Gene editing system of xanthomonas campestris and application of gene editing system

Abstract

The invention provides a gene editing system of xanthomonas campestris and application of the gene editing system, and relates to the technical field of gene engineering. The gene editing system comprises a Cpf1 protein expression vector and a crRNA expression vector, the Cpf1 protein expression vector comprises an Fncpf1 expression cassette and a lambda Red expression cassette, the Fncpf1 expression cassette is composed of a sequential promoter, an RBS sequence and a Cpf1 protein coding gene, and the lambda Red expression cassette is composed of a repressor protein AraC, a promoter Para and a lambda Red coding gene. By using the system, the gene knockout efficiency of xanthomonas campestris reaches 70%, a related gene xanK synthesized by mycoflavin is successfully knocked out, an efficient production strain of colorless xanthan gum is obtained, the modification requirements of multiple fields such as food, medicines and cosmetics on xanthomonas campestris are met, and the system has an extremely high application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene editing system for Xanthomonas brasiliensis and its application. Background Technology

[0002] Xanthomonas campestris (X. campestris) is a Gram-negative bacterium that characteristically produces the extracellular polysaccharide xanthan gum. Xanthan gum is currently the world's most produced natural polysaccharide, possessing excellent stability, rheological properties, and pseudoplasticity, and is widely used in food, pharmaceuticals, and cosmetics. Although some research has been conducted on the genome structure, pathogenic mechanism, and secretion system of X. campestris in recent years, the systematic analysis of functional genes still faces significant challenges. This is largely attributed to the lack of efficient, precise, and universal gene manipulation tools. Traditional gene knockout methods for X. campestris (such as homologous recombination and the suicide plasmid system) are inefficient and time-consuming, making it difficult to meet the needs of rapidly constructing mutant strains or manipulating multiple genes, thus limiting in-depth research on the pathogenicity, stress resistance, and metabolic network of this bacterium.

[0003] In recent years, with the gradual application of emerging gene-editing technologies such as CRISPR-Cas in bacteria, the spatiotemporal precision and efficiency of gene manipulation have been significantly improved. For example, the CRISPR-Cas9, CRISPRi (interference), and CRISPRa (activation) systems have been successfully applied in various bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Salmonella. However, research on CRISPR systems targeting this strain is currently limited, their gene-editing efficiency is low, and there is a lack of systematic research and validation of related basic components. The application of existing CRISPR systems in Xanthomonas campestris still suffers from poor adaptability and low editing efficiency, thus necessitating further optimization and technological improvements. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a system for gene editing of Xanthomonas brasiliensis, comprising a Cpf1 protein expression vector and a crRNA expression vector. This system enables highly efficient gene editing of Xanthomonas brasiliensis.

[0005] The first objective of this invention is to provide a gene editing system for *Xanthomonas campestris*, the gene editing system comprising a Cpf1 protein expression vector and a crRNA expression vector, wherein the Cpf1 protein expression vector contains an Fncpf1 expression cassette and a λRed expression cassette, wherein:

[0006] The Fncpf1 expression cassette consists of a temporal promoter, an RBS sequence, and a Cpf1 protein-coding gene, wherein the temporal promoter initiates expression during the host's logarithmic growth phase.

[0007] The λRed expression cassette consists of the repressor protein AraC and the promoter P. ara It consists of the λRed encoding gene.

[0008] Furthermore, the Cpf1 protein expression vector uses the pBBR1MCS-2 plasmid as its backbone.

[0009] Furthermore, the nucleotide sequence of the timing promoter is shown in SEQ ID NO.1.

[0010] Furthermore, the RBS sequence is shown in SEQ ID NO.2.

[0011] Furthermore, the Cpf1 protein encoding gene was optimized using codons from Xanthomonas campestris.

[0012] In one embodiment of the present invention, the nucleotide sequence of the Cpf1 protein is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0013] Furthermore, the crRNA expression vector consists of a promoter, a spacer sequence, a crRNA insertion region, and a terminator.

[0014] Furthermore, the promoter is P j23119 .

[0015] In one embodiment of the present invention, the nucleotide sequence of the crRNA expression vector is shown in SEQ ID NO.10.

[0016] A second objective of this invention is to provide the application of the above-described gene editing system in gene editing of Xanthomonas brassicae.

