CN116376726A - Identification method of target spot for improving gibberellin yield and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, and discloses an identification method of a target spot for improving gibberellin yield and application thereof. The identification method comprises the following steps: knocking out the modified genes in the original strain to obtain modified gene defective bacteria; preparing protoplast of modified gene defective bacterium, carrying out NHEJ random integration on the protoplast and the modified gene to obtain N transformation strains, and screening the N transformation strains to obtain a transformation strain with highest gibberellin yield as a target strain; and identifying an integration target point of the modified gene on the original strain by inverse PCR (polymerase chain reaction) of the target strain so as to obtain a target point for improving the gibberellin yield. The invention also uses the target strain obtained by the original strain treated by the identification method as a recombinant strain for producing gibberellin by fermentation. The identification method can identify a new target point with improved gibberellin yield, obtain a recombinant strain with high gibberellin yield, and provide a new platform for metabolic engineering transformation of gibberellin production bacteria.

Description

Identification method and application of targets for increasing gibberellin production

technical field

The invention relates to the field of genetic engineering, in particular to a method for identifying a target point for increasing gibberellin yield and its application.

Background technique

DNA double-strand break (DSB) poses a serious threat to cells, and its repair pathways mainly include non-homologous end joining (NHEJ) and homologous recombination (HR), among which HR is a delicate repair method that can convert donor DNA The fragment integrates into the target site on the genome to repair the DSB. Relatively speaking, NHEJ repair does not require the recognition of large fragments of homologous sequences, and is performed directly by connecting a few bases at the ends, which is an error-prone repair. For eukaryotes, NHEJ repair is dominant, and it can effectively repair DSBs without a template. Unlike HR, which is limited to homologous templates, it can randomly insert the target gene into different genomes. position, resulting in recombinant strains with different gene expression levels.

As the dominant host of the plant growth hormone gibberellin, Gibberella fujikura is considered to be a good cell factory for industrial fermentation production. However, it is a non-model bacterium with a complex metabolic mechanism. Relying on fermentation engineering and mutation breeding has been unable to further solve the production bottleneck. In order to obtain recombinant strains with excellent performance and stable inheritance, synthetic biology transformation technology is increasingly Much attention. Following the establishment of homologous recombination technology in Gibberella, key genes in the biological pathway of gibberellin synthesis have been identified and overexpressed. However, there is no research or report on improving the gibberellin production ability of strains by integrating genes other than the key genes of the gibberellin synthesis pathway.

Contents of the invention

The purpose of the present invention is to overcome the problems existing in the prior art, provide a kind of identification method and its application of the target point that improves gibberellin yield, this identification method can identify the new target point that gibberellin yield improves, obtain high The recombinant strain of gibberellin provides a new platform for the metabolic engineering of gibberellin-producing bacteria.

In order to achieve the above object, the first aspect of the present invention provides a method for identifying a target for increasing gibberellin production, the method for identifying comprises the following steps:

(1) Knocking out the modified gene in the starting strain to obtain the modified gene-deficient bacteria;

(2) Prepare the protoplasts of the modified gene-deficient bacteria, carry out NHEJ random integration of the protoplasts and the modified genes to obtain N transformed strains, and screen the N transformed strains to obtain the one with the highest yield of gibberellin The transformed bacterial strain is used as the target bacterial strain;

(3) identifying the integration target of the modified gene on the starting strain by inverse PCR on the target strain, so as to obtain the target for increasing the yield of gibberellin.

Preferably, the modified gene is a nitrate reductase gene.

Preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1.

Preferably, the starting strain is Gibberella Fujikura, more preferably Gibberella Fujikura with a preservation number of CCTCC NO: M2015614.

Preferably, the process of knocking out in step (1) includes: using CRISPR/Cas9 system to knock out the modified gene.

Preferably, the knockout backbone of the CRISPR/Cas9 system is pFfCas9-sgRNA plasmid, and the components of the pFfCas9-sgRNA plasmid include Cas9 gene, promoter pTRPC and strong RNA polymerase type III promoter 5s rRNA; the pFfCas9- The internally designed stuI site of the sgRNA plasmid was used to insert N20 in the nutritional selection marker niaD.

Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 9, the nucleotide sequence of the 5s rRNA is shown in SEQ ID NO: 10, and the nucleotide sequence of the N20 is shown in SEQ ID NO : 11.

Preferably, the process of random integration of NHEJ in step (2) includes: mixing the protoplasts, the modified gene with PEG6000, and STC buffer to obtain a suspension, and placing the suspension on a solid regeneration plate transformation culture.

Preferably, the culture medium of the solid regeneration plate contains: glucose 15-25g/L, yeast basal medium 5-8g/L, sodium nitrate 0.5-1g/L, agar powder 15-25g/L, sucrose 0.3-0.8 mol/L.

