Method for improving transformation efficiency of schizochytrium limacinum

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for improving the conversion efficiency of schizochytrium limacinum. According to the method, before electrotransformation, schizochytrium limacinum is subjected to enzyme treatment; the enzyme treatment comprises the following steps: before electrotransformation, treating schizochytrium limacinum by adopting an enzyme; the enzyme is protease or helicase; the enzymatic activity of the protease is more than or equal to 200U / g, and the enzymatic activity of the helicase is more than or equal to 12.5 U / g. According to the enzymolysis method applied to electric transformation of the schizochytrium limacinum, the schizochytrium limacinum is subjected to enzymolysis treatment through alkaline protease or helicase, so that the transformation efficiency of exogenous DNA (Deoxyribose Nucleic Acid) of the schizochytrium limacinum can be effectively improved, and the voltage and the concentration requirement of the exogenous DNA in the electric transformation process are reduced; the method has important application value in the field of schizochytrium limacinum related microbial engineering.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for improving the transformation efficiency of Schizochytrium. Background Art

[0002] Non-model microorganisms (such as Schizochytrium) are prone to low positive rates or even no positive transformants in electrotransformation experiments. This is because compared with model organisms, electrotransformation conditions are more difficult to explore and the concentration and purity of plasmids are more stringent. This situation also makes electrotransformation of Schizochytrium significantly more difficult, and the transformation efficiency is often not guaranteed.

[0003] There are many studies on the electrotransformation process of Schizochytrium in the prior art, but there are problems such as the high voltage used, which can easily increase the selective pressure, damage cells, and even cause cell death. Other studies have high requirements for the purity and concentration of exogenous DNA, and the experimental conditions are relatively harsh. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for improving the transformation efficiency of Schizochytrium.

[0005] High voltage during electrotransformation of microorganisms may cause a variety of problems. High voltage will generate instantaneous high temperature around the cells, which may cause thermal damage to the cells or directly damage the cell membrane, which will significantly reduce the transformation efficiency or even cell death. High voltage may also directly damage the plasmid, affect its structure, and reduce the transformation ability of the plasmid. In addition, even if some cells are successfully transformed under high voltage, they may become fragile due to excessive stress response and may face higher selective pressure in the subsequent screening process, resulting in slow growth or death. This problem is particularly evident in the electrotransformation process of non-model organisms (such as Schizochytrium), because non-model organisms are usually difficult to electrotransform, and often require higher voltages and high-purity linearized DNA fragments to improve the transformation rate.

[0006] In order to solve this problem, the present invention adopts enzymolysis to improve the conversion rate and reduce the threshold of voltage and exogenous DNA purity. However, this process is also relatively complicated. Although enzymolysis may dissolve cell walls and improve exogenous DNA permeability in theory, it may damage cells while dissolving cell walls, and even directly damage cell membranes, leading to leakage of cell contents and loss of cell activity. In this case, even if exogenous DNA successfully enters cells, cells cannot survive and replicate exogenous DNA. In addition, cells after enzymolysis become fragile, and may also produce stress reactions and produce stress proteins. These processes can affect the metabolic activity of cells, and may also interfere with the stability and expression of exogenous DNA, which in turn leads to a decrease in conversion efficiency.

[0007] After extensive research, the present invention has obtained a suitable enzymatic hydrolysis method, which can significantly improve the electrotransformation efficiency of Schizochytrium without significantly affecting its survival rate.

[0008] In a first aspect, the present invention provides a method for improving the transformation efficiency of Schizochytrium, comprising: before electrotransformation, performing an enzyme treatment on Schizochytrium; the enzyme treatment comprises: Before electrotransformation, the Schizochytrium is treated with an enzyme; the enzyme is a protease or a snail enzyme; the enzyme activity of the protease is ≥200 U / g, and the enzyme activity of the snail enzyme is ≥12.5 U / g.

[0009] Furthermore, the enzyme activity of the protease is ≥900 U / g, and the enzyme activity of the snail enzyme is ≥20 U / g.

[0010] Furthermore, the enzyme activity of the protease is ≥1800 U / g, and the enzyme activity of the snail enzyme is ≥40 U / g.

[0011] Furthermore, the enzyme activity of the protease is ≥2000U / g; the enzyme activity of the snail enzyme is ≥50U / g.