[0017] Furthermore, the gene editing is gene knockout or gene knock-in.

[0018] The third objective of this invention is to provide a gene editing method for Xanthomonas brasiliensis, wherein the above-mentioned system is transformed into the host bacterium to be modified.

[0019] Furthermore, the gene editing method specifically includes the following steps:

[0020] Step S1: Transform the Cpf1 protein expression vector into Xanthomonas oryzae and plate it onto NBG plates containing kanamycin to obtain positive colonies;

[0021] Step S2: Design a target-specific crRNA and ligate it into a crRNA expression vector;

[0022] Step S3: Select 1000 bp sequences upstream and downstream of the editing site on the genome as upstream and downstream homologous arms, ligate them into the vector already linked with crRNA, and transform them into the positive colonies described in step S1.

[0023] Step S4: Before plating, resuspend the bacterial cells in NBG liquid medium containing 100 ng / μL kanamycin and 30 ng / μL chloramphenicol and culture with shaking for 8-10 h. Then collect the bacterial cells and plating them onto NBG plates containing 100 ng / μL kanamycin and 30 ng / μL chloramphenicol.

[0024] Step S5: After single-celled bacteria have grown, colony PCR and gene sequencing are used to verify the gene editing status. For positive strains, only the crRNA expression vector can be eliminated to facilitate subsequent gene editing operations; alternatively, both the crRNA expression vector and the Cpf1 protein expression vector can be eliminated to obtain a strain without plasmids.

[0025] Step S6, the plasmid elimination method is to transfer the verified positive strain into liquid NBG medium containing 0.005% SDS and shake it for 8-12 h. If only the derivative plasmid of the crRNA expression vector needs to be eliminated, the culture can be streaked onto a kanamycin-resistant plate. If both the derivative plasmid of the crRNA expression vector and the Cpf1 protein expression vector need to be eliminated, the culture can be streaked onto an antibiotic-free plate. After a single colony grows, the plasmid-eliminated strain is selected by colony PCR.

[0026] The beneficial effects of this invention are:

[0027] This invention discloses a gene editing system for *Xanthomonas campestris*. Compared to traditional homologous recombination or suicide plasmid systems, this invention's CRISPR / Cpf1-based gene editing platform enables more efficient and precise targeted DNA cleavage and repair in *X. campestris*, significantly shortening the cycle for constructing mutant strains. Through the application of this gene editing system, this invention knocks out the xanK gene, related to xanthanin synthesis, in *Xanthomonas campestris* Xcc, successfully modifying the metabolic pathway and preventing the accumulation of xanthanin pigment. This results in a high-yield, low-impurity colorless xanthan gum engineered strain, significantly improving product purity and value. This demonstrates the enormous potential of this gene editing system in the field of *Xanthomonas campestris* modification, and its applicability in multiple fields such as food, pharmaceuticals, and cosmetics. Attached Figure Description

[0028] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0029] Figure 1 This is a schematic diagram of the structure of the Cpf1 expression vector in Embodiment 1 of the present invention;

[0030] Figure 2 This is a schematic diagram of the crRNA expression vector in Example 2 of the present invention;

[0031] Figure 3 This is a verification of gene editing efficiency in Example 3 of the present invention, where A is gene knockout efficiency and B is gene insertion efficiency;

[0032] Figure 4 This is a fermentation verification of Xanthomonas brassicae in Example 4 of the present invention, wherein A is a comparison of xanthan gum produced by Xcc and Xcc-△xanK, and B is a comparison of xanthan gum yield of Xcc and Xcc-△xanK.

[0033] Figure 5 The results show the growth status of *Xanthomonas aurantiacus* strains after gene editing using different Cpf1 values ​​in Comparative Example 1 of this invention.

[0034] Figure 6 This is the gene editing efficiency result of Xanthomonas brasiliensis after gene editing using different Cpf1 values ​​in Comparative Example 1 of this invention;

[0035] Figure 7 This is the gene editing efficiency result of Xanthomonas brasiliensis using different time-sequence promoters in Comparative Example 2 of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] (1) Strains and vectors

[0038] The plasmid was constructed in E. coli DH5α and then transformed into Xcc strain (Xanthomonas campestris pv. campestris str.) for genome integration. The vector pBBRMCS-2 used was a commercially available plasmid.

