Method for improving transformation efficiency of exogenous DNA by an engineered restriction modification system

Abstract

This invention relates to a novel engineered restriction-modification system for salt-tolerant Jeju bacteria to improve the efficiency of exogenous DNA transformation, belonging to the fields of bioengineering, metabolic engineering, and synthetic biology. This invention involves in-depth analysis of the whole-genome methylation map of Jeju bacteria to identify its unique methylation motif SAGCTS, and further identifies the corresponding restriction endonuclease Sen. Based on this, the restriction-modification system of Jeju bacteria is edited and modified, thereby improving the efficiency of exogenous DNA transformation.

Description

Technical Field

[0001] This invention relates to a novel engineered restriction modification system for salt-tolerant Jeju bacteria to improve the efficiency of exogenous DNA transformation, belonging to the fields of bioengineering, metabolic engineering and synthetic biology. Background Technology

[0002] With the rapid development of synthetic biology, the production of high-value-added chemicals and biofuels using microbial cell factories has become a research hotspot. Besides *Escherichia coli* (…),… E. coli ) and brewer's yeast ( S. cerevisiae Besides model organisms such as , many non-model microorganisms have shown great potential for industrial applications due to their unique physiological characteristics (such as high temperature resistance, high salt resistance, solvent resistance, or ability to utilize inexpensive substrates).

[0003] For example, Jeju bacteria ( Jejubacter As a salt-tolerant microorganism, *Sp.* can grow in high-salt environments and has the potential for open fermentation using unsterilized seawater, which can significantly reduce freshwater consumption and the risk of contamination. However, genetic manipulation systems for non-model microorganisms are often extremely difficult, with the main bottleneck being the difficulty for exogenous DNA to enter the cell or its rapid degradation after entry, resulting in extremely low transformation efficiency.

[0004] The primary reason lies in the naturally occurring restriction-modification (RM) system of microorganisms. The RM system typically consists of restriction endonucleases and DNA methyltransferases. The host uses methyltransferases to methylate specific sites on its own DNA. Restriction endonucleases then distinguish between their own and foreign DNA based on these methylations, cleaving and breaking down the foreign DNA to protect the organism from foreign DNA invasion. The RM system is a crucial defense mechanism for host bacteria, but the differences in RM systems between different strains hinder genetic engineering. Current solutions typically include in vitro methylation simulation or in vivo methylation modification, but these methods are cumbersome and struggle to fully mimic the complex host methylation patterns.

[0005] Therefore, there is an urgent need to develop a universal and easy-to-operate method to fundamentally remove the limitations imposed on external DNA by non-model microorganisms and establish an efficient genetic transformation platform. Summary of the Invention

[0006] This invention utilizes in-depth analysis of the whole-genome methylation map of Jejuella to identify its unique methylation motifs and further determine the restriction endonucleases corresponding to these motifs. This allows for the editing and modification of the restriction-modification system of Jejuella, thereby improving the efficiency of exogenous DNA transformation.

[0007] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0008] One of the technical solutions provided by this invention is a method for improving the transformation efficiency of Jeju Bacillus exogenous DNA, which is achieved by deleting the expression of the restriction endonuclease Sen on the Jeju Bacillus host bacteria; The term "deletion of restriction endonuclease Sen expression on Jejubacterium host bacteria" refers to a significant reduction in the expression level of restriction endonuclease Sen compared to its original level, for example, a significant reduction of at least 50%, 60%, 70%, 80%, 90%, or 100%. Gene editing on the host bacteria genome to achieve the deletion of the restriction endonuclease expression can be accomplished using conventional methods in the art, including but not limited to gene knockout, gene knock-in, point mutation, deletion mutation, and combinations thereof. Furthermore, the restriction endonuclease Sen has the amino acid sequence shown in SEQ ID NO.1; Furthermore, the gene encoding the restriction endonuclease Sen... sen The nucleotide sequence is shown in SEQ ID NO.2; Furthermore, the Jeju bacteria include, but are not limited to: Jeju bacteria L23, Jeju bacteria L23R7, Jeju bacteria DT01 IM3, etc. Furthermore, the method also includes optimizing the SAGCTS motif in the exogenous DNA into SAATTS; after optimization, it can avoid being cleaved by the restriction endonuclease Sen, and as the number of SAGCTS motifs decreases, the transformation efficiency of the exogenous DNA in Jeju Bacillus gradually increases.

