type of microbial strains screened, including temperature, time, etc. For example, when the microorganism is Schizochytrium, preferably, the culture conditions in step S3 include: temperature of 25-35°C, time of 3-100 days. The droplet culture can be carried out in the collected liquid, using static culture, the strain can grow normally in the droplet, and has good water retention and morphological stability, so that a single strain can be cultured independently in the droplet, which greatly facilitates subsequent high-throughput screening.

[0060] In the present invention, the droplet culture in step S3 is generally cultured to the third day to form a corresponding signal difference for microfluidic screening. Preferably, the droplet culture is continued to about 100 days, so that the fluorescence difference between high yield and low yield is stronger and more conducive to sorting.

[0061] According to the present invention, preferably, the screening signal used in the microfluidic sorting in step S3 is selected from at least one of fluorescence, absorbance, Raman spectroscopy and mass spectrometry; it can be specifically selected and designed according to the detection scheme of oil and fat to improve the accuracy of detection and screening.

[0062] According to the present invention, preferably, the microfluidic sorting process includes: after fluorescent staining the cultured microspheres, detecting the fluorescence intensity and setting a fluorescence screening threshold, and using a sorting chip to sort out microparticles with fluorescence intensity higher than the fluorescence screening threshold.

[0063] According to the present invention, fluorescent staining can be performed using conventional staining methods in the art. Preferably, the fluorescent staining dye uses a dimethyl sulfoxide (DMSO) solution containing Nile red. For example, after the microspheres are cultured for a period of time, a portion is taken out and placed in a culture dish, 1-3 μL of a DMSO solution with a Nile red concentration of 1-2 wt% is added, and the droplets are observed after incubation in the dark for 10-20 minutes to detect their fluorescence intensity.

[0064] According to the present invention, preferably, the sorting chip is designed as a carrier oil physical sorting chip or an electric response valve sorting chip.

[0065] According to the present invention, preferably, see Figure 2 The carrier oil physical sorting chip 1 includes a physical sorting chip body 11, a physical sorting channel 12 located on the physical sorting chip body 11, a carrier oil injection channel 13 connected to the side wall of the physical sorting channel 12, and a physical collection channel 14 connected to the side wall of the physical sorting channel 12. The carrier oil injection channel 13 and the physical collection channel 14 are arranged on both sides of the physical sorting channel 12 and are located on the same straight line, so that when the physical sorting chip body 11 detects that the fluorescence intensity of the microsphere is higher than the fluorescence screening threshold, additional carrier oil can be injected from the carrier oil injection channel 13 into the physical sorting channel 12 to push the microsphere into the physical collection channel 14. Among them, the carrier oil injection channel 13 is connected to the corresponding carrier oil supply device, and the carrier oil has a certain speed when injected into the physical sorting channel 12, so as to realize pushing the microsphere into the corresponding physical collection channel 14.

[0066] According to the present invention, preferably, see Figure 3 The electric response valve sorting chip 2 includes an electric sorting chip body 21, an electric sorting channel 22 located on the electric sorting chip body 21, an electric collection channel 23 connected to the side wall of the electric sorting channel 22, and an electric response valve 24 located in the electric sorting channel 22. The electric response valve 24 is located at the connection between the electric collection channel 23 and the electric sorting channel 22, so that when the electric sorting chip body 21 detects that the fluorescence intensity of the microsphere is higher than the fluorescence screening threshold, the electric response valve 24 is opened to push the microsphere into the electric collection channel 23. The electric response valve 24 is generally in a state of blocking the electric collection channel 23. When the fluorescence intensity of the microsphere is detected to be higher than the fluorescence screening threshold, the electric response valve 24 flips to block the electric sorting channel 22 and open the electric collection channel 23, so that the microsphere flows into the electric collection channel 23 for collection.

[0067] The second aspect of the present invention provides the use of the above method in screening high-lipid-producing Schizochytrium. The high-throughput screening method of microbial strains provided by the present invention is particularly suitable for screening high-lipid-producing strains (such as Schizochytrium, etc.).