Preferably, the transformation culture conditions include: the temperature is 25-30° C., and the time is 4-6 days.

Preferably, the screening process in step (2) includes: subculture and fermentation culture I of the transformed strain in sequence.

Preferably, the subculture medium contains: glucose 15-25g/L, yeast basal medium 5-8g/L, sodium nitrate 0.5-1g/L, agar powder 15-25g/L, sucrose 0.3-0.8mol /L.

Preferably, the subculture conditions include: the temperature is 25-30° C., and the time is 4-6 days.

Preferably, the culture medium of the fermentation culture I contains: carbon source 40-120g/L, nitrogen source 25-40g/L, soybean oil 0.8-1.6g/L, MgSO ₄ ·7H ₂ O 0.8-1.6g/L, (NH ₄ ) ₂ SO ₄ 0.2-0.5g/L, KH ₂ PO ₄ 1.5-4g/L, ZnSO ₄ 0.05-0.1g/L, MnSO ₄ 0.05-0.1g/L.

Preferably, the conditions of the fermentation culture I include: the inoculum size is 8-12% by volume, the temperature is 25-30° C., the rotation speed is 200-250 rpm, and the time is 8-12 days.

The second aspect of the present invention provides the application of the above identification method in improving the yield of gibberellin.

The third aspect of the present invention provides a recombinant strain, which is the target strain obtained by treating the starting strain with the above identification method.

Preferably, the modified gene is a nitrate reductase gene, and the integration target is a dicarboxylate transporter site.

Preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1.

Preferably, the starting strain is Gibberella Fujikura, more preferably Gibberella Fujikura with a preservation number of CCTCC NO: M2015614.

The fourth aspect of the present invention provides a preparation method of gibberellin, the preparation method comprising: inoculating the above-mentioned recombinant strain into a fermentation medium to carry out fermentation culture II.

Preferably, the conditions of the fermentation culture II include: the inoculum size is 8-12% by volume, the temperature is 25-30° C., the rotation speed is 200-250 rpm, and the time is 8-12 days.

Preferably, the fermentation medium contains: carbon source 40-120g/L, nitrogen source 25-40g/L, soybean oil 0.8-1.6g/L, MgSO ₄ ·7H ₂ O 0.8-1.6g/L, (NH ₄ ) ₂ SO ₄ 0.2-0.5g/L, KH ₂ PO ₄ 1.5-4g/L, ZnSO ₄ 0.05-0.1g/L, MnSO ₄ 0.05-0.1g/L.

Through the above technical scheme, the beneficial effects of the present invention are:

The method for identifying targets for improving gibberellin production provided by the present invention is based on the random integration method of non-terminal homologous recombination to obtain molecular transformation targets of gibberellin-producing bacteria, and introduces them into genetically deficient bacteria by using DNA random integration technology. The modified gene expression frame allows the modified gene to be randomly integrated into the chromosome of the modified gene-defective bacteria, thereby forming multiple transformants of the modified gene-deficient bacteria, which are used as a mutation library of gibberellin-producing bacteria, and the transformants are screened for gibberellin by fermentation experiments. High-yield strains with increased acid production, and the use of reverse PCR technology to identify the position of the modified gene integration in the chromosome, which is conducive to the identification of new targets for increased gibberellin production; at the same time, the target strain obtained in this identification method can be used as a high-yield gibberellin strain. Recombinant strains of mycin.

Further preferably, the present invention uses Gibberella Fujikura as the starting strain, and the nitrate reductase gene as the modified gene, which has high transformation efficiency and is more conducive to the screening of transformants. It also shows that DNA random integration technology has a good effect in Gibberella Fujikura. conversion efficiency.

Description of drawings

Fig. 1 is the map of plasmid pUC57-T7-fcc1-FfniaD in embodiment 1;

Fig. 2 is the content figure of gibberellin GA3 in the fermented liquid of 30 strains transformed bacterial strains in embodiment 2;

Fig. 3 is a process flow diagram of the method for identifying targets for increasing gibberellin production in the present invention.

Detailed ways

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

The first aspect of the present invention provides a method for identifying a target for increasing gibberellin production, referring to Figure 3, the method for identifying comprises the following steps:

(1) Knocking out the modified gene in the starting strain to obtain the modified gene-deficient bacteria;

(2) Prepare the protoplasts of the modified gene-deficient bacteria, perform nonhomologous end joining (NHEJ) random integration of the protoplasts and the modified gene to obtain N transformed strains, and transform N transformed Strains were screened to obtain the transformed strain with the highest yield of gibberellin as the target strain;

(3) identifying the integration target of the modified gene on the starting strain by inverse PCR on the target strain, so as to obtain the target for increasing the yield of gibberellin.

