CN115651926A

CN115651926A - Method for high-throughput screening of microbial strains

Info

Publication number: CN115651926A
Application number: CN202211670740.7A
Authority: CN
Inventors: 黄和; 施天穹; 王月桐; 李想; 李雅文; 郭琪
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-01-31
Anticipated expiration: 2042-12-26
Also published as: CN115651926B

Abstract

The invention relates to a biological engineering technology and discloses a method for screening microbial strains in a high-throughput manner. The method comprises the following steps: (1) Establishing a microorganism mutant library and constructing a microfluidic chip; (2) Embedding the strains in the microorganism mutant library by using a microfluidic chip to form a droplet, wherein the outer phase of the droplet is fluorinated oil containing a surfactant, the inner phase of the droplet is a mixture of a culture solution of the microorganism mutant library strains and hydrogel, and the phase transition temperature of the inner phase is the growth temperature of the microorganisms; (3) And carrying out droplet culture on the droplets, solidifying the droplets to form microspheres, and carrying out microfluidic sorting on the microspheres. The method can keep the growth shape of the microorganism, enhance the appearance of the liquid drop, effectively avoid the breakage of the microsphere and facilitate the accurate screening of the microsphere to obtain the required microorganism strain.

Description

Method for high-throughput screening of microbial strains

Technical Field

The invention relates to a biological engineering technology, in particular to a method for screening microbial strains in high flux.

Background

Gibberellin is a natural plant growth regulator, plays an important role in the growth process of plants, can promote the growth of the plants, and has an obvious relieving effect on severe environments such as drought, salt damage and the like. The filamentous fungus Gibberella fujikuroi is a main industrial strain for high yield of gibberellin, if high yield of gibberellin is required, directional evolution and transformation of Gibberella fujikuroi are required, a large number of mutant libraries are formed by a random mutagenesis method generally, and then high-throughput screening is carried out on the libraries to obtain an ideal strain for high yield of gibberellin. At present, a lot of methods for high-throughput screening of industrial fungi by using a microfluidic technology are established, for example, a Chenyu roller and the like use a droplet microfluidic device to perform high-throughput screening on bacillus licheniformis to obtain a mutant strain with high alpha-amylase yield; for example, bailiwan et al, culture a single streptomycete embedded in a droplet, and further combine with a biosensor to obtain a target strain with high erythromycin yield.

Common steps of microfluidic screening include: bacterial liquid culture, droplet generation, droplet culture and droplet sorting; the aqueous phase of the strain culture solution is used as an internal phase, the monodisperse water-in-oil droplets with picoliter size are rapidly generated in the continuous fluorinated oil phase, a surfactant is introduced into an internal and external phase solution system to keep the droplets stable and reduce fusion, and the droplets can be produced in a microfluidic device at the frequency of thousands of times per second to reach the magnitude of millions. The single mutant strain is encapsulated in droplets, each droplet corresponding to a microreactor, to allow single cell independent analysis without cross-contamination, while, within a microfluidic device, the droplets allow various forms of manipulation, including splitting, fusing, capturing, reagent injection, sorting, etc., to be accomplished by specific microchannel designs. Then, after a period of time of in-droplet culture, the thalli secrete intracellular or extracellular products, and appropriate product detection signals (such as fluorescence, absorbance, raman spectrum and the like) are selected to adapt to high-throughput sorting. Droplet sorting can be passively driven according to physical properties (e.g., size), or actively sorted by on-demand activation, such as electric fields that apply dielectrophoretic forces to the droplets, and so forth. However, the existing liquid drops are generally composed of simple microorganism culture solution, and have many disadvantages, such as poor water retention performance, and the liquid drops can volatilize and shrink after a long incubation time to influence the growth of strains; the gibberella lutescens is a filamentous fungus, a large amount of hyphae can be generated in the culture process, and the hyphae easily puncture liquid drops in the later period, so that leakage is caused, and cross contamination is caused; the conventionally used polymer chip can not be used as a medium for long-term culture of liquid drops, the liquid drops must be transferred into other glass containers, and in the transferring process, the liquid drops are easy to be disturbed and broken or even secondarily emulsified, so that the subsequent accurate screening is greatly influenced. Therefore, when the liquid drop sorting is applied to screening the gibberella bardawil, a liquid drop sorting mode capable of enhancing the appearance and the size stability of the liquid drops needs to be established urgently.

Disclosure of Invention

The invention aims to solve the problems that the prior art has poor water retention performance and can not meet the requirements of long-term culture of the gibberella barnacii and the liquid drop is easy to break and break during transfer, and provides a method for screening a microbial strain at high flux.

In order to achieve the above object, the present invention provides a method for high throughput screening of microbial strains, comprising the steps of:

(1) Establishing a microorganism mutant library and constructing a microfluidic chip;

(2) Embedding the strains in the microorganism mutant library by using a microfluidic chip to form a droplet, wherein the outer phase of the droplet is fluorinated oil containing a surfactant, the inner phase of the droplet is a mixture of a culture solution of the microorganism mutant library strains and hydrogel, and the phase transition temperature of the inner phase is the growth temperature of the microorganisms;

(3) And performing droplet culture on the droplets, solidifying the droplets to form microspheres, and performing microfluidic sorting on the microspheres.