[0012] Furthermore, the enzyme activity of the protease is ≥9000U / g; the enzyme activity of the snail enzyme is ≥40U / g; further, the enzyme activity of the protease is ≤22000U / g, and the enzyme activity of the snail enzyme is ≤2200U / g.

[0013] Furthermore, the enzyme activity of the protease is ≥9000U / g; the enzyme activity of the snail enzyme is ≥400U / g; further, the enzyme activity of the protease is ≤11000U / g; the enzyme activity of the snail enzyme is ≤1200U / g.

[0014] Furthermore, the protease is alkaline protease.

[0015] Furthermore, the snail enzyme has a product number of S8280 (purchased from solarbio).

[0016] Furthermore, the treatment of the protease includes: treating at 28-30°C, pH=9-10, and the treatment time is ≤75 minutes; the treatment of the snail enzyme includes: treating at 28-30°C, pH=6-7, and the treatment time is ≤75 minutes.

[0017] Furthermore, the electrotransformation includes: mixing exogenous DNA and Schizochytrium competent cells, transferring them to an electrotransformation device for electrotransformation after standing; and transferring them to a culture medium for recovery after electrotransformation.

[0018] Furthermore, the exogenous DNA is a linearized DNA fragment or a plasmid.

[0019] Furthermore, the concentration of the linearized DNA fragment is above 2 ng / mL, and the concentration of the plasmid is above 15 ng / mL.

[0020] Furthermore, the electroconversion device is an electric shock cup, and the parameters of the electroconversion include: 0.4~0.6KV, 20~30μF and 150~250Ω.

[0021] Furthermore, before the electrotransformation, the method further includes: preparing the Schizochytrium treated with the enzyme into a competent state.

[0022] In a second aspect, the present invention provides a gene editing method for Schizochytrium, comprising: introducing exogenous DNA using the method described.

[0023] The present invention has the following beneficial effects: The present invention studies and obtains an electrotransformation method for Schizochytrium. Before electrotransformation, alkaline protease or snail enzyme with a certain enzyme activity is used to treat Schizochytrium, which can improve the transformation efficiency (positive rate) while also having a better Schizochytrium cell survival rate.

[0024] The method provided by the present invention can significantly reduce the exogenous DNA concentration requirement during electrotransformation of Schizochytrium, and a nonlinear plasmid can be directly used during transformation (the existing electrotransformation methods of Schizochytrium generally use linearized plasmids).

[0025] The method provided by the present invention can significantly reduce the voltage intensity during the electrotransformation of Schizochytrium, which helps to improve the survival rate of Schizochytrium cells during the electrotransformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is the plasmid map of the plasmid pblue-Bleo provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art and can be implemented by conventional methods in the art, for example, by referring to reference books in the art or instructions for kits.

[0030] Unless otherwise specified, the experimental materials involved in the following examples can be purchased commercially.

[0031] Example 1

[0032] This embodiment provides a method for transforming Schizochytrium, including the following process: 1. Cultivate Schizochytrium to the logarithmic growth phase, OD 600nm =1.

[0033] The bacterial solution was divided into multiple tubes of 5 mL each, centrifuged at 4000 r / min for 10 min, washed twice with buffer, and the bacterial cells were collected.

[0034] 2. Resuspend the bacteria and add them to the multi-well plate. Set up a control group and multiple experimental groups. The control group is not treated. The experimental groups are tested with alkaline protease, cellulase, lytic enzyme and snail enzyme, and each enzyme is used in a gradient concentration, as shown in the following table: Table 1 Concentration gradient of different enzymes (unit: mass volume percentage)

[0035] As mentioned above, alkaline protease, cellulase, lytic enzyme and snail enzyme were purchased from solarbio, with alkaline protease activity of 200000U / g, cellulase activity of 3U / mg, lytic enzyme activity of 2000U / mg, snail enzyme activity of S8280, and enzyme activity of ≥50000. Alkaline protease activity of B8360, cellulase activity of C8270, and lytic enzyme activity of L8141.

[0036] The specific enzyme treatment method is as follows: alkaline protease is treated at 28°C, pH=9 for 1 hour; cellulase is treated at 28°C, pH=7 for 1 hour; lytic enzyme is treated at 28°C, pH=7 for 1 hour; snail enzyme is treated at 28°C, pH=7 for 1 hour.

[0037] 3. After treatment of the control group and each experimental group, centrifuge at 4000r / min for 10min to collect the bacteria.