The method for identifying targets for improving gibberellin production provided by the present invention is based on the random integration method of non-terminal homologous recombination to obtain molecular transformation targets of gibberellin-producing bacteria, and introduces them into genetically deficient bacteria by using DNA random integration technology. The modified gene expression frame allows the modified gene to be randomly integrated into the chromosome of the modified gene-defective bacteria, thereby forming multiple transformants of the modified gene-deficient bacteria, which are used as a mutation library of gibberellin-producing bacteria, and the transformants are screened for gibberellin by fermentation experiments. High-yield strains with increased acid production, and the use of reverse PCR technology to identify the position of the modified gene integration in the chromosome, which is conducive to the identification of new targets for increased gibberellin production; at the same time, the target strain obtained in this identification method can be used as a high-yield gibberellin strain. Recombinant strains of mycin.

According to the present invention, preferably, the modified gene is a nitrate reductase gene (niaD gene). Further preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1.

During the research process, the inventors of the present invention unexpectedly found that using the niaD gene as the modified gene, the obtained mutant strains have high transformation efficiency, which is conducive to the screening of transformants. Based on the final gibberellin yield, efficient and rapid High-yielding gibberellin strains were screened out; at the same time, specific integration targets were identified by inverse PCR, and the internal mechanism affecting gibberellin production was revealed.

According to the present invention, the starting strain may be any gibberellin-producing strain, preferably, the starting strain is Gibberella Fujikura, more preferably Gibberella Fujikura with a preservation number of CCTCC NO: M2015614. The inventors found that in this preferred embodiment, it is beneficial to further increase the yield of gibberellin synthesized by fermentation of the obtained target strain.

According to the present invention, preferably, the process of knocking out in step (1) includes: knocking out the modified gene by using CRISPR/Cas9 system. The inventors found that in this preferred embodiment, it is beneficial to improve the transformation efficiency of DNA random integration.

In the present invention, the CRISPR/Cas9 system can adopt existing compositions and methods in the prior art. Specifically, the knockout backbone of the modified gene knockout using the CRISPR/Cas9 system is the pFfCas9-sgRNA plasmid, and the pFfCas9-sgRNA The components of the plasmid include the Cas9 gene, the promoter pTRPC and the strong RNA polymerase type III promoter 5srRNA, and the internal design stuI site of the pFfCas9-sgRNA plasmid is used to insert the N20 in the nutritional screening marker niaD. Further preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 9, the nucleotide sequence of the 5s rRNA is shown in SEQ ID NO: 10, and the nucleotide sequence of the N20 is shown in SEQ ID NO: 11 shown.

In the present invention, the protoplasts of the modified gene-deficient bacteria can be prepared using existing methods. Exemplarily, the process of preparing the protoplasts of the modified genetically deficient bacteria includes: first configuring a collapse enzyme (Dryslase) and The weight ratio of the enzyme (Lysing) is 3:7 enzyme solution, the enzyme solution is filtered and sterilized, then added to the wet mycelia, placed in a shaker at 25-35°C with a rotation speed of 50-70rpm, sampling every 0.5h for microscopy Observe the lysis of mycelium and the formation of protoplasts. After enzymatic hydrolysis, filter the mycelia through lens cleaning paper, take the filtrate and centrifuge at 2500-3500rpm for 8-12min, discard the supernatant, wash the protoplast precipitate with STC buffer, and finally save it in STC in the buffer.

In the present invention, the components of the STC buffer are: sorbitol 180-185g/L, anhydrous calcium chloride 0.4-0.7g/L, Tris-base 0.1-0.15g/L, and hydrochloric acid to adjust the pH to 7-8.

According to the present invention, preferably, the process of random integration of NHEJ in step (2) includes: mixing the protoplasts, the modified gene with polyethylene glycol 6000 (PEG6000) and STC buffer and then suspending to obtain a suspension , placing the suspension on a solid regeneration plate for transformation culture.

In the present invention, the protoplasts can be mixed and suspended with the modified gene, PEG6000 and STC buffer after forming a solution with STC buffer.

According to the present invention, preferably, the medium of the solid regeneration plate contains: glucose 15-25g/L, yeast basal medium 5-8g/L, sodium nitrate 0.5-1g/L, agar powder 15-25g/L, Sucrose 0.3-0.8mol/L.

According to the present invention, preferably, the transformation culture conditions include: the temperature is 25-30° C., and the time is 4-6 days.

According to the present invention, preferably, the screening process in step (2) includes: subculture and fermentation culture I of the transformed strain in sequence. Wherein, the number of times of subculture can be one or more times, so as to obtain a transformed strain that is stably passed down.

According to the present invention, the subculture medium adopts solid regeneration medium, preferably, the subculture medium contains: glucose 15-25g/L, yeast basal medium 5-8g/L, sodium nitrate 0.5- 1g/L, agar powder 15-25g/L, sucrose 0.3-0.8mol/L.