Preferably, the creating of the microbial mutant library in step (1) employs at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis.

Preferably, the microfluidic chip in step (1) comprises a glass capillary assembly, a glass slide and a sample application needle, wherein the glass capillary assembly comprises an external phase capillary and an internal phase capillary, one end of the internal phase capillary is nested in one end of the external phase capillary, and the external phase capillary is fixed on the glass slide; the connection part of the outer phase capillary tube and the inner phase capillary tube is hermetically connected with the sample application needle, so that the sample application needle can input the outer phase into the outer phase capillary tube.

Preferably, the inner diameter of the outer phase capillary is 300 to 800 μm and the inner diameter of the inner phase capillary is 100 to 200 μm.

Preferably, the outer phase capillary adopts a glass tube subjected to hydrophobic treatment, and the hydrophobic agent subjected to hydrophobic treatment is trichlorooctadecylsilane.

Preferably, the surfactant in step (2) is a fluorosurfactant; more preferably at least one of Pico-Surfin FC40, FS-Kryjeff D900, EA surfactant and FS-008.

Preferably, the concentration of said surfactant in said fluorinated oil is from 2 to 5wt%.

Preferably, the microorganism in the step (1) is Gibberella fujikuroi, the phase transition temperature of the inner phase in the step (2) is 28-30 ℃, and the Gibberella fujikuroi strain with high yield of gibberellin is obtained by micro-fluidic sorting in the step (3) through screening signals.

Preferably, the hydrogel is selected from at least one of modified gelatin, calcium alginate gel, agarose, chitosan and dextran; more preferably methacrylic anhydrified gelatin and/or calcium alginate gel.

Preferably, the content of the hydrogel in the internal phase is 30 to 70wt%.

Preferably, the culture solution of the microbial mutant library strain in step (2) is obtained by liquid culture of the microbial mutant library strain.

Preferably, the liquid culture medium is YPD liquid culture medium, and the culture conditions at least comprise: the temperature is 28-30 ℃.

Preferably, the flow rate of the outer phase in step (2) is 3 to 8mL/h and the flow rate of the inner phase is 0.1 to 0.5mL/h.

Preferably, the temperature of the embedding is controlled at 32-40 ℃.

Preferably, the conditions of the droplet culture in step (3) include at least: the temperature is 28-30 deg.C, and the time is 2-8 days.

Preferably, the droplet culture is performed in the external phase.

Preferably, the temperature of the curing is below 28 ℃.

Preferably, the screening signal used in the microfluidic sorting in step (3) is selected from at least one of fluorescence, absorbance, raman spectroscopy and mass spectroscopy.

Through the technical scheme, the invention has the beneficial effects that:

the method for screening the microbial strains at high flux is based on the fact that a temperature-responsive hydrogel material is added into a microbial culture solution system to serve as an internal phase, so that formed liquid drops can generate gel-sol phase transition, the optimal growth temperature of microbes is taken as the gel-sol transition temperature, the liquid drops are kept in a liquid state above the gel-sol transition temperature, and the liquid drops below the gel-sol transition temperature form solid microspheres, so that the microbial strains are placed in the gel microspheres for culture, the growth of the strains is not influenced, the water retention performance and the morphological stability of the liquid drops are enhanced, the moisture of a culture medium cannot volatilize along with the extension of incubation time, and the original shape can be kept under long-term culture; the microspheres are not broken when hyphae of the strain pokes out, so that cross contamination is effectively avoided, and the subsequent transferring and sorting operation is greatly facilitated; the liquid drops are cultured in a solid state, so that fusion and substance exchange between the liquid drops are reduced, independent growth and reaction environment of a single strain are guaranteed, accuracy of subsequent detection and screening is further improved, and a brand new material solution is provided for microbial breeding. The method for screening the microbial strains at high flux is particularly suitable for screening the gibberellin bin with high yield of gibberellin, and has simple process and convenient operation.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

FIG. 1 is a flow chart of one embodiment of a method for high throughput screening of microbial strains according to the present invention;

FIG. 2 is a plot of lethality induced by the Bacillus gibsonii ARTP in example 1;

FIG. 3 is a diagram showing the growth of the culture of Gibberella fujikuroi in example 1 in the droplet;

FIG. 4 is a graph of the average particle size of droplets of the outer and inner phases at different flow rates in example 2;

FIG. 5 is a phase transition performance plot of the internal phase at different concentrations and temperatures in example 3;

FIG. 6 is a droplet break-up diagram in comparative example 1;

FIG. 7 is a schematic diagram of an embodiment of a microfluidic chip according to the present invention.

Description of the reference numerals

a, carrying a glass slide; b, a sample application needle;

c an external phase capillary; d internal phase capillaries.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.