[0009] The second technical solution provided by this invention is a Jeju Bacillus engineered strain with high efficiency in transforming exogenous DNA. The engineered strain is obtained by deleting the expression of the restriction endonuclease Sen on the Jeju Bacillus host strain. The restriction endonuclease deletion expression method can be implemented by conventional means in the art, including but not limited to gene knockout, gene knock-in, point mutation, deletion mutation and their combination; Furthermore, the restriction endonuclease Sen has the amino acid sequence shown in SEQ ID NO.1; Furthermore, the restriction endonuclease encodes a gene sen The nucleotide sequence is shown in SEQ ID NO.2; Furthermore, the Jeju bacteria include, but are not limited to: Jeju bacteria L23, Jeju bacteria L23R7, Jeju bacteria DT01 IM3, etc. Preferably, the Jejubacterium engineered strain is Jejubacterium L23R7 as the starting strain, which is modified to target the restriction endonuclease encoding gene on the genome. senObtained by knocking out.

[0010] The third technical solution provided by this invention is the application of the Jeju Bacillus engineered strain described in the second technical solution, particularly its application as a chassis bacterium, such as in the construction of strains that produce high levels of isobutanol (IBOH) or 2,3-butanediol (2,3-BDO). Plasmids containing the production pathways of the above products are introduced into the chassis bacterium to construct production strains and use them for the production of the products; more particularly, its application as a chassis bacterium tolerant to high-salt environments.

[0011] Beneficial effects: 1. This invention is the first to discover the methylation motif SAGCTS in Jeju Bacillus and the restriction endonuclease Sen that recognizes and cuts the SAGCTS motif. By disrupting the restriction modification system of Jeju Bacillus, the conversion efficiency of exogenous DNA is improved, thus expanding the scope of gene editing using Jeju Bacillus as a chassis bacterium.

[0012] 2. This invention obtains engineered strains with significantly improved exogenous DNA transformation efficiency by knocking out the gene encoding the restriction endonuclease Sen in different Jeju bacilli. Experiments have confirmed that, compared with the original strain, sen The transformation efficiency of the gene-deficient strains to exogenous plasmids was increased by 50-231 times, successfully overcoming the main genetic manipulation obstacles of this type of industrial chassis strain.

[0013] 3. This invention also provides a method to improve the transformation efficiency of exogenous DNA in Jeju Bacillus, namely, optimizing the SAGCTS motif to SAATTS, which avoids cleavage by the restriction endonuclease Sen. Experimental results show that as the number of SAGCTS motifs decreases, the transformation efficiency of plasmid pRed-X gradually increases. The transformation efficiency of plasmid with all 21 SAGCTS motifs eliminated in L23R7 strain was 110 times higher than that of plasmid without the motifs eliminated. Attached Figure Description

[0014] Picture 1 Transformation efficiency of plasmids optimized from SAGCTS motifs to SAATTS in Jeju Bacillus was tested.

[0015] Picture 2 To knock out sen Tests on the effect of genes on the growth of bacterial strains.

[0016] Picture 3 pRed-X and pDT192 plasmids were respectively encode in L23R7 and L23R7Δ sen Comparison of transformation efficiencies among strains.

[0017] Picture 4pRed-X and pDT192 plasmids were respectively stored in DT01 IM3 and DT01 IM3Δ. sen Comparison of transformation efficiencies among strains. Detailed Implementation

[0018] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0019] The Jeju bacteria involved in this invention Jejubacter sp. L23 is prior art, published in the literature "Rapidly engineering an osmotic-pressure-tolerant gut bacterium for efficient non-sterile production of bulk chemicals", Chemical Engineering Journal 491 (2024)152076. It was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 25721.

[0020] The Jeju Bacillus L23R7 strain involved in this invention is prior art. This strain is based on L23 as the starting strain and involves knockout. frdBC, adhE, fnr, pta, pflB, ldhA as well as endA The gene was obtained later. Specific information about the L23R7 strain has been published in the literature "Rapidly engineering an osmotic-pressure-tolerant gut bacterium for efficient non-sterile production of bulk chemicals", Chemical Engineering Journal 491 (2024) 152076.

[0021] The Jeju bacteria involved in this invention Jejubactersp. DT01 IM3 is prior art and was deposited on September 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 28463.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] The primers used in the embodiments of this invention are shown in Table 1; the PCR system for fragment amplification is shown in Table 2. Table 1 shows the primer sequences used.

[0024] Table 2 shows the gene amplification PCR system (PrimeSTAR Max DNA polymerase).

[0025] The present invention will be further explained and illustrated below through specific embodiments.