[0038] 4. The bacterial cells of the control group and each experimental group were plated and counted, and the remaining cells were prepared as competent cells as follows: (1) Wash the cells twice with 5 mL of 0℃ precooled sterile water and centrifuge at 4000 rpm for 10 min.

[0039] (2) Resuspend in 5 mL of 0.5 M sorbitol solution precooled at 0°C and centrifuge at 4000 rpm for 10 min.

[0040] (3) Resuspend the bacterial pellet in 5 mL of 0.5 M sorbitol solution precooled at 0°C, divide into 100 μL aliquots, and freeze at -80°C.

[0041] 5. Construction of exogenous DNA for electroporation

[0042] The experimental object is the strain Schizochytrium sp. ATCC 20888 (this strain is disclosed in many patent documents). The resistance fragment is from the plasmid pGAPZαA (commercial), which contains the promoter TEF1, the bleomycin resistance sequence bleoR, and the terminator CYC1 (all public fragments). The electroporation plasmid pblue-Bleo was constructed by cloning. The plasmid map is shown in Figure 1 According to the sequencing results, the neutral site was selected as the homologous recombination site of the transformed fragment, and 1000 bp homologous arm sequences were constructed upstream and downstream (corresponding to upstream and downstream in the plasmid).

[0043] The linearized fragment of plasmid pblue-Bleo can be obtained by double digestion with PstI and XbaI (the fragment size obtained by double digestion is about 3100 bp).

[0044] 6. Electrical conversion

[0045] (1) Use 200 ng / mL of the linearized fragment from the previous step for electroporation. After diluting 10 times, the final concentration of exogenous DNA in the electroporation system is 20 ng / mL.

[0046] (2) Add 10 μL of the linearized fragment to be transformed into 100 μL of Schizochytrium competent cells, mix gently, let stand in an ice bath for 5 min, then transfer to an ice-precooled electroporation cup and let stand for 10 min. The electroporation parameters are 0.5 KV, 25 μF, and 200 Ω.

[0047] (3) After electroporation, add 1 mL of culture medium and resuscitate at 28°C and 200 r / min for 1 h.

[0048] (4) Spread the transformed bacterial solution on a resistant plate and culture at 28°C.

[0049] 7. Statistics of survival rate and positive rate

[0050] The survival rate and positive rate of Schizochytrium were calculated by the following formula: Survival rate of Schizochytrium (%) = (number of cells after enzyme treatment / number of cells before enzyme treatment) × 100.

[0051] The positive rate of Schizochytrium (%) = (number of positive clones after transformation / number of cells before enzyme treatment) × 100.

[0052] The results are shown in the following table, where a positive rate greater than 1% indicates good effect, a positive rate between 0.5%-1% indicates average effect, 0.1-0.5% indicates poor effect, and a positive rate below 0.1% indicates poor effect.

[0053] Table 2 Survival rate and positive rate of Schizochytrium in the control group and different experimental groups

[0054] As shown in the above results, cellulase has the worst effect on Schizochytrium. It cannot improve the positive rate at multiple concentrations, and even reduces the positive rate at concentrations of 0.1%, 2% and 4%. The effect of lysin on improving the positive rate is not obvious, and the survival rate of Schizochytrium will be significantly reduced due to the excessive enzymatic effect on the cell wall. In contrast, alkaline protease and snail enzyme treatment have better effects. The positive rate reached 1.08% under 5% alkaline protease conditions, but higher concentrations of alkaline protease will cause the cell wall to be completely enzymatically degraded and die. After treatment with 1% and 2% snail enzyme, the conversion survival rate and conversion rate remained at a high level, indicating that it has the most obvious improvement in cell wall permeability, among which the positive rate of 2% snail enzyme treatment reached 2.23%.

[0055] Example 2

[0056] Based on the results of Example 1, in order to further explore the minimum treatment concentration and the longest treatment time of alkaline protease and snail enzyme, the concentration gradient used in the present invention is as follows: Table 3 Concentration gradient of different enzymes (unit: mass volume percentage)

[0057] The specific enzyme treatment methods are as follows: alkaline protease is treated at 28°C, pH = 9 for 1h, 3h, and 5h. Snail enzyme is treated at 28°C, pH = 7 for 1h, 3h, and 5h. The other conditions are unchanged compared to Example 1. The survival rate and positive rate are shown in the following table.