The invention provides a method for screening microbial strains in high flux, which comprises the following steps:

(1) Establishing a microorganism mutant library and constructing a micro-fluidic chip;

In the invention, the growth temperature of the microorganism refers to the temperature required by the normal growth of microorganism strains, and the phase transition temperature of the internal phase is set to the temperature required by the growth of the microorganism strains, so that the internal phase is not solidified during the liquid drop culture, but is cooled and solidified to form the solid microspheres after the liquid drop culture is finished. The hydrogel system formed by the inner phase adopts gel components with the factors of molecular weight, mass concentration and the like to determine the phase transition temperature of the inner phase, and preferably, the hydrogel system with the phase transition temperature at the optimal growth temperature of the screened microbial strains is obtained as an ideal gel system through debugging and exploration, for example, the optimal growth temperature of the Gibberella fujikuroi is 28-30 ℃, and the ideal hydrogel system with the phase transition temperature of 28-30 ℃ is obtained as the inner phase through debugging and exploration.

In the research process, the inventor of the present invention unexpectedly finds that the hydrogel has excellent dynamic responsiveness, that is, the formation and dissociation of a three-dimensional network can be controlled by external stimulation, the gel state in a macroscopic solid state is converted into a sol state in a flowing state, and reversible phase transition of gel-sol can be performed by controlling temperature, however, the characteristic of the hydrogel has not been applied to droplet microfluidic high-throughput screening of microbial strains. Therefore, based on the reversible phase transition characteristic of the hydrogel gel-sol, the hydrogel material is added into a microorganism culture solution system to serve as an internal phase, so that the formed liquid drop can generate gel-sol phase transition, the optimal growth temperature of a microorganism strain is taken as the transition temperature of two phases of the gel-sol, the liquid drop is kept in a liquid state above the gel-sol transition temperature, the liquid drop below the gel-sol transition temperature forms solid microspheres, the microorganism strain is placed in the gel microspheres for culture, the growth of the strain can be kept, the water retention performance and the form stability of the liquid drop can be enhanced, the water in a culture medium cannot volatilize along with the extension of incubation time, and the original shape can be kept under the long-term culture; after the liquid drops are transformed into solid microspheres through phase transformation, the microspheres are stabbed by hyphae of the strains, so that the microspheres are not broken, cross contamination is effectively avoided, and the subsequent transfer and sorting operations are greatly facilitated; the liquid drops are cultured in a non-liquid state, so that fusion and substance exchange between the liquid drops are reduced, independent growth and reaction environment of a single strain are guaranteed, accuracy of subsequent detection and screening is further improved, and a brand new material solution is provided for microbial breeding.

The method for screening microbial strains at high flux provided by the invention can be suitable for screening any microbial strain, detection signals and parameters of micro-fluidic sorting in the step (3) can be set according to the purpose of screening, for example, the purpose of screening strains with high yield of a certain product is realized, the detection signals of micro-fluidic sorting can be determined according to the detection method of the product, illustratively, strains with high yield of gibberellin need to be screened, signals for detecting fluorescence, absorbance, raman spectrum, mass spectrum and the like of the gibberellin can be used as a signal detection strategy of micro-fluidic sorting, and strain screening with high yield of gibberellin can be realized through signal comparison. The method for screening the microbial strains at high flux provided by the invention relies on a microfluidic technology, adopts a microfluidic chip to prepare liquid drops, has simple channels, few building steps, no need of complex machining process, simple process and convenient operation, and is particularly suitable for screening the gibberellic disease with high yield of gibberellin.

The method provided by the invention has the obvious interdisciplinary characteristics, provides a material solution for the problem of the droplet microfluidic high-throughput screening in the biological field, perfects the material system formula in the current industrial application, and does not relate to the modification of a hardware mechanical device, so that the method can be adapted to the existing process and realize large-scale popularization.

According to the invention, preferably, the microorganism is saccharomycete and/or gibberella, and further preferably gibberella barnacii, and the micro-fluidic sorting is carried out to obtain the gibberella barnacii strain with high yield of gibberellin by screening signals.

According to the present invention, preferably, the screening signal used in the microfluidic sorting in the step (3) is at least one selected from fluorescence, absorbance, raman spectrum and mass spectrum, so as to improve the accuracy of detection screening.

According to the present invention, the establishment of the microbial mutant library in step (1) can be carried out by subjecting a microbial strain to mutagenesis by a conventional microbial mutagenesis method to obtain a mutant library comprising a plurality of microbial strains. Preferably, the creating of the microbial mutant library in step (1) employs at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis. Wherein, the ultraviolet irradiation, the plasma mutagenesis and the chemical mutagenesis can be carried out by adopting a commercial multifunctional plasma mutagenesis system.

According to the present invention, preferably, referring to fig. 7, in step (1), the microfluidic chip comprises a glass capillary assembly, a glass slide a and a sample application needle b, wherein the glass capillary assembly comprises an external phase capillary c and an internal phase capillary d, one end of the internal phase capillary d is nested in one end of the external phase capillary c, and the external phase capillary c is fixed on the glass slide a; the connection of the outer phase capillary c and the inner phase capillary d is hermetically connected with the sample application needle b, so that the sample application needle b can input the outer phase into the outer phase capillary c. When the micro-fluidic chip is used, an external phase is input into an external phase capillary tube c from a sample application needle b, and an internal phase is input into the external phase capillary tube c from one end, far away from the external phase capillary tube c, of an internal phase capillary tube d to form a liquid drop with the external phase wrapping the internal phase to package monospores.