[0068] As a relatively preferred embodiment of the method for high-throughput screening of microbial strains in the present invention, taking Schizochytrium as an example, see Figure 1 , including the following steps:

[0069] S1. Mutate Schizochytrium by at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis to obtain a Schizochytrium mutant library. Place the strains of the Schizochytrium mutant library in a liquid culture medium, and culture them at a temperature of 25-35° C. for 2-5 days to obtain a culture solution of the strains of the microbial mutant library (calculate the ideal biomass to ensure the subsequent single-encapsulation of the strains, OD 600 is about 0.6); construct an electrospray device, which includes a peristaltic pump, a syringe fixed on the peristaltic pump, and a capillary connected to the needle of the syringe through a PE tube, and the inner diameter of the capillary is 40-100 μm;

[0070] S2, add 1 μL of OD 600 The culture fluid of about 0.6% is mixed with 1 mL of a gelling agent solution with a gelling agent concentration of 1-3% by weight to obtain a dispersion, the dispersion is loaded into a syringe of an electrospray device, the wire of a high-voltage power supply of the electrospray device is connected to the needle of the syringe, the ground wire is placed at the collection point of the droplets, the flow rate of the dispersion in the syringe is set to be less than or equal to 5 mL / h, the voltage is 4-20 kV, the distance from the outlet tip of the capillary to the surface of the collecting liquid is 0-5 cm, and electrospraying is performed to form monodisperse droplets;

[0071] S3. Collect the monodisperse droplets in a glass bottle containing excess collection liquid (cross-linker concentration is 2-5 wt %), cross-link and solidify the gelling agent and the cross-linker, solidify the droplets to form microspheres, culture the droplets at a temperature of 25-35°C for 3-100 days, and then stain the microspheres using fluorescence staining, detect their fluorescence intensity and set a fluorescence screening threshold, and use a sorting chip to sort out the particles with fluorescence intensity higher than the fluorescence screening threshold to achieve screening of high-oil-yielding strains.

[0072] The present invention will be described in detail below through examples.

[0073] In the following examples, the wild-type strain of Schizochytrium was purchased from Shanghai Mingjin Biotechnology Co., Ltd. with the number ATCC20888, sodium alginate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with the number 9005-38-3, and calcium chloride was purchased from Xilong Chemical Co., Ltd. with the number 10043-52-4.

[0074] Unless otherwise specified, other raw materials and reagents are conventional commercial products, and the detection method adopts the conventional method in the art.

[0075] In the following examples, unless otherwise specified, the room temperature is 25±2°C;

[0076] The formula of seed solution is: sodium sulfate 20g / L, magnesium sulfate heptahydrate 4.6g / L, ammonium sulfate 4.8g / L, sodium chloride 1.25g / L, potassium chloride 1g / L, monosodium glutamate 20g / L, glucose 50g / L, dipotassium hydrogen phosphate 4g / L, yeast powder 4g / L, vitamin B11mL, vitamin B61mL, vitamin B 12 1mL.

[0077] The formula of the fermentation liquid is sodium sulfate 12g / L, magnesium sulfate heptahydrate 2g / L, ammonium sulfate 4g / L, potassium sulfate 0.7g / L, potassium chloride 0.5g / L, calcium chloride 0.15g / L, sodium glutamate 20g / L, glucose 100g / L, potassium dihydrogen phosphate 4g / L, yeast powder 4g / L, vitamin B11mL, vitamin B61mL, vitamin B 12 1mL.

[0078] In the following embodiments, the structure of the carrier oil physical separation chip 1 is shown in Figure 2 , including a physical sorting chip body 11, a physical sorting channel 12 located on the physical sorting chip body 11, a carrier oil injection channel 13 connected to the side wall of the physical sorting channel 12, and a physical collection channel 14 connected to the side wall of the physical sorting channel 12. The carrier oil injection channel 13 and the physical collection channel 14 are arranged on both sides of the physical sorting channel 12 and are located on the same straight line, so that when the physical sorting chip body 11 detects that the fluorescence intensity of the microspheres is higher than the fluorescence screening threshold, additional carrier oil can be injected from the carrier oil injection channel 13 into the physical sorting channel 12 to push the microspheres into the physical collection channel 14.

