阅读说明：本技术 一种高通量筛选高产槐糖脂菌株的方法 (Method for high-throughput screening of high-yield sophorolipid strains ) 是由 储炬 李前会 田锡炜 杭海峰 夏建业 庄英萍 李璟曦 张嘉兴 韩思宇 徐文静 杨 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种高通量筛选高产槐糖脂菌株的方法,所述方法包括：菌种活化的步骤；常压室温等离子体与亚硝酸钠组合诱变的步骤；松弛培养的步骤；多重压力培养的步骤；其中,所述菌种为假丝酵母菌；所述多重压力培养的步骤中采用碘乙酸和丙二酸结合进行双压力培养筛选。本发明的方法中采用常压室温等离子体与亚硝酸钠组合诱变,并采用松弛培养与碘乙酸和丙二酸双压力结合进行筛选,增加菌株的突变率以及正突变率,增加得到高产菌株的概率,进而得到遗传稳定性量好的高产菌株。(The invention discloses a method for screening high-yield sophorolipid strains in a high-throughput manner, which comprises the following steps: activating strains; carrying out combined mutagenesis on the normal-pressure room-temperature plasma and sodium nitrite; a step of relaxation culture; multiple pressure culture; wherein the strain is candida; in the step of the multiple pressure culture, iodoacetic acid and malonic acid are combined to carry out double pressure culture screening. According to the method, normal-pressure room-temperature plasma and sodium nitrite are combined for mutagenesis, relaxation culture and iodoacetic acid and malonic acid dual-pressure combination are adopted for screening, the mutation rate and positive mutation rate of the strain are increased, the probability of obtaining high-yield strains is increased, and the high-yield strains with good genetic stability are obtained.)

1. A method for high throughput screening of high yielding sophorolipid strains, said method comprising:

activating strains;

carrying out combined mutagenesis on the normal-pressure room-temperature plasma and sodium nitrite;

a step of relaxation culture;

multiple pressure culture;

wherein the strain is candida;

in the step of the multiple pressure culture, iodoacetic acid and malonic acid are combined to carry out double pressure culture screening.

2. The method for high-throughput screening of sophorolipid-producing strains according to claim 1, wherein the total mortality rate of the combined mutagenesis of sodium nitrite and the atmospheric pressure room temperature plasma is 88-92%.

3. The method for high-throughput screening of sophorolipid-producing strains according to claim 1 or 2, wherein the step of activating the bacterial species comprises: inoculating glycerol tube strain to activating culture medium, shake-culturing at 25 deg.C at 220r/min for 48 hr to activate strain, diluting to make strain concentration 10⁷And (4) obtaining activated bacteria liquid.

4. The method for high-throughput screening of sophorolipid-producing strains according to claim 1 or 2, wherein the step of the combined mutagenesis by the atmospheric pressure room temperature plasma and sodium nitrite comprises: and mixing the activated bacterial liquid with a sodium nitrite solution to obtain a mixed bacterial liquid of 5mg/L sodium nitrite, culturing at 25 ℃ for 10min at 220r/min, washing, resuspending, diluting, coating the bacterial liquid, and mutagenizing for 18-20 s on an ARTP instrument.

5. The method for high-throughput screening of sophorolipid-producing strains according to claim 1, wherein the step of relaxation culture comprises: and inoculating the combined mutagenized bacterial liquid into a pore plate seed culture medium for relaxation culture for 48 h.

6. The method for high-throughput screening of sophorolipid-producing strains according to claim 1, wherein the step of multiplex pressure cultivation comprises: transferring the bacteria liquid after the relaxation culture into a 96-well plate for culturing for 24h, selecting the strain with higher bacteria concentration, transferring the strain into a 24-well plate for culturing for 24h, selecting the strain with higher bacteria concentration again, transferring the strain into a 24-well plate for culturing for 24h, then selecting the strain with higher bacteria concentration for dilution, coating the diluted strain on a solid plate, culturing for three days, selecting the strain with larger bacterial colony for storage;

wherein a well plate seed liquid pressure culture medium containing 0.02g/L of iodoacetic acid is added into each of the 96-well plate and the 24-well plate; the solid plate was supplemented with a solid pressure medium containing 10g/L malonic acid.