In the present invention, the external phase capillary c is fixed on the slide glass a by assembling and fixing with a colloid such as a quick-drying adhesive. In order to further improve the uniformity of the droplets formed by the microfluidic chip, the inner phase capillary d and the outer phase capillary c are preferably coaxially nested.

According to the present invention, the size of the droplets and microspheres can be adjusted by changing the inner diameters of the outer phase capillary c and the inner phase capillary d, or by changing the flow rates of the inner phase and the outer phase. The outer phase capillary tube c and the inner phase capillary tube d in the invention can be obtained commercially, and also can be drawn into two glass capillary tubes with different sizes by using an acetylene burner. In order to form droplets with uniform size via the microfluidic chip, it is preferable that the inner diameter of the outer phase capillary c is 300 to 800 μm and the inner diameter of the inner phase capillary d is 100 to 200 μm.

According to the present invention, preferably, the flow rate of the outer phase in step (2) is 3 to 8mL/h and the flow rate of the inner phase is 0.1 to 0.5mL/h. The outer phase and the inner phase can be pumped into the microfluidic chip at certain flow rates by liquid pumps (such as peristaltic pumps) respectively, so that the flow rates of the outer phase and the inner phase can be controlled conveniently.

According to the present invention, it is preferable that the outer phase capillary c is a glass tube subjected to a hydrophobic treatment, and the hydrophobic agent of the hydrophobic treatment is trichlorooctadecylsilane (OTS), so that the inner wall of the outer phase capillary c has hydrophobicity to avoid adverse effects on the formation of liquid droplets. Specifically, the outer phase capillary c may be subjected to a hydrophobic treatment by using an evaporation hydrophobic method, and the hydrophobic process is, for example: mixing dichloromethane and trichlorooctadecylsilane (OTS) according to the volume ratio of 100 to prepare a hydrophobic agent, placing the external phase capillary c and the hydrophobic agent in a closed container together, then placing the closed container in an oven at about 65 ℃ for 2h, repeating the operation for 3-5 times, and drying the external phase capillary c. The sufficient hydrophobic treatment of the external phase capillary c can reduce the viscosity between the droplet and the glass tube wall of the external phase capillary c, and improve the droplet generation and the stability of long-term culture.

According to the present invention, preferably, the surfactant in step (2) is a fluorine-containing surfactant; preferably at least one of Pico-Surfin FC40, FS-Kryjeff D900, EA surfactant and FS-008. In this preferred embodiment, the outer phase and the inner phase are advantageously wrapped, and the outer phase and the inner phase can be better matched with each other, thereby improving the form stability of the droplets. The surfactants described in the present invention are commercially available.

According to the invention, preferably, the concentration of said surfactant in said fluorinated oil is between 2 and 5% by weight. In this preferred embodiment, the droplets are less likely to fuse during longer incubation, which is advantageous for further improvement of droplet stability.

According to the present invention, in order to be suitable for screening of gibberella barnacii and form an ideal hydrogel system with a phase transition temperature of 28-30 ℃, the hydrogel is preferably at least one selected from the group consisting of modified gelatin, calcium alginate gel, agarose, chitosan and dextran. The modified gelatin may be at least one selected from acrylic acid modified gelatin, caffeic acid modified gelatin and methacrylic acid anhydrified gelatin, and preferably, the hydrogel is methacrylic acid anhydrified gelatin and/or calcium alginate gel. Under the preferred embodiment, the phase change performance of the hydrogel system is improved, and the stability and the accuracy of the microorganism screening process are improved. The hydrogels of the present invention can be prepared by methods conventional in the art or can be obtained commercially.

According to the present invention, it is further preferred that the content of the hydrogel in the internal phase is 30 to 70wt%, which is more advantageous for obtaining an ideal hydrogel system with a phase transition temperature of 28 to 30 ℃.

According to the present invention, preferably, the culture solution of the library strain of the microbial mutants in step (2) is obtained by subjecting the library strain of the microbial mutants to liquid culture. Specifically, the microbial mutant library strains are inoculated into corresponding liquid culture media for liquid culture, and ideal biomass is calculated to ensure subsequent strain single-package.

According to the present invention, the liquid medium of the strain is not particularly limited, and a medium capable of providing nutrients necessary for growth of the microorganism species may be selected according to the microorganism species, and for example, when the microorganism species is gibberella alvarezii, the liquid medium contains a carbon source and a nitrogen source, and specifically a medium containing glucose, peptone, and yeast extract may be used, and the liquid medium includes: 15-25g/L of glucose, 15-25g/L of peptone and 5-15g/L of yeast extract. Preferably, the liquid culture medium is YPD liquid culture medium, and the culture conditions at least comprise: the temperature is 28-30 ℃, and the specific culture time can be controlled according to the ideal biomass to be realized and the usage amount of the external phase and the internal phase in the microfluidic chip.