According to the present invention, preferably, the subculture conditions include: the temperature is 25-30° C., and the time is 4-6 days.

According to the present invention, before the fermentation culture I is carried out, the transformed bacterial strain obtained by subculture can be inoculated into the seed medium I for activation culture I, so as to effectively activate and promote the growth of the transformed bacterial strain, and improve the production of gibberella by the transformed bacterial strain. Accurate characterization of prime capabilities. Condition parameters such as temperature and time of the activation culture I can be selected according to the type of the starting strain so as to be suitable for the growth of the transformed strain. Preferably, the conditions of the activation culture I include: a temperature of 25-30° C. and a time of 40-60 h.

According to the present invention, the components of the medium of the seed medium I and the fermentation culture I can be selected according to the type of starting strain, and generally contain nutrients such as carbon source, nitrogen source, and metal salt. Preferably, the culture medium of fermentation culture I described in step (2) contains: carbon source 40-120g/L, nitrogen source 25-40g/L, soybean oil 0.8-1.6g/L, MgSO ₄ ·7H ₂ O 0.8- 1.6g/L, (NH ₄ ) ₂ SO ₄ 0.2-0.5g/L, KH ₂ PO ₄ 1.5-4g/L, ZnSO ₄ 0.05-0.1g/L, MnSO ₄ 0.05-0.1g/L.

In the present invention, the carbon source may be one or more of glucose, fructose, sucrose and the like, and the nitrogen source may be one or more of peptone, beef extract, yeast powder, and yeast extract.

According to the present invention, the condition parameters such as temperature and time of the fermentation culture I can be selected according to the type of the starting strain, so as to be suitable for the large-scale proliferation of the transformed strain. Preferably, the conditions of the fermentation culture I include: the inoculum size is 8-12% by volume, the temperature is 25-30° C., the rotation speed is 200-250 rpm, and the time is 8-12 days.

The second aspect of the present invention provides the application of the above identification method in improving the yield of gibberellin. Based on the identification method provided by the invention, the gene action target of gibberellin-producing strains to increase the yield of gibberellin can be obtained, providing a research basis for the transformation of engineering bacteria that efficiently produce gibberellin.

Through the identification method provided by the present invention, the target strain that expresses the modified gene on the integration target can be obtained. Based on this, the third aspect of the present invention provides a recombinant strain. The recombinant strain is the starting strain treated by the above-mentioned identification method obtained target strains.

According to the present invention, preferably, the modified gene is a nitrate reductase gene, and the integration target is a dicarboxylate transporter site. The inventors found that in this preferred embodiment, it is beneficial to further increase the yield of gibberellin synthesized by fermentation of the obtained target strain.

According to the present invention, preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1.

According to the present invention, preferably, the starting strain is Gibberella Fujikura, more preferably Gibberella Fujikura with a preservation number of CCTCC NO: M2015614. The inventors found that in this preferred embodiment, it is beneficial to better increase the yield of gibberellin, which provides a new platform for the metabolic engineering of Gibberella fujikura.

The fourth aspect of the present invention provides a preparation method of gibberellin, the preparation method comprising: inoculating the above-mentioned recombinant strain into a fermentation medium to carry out fermentation culture II.

According to the present invention, prior to the fermentation culture II, the recombinant strain can be inoculated into the seed medium II for activation culture II, so as to effectively activate and promote the growth of the recombinant strain. Condition parameters such as temperature and time of the activation culture II can be selected according to the type of the recombinant strain, so as to be suitable for the growth of the recombinant strain. Preferably, the conditions for the activation culture II include: a temperature of 25-30° C. and a time of 40-60 h.

According to the present invention, the components of the seed medium II and the fermentation medium can be selected according to the type of the recombinant strain, and generally contain nutrients such as carbon source, nitrogen source, and metal salt. Preferably, the fermentation medium contains: carbon source 40-120g/L, nitrogen source 25-40g/L, soybean oil 0.8-1.6g/L, MgSO ₄ ·7H ₂ O 0.8-1.6g/L, (NH ₄ ) ₂ SO ₄ 0.2-0.5g/L, KH ₂ PO ₄ 1.5-4g/L, ZnSO ₄ 0.05-0.1g/L, MnSO ₄ 0.05-0.1g/L.

According to the present invention, the condition parameters such as temperature and time of the fermentation culture II can be selected according to the type of the recombinant strain, so as to be suitable for the large-scale proliferation of the recombinant strain. Preferably, the conditions of the fermentation culture II include: the inoculum size is 8-12% by volume, the temperature is 25-30° C., the rotation speed is 200-250 rpm, and the time is 8-12 days.