7. The method for high-throughput screening of sophorolipid-producing strains according to claim 1, wherein the method for high-throughput screening of sophorolipid-producing strains further comprises a preliminary screening step; the primary screening method comprises the following steps: transferring the selected bacterial strain with larger bacterial colony into a 96-pore plate for two days in a non-pressure culture mode, transferring the bacterial strain into a 24-pore plate in a non-pressure fermentation culture medium for three days in a culture mode, measuring the yield of sophorolipid in the pore plate by an iodine method, selecting the bacterial strain with higher yield, and screening out the bacterial strain with high yield of sophorolipid according to the yield of sophorolipid.

8. The method for high-throughput screening of sophorolipid-producing strains according to claim 3 or 5, wherein the activation medium and the well plate seed medium each comprise: 50g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor.

9. The method for high-throughput screening of sophorolipid-producing strains according to claim 6, wherein the well plate seed liquid pressure medium comprises: 50g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor.

10. The method for high-throughput screening of sophorolipid-producing strains according to claim 6, wherein the solid pressure medium comprises: 20g/L glucose, 10g/L yeast powder, 20g/L peptone and 20g/L agar; the pH is natural.

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a method for screening high-yield sophorolipid strains in a high-throughput manner.

Background

Sophorolipid is a glycolipid biosurfactant, and although there are many microorganisms capable of synthesizing sophorolipid, Candida bombicola is the most important production strain in research reports. Sophorolipids have attracted considerable attention in industrial production and scientific research and are considered to be one of the most promising bioactive agents because they are environmentally friendly. Due to the characteristics of sophorolipid, high oxygen consumption and high viscosity in the fermentation production process lead to increase of production cost, so that it is necessary to screen high-yield sophorolipid producing strains.

Rational genetic engineering breeding methods are advantageous methods for strain improvement, but they have several major disadvantages: (1) cell metabolism is a very complex adaptive network, and changing one or two genes may not achieve the required goal; (2) the production and sale of related products of some genetic engineering strains are limited by related application fields. Therefore, the traditional mutation breeding is still an indispensable method for obtaining excellent-performance production strains, and the rapid development of high-throughput screening technology is combined, so that the availability of high-performance strains is greatly improved. Traditional mutagenesis approaches are generally divided into physical and chemical mutagenesis, physical mutagenesis including: ultraviolet mutagenesis, irradiation with various rays, microwave irradiation, and the like. Chemical mutagenesis comprises: nitrosoguanidine, nitrous acid, diethyl sulfate, ethylene imine, mustard gas, and nucleobase analogs. Hafez et al, by means of physical and chemical mutagenesis, raised the enzyme activity of Streptomyces griseus E44G in producing chitin by 1.39 times. The Atmospheric Room Temperature Plasma (ARTP) is a newly developed whole-cell mutagenesis tool based on radio frequency atmospheric pressure luminous discharge plasma, induced DNA damage is repaired by an SOS system subsequently, DNA change is caused, and the operation is convenient and safe.

In the process of mutagenesis, mutagenesis is a means, screening is a key, and a reasonable screening model provides convenience for finally obtaining high-yield bacteria. During mutagenesis of a strain, the use of a single mutagenesis method can cause the mutagen to produce a "fatigue effect", to be slowly metabolized, and the like; and the strain screened by single mutagenesis is easy to degenerate. Generally, the traditional screening process is to directly coat the strain subjected to single mutagenesis on a stressed plate. In the traditional high-throughput screening method, for example, the research results of Zhougang and the like show that the strain obtained by the traditional mutagenesis method has slow growth and is easy to degenerate, and the screening efficiency is not high; zhougang and the like only adopt single screening pressure in the process of high-throughput screening, and finally the obtained high-yield strain is easy to degenerate in the actual fermentation process.

Therefore, it is highly desirable to provide a method for screening high-yielding sophorolipid strains at high throughput, so as to increase the mutation rate and positive mutation rate of the strains, increase the probability of obtaining high-yielding strains, and obtain high-yielding strains with good genetic stability.

