A method for improving transformation efficiency of schizochytrium sp.

Abstract

The present application relates to the field of microbial technology, and more particularly to a method for improving transformation efficiency of schizochytrium sp. The method comprises the following steps: before the electrotransformation, the schizochytrium sp. is subjected to enzyme treatment; the enzyme treatment comprises the following steps: before the electrotransformation, the schizochytrium sp. is subjected to enzyme treatment; the enzyme is protease or snailase; the enzyme activity of the protease is greater than or equal to 200 U / g, and the enzyme activity of the snailase is greater than or equal to 12.5 U / g. The present application studies an enzymatic hydrolysis method applied to the electrotransformation of schizochytrium sp., and the transformation efficiency of exogenous DNA of schizochytrium sp. can be effectively improved by subjecting the schizochytrium sp. to enzymatic hydrolysis treatment by alkaline protease or snailase, and the voltage size and the concentration requirement of exogenous DNA in the electrotransformation process are reduced, which has important application value in the field of microbial engineering related to schizochytrium sp.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for improving the transformation efficiency of Schizochytrium. Background Technology

[0002] Non-model microorganisms (such as Schizochytrium) often exhibit low positive rates or even no positive transformants during electroporation experiments. This is because, compared to model organisms, electroporation conditions are more difficult to determine, and the requirements for plasmid concentration and purity are more stringent. This situation significantly increases the difficulty of electroporation of Schizochytrium, often resulting in unreliable transformation efficiency.

[0003] Existing technologies include several studies involving the electroporation process of Schizochytrium fungi, but these studies suffer from drawbacks such as the use of high voltages, which can easily lead to increased selective stress, cell damage, and even cell death. Other studies also require high purity and concentration of exogenous DNA, resulting in demanding experimental conditions. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for improving the transformation efficiency of Schizochytrium.

[0005] High voltages during microbial electroporation can lead to various problems. High voltage generates instantaneous high temperatures around cells, potentially causing thermal damage or directly damaging the cell membrane, significantly reducing transformation efficiency or even causing cell death. High voltage can also directly damage plasmids, affecting their structure and reducing their transforming ability. Furthermore, even if some cells are successfully transformed under high voltage, they may become vulnerable due to excessive stress, facing higher selective pressure during subsequent selection processes, leading to slow growth or death. This problem is particularly pronounced in the electroporation of non-model organisms (such as Schizochytrium), as these organisms are generally more difficult to electroporate and often require higher voltages and highly purified linearized DNA fragments to improve transformation efficiency.

[0006] To address this issue, this invention employs enzymatic hydrolysis to improve conversion efficiency and lower the thresholds for voltage and exogenous DNA purity. However, this process is also complex. While enzymatic hydrolysis theoretically may dissolve the cell wall and increase the permeability of exogenous DNA, it can also damage the cell, even directly damaging the cell membrane, leading to leakage of cell contents and loss of cell viability. In such cases, even if the exogenous DNA successfully enters the cell, the cell cannot survive and replicate it. Furthermore, cells treated with enzymatic hydrolysis become fragile and may exhibit stress responses, producing stress proteins. These processes can affect cellular metabolic activities and may interfere with the stability and expression of exogenous DNA, ultimately leading to a decrease in conversion efficiency.

[0007] Through extensive research, this invention has developed a suitable enzymatic hydrolysis method that can significantly improve the electroconversion 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: enzymatic treatment of Schizochytrium before electroconversion; said enzyme treatment comprising:

[0009] Before electroconversion, 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.

[0010] Furthermore, the enzyme activity of the protease is ≥900 U / g, and the enzyme activity of the snail enzyme is ≥20 U / g.

[0011] Furthermore, the enzyme activity of the protease is ≥1800 U / g, and the enzyme activity of the snail enzyme is ≥40 U / g.

[0012] Furthermore, the enzyme activity of the protease is ≥2000 U / g; the enzyme activity of the snail enzyme is ≥50 U / g.

[0013] Further, the enzyme activity of the protease is ≥9000 U / g; the enzyme activity of the snail enzyme is ≥40 U / g; even further, the enzyme activity of the protease is ≤22000 U / g, and the enzyme activity of the snail enzyme is ≤2200 U / g.

[0014] Further, the enzyme activity of the protease is ≥9000 U / g; the enzyme activity of the snail enzyme is ≥400 U / g; even further, the enzyme activity of the protease is ≤11000 U / g; and the enzyme activity of the snail enzyme is ≤1200 U / g.