According to the present invention, preferably, the fermentation medium contains: carbon source 40-120g/L, nitrogen source 25-40g/L, soybean oil 0.8-1.6g/L, MgSO ₄ ·7H ₂ O 0.8-1.6g/L , (NH ₄ ) ₂ SO ₄ 0.2-0.5g/L, KH ₂ PO ₄ 1.5-4g/L, ZnSO ₄ 0.05-0.1g/L, MnSO ₄ 0.05-0.1g/L. The inventors found that in this preferred embodiment, it is beneficial to better increase the yield of gibberellin.

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

The experimental methods used in the following examples, unless otherwise specified, are conventional methods, and the reagents, methods and equipment used, unless otherwise specified, are conventional reagents, methods and equipment in the art.

In the following examples, the components of the STC buffer are: 182g/L sorbitol, 0.555g/L anhydrous calcium chloride, 0.121g/L Tris-base, hydrochloric acid to adjust the pH to 7.5;

The components of the solid regeneration medium are: glucose 20g/L, yeast basal medium 6g/L, sodium nitrate 0.8499g/L, agar powder 20g/L, sucrose 0.5mol/L, pH adjusted to 6.5;

The components of the seed medium are: glucose 80g/L, yeast extract 30g/L, MgSO ₄ ·7H ₂ O 1.2g/L, KH ₂ PO ₄ 3g/L, ZnSO ₄ 0.08g/L, MnSO ₄ 0.08g/L L;

The components of the fermentation medium are: glucose 80g/L, yeast extract 30g/L, soybean oil 1.2g/L, MgSO ₄ 7H ₂ O 1.2g/L, (NH ₄ ) ₂ SO ₄ 0.3g/L, KH ₂ PO ₄ 3g/L, ZnSO ₄ 0.08g/L, MnSO ₄ 0.08g/L.

Example 1

(1) Using Fusarium fujikuroi NJtech02 (provided by the research group of Professor Ji Xiaojun of Nanjing University of Technology, the preservation number is CCTCC NO: M 2015614, which has been disclosed in the patent application CN105441340A) as the starting strain, using CRISPR/Cas9 technology The modified gene is knocked out to obtain a nitrate reductase gene knockout bacterium; wherein, the knockout backbone of the CRISPR/Cas9 system is a pFfCas9-sgRNA plasmid, and the components of the pFfCas9-sgRNA plasmid include a Cas9 gene, a promoter pTRPC and a strong RNA polymerization Enzyme type III promoter 5s rRNA, and the internal design stuI site of the plasmid is used to insert N20 in the nutritional screening marker niaD; the nucleotide sequence of sgRNA is shown in SEQ ID NO: 9, the nucleotide sequence of 5s rRNA As shown in SEQ ID NO: 10, the nucleotide sequence of N20 is shown in SEQ ID NO: 11;

(2) The pUC57-T7-fcc1 plasmid (nucleotide sequence shown in SEQ ID NO: 2) was single-digested with EcoRI to obtain a linearized plasmid backbone, and the primer pair FfniaD-PCR-F (nucleotide sequence shown in shown in SEQ ID NO: 3) and FfniaD-PCR-R (nucleotide sequence shown in SEQ ID NO: 4), a fragment of 4074bp was amplified from Gibberella fujikura genome (CCTCC NO: M 2015614), The two sides of the fragment have 22bp homologous regions with the linearized plasmid backbone. Under the action of Clone ExpressUltra OneStep Cloning Kit cloning enzyme (purchased from Nanjing Novizan Biotechnology Co., Ltd.), Gibson assembly was completed to obtain the connecting solution, and 20 μL of the ligated The solution was introduced into Escherichia coli DH5α competent, spread on LB plates (containing a final concentration of ampicillin resistance of 100 μg/mL) and cultivated overnight, and the positive clones on the plates were selected using the primer pair pUC-tongyong-F (nucleotide sequence such as SEQ ID NO: 5) and pUC-tongyong-R (nucleotide sequence shown in SEQ ID NO: 6) for PCR verification, select the plasmid corresponding to the correct nucleotide band and send it to Nanjing Qingke Company for SangerDNA Sequencing finally obtained the plasmid pUC57-T7-fcc1-FfniaD (the nucleotide sequence of niaD is shown in SEQ ID NO: 1, and the Ff prefix indicates that the gene is the endogenous gene of Gibberella fujikura), and its plasmid map is shown in Figure 1 shown;

FfniaD-PCR-F: ttgtaaaacgacggccagtgaaGAGAGTTGCAGACTATAAGACAT ATG (SEQ ID NO: 3);

FfniaD-PCR-R: ttcgcgaggtaccgagctcgaaATATTGTTTGTTCATATGTACGTT CC (SEQ ID NO: 4);

pUC-tongyong-F:tcttcgctattacgccagct (SEQ ID NO: 5);

pUC-tongyong-R: caggaaacagctatgaccat (SEQ ID NO: 6);

