Construction and application of bacillus licheniformis recombination system mediated by phage recombinase protein recT

Abstract

The application discloses a construction and application of a Bacillus licheniformis recombination system mediated by a phage recombinase protein RecT, and belongs to the technical field of genetic engineering. The application mines and identifies a recombination protein RecT from a Bacillus phage 049ML001, and constructs a homologous recombination system of a recombination enzyme conditional expression by using a rhamnose inducible promoter Prha. Further, the recombination efficiency of the system is improved by optimizing the action condition of the recombination enzyme. The application firstly constructs a phage recombinase protein mediated recombination system in the Bacillus licheniformis and realizes gene knockout, and provides an effective technical means for metabolic engineering modification of the Bacillus licheniformis.

Description

Technical Field

[0001] This invention relates to the construction and application of a Bacillus licheniformis recombination system mediated by the phage recombinant protein RecT, belonging to the field of genetic engineering technology. Background Technology

[0002] Bacillus licheniformis is a Gram-positive bacterium with high application value due to its advantages such as simple fermentation conditions, abundant enzyme system, high enzyme production, and food-grade strains. Currently, Bacillus licheniformis is widely used in industry to produce peptide antibiotics (such as bacitracin and proteins), organic acids, and polymers (such as citric acid, inosinic acid, and polyglutamic acid). In addition, Bacillus licheniformis has important applications in aquaculture, agriculture, biomedicine, and pharmaceuticals.

[0003] Currently, metabolic engineering based on artificially designed genetic perturbations, using genomic sequence information as a blueprint, has become an effective strategy for studying the complex metabolic pathways of Bacillus licheniformis and for modifying strains to construct related phenotypes. The classic gene editing strategy involves homologous substitution of the target gene with a transformed mutant cassette fragment, i.e., homologous recombination (HR), to achieve the knockout or integration of the target fragment into the microbial genome. For example, Escherichia coli utilizes its own RecA recombination system to achieve the knockout and integration of large chromosomal fragments. With the discovery of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, CRISPR-Cas9-based gene editing tools have become a major focus in recent years. Li Kaifeng et al. used the CRISPR-cas9n system, a protein mutant of cas9, to knock out the yvmC gene in Bacillus licheniformis. After cas9n cuts the DNA single strand to create breaks, the insertion of the target gene is completed by integration of homologous complementary fragments (Li KF, Cai DB, Wang ZQ, He ZL, and Chen SW, 2018, Development of an efficient genome editing tool in Bacillus licheniformis using CRISPR-Cas9 nickase, Appl. Environ. Microbiol., 84(6):e02608-17.). In this technique, the efficiency of homologous recombination is the rate-limiting step in gene editing. Li Zongwen used a temperature-sensitive plasmid as a vector to construct a knockout plasmid-mediated homologous double crossover to inactivate the α-amylase gene. Subsequently, the resistance marker used for selection was deleted using FLP / FRT recombinase (Li Zongwen, Li Youran, Gu Zhenghua, Ding Chongyang, Zhang Liang, Xu Sha, Shi Guiyang, 2019, Construction and validation of Bacillus licheniformis FLP / FRT gene editing system, Chinese Journal of Biotechnology, 35(3): 458-471.). This gene editing method still relies on the homologous recombination mechanism, therefore, recombination efficiency is the key to the success of existing gene editing technologies.

[0004] However, existing gene knockout methods for Bacillus licheniformis mainly rely on the bacterial endogenous recombination system, which has extremely low knockout efficiency. It is almost impossible to achieve gene knockout or introduction of exogenous genes by relying on Bacillus licheniformis itself, which limits its strain selection and industrial application. There is an urgent need to develop simple and efficient upstream modification tools. Summary of the Invention

[0005] This invention provides a RecT-mediated heterologous recombination system for Bacillus licheniformis, which can efficiently achieve gene knockout and provides an effective technical means for the metabolic engineering of Bacillus licheniformis.

[0006] This invention provides a recombinant plasmid containing the rhamnose-inducible promoter P. rha Recombinant protein RecT derived from Bacillus phage 049ML001; the rhamnose-inducible promoter P rha Regulate the expression of the recombinant protein RecT.

[0007] In one embodiment, the rhamnose-inducible promoter P rha The nucleotide sequence is shown in SEQ ID NO.1.

[0008] In one embodiment, the recombinant protein RecT contains the amino acid sequence shown in SEQ ID NO.2.