According to the invention, the microfluidic chip is kept at a constant temperature for embedding to generate the liquid drops, and preferably, the embedding temperature is controlled to be 32-40 ℃ so as to ensure the stable generation of the liquid drops. The liquid drop realizes phase transition at the gel-sol transition temperature, keeps solid below the gel-sol transition temperature and keeps liquid above the gel-sol transition temperature, can improve the stability of the liquid drop and is not easy to fuse in the culture process.

According to the present invention, preferably, the conditions of the droplet culture in step (3) include at least: the temperature is 28-30 deg.C, and the time is 2-8 days. The liquid drop culture adopts static culture, the bacterial strains can normally grow in the liquid drops, and the liquid drop culture has good water retention performance and morphological stability, so that the single bacterial strains can be independently cultured in the liquid drops, and great convenience is brought to subsequent high-throughput screening.

According to the invention, preferably, the droplet culture is carried out in the external phase, and illustratively, a certain volume of the external phase is arranged in a glass bottle, and an external phase capillary tube c extends into the glass bottle below the liquid level of the glass bottle, so that the droplet formed in the step (2) is input into the glass bottle for droplet culture, and the droplet is grown below the liquid level of the oil phase, and the independent growth environment of the microbial strains is ensured.

According to the invention, the temperature of the curing is preferably below 28 ℃. The solidification is generally carried out by cooling after the droplet culture, so that the droplets are completely solidified to form microspheres for subsequent microfluidic separation.

As a relatively preferred embodiment of the method for screening microorganism strains in high throughput in the present invention, the method takes the red-blood bin as an example, and referring to FIG. 1, the method comprises the following steps:

(1) Mutagenizing the gibberella bardii by at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis to obtain a gibberella bardii mutant library, placing strains of the gibberella bardii mutant library in a YPD liquid culture medium, and culturing at 28-30 ℃ to obtain a culture solution of the strains of the microorganism mutant library (calculating ideal biomass to ensure that subsequent strains are singly wrapped); constructing a micro-fluidic chip, wherein the micro-fluidic chip comprises a glass capillary assembly, a glass slide a and a point sampling needle b, the glass capillary assembly comprises an external phase capillary c and an internal phase capillary d, one end of the internal phase capillary d is coaxially nested in one end of the external phase capillary c, and the external phase capillary c is fixed on the glass slide a by adopting quick-drying glue; the joint of the external phase capillary tube c and the internal phase capillary tube d is hermetically connected with the sample application needle b so that the sample application needle b can input an external phase into the external phase capillary tube c, the external phase capillary tube c adopts a glass tube which is subjected to hydrophobic treatment by trichlorooctadecylsilane, the inner diameter of the external phase capillary tube c is 300-800 mu m, and the inner diameter of the internal phase capillary tube d is 100-200 mu m;

(2) Taking fluorinated oil containing fluorosurfactant (concentration of surfactant is 2-5 wt%) as external phase, taking mixture of culture solution of microorganism mutant library strain and hydrogel (content of hydrogel is 30-70wt%, and proportion of hydrogel is adjusted to make phase transition temperature of mixture be 28-30 ℃) as internal phase; keeping the micro-fluidic chip at a constant temperature of 32-40 ℃, inputting an external phase into an external phase capillary tube c from a sample application needle b at a flow rate of 3-8mL/h by using an external phase peristaltic pump, inputting an internal phase into the external phase capillary tube c from one end, far away from the external phase capillary tube c, of an internal phase capillary tube d at a flow rate of 0.1-0.5mL/h by using an internal phase peristaltic pump, and embedding strains in a microbial mutant library to form a droplet for encapsulating monospores;

(3) Collecting the liquid drops in a glass bottle containing excessive external phase, carrying out liquid drop culture at the temperature of 28-30 ℃ for 2-8 days, placing the glass bottle at room temperature for cooling, completely solidifying the liquid drops to form microspheres, pumping the microspheres into a microfluidic sorting device, and selecting a proper signal detection strategy (at least one of fluorescence, absorbance, raman spectrum and mass spectrum) to realize strain screening of high-yield gibberellin.

The present invention will be described in detail below by way of examples.

In the following examples, fluorescence signals were detected using a beckmann coulter CytoFLEX SRT flow sorter, and the particle size of droplets was measured by a light microscopy measurement method; the Gibberella fujikuroi is purchased from Shanghai Haosheng industries, inc., and is numbered as FS-J5114, the gelatin is purchased from Mecang, and is numbered as 9000-70-8, the Pico-Surfin FC40 is purchased from Shilianbo research (Beijing) science and technology Inc., and is numbered as C012, the fluorinated oil (electronic fluorinated liquid) is purchased from Huamei subfamily Inc., of Shenzhen, and is numbered as KEY-128, and the calcium chloride, the EDTA disodium, the sodium hydroxide and the sodium alginate are all purchased from Shanghai Aladdin, and other raw materials and reagents are conventional commercial products without special description.

In the following examples, room temperature was 25. + -. 2 ℃ unless otherwise specified;

the YPD liquid culture medium comprises the following components: 20g/L of glucose, 20g/L of peptone and 10g/L of yeast extract.