Disclosure of Invention

The invention aims to provide a method for screening high-yield sophorolipid strains in a high-throughput manner, which is characterized in that ARTP mutagenesis and sodium nitrite are combined for mutagenesis, relaxation culture and combined pressure culture screening are introduced, so that the positive mutation rate of the strains can be effectively improved, and the probability of the positive mutant strains in the screening process is improved.

In order to achieve the purpose, the invention adopts the following technical scheme.

The invention provides a method for screening high-yield sophorolipid strains in a high-throughput manner, which comprises the following steps:

activating strains;

a step of combined mutagenesis of Atmospheric Room Temperature Plasma (ARTP) and sodium nitrite;

a step of relaxation culture;

multiple pressure culture;

wherein the strain is candida; in the step of the multiple pressure culture, iodoacetic acid and malonic acid are combined to carry out double pressure culture screening.

Further, the total lethality of the sodium nitrite and the ARTP combined mutagenesis is 88-92%.

Further, the total lethality of the sodium nitrite in combination with the ARTP mutagenesis was 90%.

Further, the strain activation step comprises: inoculating glycerol tube strain to activating culture medium, shake-culturing at 25 deg.C at 220r/min for 48 hr to activate strain, diluting to make strain concentration 10⁷And (4) obtaining activated bacteria liquid.

Further, the step of the combined mutagenesis of the normal pressure room temperature plasma (ARTP) and the sodium nitrite comprises the following steps: and mixing the activated bacterial liquid with a sodium nitrite solution to obtain a mixed bacterial liquid of 5mg/L sodium nitrite, culturing at 25 ℃ for 10min at 220r/min, washing, resuspending, diluting, coating a proper amount of bacterial liquid, and mutagenizing for 18-20 s on an ARTP instrument.

Further, the step of relaxation culture comprises: and inoculating the combined mutagenized bacteria liquid into a 96-well plate seed culture medium without pressure for relaxation culture for 48 hours.

Further, the step of multiple pressure culturing comprises: transferring the bacteria liquid after the relaxation culture into a 96-well plate for 24h, selecting a strain with higher bacteria concentration (OD) to transfer into a 24-well plate for 24h, then, selecting a strain with higher bacteria concentration (OD) for dilution, coating the diluted strain on a solid plate, culturing for three days, and selecting a strain with larger bacterial colony for storage;

wherein a well plate seed liquid pressure culture medium containing 0.02g/L of iodoacetic acid is added into each of the 96-well plate and the 24-well plate; the solid plate was supplemented with a solid pressure medium containing 10g/L malonic acid.

Further, the step of multiple pressure culturing comprises: transferring the bacteria liquid after the relaxation culture into a 96-well plate for 24h, selecting the bacterial strain with higher bacterial concentration (OD) to transfer into two 24-well plates for 24h, selecting the bacterial strain with higher bacterial concentration (OD) again to transfer into one 24-well plate for 24h, then selecting 5 bacterial strains with higher bacterial concentration (OD) for dilution, coating the diluted bacterial strains on a solid plate, culturing for three days, and selecting the bacterial strain with larger bacterial colony for storage.

Further, the method for screening the high-yield sophorolipid strain in high flux also comprises a primary screening step;

the primary screening method comprises the following steps: and transferring the selected bacterial strain with larger bacterial colony into a 96-pore plate for two days in a non-pressure culture mode, transferring the bacterial strain into a 24-pore plate in a non-pressure fermentation culture medium for three days in a culture mode, measuring the yield of sophorolipid in the pore plate by an iodine method, and selecting the bacterial strain with higher yield of sophorolipid.

Further, the activation medium comprises: 50g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor.

Further, the well plate seed medium comprises: 50g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor.

Further, the well plate seed liquid pressure medium comprises: 50g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor.

Further, the solid pressure medium includes: 20g/L glucose, 10g/L yeast powder, 20g/L peptone and 20g/L agar; the pH is natural.

In the present invention, the shake flask fermentation can be cultured by a conventional method.