[0009] In one embodiment, the gene encoding the recombinant protein RecT is shown in SEQ ID NO.3.

[0010] In one embodiment, the recombinant plasmid uses the Escherichia coli-Bacillus subtilis shuttle plasmid pHY-PLK300 as the starting plasmid.

[0011] This invention provides the application of RecT, a recombinant protein derived from Bacillus phage 049ML001, in gene editing in Bacillus licheniformis.

[0012] In one implementation, the gene editing includes, but is not limited to, gene knockout.

[0013] In one implementation, the application includes the following steps:

[0014] (1) Construct a gene knockout cassette for the target gene to be knocked out; the gene knockout cassette contains an upstream fragment of the target gene, an anti-resistance gene, and a downstream fragment of the target gene;

[0015] (2) Link the gene knockout cassette to the rhamnose inducible promoter P rha Upstream;

[0016] (3) Transform the recombinant plasmid constructed in step (2) into the host cell to obtain recombinant bacteria;

[0017] (4) The recombinant bacteria obtained in step (3) are cultured in a medium containing antibiotics corresponding to the resistance gene for a period of time, and rhamnose is added to induce the expression of the recombinant protein RecT.

[0018] In one embodiment, the target gene is the α-amylase gene amyL, and the amylase gene knockout cassette for knocking out the target gene has the nucleotide sequence shown in SEQ ID NO.4.

[0019] The specific methods for gene knockout in the above-mentioned recombinant system are as follows:

[0020] (1) The recombinant strain was transferred to 15 mL of tetracycline-resistant LB medium and cultured at 37 °C and 200 rpm until a sufficient bacterial concentration was reached.

[0021] (2) Adding rhamnose to induce the promoter P rha The recombinase RecT was expressed and cultured at 37°C and 200 rpm for a period of time.

[0022] (3) Dilute the bacterial solution with sterile water 10 -5 -10 -7 The resulting plating was then spread onto knockout-resistant plates, which were then incubated at 37°C until single colonies appeared.

[0023] (4) Select a single colony for colony PCR and screen for strains that successfully knock out the target gene.

[0024] In one embodiment, step (4) involves culturing the recombinant strain in LB medium containing antibiotics at 30–37°C for 2–10 h, then adding 5–20 g / L of rhamnose to induce the recombinase RecT, and continuing to culture at 28–37°C for 12–36 h.

[0025] In one implementation, step (4) is repeated 1 to 3 times.

[0026] The present invention also claims protection for the application of the recombinant plasmid or the phage recombinant protein RecT-mediated Bacillus licheniformis heterologous recombination system in Bacillus licheniformis gene knockout.

[0027] Beneficial Effects: This invention is the first to utilize a RecT-mediated recombination system in Bacillus licheniformis for gene editing, and significantly improves the gene editing efficiency of Bacillus licheniformis through optimization of recombinase activity conditions. This technology introduces a heterologous recombinase, circumventing the bacteria's own tight regulation of gene recombination and significantly improving recombination efficiency. The recombination efficiency of this system is 10 times higher than that of Bacillus licheniformis itself. -6 Increased by 10 5 The constructed gene knockout system was applied to the knockout of the α-amylase gene amyL, and the recombination efficiency reached 5.56%–16.67%. Attached Figure Description

[0028] Figure 1: Knockout plasmid pKA.

[0029] Figure 2 : pKAR, a plasmid for knocking out the amyL gene with a RecT expression cassette.

[0030] Figure 3 : Schematic diagram of the double crossover principle of knocking out the amyL gene in the recombination system.

[0031] Figure 4 : PCR electrophoresis image of colonies with the amyL gene knocked out in the recombinant system.

[0032] Figure 5 : Efficiency diagram of knocking out the amyL gene in the recombinant system.

[0033] Figure 6 : Optimization of the conditions for recombination system operation; A: Effect of induction time on recombination efficiency; B: Effect of inducer concentration on recombination efficiency; C: Effect of culture time on recombination efficiency; D: Effect of passage number on recombination efficiency. Detailed Implementation

[0034] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl (solid medium with 1.5% agar powder).

[0035] The recombination efficiency is calculated as follows: (Number of positive transformants ÷ Number of single colonies picked) × 100%.

[0036] The primer sequences involved in the examples are shown in Table 1.

[0037] Table 1 Primers and Sequences

[0038]

[0039] Note: The restriction enzyme sites carried by the primers are marked with a horizontal line at the bottom.