[0026] Example 1: Discovery of the restriction transformation barrier and identification of the specific RM system in Jeju Bacillus L23R7 1. Discovery and localization of the transformation barrier: In the early stages of L23R7 genetic manipulation, it was found that it was extremely difficult for exogenous DNA to enter this strain. Experiments showed that DNA derived from... E. coli The electroconversion efficiency (2.6 kV, 25 μF, 200 Ω) of 1000 ng pRed-X plasmid from JM109 into L23R7 was extremely low (only about 11 CFUs), and about 12.5% ​​of the colonies contained erroneous plasmids that might have been degraded or truncated. In contrast, the 1000 ng pRed-X plasmid derived from L23R7 itself showed a much higher conversion efficiency (about 278 CFUs). Agarose gel electrophoresis analysis directly confirmed that the pRed-X plasmid derived from L23R7 itself... E. coli The plasmid from JM109 was severely degraded after entering L23R7, while the plasmid from L23R7 remained intact. This strongly suggests that Jeju bacteria such as L23R7 have a defense system that can recognize “non-self” DNA.

[0027] To explore the nature of this barrier, we first attempted to analyze the effects of plasmid size and replicon type. While larger plasmids generally have lower transformation efficiency, an anomaly was observed in L23R7: plasmids with similar ColE1 replicons (approximately 10 kb) exhibited transformation efficiencies differing by two orders of magnitude, with low efficiency producing about 10 transformants and high efficiency producing about 1000. Furthermore, plasmids with the same p15A replicon showed a 30.7-fold decrease in transformant numbers simply due to size differences (8.7 kb vs. 13.4 kb), far exceeding the normal range. These anomalous data suggest that physical parameters are not the decisive factor; rather, specific DNA sequence characteristics on the plasmid are key to triggering host defense.

[0028] 2. Methylation Analysis and Motif Mining Based on Single-Molecular Real-Time Sequencing (SMRT): To break this defense mechanism at the genomic level, PacBio SMRT sequencing technology was used to perform in-depth analysis of the whole-genome methylation profile of L23R7. E. coli Unlike other strains, L23R7 exhibits a unique methylation modification pattern.

[0029] Through comparative analysis, the inventors discovered that the plasmid derived from L23R7 exhibits an extremely high level (nearly 100%) of m on the specific sequence SAGCTS. 4 C-methylation modification, derived from E. coli The JM109 plasmid completely lacked this modification. Further whole-genome scanning revealed two sites in the L23R7 genome with extremely high methylation probabilities: the SAGCTS and CGTAACB sites, with methylation probabilities of 99.6% and 99.9%, respectively. The methylation modification patterns were m... 4 C and m 6 A, with 14285 and 1771 modifications detected on the genome, respectively, indicates that these are key protective features of the host itself.

[0030] 3. Comparative Genomics Analysis and Specific Screening: The discovery of methylation sites alone is insufficient to identify functional genes, as bacteria typically possess multiple restriction endonuclease (RM) systems. A database of restriction endonucleases and methyltransferases was created using Diamond software to extract all restriction endonucleases and methyltransferases from the REBASE database. Protein sequences obtained from the translation of the complete genome nucleic acid sequences of six species (Jejubacterium L23R7, Pseudomonas aeruginosa, legumes, Escherichia coli JM109, Escherichia coli MG1655, and Escherichia coli Nissle 1917) were then compared with the enzyme sequence database using Diamond software to preliminarily determine all restriction modification systems and corresponding recognition sequences for these six species. Through comparative analysis, four L23R7-specific methylation motifs were identified at the recognition sites of all restriction endonucleases and methyltransferases from the other five species: SAGCTS, CCAAAC, CCWGG, and GCCGGC. The corresponding restriction endonucleases for these sites were Sen, PglX, Vsr, and Ngomiv, respectively.

[0031] The methylated sequences obtained by the above methods all contain SAGCTS. In addition, it is necessary to accurately identify the one that caused the transformation failure from the potential restriction endonucleases (Sen, PglX, Vsr and Ngomiv) corresponding to these four sequences (SAGCTS, CCAAAC, CCWGG and GCCGGC).

[0032] Example 2: Plasmid transformation efficiency test based on SAGCTS motif optimization In Example 1, methylation analysis of plasmids from different sources and genomic-level methylation analysis both showed that the SAGCTS site was methylated with a very high probability. Furthermore, analysis of differences in restriction endonuclease recognition sites among different species revealed the presence of a strain-specific restriction endonuclease, Sen (amino acid sequence shown in SEQ ID NO. 1), in L23R7 that recognizes the SAGCTS site.

[0033] To further verify the correlation between SAGCTS and transformation efficiency in strain L23R7, the sequences of different plasmids were scanned. It was found that plasmids with low transformation efficiency contained a large number of SAGCTS sites (>20), while plasmids with high transformation efficiency had fewer such sites, thus establishing a negative correlation between the number of sites and transformation efficiency.