[0058] Table 4 Survival rate at different enzyme concentrations

[0059] Table 5 Positive rate of different enzyme concentrations

[0060] As can be seen from the above results, reducing the enzyme concentration and extending the treatment time will lead to a decrease in conversion efficiency. After the treatment time is too long to 3h, the cells may be damaged, resulting in a significant decrease in survival rate. The survival rate will be 0 after 5h. It can be seen that 5h is the maximum time for enzyme treatment. When the alkaline protease concentration is 0.05%-0.1%, the positive rate is similar to that of the no enzyme treatment after 3h treatment, indicating that 0.05%-0.1% is the lowest effective alkaline protease concentration. Similarly, when the snail enzyme concentration is 0.01%-0.025%, the conversion rate is similar to that of the no enzyme treatment after 3h treatment, indicating that 0.01%-0.025% is the lowest effective snail enzyme concentration.

[0061] Example 3

[0062] This example further verifies the conversion efficiency of low-concentration exogenous DNA. The steps are the same as those in Example 1, except that: 1. The experimental groups only used 5% alkaline protease, 1% snail enzyme and 2% snail enzyme.

[0063] 2. The exogenous DNA used for electrotransformation was adjusted from 200 ng / mL linearized fragment to 50 ng / mL linearized fragment, or 200 ng / mL plasmid pblue-Bleo without linearization enzyme digestion. Both were diluted tenfold during electrotransformation.

[0064] The results are shown in the following table: Table 6 Conversion rates of the control group and different experimental groups (low concentration of exogenous DNA)

[0065] It can be seen from the above results that according to the transformation method provided by the present invention (alkaline protease and snailase treatment), even low-concentration linearized DNA fragments and circular DNA (plasmids) still maintain a high conversion rate and can stably obtain positive transformants, which shows that the method provided by the present invention significantly improves the efficiency of genetic transformation of Schizochytrium.

[0066] Example 4

[0067] The present invention further applied the method shown in Example 1 to Schizochytrium sp. HX-308, and obtained the following results: Table 7 Survival rate and positive rate of Schizochytrium in the control group and different experimental groups

[0068] The present invention further applies the method shown in Example 1 to Aurantiochytrium limacinum, and obtains the following experimental results: Table 8 Survival rate and positive rate of Schizochytrium in the control group and different experimental groups

[0069] It can be seen from the results that the method provided in this embodiment shows a similar improvement effect and trend as Schizochytrium sp. ATCC 20888 when applied to other Schizochytrium sp. ATCC 20888, which shows that the method provided by the present invention can be applied to Schizochytrium sp. ATCC 20888, and is not limited to Schizochytrium sp. ATCC 20888.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the transformation efficiency of Schizochytrium, characterized in that: include: Before electrotransformation, the Schizochytrium is subjected to an enzyme treatment; the enzyme treatment comprises: Before electrotransformation, the Schizochytrium is treated with an enzyme; the enzyme is a protease or a snail enzyme; the enzyme activity of the protease is ≥200 U / g, and the enzyme activity of the snail enzyme is ≥12.5 U / g.

2. The method according to claim 1, characterized in that The protease is alkaline protease.

3. The method according to claim 1 or 2, characterized in that: The enzyme activity of the protease is ≥2000U / g; the enzyme activity of the snail enzyme is ≥50U / g.

4. The method according to any one of claims 1 to 3, characterized in that: The treatment of the protease comprises: treating at 28-30°C, pH=9-10, and the treatment time is ≤75 minutes; the treatment of the snail enzyme comprises: treating at 28-30°C, pH=6-7, and the treatment time is ≤75 minutes.

5. The method according to any one of claims 1 to 4, characterized in that: The electrotransformation comprises: mixing exogenous DNA and Schizochytrium competent cells, transferring to an electrotransformation device for electrotransformation after standing; and transferring to a culture medium for recovery after electrotransformation.

6. The method according to claim 5, characterized in that The exogenous DNA is a linearized DNA fragment or a plasmid.

7. The method according to claim 6, characterized in that The concentration of the linearized DNA fragment is above 2 ng / mL, and the concentration of the plasmid is above 15 ng / mL.

8. The method according to any one of claims 1 to 7, characterized in that: Before the electrotransformation, the method further includes: preparing the Schizochytrium treated with the enzyme into a competent state.

9. A gene editing method for Schizochytrium, characterized in that: include: The exogenous DNA is introduced by the method according to any one of claims 1 to 8.

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