[0040] Example 1: Construction of the knockout plasmid pKA

[0041] The α-amylase gene amyL (Genbank accession number: CP005965.1, REGION: 723302..724840) from *B. licheniformis* CICIM B1391 was selected as the gene to be knocked out. Using plasmid pNZTT-AFKF (published in the paper "Construction and Validation of the *B. licheniformis* FLP / FRT Gene Editing System") as a template, the amyL knockout cassette fragment (SEQ ID NO. 4) was amplified using primers amyL-HindⅢ-F and amyL-EcoRI-R. After purification, it was double-digested with HindⅢ and EcoRI. The digested product was ligated to plasmid pHY-PLK300, which had undergone the same double digestion, to construct the α-amylase gene knockout plasmid pKA. Figure 1 ).

[0042] Example 2: Construction of the knockout plasmid pKAR with the RecT expression cassette

[0043] The RecT recombinase gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. The RecT recombinase gene fragment shown in SEQ ID NO.3 was amplified using primers RecT-XhoⅠ-F and RecT-EcoRⅠ-R. Using B. licheniformis CICIMB1391 as a template, primer P... rha -EcoRⅠ-F、P rha -XhoⅠ-R amplifies the rhamnose promoter P rha The fragment (shown in SEQ ID NO.1) will contain P rha The fragment and the RecT gene fragment were subjected to overlap extension PCR to obtain the RecT expression cassette. After purification, it was digested with EcoRI and ligated into the plasmid pKA constructed in Example 1, which had been digested with the same single enzyme, to obtain the α-amylase gene knockout plasmid pKAR containing the RecT expression cassette. Figure 2 ).

[0044] Example 3: Knocking out the amyL gene in Bacillus licheniformis using a knockout plasmid

[0045] The α-amylase gene knockout plasmid pKAR constructed in Example 2 was transformed into Bacillus licheniformis CICIM B1391 to obtain recombinant Bacillus licheniformis. The recombinant Bacillus licheniformis was then transferred to 15 mL LB medium supplemented with tetracycline resistance and cultured at 37°C and 200 rpm for 8 h. Rhamnose was then added to a final concentration of 10 g / L, and cultured at 37°C for an induction of P... rha The recombinase RecT was expressed and cultured at 37°C and 200 rpm for a period of time. The bacterial culture was then diluted 10% with sterile water. -5 -10-7 Spread the bacterial culture onto kanamycin-resistant plates and incubate them at 37°C until single colonies appear. Simultaneously, transfer 500 μL of the bacterial culture to a fresh 15 mL LB medium supplemented with tetracycline resistance, subculture under the same conditions, dilute, and spread onto kanamycin-resistant plates.

[0046] A certain number of single colonies were picked from the plate, and colony PCR was performed using the knockout verification primers amyL-YZ-F / amyL-YZ-R to verify the colonies and screen for positive transformants. During homologous recombination, one homologous arm successfully replaces a homologous fragment on the genome, integrating the entire recombinant plasmid into the bacterial genome; this process is called a single crossover. When the other homologous arm recombines again, inserting the artificially designed target fragment into the target site in the genome, a double crossover is completed, resulting in successful homologous recombination and gene knockout. The principle is as follows: Figure 3 As shown. Strains that failed to recombine will produce a 1947bp band of the original strain genome or no band after colony PCR. When the colony PCR yields a 2649bp band, consistent with the theoretical size, it indicates that the amyL gene has been successfully knocked out. Figure 4 ).

[0047] Neither the knockout plasmid pKA (lacking the recombinase RecT) nor the plasmid pKAR (without rhamnose to induce RecT expression) produced positive transformants with successful amyL gene knockout during recombination, resulting in a recombination efficiency of 0. The recombination system of this invention achieves a direct knockout efficiency of 5.56% (…). Figure 5 ).

[0048] Example 4: The effect of promoter induction time on recombination efficiency

[0049] The recombinant Bacillus licheniformis constructed in Example 2 was transferred to 15 mL of tetracycline-resistant LB medium and cultured at 37°C and 200 rpm for 2, 4, 6, 8, and 10 h, respectively. Rhamnose was then added to a final concentration of 10 g / L to induce recombinase expression, followed by further culture for 12 h. The bacterial culture was then diluted and plated on kanamycin-resistant plates, and colony PCR was used to screen for successfully recombinant strains. The highest number of successfully knocked-out transformants were obtained when rhamnose was added 8 h into the culture time, with a calculated recombination efficiency of 6.25%. Figure 6 A).