Example 1

(1) Culturing Gibberella fujikuroi in YPD liquid culture medium for 48 hr, and collecting OD ₆₀₀ Carrying out normal-pressure room temperature plasma (ARTP) mutagenesis on bacterial liquid of =0.6, specifically, mixing the bacterial liquid with 5% glycerol solution, coating 10 mu L of the mixed bacterial liquid on an iron sheet, setting the power of an ARTP mutagenesis instrument to be 120W, setting the air flow to be 10SLM, selecting 20s, 30s, 40s, 50s and 60s as gradient time, sequentially carrying out mutagenesis, after the mutagenesis is finished, putting the iron sheet into an EP (EP) tube filled with 1mL of sterile physiological saline, marking, diluting and coating the solution of each gradient on a culture dish, and respectively constructing 5 parallel groups; the lethality obtained in different mutagenesis time is shown in figure 2, the mutagenesis time with the highest lethality is found to be 40s, the gibberellic bin fungi is mutagenized according to the highest lethal mutagenesis time, the mutagenized mutant strain is inoculated into a YPD liquid culture medium, and the culture solution of the mutant library strain of the gibberellic bin fungi is obtained after the mutagenized mutant strain is cultured for 10 hours at the temperature of 29 ℃; assembling the oil-in-water O/W single-emulsion microfluidic chip: drawing two glass capillaries with different sizes by using an acetylene burner as an outer phase capillary tube c and an inner phase capillary tube d, wherein the inner diameter of the inner phase capillary tube d is 150 mu m, the inner diameter of the outer phase capillary tube c is 580 mu m, performing hydrophobic treatment on the outer phase capillary tube c by using trichlorooctadecylsilane (OTS) by adopting an evaporation hydrophobic method (dichloromethane and trichlorooctadecylsilane (OTS) are mixed according to the volume ratio of 100 to prepare a hydrophobic agent, and placing the glass capillary tube and the hydrophobic agent togetherPlacing the capillary tube in a closed container, then placing the container in an oven at about 65 ℃ for 2h, repeating the operation for 4 times), coaxially nesting one end of an inner phase capillary tube d in one end of an outer phase capillary tube c, fixing the outer phase capillary tube c on a glass slide a by using quick-drying glue, and hermetically connecting the joint of the outer phase capillary tube c and the inner phase capillary tube d with a sample application needle b to form a microfluidic chip;

(2) Preparation of methacrylic anhydrified gelatin: adding 20g of gelatin into preheated 200mL of water, heating and stirring for about 1h in a water bath on a 60 ℃ heating table to obtain a gelatin solution, preparing 50mL of a 10wt% sodium carbonate solution (the whole system is coated with tinfoil to prevent light), heating in the water bath on the 60 ℃ heating table, slowly adding the gelatin solution by using a dropper, adding 4mL of methacrylic anhydride into the solution, and reacting for 0.5h to obtain a milky solution; measuring the pH, and finishing the reaction if the pH is 8-9; if the pH value is lower than 8, adding a proper amount of NaOH solution to adjust the pH value to be more than 8, filling the solution into a dialysis bag, dialyzing for 6 days in a dark place in the whole process, and after the dialysis is finished, putting the dialysis bag into a freeze dryer until the dialysis bag is completely freeze-dried to obtain methacrylic anhydrized gelatin as a hydrogel material;

(3) Selecting fluorinated oil with Pico-Surfin FC40 (the content of Pico-Surfin FC40 in the fluorinated oil is 2 wt%) as an external phase, and mixing the hydrogel material obtained in the step (2) with the culture solution obtained in the step (1) to form a mixture (the content of the hydrogel material is 60 wt%) as an internal phase; keeping the micro-fluidic chip at a constant temperature of 40 ℃, respectively sucking 1mL of each of the inner phase and the outer phase by using a syringe, installing the syringe of the outer phase on an outer-phase peristaltic pump, connecting the syringe with a sample application needle b through a polyethylene tube, inputting the outer phase into an outer-phase capillary tube c from the sample application needle b at a flow rate of 4mL/h, installing the syringe of the inner phase on an inner-phase peristaltic pump, connecting the syringe with one end of an inner-phase capillary tube d away from the outer-phase capillary tube c through the polyethylene tube, inputting the inner phase into the outer-phase capillary tube c from an inner-phase capillary tube d at a flow rate of 0.4mL/h, and embedding the strains in the trichoderma gambosum mutant library to form a liquid drop for packaging single spores;

(4) Collecting the liquid drops in the step (3) in a glass bottle containing excessive external phase, then standing and culturing for 7 days at the temperature of 29 ℃ (as shown in figure 3), placing the glass bottle at room temperature for cooling, and completely solidifying the liquid drops into microspheres; pumping a culture solution containing microspheres into an inlet corresponding to the microfluidic sorting device, aligning laser to a detection point of the sorting device, collecting a fluorescence signal of each droplet, and performing voltage-applying sorting on the droplet with the highest fluorescence signal intensity to enable the droplet to flow into a collecting channel;

and screening one or more liquid drops with higher signal intensity, respectively carrying out shake flask fermentation to screen out gibberellin high-yield strains, wherein the yield of gibberellin in the screened gibberellin high-yield strains is 4.2g/L at most, and is improved by 1.9 times compared with that of wild strains.