[0079] The structure of the electric response valve sorting chip 2 is shown in Figure 3 , including an electric sorting chip body 21, an electric sorting channel 22 located on the electric sorting chip body 21, an electric collecting channel 23 connected to the side wall of the electric sorting channel 22, and an electric response valve 24 located in the electric sorting channel 22. The electric response valve 24 is located at the connection between the electric collecting channel 23 and the electric sorting channel 22, so that when the electric sorting chip body 21 detects that the fluorescence intensity of the microspheres is higher than the fluorescence screening threshold, the electric response valve 24 is opened to push the microspheres into the electric collecting channel 23.

[0080] Example 1

[0081] S1. Cultivate the wild-type strain of Schizochytrium in liquid culture medium at 30°C for 3 days and take the OD 600= 0.6 was subjected to atmospheric pressure room temperature plasma (ARTP) mutagenesis. Specifically, the bacterial solution was mixed with 5% glycerol solution, 10 μL was applied on an iron sheet, the ARTP mutagenizer power was set to 120 W, the airflow was 10 SLM, and the gradient time was 10 s for mutagenesis. The induced mutant strain was inoculated into a liquid culture medium and cultured at 30 ° C for 2-5 days to obtain a culture solution of the Schizochytrium mutant library strain. 1 mL of the culture solution was taken for microscopic examination to obtain the OD 600 = 0.6 of the culture medium; construct an electrospray device, the electrospray device comprising a peristaltic pump, a syringe fixed on the peristaltic pump, and a capillary connected to the needle of the syringe through a PE tube, the inner diameter of the capillary being 50 μm;

[0082] S2, add 1 μL of OD 600 A culture solution with a concentration of about 0.6 and 1 mL of a solution with a sodium alginate concentration of 2% by weight are uniformly mixed to obtain a dispersion, and the dispersion is loaded into a syringe of an electrospray device, the wire of a high-voltage power supply of the electrospray device is connected to the needle of the syringe, and a ground wire is placed at a droplet collection point, and the flow rate of the dispersion in the syringe is set to 0.2 mL / h, the voltage is 7 kV, and the distance from the outlet tip of the capillary to the surface of the collecting liquid is 2 cm, and electrospraying is performed to form monodisperse droplets;

[0083] S3. The monodisperse droplets were collected in a glass bottle containing excess collection liquid (calcium chloride concentration was 3 wt %). The sodium alginate and calcium chloride were cross-linked and solidified. The droplets solidified to form microspheres. The droplets were cultured for a long time at 30°C for 100 days. The morphology of the droplets on the 0th day and the 72nd day of culture was as follows: Figure 4 As shown; take out a portion every 9 days and put it into a culture dish, add 2μL of Nile red dye with a concentration of 1% by weight, incubate in the dark for 15 minutes, observe the microspheres, detect their fluorescence intensity, select the microspheres of the same batch with moderate fluorescence intensity difference (cultured for 72 days) and set the fluorescence screening threshold (specifically 36000RFU); inject the droplet into the carrier oil physical sorting chip 1, aim the laser at the detection point of the physical sorting channel 12, and collect the fluorescence signal of each microsphere. When the microsphere with fluorescence intensity higher than the fluorescence screening threshold is detected, the system immediately injects it into the physical sorting channel 12 from the carrier oil injection channel 13 to push the microsphere into the physical collection channel 14.

[0084] One or several droplets with higher fluorescence signal intensity in the physical collection channel 14 were screened out, and shake flask fermentation was performed to screen out high-oil-yielding strains. The highest oil yield of the screened high-oil-yielding strains was 19.49 g / L, which was about 1.45 times higher than that of the wild-type strain.