[0015] Furthermore, the protease is an alkaline protease.

[0016] Furthermore, the snail enzyme has the product number S8280 (purchased from Solarbio).

[0017] Furthermore, the treatment of the protease includes: treatment at 28~30℃ and pH=9~10 for a treatment time ≤75 minutes; the treatment of the snail enzyme includes: treatment at 28~30℃ and pH=6~7 for a treatment time ≤75 minutes.

[0018] Furthermore, the electroconversion includes: mixing exogenous DNA and Schizochytrium competent cells, allowing them to stand, and then transferring them to an electroconversion device for electroconversion; after electroconversion, transferring them to a culture medium for recovery.

[0019] Furthermore, the exogenous DNA is a linearized DNA fragment or a plasmid.

[0020] Furthermore, the concentration of the linearized DNA fragment is above 2 ng / mL, and the concentration of the plasmid is above 15 ng / mL.

[0021] Furthermore, the electroconversion device is an electroconvulsive cup, and the parameters of the electroconversion include:

[0022] 0.4~0.6KV, 20~30μF and 150~250Ω.

[0023] Furthermore, prior to the electroconversion, the method further includes preparing the Schizochytrium fungi treated with the enzyme into competent cells.

[0024] Secondly, the present invention provides a gene editing method for Schizochytrium fungi, comprising: introducing exogenous DNA using the method described above.

[0025] The present invention has the following beneficial effects:

[0026] This invention provides an electroconversion method for Schizochytrium. By treating Schizochytrium with alkaline protease or snail enzyme with a certain enzyme activity before electroconversion, the conversion efficiency (positive rate) can be improved while also achieving a better Schizochytrium cell survival rate.

[0027] The method provided by this invention can significantly reduce the required concentration of exogenous DNA during electroporation of Schizochytrium, and can directly use nonlinear plasmids during transformation (existing electroporation methods for Schizochytrium generally use linear plasmids).

[0028] The method provided by this invention can significantly reduce the voltage intensity during the electroporation of Schizochytrium, which helps to improve the cell survival rate of Schizochytrium during electroporation. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is the plasmid map of the plasmid pblue-Bleo provided in Example 1 of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art and can be implemented by conventional methods in the art, such as referring to reference books or kit instructions in the art.

[0033] Unless otherwise specified, all experimental materials used in the following examples are commercially available.

[0034] Example 1

[0035] This embodiment provides a method for transforming Schizochytrium fungi, including the following steps:

[0036] 1. When Schizochytrium is cultured to the logarithmic growth phase, OD 600nm =1.

[0037] Dispense 5 mL of bacterial culture into multiple tubes, centrifuge at 4000 rpm for 10 min, wash twice with buffer, and collect the bacterial cells.

[0038] 2. Resuspend the bacterial cells in multi-well plates and set up a control group and multiple experimental groups. The control group received no treatment. The experimental groups were treated with alkaline protease, cellulase, lysozyme, and snailase, respectively, with each enzyme used in a gradient concentration, as shown in the table below:

[0039] Table 1. Concentration gradient of different enzymes (unit: mass-volume percentage)

[0040] alkaline protease gradient 1% 5% 10% 20% Cellulase gradient 0.1% 2% 3% 4% Snail enzyme gradient 0.1% 2% 3% 4% Lysozyme gradient 0.1% 2% 3% 4%

[0041] The alkaline protease, cellulase, lysozyme, and snail enzyme mentioned above were all purchased from SolarBio. The alkaline protease activity was 200,000 U / g, the cellulase activity was 3 U / mg, the lysozyme activity was 2,000 U / mg, and the snail enzyme activity was ≥50,000 U / g (product number S8280). The alkaline protease activity was B8360, the cellulase activity was C8270, and the lysozyme activity was L8141.

[0042] The specific enzyme treatment methods are as follows: alkaline protease is treated at 28℃ and pH=9 for 1 hour; cellulase is treated at 28℃ and pH=7 for 1 hour; lysozyme is treated at 28℃ and pH=7 for 1 hour; and snail enzyme is treated at 28℃ and pH=7 for 1 hour.

[0043] 3. After treatment of the control group and each experimental group, centrifuge at 4000 r / min for 10 min and collect the bacterial cells.