[0034] Therefore, we designed a method to optimize SAGCTS sites into SAATTS sites, making them unrecognizable and cleavable by the restriction endonuclease Sen. The 21 SAGCTS sites on the pRed-X plasmid derived from *E. coli* JM109 were divided into 7 groups. The first group optimized 3 SAGCTS sites, the second group optimized 6 SAGCTS sites, and so on, with the seventh group optimizing all 21 SAGCTS sites. The plasmids constructed after optimization for each group were named pRed-X-1, ..., pRed-X-7, respectively. These 7 optimized plasmids were then transformed into strain L23R7. The results showed that the more SAGCTS motifs optimized, the higher the transformation efficiency. When all 21 motifs were optimized, the transformation efficiency was 110 times higher than that of the unoptimized plasmids (e.g., ...). Picture 1 (As shown).

[0035] The above results indicate that the low transformation efficiency of pRed-X plasmid is due to the presence of multiple SAGCTS sites on the plasmid. It can be concluded that the presence of the restriction endonuclease Sen, which recognizes SAGCTS sites, leads to the low transformation efficiency of plasmid pRed-X.

[0036] Example 3 sen Gene-deleted strain L23R7Δ sen Construction and performance testing 1. To further determine the effect of the restriction endonuclease Sen on the transformation efficiency of exogenous DNA from Jeju bacteria, this example involved knocking out its coding gene. sen Verification was performed using (as shown in SEQ ID NO.2). The details are as follows: Using the L23R7 genome as a template, primers 5HA-F and 5HA-R were designed for amplification, yielding a 474 bp donor DNA 5HA ( sen (the upstream homologous arm), the nucleotide sequence is shown in SEQ ID NO.3; Using the L23R7 genome as a template, primers 3HA-F and 3HA-R were designed for amplification, yielding a 494 bp donor DNA 3HA ( sen (downstream homologous arm), the nucleotide sequence is shown in SEQ ID NO.4; Primers sgRNA-F and sgRNA-R were designed, and the pRed-X plasmid derived from E. coli JM109 was used as a template for amplification (the complete nucleotide sequence of the plasmid is shown in SEQ ID NO.5) to obtain an SGPRO fragment of size 770 bp. Primers GJ-F and GJ-R were designed and amplified using plasmid pRed-X as a template to obtain a 12800 bp GJ fragment (a fragment of linearized plasmid pRed-X). The Gibson enzyme was used to perform multi-fragment ligation of four fragments: 5HA, 3HA, SGPRO, and GJ to construct... sen gene knockout plasmid pRed-X- sen .

[0037] plasmid pRed-X- sen Electroporation was used to transform the L23R7 host strain, which underwent gene editing, and then... (The sentence is incomplete and requires more context to translate accurately.) R Antibiotics are screened for resistance. Confirmed by colony PCR and sequencing. sen The gene has been successfully knocked out, resulting in the mutant strain L23R7Δ. sen .

[0038] 2. In order to further determine sen The effect of gene knockout on the normal growth performance of the strain, using the mutant strain L23R7Δ sen Growth curves were determined for L23R7.

[0039] The culture was carried out in standard LB medium at 37 °C and 220 rpm for 12 h, and the growth OD was measured every 2 h. 600 The growth status of the two strains was compared. The results showed that the OD values ​​of the two strains differed throughout the entire culture period. 600 There was no significant difference, indicating that the mutant strain was knocked out. sen The gene will not affect the growth of the strain (e.g.) Picture 2 (As shown).

[0040] Example 4: Determination of exogenous DNA transformation efficiency in restriction endonuclease knockout strains and wild-type strains 1. Validation in Jeju bacteria L23R7 Host: Preparation of wild-type strain L23R7 and mutant strain L23R7Δ sen competent cells.

[0041] Exogenous DNA: pRed-X plasmid (containing the Jeju Bacillus-recognizable replicon p15A and the resistance marker Kan) R (The sequence is shown in SEQ ID NO.5) and pDT192 plasmid (containing the Jeju Bacillus-recognizable replicon p15A and the resistance marker Amp). R The sequence is shown in SEQ ID NO.6, and this plasmid contains the isobutanol biosynthesis pathway.

[0042] Equal amounts of 500 ng shuttle plasmids pRed-X and pDT192 were used to transform two host cell lines (competent cell concentration was approximately 1 × 10⁻⁶). 10CFU / mL; Electroporation parameters: voltage 2.6 kV, capacitance 25 μF, resistance 200 Ω), after 1 h of recovery in LB medium, all bacterial culture was centrifuged and resuspended in 100 μL of fresh LB medium before being spread onto LB solid medium culture dishes containing the corresponding antibiotics.