[0085] Example 2

[0086] High-throughput screening of Schizochytrium high-yield DHA was carried out according to the method of Example 1, except that the carrier oil physical sorting chip 1 in step S3 was replaced by the electric response valve sorting chip 2, the laser was aimed at the detection point of the electric sorting channel 22, and the fluorescence signal of each microsphere was collected. When a microsphere with a fluorescence intensity higher than the fluorescence screening threshold was detected, the system immediately opened the electric response valve 24 to push the microsphere into the electric collection channel 23.

[0087] One or several droplets with higher fluorescence signal intensity in the electrokinetic collection channel 23 were screened out, and shake flask fermentation was performed to screen out DHA high-yielding strains. The highest DHA yield of the screened DHA high-yielding strain was 16.13 g / L, which was about 1.2 times higher than that of the wild-type strain.

[0088] Example 3

[0089] High-throughput screening of Schizochytrium high-yield DHA was carried out according to the method of Example 1, except that the inner diameter of the capillary in step S1 was replaced with 75 μm, the voltage in step S2 was replaced with 5 kV, and the flow rates of the dispersion in the syringe were set to 0.2 mL / h, 0.4 mL / h, 0.6 mL / h, 0.8 mL / h, 1.0 mL / h, 1.2 mL / h, 1.4 mL / h, 1.6 mL / h, 1.8 mL / h, 2.0 mL / h, and 2.2 mL / h, respectively. The effects of different flow rates on the droplet size are shown in Figure 2. Figure 5 As shown in (a), it can be seen that as the flow rate of the dispersion increases, more droplets gather at the nozzle, so the overall trend of the droplet particle size is increasing.

[0090] Example 4

[0091] The high-throughput screening of Schizochytrium high-yield DHA was carried out according to the method of Example 1, except that the inner diameter of the capillary in step S1 was replaced with 75 μm, and the voltage in step S2 was set to 4 kV, 5 kV, 6 kV, 7 kV, 8 kV, and 9 kV, respectively. The effects of different voltage conditions on the droplet size are shown in FIG. Figure 5 As shown in (b), as the electric field strength increases, the electrostatic repulsion of the liquid surface on the Taylor cone becomes stronger and the particle size of the droplet gradually decreases.

[0092] Example 5

[0093] The high-throughput screening of Schizochytrium high-yield DHA was carried out according to the method of Example 1, except that the voltage in step S2 was replaced with 5 kV, and the inner diameter of the capillary in step S1 (i.e., the channel diameter of the dispersion liquid) was set to 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, and 100 μm, respectively. The effects of different capillary inner diameters on the droplet size are shown in FIG. Figure 5As shown in (c), as the inner diameter of the capillary increases, the overall trend of the droplet size increases.

[0094] Example 6

[0095] High-throughput screening of Schizochytrium high-yield DHA was carried out according to the method of Example 1, except that the inner diameter of the capillary in step S1 was replaced with 44 μm, the voltage in step S2 was replaced with 5 kV, and the distances from the outlet tip of the capillary to the surface of the collected liquid were set to 0.5 cm, 1.0 cm, 1.5 cm, 2.0 cm, 2.5 cm, 3.0 cm, 3.5 cm, 4.0 cm, 4.5 cm, and 5.0 cm, respectively. The effects of different voltage conditions on the droplet size are shown in Figure 2. Figure 5 As shown in (d), as the distance from the outlet tip of the capillary to the surface of the collecting liquid increases, the droplet size first increases and then decreases. This is because within a certain distance range, the droplets are mainly affected by the electric field force. The longer the distance, the smaller the electric field force, and the larger the droplet size. When the distance from the outlet tip of the capillary to the surface of the collecting liquid exceeds a certain limited range, the droplets are mainly affected by gravity. The longer the distance, the greater the force on the droplets, and the smaller the droplet size.