[0050] Example 5: Effect of inducer concentration on recombination efficiency

[0051] The recombinant Bacillus licheniformis constructed in Example 2 was transferred to 15 mL of tetracycline-resistant LB medium and cultured at 37°C and 200 rpm for 8 h. Then, rhamnose was added at final concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L to induce the promoter. After adding the inducer, the culture was continued at 37°C and 200 rpm for 12 h. The bacterial culture was diluted and plated on kanamycin-resistant plates. Colony PCR was used to screen for successfully recombinant strains. When the inducer concentration was below 1%, no positive transformants were screened; when the inducer concentration was 1.5%, the most positive transformants were screened, and the calculated recombination efficiency was 6.94%. Figure 6 B).

[0052] Example 6: Effect of strain culture time on recombination efficiency

[0053] The recombinant Bacillus licheniformis constructed in Example 2 was transferred to 15 mL of tetracycline-resistant LB medium and cultured at 37°C and 200 rpm for 8 h. Then, a rhamnose promoter with a final concentration of 15 g / L was added for induction. After adding the inducer, the culture time at 37°C and 200 rpm was controlled for 12, 18, 24, 30, and 36 h, respectively. The bacterial suspension was diluted and plated on kanamycin-resistant plates for colony PCR screening of successfully recombinant strains. The highest number of positive transformants were obtained when the culture time after induction was 24 h, and the recombinant efficiency was calculated to be 9.64%. Figure 6 C).

[0054] Example 7: The effect of passage number on recombination efficiency

[0055] The recombinant Bacillus licheniformis constructed in Example 2 was transferred to 15 mL of tetracycline-resistant LB medium and cultured at 37°C and 200 rpm for 8 h. Then, 1.5% rhamnose was added to induce P. rha Recombinase RecT was expressed, and the culture was continued at 37℃ and 200 rpm for 24 h. Then, 500 μL of the bacterial culture was transferred to 15 mL of fresh LB medium supplemented with tetracycline resistance. The culture was subcultured three times under the same conditions. After each subculture, the bacterial culture was diluted and plated on kanamycin-resistant plates. Colony PCR was used to screen for successfully recombinant strains. The results showed that the recombination efficiency was highest at three subcultures, reaching 16.67%. Figure 6 D).

[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of the recombinant protein RecT, with the amino acid sequence shown in SEQ ID NO.2, in gene editing in Bacillus licheniformis, characterized in that, Includes the following steps: (1) Construct a gene knockout cassette for the target gene to be knocked out; the gene knockout cassette contains an upstream fragment of the target gene, an anti-resistance gene, and a downstream fragment of the target gene; (2) Link the gene knockout cassette constructed in step (1) to the rhamnose-inducible promoter P shown in SEQ ID NO.1 on the plasmid. rha Upstream of; the plasmid contains a gene encoding the recombinant protein RecT and is promoted by the rhamnose-inducible promoter P. rha Regulation of expression; (3) Transform the recombinant plasmid constructed in step (2) into the host cell to obtain recombinant bacteria; (4) The recombinant bacteria obtained in step (3) are cultured in a medium containing antibiotics corresponding to the resistance gene for a period of time, and rhamnose is added to induce the expression of the recombinant protein RecT.

2. The application according to claim 1, characterized in that, The final concentration of rhamnose is 5–20 g / L.

3. The application according to claim 1, characterized in that, Step (4) involves culturing the recombinant bacteria in LB medium containing antibiotics at 30–37°C for 2–10 h, then adding 5–20 g / L of rhamnose, and continuing to culture at 28–37°C for 12–36 h.

4. The application according to any one of claims 1 to 3, characterized in that, Repeat step (4) 1 to 3 times.

5. The application of recombinant plasmids in improving the recombination efficiency of Bacillus licheniformis, characterized in that, The recombinant plasmid was derived from the Escherichia coli-Bacillus subtilis shuttle plasmid pHY-PLK300 and contained the rhamnose-inducible promoter P. rha and the recombinant protein-coding gene RecT; the rhamnose-inducible promoter P rha Regulates the expression of the recombinant protein encoding gene RecT; the rhamnose inducible promoter P rha The nucleotide sequence of the recombinant protein encoding gene RecT is shown in SEQ ID NO.1; the nucleotide sequence of the recombinant protein encoding gene RecT is shown in SEQ ID NO.3.

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