Example 2

High-throughput screening of gibberellin production by Gibberella fujikuroi was performed according to example 1, except that the flow rate of the external phase was controlled to be 5mL/h and the flow rates of the internal phase were set to be: 0.1mL/h, 0.2mL/h, 0.3mL/h, 0.4mL/h, 0.5mL/h; the particle size of the monodisperse emulsion droplets was found to increase with increasing internal phase flow rate as measured by optical microscopy, as shown in figure 4;

controlling the flow rate of the inner phase in the step (3) to be 0.5mL/h, and setting the flow rates of the outer phase to be respectively as follows: 5mL/h, 6mL/h, 7mL/h, 8mL/h, 9mL/h, 10mL/h; the size of the monodisperse emulsion droplets was found to decrease with increasing flow rate of the external phase as measured by optical microscopy as shown in figure 4.

The results show that the size of the liquid drop can be adjusted by changing the flow velocity of the internal phase and the external phase, when the microfluidic sorting device is used for screening in the step (4), the size of the liquid drop has an important influence on the sorting efficiency, and the liquid drop with larger size cannot flow into the collecting port under the action of voltage; therefore, the preparation of droplets with smaller size is important for improving the high-throughput screening of the gibberella barnacii, and the size of the droplets needs to be controlled by controlling the flow rate of the internal phase and the external phase or the inner diameter of the microchip, so that the high-throughput screening efficiency of the gibberella barnacii is improved.

Based on this, the flow rate of the outer phase was set to 5mL/h, the flow rate of the inner phase was set to 0.5mL/h, and the particle size of the droplets was controlled to be 170-200. Mu.m.

Example 3

High throughput screening of gibberellin production from Gibberella fujikunii was performed according to the method of example 1, except that the content of Pico-Surfin FC40 in the fluorinated oil was 2wt%; the concentration of the hydrogel material is set to be 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt% and 70wt% in a mixture formed by mixing the hydrogel material with a culture solution of a gibberella gambieri mutant library strain; the phase transition temperature of the internal phase of the hydrogels with different concentrations is shown in fig. 5, the phase transition temperature of the internal phase is between 28 ℃ and 30 ℃ when the concentration of the hydrogel is between 55wt% and 70wt%, the hydrogel concentration is in the range of 55wt% to 70wt%, and the higher the hydrogel concentration is, the higher the stability of microsphere transformation of the generated liquid drops at the optimum culture temperature of gibberella bardii is at 28 ℃ to 30 ℃.

Example 4

High-throughput screening of high-yield gibberellins of the gibelia canescens is carried out according to the method in the embodiment 1, except that in the step (3), the external phase is replaced by fluorinated oil added with FS-008 (the content of FS-008 in the fluorinated oil is 4 wt%), and the FS-008 is purchased from Shanghai Shilan chemical engineering Limited and is numbered 84166-37-0; the flow rate of the outer phase was set to 5mL/h and the flow rate of the inner phase was set to 0.5mL/h.

And screening one or more liquid drops with higher signal intensity, respectively carrying out shake flask fermentation to screen out gibberellin high-yield strains, wherein the yield of gibberellin in the screened gibberellin high-yield strains is 3.5g/L at most and is improved by 1.41 times compared with the original strains.

Example 5

High throughput screening of high yield gibberellins of gibberella gambieri was performed according to the method of example 1, except that the hydrogel material in step (2) was replaced with calcium alginate gel, specifically: mixing calcium chloride (100X 10) ^-3 M) solution with disodium EDTA (100X 10) ^-3 M) mixing the solutions in equal proportion to form a calcium-EDTA complex, and adjusting the pH value of the solution to 7 by using sodium hydroxide; mixing calcium-EDTA complex aqueous solution (50 × 10) ^-3 M) and sodium alginate aqueous solution (2 wt%) are prepared into a uniform mixture according to the following ratio of 9; the flow rate of the outer phase was set to 5mL/h and the flow rate of the inner phase was set to 0.5mL/h.

And screening one or more liquid drops with higher signal intensity, respectively carrying out shake flask fermentation to screen out gibberellin high-yield strains, wherein the yield of gibberellin in the screened gibberellin high-yield strains is 3.15g/L at most and is improved by 1.17 times compared with the original strains.

Comparative example 1

(1) Culturing Gibberella fujikuroi in YPD liquid culture medium for 48 hr, and collecting OD ₆₀₀ Carrying out normal-pressure room-temperature plasma (ARTP) mutagenesis on the bacterial liquid of =0.6 for 40s, inoculating the mutagenized mutant strain into a YPD liquid culture medium, and culturing at 29 ℃ for 10h to obtain a culture solution of a gibsonia alnoides mutant library strain; assembling the oil-in-water O/W single-emulsion microfluidic chip: drawing two glass capillaries with different sizes by using an acetylene blowtorch to serve as an outer-phase capillary tube c and an inner-phase capillary tube d, wherein the inner diameter of the inner-phase capillary tube d is 150 micrometers, the inner diameter of the outer-phase capillary tube c is 580 micrometers, performing hydrophobic treatment on the outer-phase capillary tube c by using trichlorooctadecylsilane (OTS), coaxially nesting one end of the inner-phase capillary tube d into one end of the outer-phase capillary tube c, fixing the outer-phase capillary tube c on a glass slide a by using quick-drying adhesive, and hermetically connecting the joint of the outer-phase capillary tube c and the inner-phase capillary tube d with a sample application needle b to form a microfluidic chip;