[0096] Example 7

[0097] High-throughput screening of Schizochytrium for high lipid production was performed according to the method of Example 1, except that the solution with a sodium alginate concentration of 2 wt % in step S2 was replaced by a solution with a sodium alginate concentration of 4 wt %.

[0098] One or several droplets with higher fluorescence signal intensity in the electrokinetic collection channel 23 were screened out, and shake flask fermentation was performed to screen out high-oil-yielding strains. The highest oil yield of the screened high-oil-yielding strains was 15.05 g / L, which was about 1.12 times higher than that of the wild-type strain.

[0099] Example 8

[0100] High-throughput screening of Schizochytrium high-yield lipids was carried out according to the method of Example 1, except that the solution with a sodium alginate concentration of 2 wt % in step S2 was replaced by a chitosan solution with a concentration of 2 wt %, and correspondingly, the collection solution in step S3 was replaced by a FeCl3 aqueous solution with a concentration of 3 wt %.

[0101] One or several droplets with higher fluorescence signal intensity in the electrokinetic collection channel 23 were screened out, and shake flask fermentation was performed to screen out high-oil-yielding strains. The highest oil yield of the screened high-oil-yielding strains was 16.40 g / L, which was about 1.22 times higher than that of the wild-type strain.

[0102] Example 9

[0103] The high-throughput screening of Schizochytrium high-yield oil was carried out according to the method of Example 1, except that the carrier oil physical sorting chip 1 in step S3 was replaced by a dielectrophoresis sorting chip (for details, see Wen N, Zhao Z, Fan B, et al .Development of droplet microfluidics enabling high-throughput single-cell analysis [J]. Molecules, 2016, 21(7): 881).

[0104] One or several droplets with higher fluorescence signal intensity were screened out, and shake flask fermentation was performed to screen out high-oil-yielding strains. The highest oil yield of the screened high-oil-yielding strains was 15.45 g / L, which was about 1.15 times higher than that of the wild-type strain.

[0105] Comparative Example 1

[0106] S1. Cultivate the wild-type strain of Schizochytrium in liquid culture medium at 30°C for 3 days and take the OD 600 = 0.6 of the bacterial solution for atmospheric pressure room temperature plasma (ARTP) mutagenesis, specifically, the bacterial solution was mixed with 5% glycerol solution, 10 μL was applied on an iron sheet, the ARTP mutagenizer power was set to 120 W, the airflow was 10 SLM, and the gradient time was 10 s for mutagenesis, the mutated strains after mutagenesis were inoculated into liquid culture medium, and cultured at 25 ° C for 2-5 days to obtain the culture liquid of the Schizochytrium mutant library strain; Assemble the microfluidic chip: Use an acetylene blowtorch to draw two glass capillaries of different sizes The diameter of the inner glass capillary is 100 μm, and the diameter of the outer glass capillary is 500 μm. The outer glass capillary is placed at a suitable position on a glass slide, and then the inner glass capillary is inserted into the outer glass capillary, so that the tip of the inner glass capillary penetrates into the tip of the outer glass capillary but does not exceed the tip of the outer glass capillary. Then, the relative positions of the two capillaries are adjusted under a microscope so that their central axes overlap with each other and present a coaxial arrangement mode. They are fixed with quick-drying glue to form a microfluidic chip.

[0107] S2, select soybean oil with added surfactant Span 80 (the content of Span 80 in soybean oil is 2wt%) as the external phase, mix the culture solution obtained in step S1 with fluorescein as the internal phase; extract the internal phase into a 5mL syringe, extract the external phase into a 10mL syringe, and then load them onto two peristaltic pumps respectively, the 5mL syringe is connected to the side wall of the internal phase glass capillary through a polyethylene tube, and the 10mL syringe is connected to the side wall of the external phase glass capillary through a polyethylene tube, set the internal phase flow rate to 0.3mL / h, and the external phase flow rate to 3mL / h, start the two peristaltic pumps, and embed the strains in the Schizochytrium mutant library to form core-shell structure droplets;