[0044] 4. Cells from the control group and each experimental group were plate-coated for counting. The remaining cells were prepared as competent cells, as follows:

[0045] (1) Wash the bacterial cells twice with 5 mL of sterile water pre-cooled at 0℃, and centrifuge at 4000 r / min for 10 min.

[0046] (2) Resuspend in 5 mL of 0.5 M sorbitol solution pre-cooled at 0 °C, and centrifuge at 4000 r / min for 10 min.

[0047] (3) Resuspend the bacterial precipitate in 5 mL of 0.5 M sorbitol solution pre-cooled at 0℃, dispense 100 μL, and freeze at -80℃.

[0048] 5. Constructing exogenous DNA for electroporation

[0049] The experimental subject was the bacterial strain *Schizochytrium* sp. ATCC 20888 (disclosed in multiple patent documents). The resistance fragment was derived from plasmid pGAPZαA (commercially available), containing the promoter TEF1, the bleomycin resistance sequence bleoR, and the terminator CYC1 (all publicly available fragments). The electroporation plasmid pblue-Bleo was constructed by cloning; the plasmid map is shown below. Figure 1 As shown. Based on the sequencing results, neutral sites were selected as homologous recombination sites for the transformed fragments, and 1000bp homologous arm sequences were constructed upstream and downstream (corresponding to the upstream and downstream in the plasmid).

[0050] Linearized fragments of plasmid pblue-Bleo can be obtained by double digestion with PstI and XbaI (the fragment size obtained by double digestion is about 3100bp).

[0051] 6. Electroconversion

[0052] (1) The linearized fragment of 200 ng / mL from the previous step was used for electroporation. After diluting it 10 times, the final concentration of exogenous DNA in the electroporation system was 20 ng / mL.

[0053] (2) Add 10 μL of the linearized fragment to be transformed to 100 μL of Schizochytrium competent cells, mix gently, incubate on ice for 5 min, and then transfer to an ice-cold electroporation cuvette and incubate for 10 min. The electroporation parameters are 0.5 kV, 25 μF, and 200 Ω.

[0054] (3) After electric shock, add 1 mL of culture medium and revive at 28℃ and 200 r / min for 1 h.

[0055] (4) Spread the transformed bacterial culture on a resistant plate and incubate at 28°C.

[0056] 7. Survival rate and positive rate statistics

[0057] The survival rate and positive rate of Schizochytrium were statistically analyzed using the following formula:

[0058] The survival rate of Schizochytrium (%) = (number of cells after enzyme treatment / number of cells before enzyme treatment) × 100.

[0059] Schizochytrium positive rate (%) = (number of positive clones after transformation / number of cells before enzyme treatment) × 100.

[0060] The results are shown in the table below. A positive rate greater than 1% indicates good results, a positive rate between 0.5% and 1% indicates moderate results, a positive rate between 0.1% and 0.5% indicates poor results, and a positive rate below 0.1% indicates very poor results.

[0061] Table 2. Survival rate and positive rate of Schizochytrium in the control group and different experimental groups.

[0062]

[0063] The results above show that cellulase was the least effective treatment for Schizochytrium, failing to improve the positive rate at various concentrations and even decreasing it at concentrations of 0.1%, 2%, and 4%. Lysozyme had little effect on improving the positive rate and, due to its excessive enzymatic effect on cell wall lysis, significantly reduced the survival rate of Schizochytrium. In contrast, alkaline protease and snail enzyme treatments were more effective. The positive rate reached 1.08% at 5% alkaline protease, but higher concentrations led to complete enzymatic lysis of the cell wall, resulting in cell death. Treatment with 1% and 2% snail enzyme maintained high transformation survival and transformation rates, indicating that they most significantly improved cell wall permeability, with the positive rate reaching 2.23% at 2% snail enzyme treatment.

[0064] Example 2

[0065] Based on the results of Example 1, in order to further explore the minimum treatment concentration and maximum treatment time of alkaline protease and snail enzyme, the following concentration gradient was used:

[0066] Table 3 Concentration gradients of different enzymes (unit: mass-volume percentage)

[0067] alkaline protease gradient 0.05% 0.1% 0.5% Snail enzyme gradient 0.01% 0.025% 0.05%

[0068] The specific enzyme treatment methods are as follows: alkaline protease was treated at 28°C and pH=9 for 1 hour, 3 hours, and 5 hours. Snail enzyme was treated at 28°C and pH=7 for 1 hour, 3 hours, and 5 hours. All other conditions remained unchanged compared to Example 1. Survival rate and positive rate are shown in the table below.