(3) Use primers FfniaD-PCR-F and FfniaD-PCR-R to amplify the FfniaD fragment, recover DNA from the PCR reaction solution, and concentrate to obtain a high-concentration linear FfniaD nucleotide fragment;

(4) Preparation of Gibberella fujikura protoplasts: first prepare an enzyme solution with a weight ratio of Dryslase and Lysing of 3:7, add 1 g of wet mycelia to 10 mL of the enzyme solution after filtering and sterilizing placed in a shaker at 30°C with a rotation speed of 60rpm, and samples were taken every 0.5h to observe the lysis of mycelium and the formation of protoplasts under a microscope. After enzymolysis, the mycelium was filtered through lens-cleaning paper, and the filtrate was centrifuged at 3000rpm for 10min and discarded. Clear, wash the protoplast precipitate with STC buffer, and finally save it in STC buffer as the protoplast solution;

Take 10 μL of the high-concentration linear FfniaD nucleotide fragment obtained in step (3) and add it to the protoplast solution, and add 1 mL of PEG6000 to stand on ice for 20 minutes, then add 2 mL of STC buffer, suspend and spread on solid regeneration medium On the plate, culture at a temperature of 28° C. for 5 days and wait for the generation of transformants.

Example 2

The transformant obtained in Example 1 was subcultured for 15 times on a solid regeneration medium (each time at a temperature of 28° C. for 48 hours), and finally a transformed bacterial strain that was stably passed down was obtained, and 30 transformed bacterial strains were selected for inoculation. Into 30mL seed culture medium, cultured at a temperature of 28°C for 48h to obtain a seed liquid; inoculate each seed liquid into a 40mL fermentation medium with an inoculum size of 10% by volume, and inoculate the fermentation medium at a temperature of 28°C and a rotation speed of 220rpm After continuous cultivation in a shaker for 9 days, the fermentation broth was obtained. The fermentation broth was centrifuged at 12,000rpm for 5 minutes to obtain the supernatant. After the supernatant was filtered through a 0.22μm water-based membrane, it was put into a liquid-phase sampling bottle for HPLC analysis to detect each transformation The yield of gibberellin GA3 in the fermentation broth corresponding to the strain.

The content of gibberellin GA3 in the fermented liquid of 30 strains of transformed strains is shown in Figure 2 and Table 1, with departure bacterial strain as contrast (Control), obtain altogether the transformation strain of 6 strains yields that do not improve (with the GA3 output of departure strains) The difference between them is less than 5mg/g DCW), 9 transformed strains with increased yield (compared to the starting strain, GA3 production increased by more than 5mg/g DCW) and 15 transformed strains with reduced yield (relative to the starting strain, GA3 yield decreased 5mg/g DCW or more), according to the results, it can be speculated that there are 6 new targets that do not affect the production performance of the bacteria and 9 targets that are beneficial to the synthesis of GA3. It can be seen from this that the DNA random integration platform constructed by the present invention can efficiently run in Gibberella Fujikura, which is convenient for subsequent target modification.

Table 1

Example 3

The No. 29 transformed strain (as the target strain) with the highest GA3 yield in Example 2 was streaked on a YPD plate, and the genome of Gibberella was extracted by a fungal genome DNA extraction kit (purchased from Beijing Suolaibao Technology Co., Ltd.); Carry out enzyme digestion reaction (3 μL genomic DNA) in a 10 μL system, and select the isotail enzyme combination NheI/XbaI (this enzyme includes a 6bp degenerate site, which can increase the probability of circularization);

Inactivate the enzyme cutting solution by heat shock at 80°C. At this time, the endonuclease no longer has the function of cutting nucleotide sequences. Add 10 μL of the enzyme cutting solution to 100 μL T4 ligase self-serialization system, and the linearized fragments complete self-circulation. The self-ligation product was obtained; the self-ligation product was directly used as a template, and the primer pair niaD-PCR-F1 (nucleotide sequence as shown in SEQ ID NO: 7) was designed in the niaD gene with the primer sequence in the 50 μL KOD amplification system. shown) and niaD-PCR-R2 (nucleotide sequence shown in SEQ ID NO: 8) for inverse PCR (KOD amplification system consists of: 1 μL KOD, 25 μL 2xBuffer, 10 μL 1M dNTP, 1.5 μL niaD-PCR-F1, 1.5 μL niaD-PCR-R1), KOD FX extension for 3min, 40 cycles, run the gel for gel electrophoresis analysis, and recover the DNA fragment from the single-segment gel tapping, and send it to Nanjing Qingke Company for Sanger DNA sequencing, and compare the sequence with Gibberella Comparing the genome sequences, it was found that the niaD target was integrated at the position of XP_023429025.1, and the XP_023429025.1 target encoded the dicarboxylic acid transporter carboxylic acid transport, which may increase the production of GA3 by affecting the efflux of precursor substances and other factors.

niaD-PCR-F1: CATCAACGCATACCAGCAATTG (SEQ ID NO: 7);

niaD-PCR-R2:GTCAATTGCTGGTATGCGTTGA (SEQ ID NO: 8).