In one embodiment of the invention, the positive mutation rate of the ARTP sodium nitrite combined mutagenesis can be obtained by the following method: firstly, treating the activated seeds with sodium nitrite with proper concentration, and then carrying out ARTP mutagenesis by using sterile water for heavy suspension; diluting the mutagenized bacterial liquid by a proper multiple, coating the bacterial liquid, culturing for three days, selecting a larger single colony, inoculating the single colony into a 96-seed empty plate, culturing for two days, transferring the single colony to a 24-pore plate, fermenting and culturing for three days, and detecting after the culture is finished; calculating the positive mutation rate of ARTP sodium nitrite combined mutagenesis.

In one embodiment of the present invention, the culture medium used may be a conventional culture medium. For example, the following shake flask fermentation medium, well plate fermentation medium and fermentation medium were used:

the shake flask fermentation medium comprises: 150g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor and 6g/L CaCO₃50g/L rapeseed oil;

the pore plate fermentation medium comprises: 150g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor and 6g/L CaCO₃50g/L of triolein;

the fermentation medium comprises: 100g/L glucose, 1g/L KH₂PO₄4g/L of (NH)₄)₂SO₄0.5g/L MgSO₄·7H₂O, 10g/L corn steep liquor 10; the amount of glucose solution was weighed to 500g during sterilization.

In the present invention, the calculation formula of the mutation rate (QM) and the positive mutation rate (QT) is as follows:

in the formula: h represents the total number of selected colonies; m represents the total number of mutant bacteria; t represents the number of colonies with higher yield than the developing strain. In the present invention, a strain with sophorolipid yield exceeding 5% was taken as an effective strain for the positive mutant strain, because the error of parallelism was about 3%.

In the invention, the sophorolipid content is determined: in the primary screening process, the content of sophorolipid is rapidly detected by an iodine method, pure sophorolipid solutions with different concentration gradients are prepared, and a standard curve is drawn. And in the secondary screening process, a DNS method is adopted for detection, and the content of sophorolipid is determined.

In the present invention, data processing and analysis may be performed using origin8 software.

The invention has the beneficial effects that:

according to the invention, normal-pressure room-temperature plasma and sodium nitrite are adopted for combined mutagenesis, relaxation culture and pressure culture are combined, and iodoacetic acid and malonic acid are adopted for double pressure culture screening, so that the mutation rate and positive mutation rate of the strain can be increased, the probability of obtaining high-yield strains is increased, and further, high-yield strains with good genetic stability are obtained. The combined mutagenesis has a synergistic effect, can effectively improve the positive mutation rate of the strain, and ensures that the obtained strain has better genetic stability and smaller yield change between generations; the loose culture can improve the probability of positive mutant strains in the screening process and solve the problem of low screening efficiency due to slow growth of the strains; the combination pressure can also increase the mutation rate and positive mutation rate in the screening process, increase the probability of obtaining high-yield strains, and simultaneously, the obtained high-yield strains have good genetic stability.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a graph I showing the sophorolipid production by the strain of test example 1 of the present invention.

FIG. 2 is a graph II showing the sophorolipid production by the strain of test example 1 of the present invention.

FIG. 3 is a graph I of sophorolipid production by the strain in test example 2 of the present invention.

FIG. 4 is a graph II showing the sophorolipid production by the strain in test example 2 of the present invention.

FIG. 5 is a graph I showing the sophorolipid production by the strain in test example 3 of the present invention.

FIG. 6 is a graph II showing the sophorolipid production by the strain in test example 3 of the present invention.

FIG. 7 is a graph comparing the yield of sophorolipid in shake flask experiments of the high producing strain and the starting strain of the present invention.

FIG. 8 is a schematic diagram showing the genetic stability of the highly productive strain of the present invention.

FIG. 9 is a graph comparing the production of sophorolipid in the 5L fermenter experiment of the high producing strain and the starting strain of the present invention.

FIG. 10 is a graph showing CER in 5L fermentors of the high-producing strain of the present invention and the starting strain.

FIG. 11 is a graph comparing the sophorolipid yields in 5L fermentors of the high-producing strain and the starting strain of the present invention.

FIG. 12 is a process diagram of the method for high throughput screening of sophorolipid-producing strains according to example 2 of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.