(2) Selecting fluorinated oil added with Pico-Surfin FC40 (the content of the Pico-Surfin FC40 in the fluorinated oil is 3 wt%) as an external phase, and directly taking the culture solution obtained in the step (1) as an internal phase; respectively sucking 1mL of the internal phase and 1mL of the external phase by using a syringe, wherein the syringe of the external phase is arranged on an external phase peristaltic pump and is connected with a sample application needle b through a polyethylene tube, the external phase is input into an external phase capillary tube c from the sample application needle b at the flow rate of 4mL/h, the syringe of the internal phase is arranged on an internal phase peristaltic pump and is connected with one end, away from the external phase capillary tube c, of an internal phase capillary tube d through the polyethylene tube, the internal phase is input into the external phase capillary tube c from the internal phase capillary tube d at the flow rate of 0.4mL/h, and the bacterial strains in the trichoderma gambieri mutant library are embedded to form liquid drops for packaging monospores; subsequently, it is found that the phenomena of microsphere cracking and the like exist in the processes of droplet transfer and screening, and the stability is poor, as shown in fig. 6;

(3) Collecting the liquid drops in the step (2) in a glass bottle containing excessive external phase, standing and culturing for 7 days at the temperature of 29 ℃, pumping the culture solution containing the liquid drops into an inlet corresponding to the microfluidic sorting device, aligning laser to a detection point of the sorting device, collecting a fluorescence signal of each liquid drop, and performing voltage-adding sorting on the liquid drop with the highest fluorescence signal intensity to enable the liquid drop to flow into a collecting channel.

And screening one or more liquid drops with higher signal intensity, respectively carrying out shake flask fermentation to screen out gibberellin high-yield strains, wherein the yield of gibberellin in the screened gibberellin high-yield strains is 1.9g/L at most and is improved by 31 percent compared with the original strains.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A method for screening microbial strains at high flux is characterized by comprising the following steps:

(3) And carrying out droplet culture on the droplets, solidifying the droplets to form microspheres, and carrying out microfluidic sorting on the microspheres.

2. The method of claim 1, wherein the creating of the library of mutants of a microorganism in step (1) employs at least one of ultraviolet irradiation, plasma mutagenesis and chemical mutagenesis.

3. The method of claim 1, wherein the microfluidic chip in step (1) comprises a glass capillary assembly, a glass slide and a sample application needle, the glass capillary assembly comprises an outer phase capillary and an inner phase capillary, one end of the inner phase capillary is nested in one end of the outer phase capillary, the outer phase capillary is fixed on the glass slide, and the connection of the outer phase capillary and the inner phase capillary is hermetically connected with the sample application needle, so that the sample application needle can input the outer phase into the outer phase capillary;

the inner diameter of the external phase capillary tube is 300-800 μm, and the inner diameter of the internal phase capillary tube is 100-200 μm.

4. The method of claim 3, wherein the external phase capillary is a glass tube that has been subjected to a hydrophobic treatment, and the hydrophobic agent of the hydrophobic treatment is trichlorooctadecylsilane.

5. The method according to any one of claims 1 to 4, wherein the surfactant in step (2) is at least one of Pico-Surfin FC40, FS-Kryjeff D900, EA surfactant and FS-008;

the concentration of said surfactant in said fluorinated oil is from 2 to 5wt%.

6. The method according to any one of claims 1 to 4, wherein the microorganism in step (1) is Gibberella fujiri, the phase transition temperature of the internal phase in step (2) is 28-30 ℃, and the microfluidic sorting in step (3) is performed to obtain Gibberella fujiri strain with high yield of gibberellin through screening signals;

the hydrogel in the step (2) is methacrylic anhydride-modified gelatin and/or calcium alginate gel, and the content of the hydrogel in the internal phase is 30-70wt%.

7. The method according to claim 6, wherein the culture solution of the microbial mutant library strain in the step (2) is obtained by subjecting the microbial mutant library strain to liquid culture;

the YPD liquid culture medium is adopted as the culture medium of the liquid culture, and the culture conditions at least comprise: the temperature is 28-30 ℃.

8. The method according to claim 6, wherein the flow rate of the outer phase in step (2) is 3 to 8mL/h and the flow rate of the inner phase is 0.1 to 0.5mL/h;

the embedding temperature is controlled at 32-40 ℃.

9. The method according to claim 6, wherein the conditions of the droplet culture in step (3) comprise at least: the temperature is 28-30 ℃, and the time is 2-8 days;

the droplet culture is carried out in the external phase;

the temperature of the curing is below 28 ℃.

10. The method of any one of claims 1 to 4, wherein the step (3) employs a screening signal selected from at least one of fluorescence, absorbance, raman spectroscopy, and mass spectroscopy.