Example 4

The target strain in Example 3 was used as a recombinant strain producing Gibberella, inoculated into 30 mL of seed culture medium, and cultivated at a temperature of 28° C. for 48 hours to obtain a seed liquid; the seed liquid was inoculated to 40 mL with an inoculation amount of 10% by volume In the fermentation medium, the fermentation broth was obtained after continuous cultivation in a shaker at a temperature of 28°C and a rotation speed of 220rpm for 9 days, and the fermentation broth was centrifuged at 12000rpm for 5min to obtain a supernatant, which was filtered through a 0.22μm aqueous membrane and packed Put it into the liquid phase sampling bottle, carry out HPLC analysis, detect the gibberellin GA3 output in the fermented liquid of recombinant bacterial strain, the result is shown in Table 2.

Example 5

The target strain in Example 3 was used as a recombinant strain producing Gibberella, inoculated into 30 mL of seed culture medium, and cultivated for 60 h at a temperature of 25° C. to obtain seed liquid; the seed liquid was inoculated to 40 mL with an inoculum size of 8% by volume Fermentation medium (the components of the fermentation medium are replaced by: glucose 40g/L, yeast extract 40g/L, soybean oil 1.6g/L, MgSO ₄ 7H ₂ O 1.6g/L, (NH ₄ ) ₂ SO ₄ 0.2g /L, KH ₂ PO ₄ 1.5g/L, ZnSO ₄ 0.1g/L, MnSO ₄ 0.1g/L), in a shaker with a temperature of 25°C and a rotation speed of 250rpm, the fermentation broth was obtained after continuous cultivation for 12 days. The fermentation broth was centrifuged at 12000rpm for 5min to obtain the supernatant. After the supernatant was filtered through a 0.22 μm aqueous membrane, it was put into a liquid phase sampling bottle, and HPLC analysis was performed to detect the gibberellin GA3 output in the fermentation broth of the recombinant strain. The result See Table 2.

Example 6

The target strain in Example 3 was used as a recombinant strain producing Gibberella, inoculated into 30 mL of seed culture medium, and cultivated for 40 h at a temperature of 30° C. to obtain seed liquid; the seed liquid was inoculated to 40 mL with an inoculum size of 12% by volume Fermentation medium (the components of the fermentation medium are replaced by: glucose 120g/L, yeast extract 25g/L, soybean oil 0.8g/L, MgSO ₄ 7H ₂ O 0.8g/L, (NH ₄ ) ₂ SO ₄ 0.5g /L, KH ₂ PO ₄ 4g/L, ZnSO ₄ 0.05g/L, MnSO ₄ 0.05g/L), in a shaker with a temperature of 30°C and a rotation speed of 200rpm, the fermentation broth was obtained after continuous cultivation for 8 days, and the fermentation The liquid was centrifuged at 12000rpm for 5min to obtain the supernatant, and the supernatant was filtered through a 0.22μm aqueous membrane, then put into a liquid-phase sampling bottle, and analyzed by HPLC to detect the gibberellin GA3 output in the fermentation broth of the recombinant strain, the results are shown in Table 2.

Example 7

According to the method of Example 4, gibberellin was fermented and prepared, the difference was that the components of the fermentation medium were replaced by: glucose 81.2g/L, yeast extract 30g/L, MgSO ₄ 7H ₂ O 1.2g/L, ( NH ₄ ) ₂ SO ₄ 0.3g/L, KH ₂ PO ₄ 3g/L, ZnSO ₄ 0.08g/L, MnSO ₄ 0.08g/L.

Example 8

According to the method of Example 4, gibberellin was fermented and prepared, the difference was that the components of the fermentation medium were replaced by: glucose 80g/L, yeast extract 30g/L, soybean oil 3g/L, MgSO ₄ ·7H ₂ O 1.2g /L, (NH ₄ ) ₂ SO ₄ 0.3g/L, KH ₂ PO ₄ 3g/L, ZnSO ₄ 0.08g/L, MnSO ₄ 0.08g/L.

Comparative example 1

The gibberellin was prepared by fermentation according to the method of Example 4, except that the recombinant strain was replaced with the starting strain (the preservation number is CCTCC NO: M2015614). The results are shown in Table 2.

Table 2

serial number Gibberellin GA3 yield in fermentation broth (mg/g DCW) Example 4 105.7 Example 5 98.5 Example 6 120.5 Example 7 92.3 Example 8 90.8 Comparative example 1 60.2

The preferred embodiments of the present invention have been described in detail above, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.