[0043] After static incubation at 37 ℃ for 12 hours, the transformants were counted. The wild-type strain L23R7 plate produced only 9 transformants of plasmids pRed-X and pDT192, respectively. Meanwhile, the L23R7Δ... sen On the plate, the transformants of plasmids pRed-X and pDT192 were 2079 and 2165, respectively.

[0044] L23R7Δ sen The number of transformants was increased by 231 times and 90 times compared to wild-type L23R7 (e.g. Picture 3 (As shown).

[0045] 2. Validation in Jeju Bacillus DT01 IM3 Host: Preparation of wild-type strain DT01 IM3 and mutant strain DT01 IM3Δ sen competent cells (using the method described in Example 3) from strain DT01 IM3 sen Gene knockout yielded the mutant strain DT01 IM3Δ sen ).

[0046] Exogenous DNA: pRed-X plasmid (containing the Jeju Bacillus-recognizable replicon p15A and the resistance marker Kan) R (The sequence is shown in SEQ ID NO.5) and pDT192 plasmid (containing the Jeju Bacillus-recognizable replicon p15A and the resistance marker Amp). R (The sequence is shown in SEQ ID NO.6).

[0047] Equal amounts of 500 ng shuttle plasmids pRed-X and pDT192 were used to transform two cell lines (competent cell concentration was approximately 1 × 10⁻⁶). 10 CFU / mL; Electroporation parameters: voltage 2.6 kV, capacitance 25 μF, resistance 200 Ω), after 1 h of recovery in LB medium, all bacterial culture was centrifuged and resuspended in 100 μL of fresh LB medium before being spread onto LB solid medium culture dishes containing the corresponding antibiotics.

[0048] After static incubation at 37 ℃ for 12 hours, the transformants were counted. On the wild-type strain DT01 IM3 plate, only 11 transformants of plasmids pRed-X and pDT192 were found, and 36 were found, respectively. However, on the DT01 IM3Δ... senOn the plate, the transformants of plasmids pRed-X and pDT192 were 1713 and 1965, respectively.

[0049] DT01 IM3Δ sen The number of transformants was increased by 156 times and 54.6 times compared to wild-type DT01 IM3 (e.g., Picture 4 (As shown).

[0050] The above results indicate that knockout is effective against different Jeju bacteria. sen The gene can effectively remove the host's restriction on foreign DNA, and this method is universally applicable to Jeju bacteria.

[0051] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A method for improving the transformation efficiency of Jeju bacteria exogenous DNA, characterized in that, The method is achieved by deleting the expression of the restriction endonuclease Sen on the host bacterium Jejubacterium; The restriction endonuclease Sen has the amino acid sequence shown in SEQ ID NO.

1.

2. The method for improving the transformation efficiency of Jeju bacteria exogenous DNA as described in claim 1, characterized in that, The methods for achieving the absence of expression of the restriction endonuclease include, but are not limited to, gene knockout, gene knock-in, point mutation, deletion mutation, and combinations thereof.

3. The method for improving the transformation efficiency of Jeju bacteria exogenous DNA as described in claim 1, characterized in that, The method also includes optimizing the SAGCTS motif in exogenous DNA into SAATTS; after optimization, it can avoid being cleaved by the restriction endonuclease Sen.

4. A strain of Jeju Bacillus engineered bacteria with highly efficient ability to transform exogenous DNA, characterized in that, The engineered bacteria were obtained by deleting the expression of the restriction endonuclease Sen on the Jeju Bacillus host bacteria. The restriction endonuclease Sen has the amino acid sequence shown in SEQ ID NO.

1.

5. The Jeju Bacillus strain with highly efficient exogenous DNA transformation capability as described in claim 4, characterized in that, The hosts of Jeju Bacillus include, but are not limited to: Jeju Bacillus L23, Jeju Bacillus L23R7, and Jeju Bacillus DT01 IM3.

6. The Jeju Bacillus strain with highly efficient exogenous DNA transformation capability as described in claim 4, characterized in that, The engineered Jejubacterium strain uses Jejubacterium L23R7 or DT01 IM3 as the host bacterium, and targets restriction endonuclease-encoding genes on the host genome. sen Obtained by knocking out.

7. The application of the Jeju Bacillus engineered strain as described in claim 4.

8. The application as described in claim 7, characterized in that, It is used as a chassis bacteria.

9. The application as described in claim 8, characterized in that, A plasmid containing the production pathway of the target product is introduced into the chassis bacteria to construct a production strain and used for the production of the product.

10. The application as described in claim 9, characterized in that, The target product includes isobutanol and 2,3-butanediol.

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