[0108] S3, collecting the droplets in step S2 in a glass bottle containing an excess of the above-mentioned external phase, placing the glass bottle under ultraviolet light to irradiate the droplets for 40 seconds, and the droplets are completely solidified into microspheres; then culturing at 30°C for 72 days to obtain a microsphere culture solution, and pumping the microsphere culture solution into a dielectrophoresis sorting chip (for details, see Wen N, Zhao Z, Fan B, et al .Development of droplet microfluidics enabling high-throughput single-cell analysis [J]. Molecules, 2016, 21(7): 881) The laser is aimed at the detection point of the sorting device, and the fluorescence signal of each droplet is collected. The droplet with the highest fluorescence signal intensity is sorted by applying voltage, so that it flows into the collection channel.

[0109] One or several droplets with higher signal intensity were screened out, and shake flask fermentation was performed to screen out high-oil-yielding strains. The highest oil yield of the screened high-oil-yielding strains was 14.51 g / L, which was about 1.08 times higher than that of the wild-type strain.

[0110] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for high-throughput screening of microbial strains by electrospray single emulsion, characterized in that: The method comprises the following steps: S1. Establishing a microbial mutant library and obtaining a culture solution of the microbial mutant library; S2, mixing the culture solution with a solution containing a gelling agent to obtain a dispersion, and electrospraying the dispersion to form monodisperse droplets; S3, contacting the monodisperse droplets with a collection solution containing a crosslinking agent to solidify them to form microspheres, and culturing the microspheres and performing microfluidic sorting; The gelling agent and the cross-linking agent can undergo a cross-linking and curing reaction when in contact; the OD of the culture solution in step S1 is 600 is 0.5-0.7, the volume ratio of the culture solution to the solution containing the gelling agent in step S2 is 1:800-1200; the concentration of the gelling agent in the solution containing the gelling agent is 1-3% by weight, and the concentration of the cross-linking agent in the collected solution is 2-5% by weight; the gelling agent is sodium alginate and / or chitosan, and the cross-linking agent is calcium chloride and / or ferric chloride; The microfluidic sorting process includes: after fluorescent staining the cultured microspheres, detecting the fluorescence intensity thereof and setting a fluorescence screening threshold, and using a sorting chip to sort out microparticles with a fluorescence intensity higher than the fluorescence screening threshold; the sorting chip is designed as an electric response valve sorting chip; The electric response valve sorting chip includes an electric sorting chip body, an electric sorting channel located on the electric sorting chip body, an electric collection channel connected to the side wall of the electric sorting channel, and an electric response valve located in the electric sorting channel. The electric response valve is located at the connection between the electric collection channel and the electric sorting channel, so that when the electric sorting chip body detects that the fluorescence intensity of the microsphere is higher than the fluorescence screening threshold, the electric response valve can be opened to push the microsphere into the electric collection channel.

2. The method according to claim 1, characterized in that The establishment of the microbial mutant library in step S1 adopts at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis.

3. The method according to claim 1 or 2, characterized in that: The channel diameter of the dispersion liquid in step S2 is 40-100 μm; The electrospraying conditions include: a voltage of 4-20 kV, a flow rate of the dispersion liquid less than or equal to 3 mL / h, and a distance between the droplet outlet of the channel and the collection liquid of 0-5 cm.

4. The method according to claim 1 or 2, characterized in that: It is characterized in that The culture conditions in step S3 include: a temperature of 25-35° C. and a culture time of 3-100 days.

5. The method according to claim 1 or 2, characterized in that: The screening signal used in the microfluidic sorting in step S3 is selected from at least one of fluorescence, absorbance, Raman spectroscopy and mass spectrometry.

6. The method according to claim 1, characterized in that The fluorescent dye is a dimethyl sulfoxide solution containing Nile red.

7. Use of the method according to any one of claims 1 to 6 in screening Schizochytrium with high lipid production.

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