Claims (10)

1. A method for identifying a target for increasing gibberellin production, the method comprising the steps of:

(1) Knocking out the modified genes in the original strain to obtain modified gene defective bacteria;

(2) Preparing protoplast of the modified gene defective bacterium, carrying out NHEJ random integration on the protoplast and the modified gene to obtain N transformation strains, and screening the N transformation strains to obtain a transformation strain with highest gibberellin yield as a target strain;

(3) And identifying an integration target point of the modified gene on the original strain by inverse PCR (polymerase chain reaction) of the target strain so as to obtain a target point for improving gibberellin yield.

2. The method of claim 1, wherein the engineered gene is a nitrate reductase gene;

preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1 is shown in the specification;

preferably, the starting strain is gibberella cappa, more preferably gibberella cappa with a preservation number of CCTCC NO: M2015614.

3. The method of claim 1 or 2, wherein the knocking out in step (1) comprises: knocking out the modified gene by using a CRISPR/Cas9 system;

preferably, the knockout backbone of the CRISPR/Cas9 system is a pFfCas9-sgRNA plasmid, and the components of the pFfCas9-sgRNA plasmid include Cas9 gene, promoter pTRPC and strong RNA polymerase type III promoter 5s rRNA; the internal design stuI site of the pFCas 9-sgRNA plasmid is used for inserting N20 in a nutrition screening marker niaD;

preferably, the nucleotide sequence of the sgRNA is as set forth in SEQ ID NO:9, the nucleotide sequence of the 5s rRNA is shown as SEQ ID NO:10, the nucleotide sequence of the N20 is shown as SEQ ID NO: 11.

4. The assay of claim 1 or 2, wherein the process of random integration of NHEJ in step (2) comprises: mixing the protoplast and the modified gene with PEG6000 and STC buffer solution, suspending to obtain a suspension, and placing the suspension on a solid regeneration flat plate for transformation culture;

preferably, the medium of the solid regeneration plate contains: 15-25g/L of glucose, 5-8g/L of yeast basic culture medium, 0.5-1g/L of sodium nitrate, 15-25g/L of agar powder and 0.3-0.8mol/L of sucrose;

preferably, the conditions of the transformation culture include: the temperature is 25-30deg.C, and the time is 4-6 days.

5. The identification method according to claim 1 or 2, wherein the screening in step (2) comprises: carrying out subculture and fermentation culture I on the transformed strain in sequence;

preferably, the subcultured medium contains: 15-25g/L of glucose, 5-8g/L of yeast basic culture medium, 0.5-1g/L of sodium nitrate, 15-25g/L of agar powder and 0.3-0.8mol/L of sucrose;

preferably, the conditions of subculture include: the temperature is 25-30deg.C, and the time is 4-6 days.

6. The method according to claim 5, wherein the medium for fermentation culture I comprises: 40-120g/L of carbon source, 25-40g/L of nitrogen source, 0.8-1.6g/L of soybean oil and MgSO ₄ ·7H ₂ O0.8-1.6g/L，(NH ₄ ) ₂ SO ₄ 0.2-0.5g/L，KH ₂ PO ₄ 1.5-4g/L，ZnSO ₄ 0.05-0.1g/L，MnSO ₄ 0.05-0.1g/L；

Preferably, the conditions of the fermentation culture I include: the inoculation amount is 8-12 vol%, the temperature is 25-30 ℃, the rotating speed is 200-250rpm, and the time is 8-12 days.

7. Use of the identification method of any one of claims 1 to 6 to increase gibberellin production.

8. A recombinant strain, characterized in that the recombinant strain is a target strain obtained by treating a starting strain by the identification method according to any one of claims 1 to 6.

9. The recombinant strain of claim 8, wherein the engineered gene is a nitrate reductase gene and the integration target is a dicarboxylic acid transporter site;

preferably, the nucleotide sequence of the nitrate reductase gene is shown in SEQ ID NO:1 is shown in the specification;

preferably, the starting strain is gibberella cappa, more preferably gibberella cappa with a preservation number of CCTCC NO: M2015614.

10. A preparation method of gibberellin is characterized by comprising the following steps: inoculating the recombinant strain of claim 8 or 9 into a fermentation medium for fermentation culture II;

preferably, the conditions of the fermentation culture II include: the inoculation amount is 8-12 vol%, the temperature is 25-30 ℃, the rotating speed is 200-250rpm, and the time is 8-12 days;

preferably, the fermentation medium contains: 40-120g/L of carbon source, 25-40g/L of nitrogen source, 0.8-1.6g/L of soybean oil and MgSO ₄ ·7H ₂ O 0.8-1.6g/L，(NH ₄ ) ₂ SO ₄ 0.2-0.5g/L，KH ₂ PO ₄ 1.5-4g/L，ZnSO ₄ 0.05-0.1g/L，MnSO ₄ 0.05-0.1g/L。

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