[0069] Table 4 Survival rates at different enzyme concentrations

[0070]

[0071] Table 5 Positive rates at different enzyme concentrations

[0072]

[0073] The results above show that reducing enzyme concentration and extending treatment time leads to decreased transformation efficiency. After 3 hours of treatment, cells may be damaged, resulting in a significant decrease in survival rate. After 5 hours, the survival rate will be 0. Therefore, 5 hours is the maximum enzyme treatment time. When the alkaline protease concentration is 0.05%-0.1%, the positive rate after 3 hours of treatment is similar to that without enzyme, indicating that 0.05%-0.1% is the minimum effective alkaline protease concentration. Similarly, when the snail enzyme concentration is 0.01%-0.025%, the transformation rate after 3 hours of treatment is similar to that without enzyme, indicating that 0.01%-0.025% is the minimum effective snail enzyme concentration.

[0074] Example 3

[0075] This embodiment further verifies the transformation efficiency of low-concentration exogenous DNA. The steps are the same as in Example 1, except that:

[0076] 1. The experimental group used only 5% alkaline protease, 1% snail enzyme and 2% snail enzyme.

[0077] 2. The exogenous DNA used for electroporation was adjusted from a 200 ng / mL linearized fragment to a 50 ng / mL linearized fragment, or the 200 ng / mL plasmid pblue-Bleo that had not undergone linearization enzyme digestion was used. Both were diluted tenfold before electroporation.

[0078] The results are shown in the table below:

[0079] Table 6. Conversion rates (low concentration of exogenous DNA) of the control group and different experimental groups

[0080]

[0081] As can be seen from the results above, the transformation method provided by this invention (treatment with alkaline protease and snail enzyme) still maintains a high transformation rate even with low concentrations of linearized DNA fragments and circular DNA (plasmids), and can stably obtain positive transformants. This indicates that the method provided by this invention significantly improves the efficiency of genetic transformation of Schizochytrium.

[0082] Example 4

[0083] The present invention further applies the method shown in Example 1 to Schizochytrium sp. HX-308, and obtains the following results:

[0084] Table 7. Survival rate and positive rate of Schizochytrium in the control group and different experimental groups.

[0085]

[0086] The present invention further applies the method shown in Example 1 to the fungus Aurantiochytrium limacinum, and obtains the following experimental results:

[0087] Table 8. Survival rate and positive rate of Schizochytrium in the control group and different experimental groups.

[0088]

[0089] The results show that the method provided in this embodiment exhibits a similar improvement effect and trend as Schizochytrium sp. ATCC 20888 when applied to other Schizochytrium species. This indicates that the method provided by the present invention can be applied to Schizochytrium species, and is not limited to Schizochytrium sp. ATCC 20888.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 fungi, characterized in that, include: Prior to electroconversion, the Schizochytrium fungi were subjected to enzyme treatment; the enzyme treatment included: Before electroconversion, Schizochytrium fungi is treated with enzymes; the enzymes are proteases or snail enzymes; the enzyme activity of the proteases is ≥200 U / g and ≤20000 U / g; the enzyme activity of the snail enzymes is ≥50 U / g and ≤2200 U / g. The protease is an alkaline protease, and the treatment includes: treatment at 28~30℃ and pH=9~10 for 60 minutes ≤ treatment time ≤ 75 minutes; The snail enzyme treatment includes: treatment at 28~30℃ and pH=6~7 for 60 minutes ≤ treatment time ≤ 75 minutes.

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

3. The method according to any one of claims 1-2, characterized in that, The electroconversion process includes: mixing exogenous DNA and Schizochytrium competent cells, allowing them to stand, and then transferring them to an electroconversion device for electroconversion; after electroconversion, transferring them to a culture medium for recovery.

4. The method according to claim 3, characterized in that, The exogenous DNA is a linearized DNA fragment or plasmid.

5. The method according to claim 4, 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.

6. The method according to any one of claims 1, 2, 4 or 5, characterized in that, Prior to the electroconversion, the method further includes preparing competent cells of the Schizochytrium fungi treated with the enzyme.

7. The method according to claim 3, characterized in that, Prior to the electroconversion, the method further includes preparing competent cells of the Schizochytrium fungi treated with the enzyme.

8. A gene editing method for Schizochytrium fungi, characterized in that, include: The method described in any one of claims 1-7 is used to introduce exogenous DNA.

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