[0039] (2) Culture medium

[0040] E. coli was cultured in LB medium (containing 10 g tryptone, 5 g yeast extract and 10 g NaCl per liter).

[0041] Xcc was cultured in NBG medium (containing 18 g of nutrient broth and 10 g of glucose per liter).

[0042] Example 1: Construction of Cpf1 expression vector pBBRCpf1

[0043] The structure of the Fncpf1 expression vector pBBRCpf1 is as follows: Figure 1 As shown,

[0044] The commercial plasmid pBBR1MCS-2 was used as a vector, containing the Fncpf1 expression cassette and the λRed expression cassette.

[0045] The Fncpf1 expression cassette is generated by the genomic time-series promoter P. 1-23 It consists of the RBS sequence and the Fncpf1 gene sequence. The time-sequential promoter P... 1-23 Derived from the X. campestris genome, it is not expressed during the lag phase and is initiated during the logarithmic growth phase. Its nucleotide sequence is shown in SEQ ID NO.1. The nucleotide sequence of the ribosome binding site (RBS) is shown in SEQ ID NO.2. Cpf1 is codon-optimized (Opt-Cpf1), and its nucleotide and amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0046] The λRed expression cassette includes the repressor protein AraC and the promoter P. ara The sequences of the λRed gene, wherein the nucleotide and amino acid sequences of λRed are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively; the nucleotide and amino acid sequences of the repressor protein AraC are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; and the promoter P... ara The nucleotide sequence is shown in SEQ ID NO.9.

[0047] The recombinant plasmid pBBRCpf1 was transformed into X. campestris strain to obtain an engineered strain carrying the pBBRCpf1 plasmid. This strain was then further prepared into competent cells for subsequent transformation of a second plasmid and gene editing operations.

[0048] Example 2: Construction of crRNA expression vector pETHR

[0049] The structure of the expression vector pETHR is as follows: Figure 2 As shown, a crRNA expression cassette was added to the pET28a plasmid as the basic framework.

[0050] crRNA expression cassette is generated by promoter P j23119 The pETHR plasmid consists of a spacer sequence, a crRNA insertion region, and a terminator sequence. The crRNA insertion region contains Eco31I restriction sites at both ends. The pETHR plasmid sequence is shown in SEQ ID NO.10.

[0051] Taking xanK gene knockout as an example, a 23 bp crRNA sequence was selected from xanK. First, primer sequence annealing was used to obtain a crRNA sequence with sticky ends. Then, the expression vector pETHR was digested with Eco31I enzyme to obtain a linear vector. The crRNA sequence and the linear vector were ligated using T4 ligase. The ligation product was transformed into *E. coli* to obtain a crRNA plasmid. Homologous arms were added to this plasmid template. The upstream and downstream 1000 bp segments of the edited fragment were used to construct the upstream and downstream homologous arms, which were then ligated into the plasmid vector to obtain the plasmid pETHR-△xanK. The 23 bp crRNA sequence is shown in SEQ ID NO.11, and the upstream and downstream homologous arm sequences are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0052] Example 3: Gene editing in Xanthomonas brasiliensis based on the CRISPR / Cpf1 system

[0053] First, plasmid pBBRCpf1 was transformed into *Xanthomonas oryzae* Xcc, the gene to be edited. When using plasmid pBBRCpf1 for gene editing, the corresponding crRNA and homologous arms were designed according to the method described in Example 2 and ligated into plasmid pETHR. The xanK and gum genes were selected as target genes for knockout to verify the knockout efficiency of the system. Six insertion sites (S1-S6, CRrna as shown in SEQ ID NO. 14-19) were selected on the genome, and the fluorescent protein sfGFP was inserted as a verification gene for insertion efficiency. The gene knockout efficiency was verified to reach 70%. Figure 3 A), gene insertion efficiency is slightly lower, around 60% ( Figure 3 B).

[0054] Example 4: Fermentation verification of colorless xanthan gum producing strain Xcc-△xanK

[0055] The xanK gene knockout strain Xcc-ΔxanK obtained in Example 3 was verified by fermentation.

[0056] The strain was inoculated into shake tubes containing 2 mL of NBG medium and cultured at 30 °C and 200-220 rpm for 10-15 h to obtain seed culture. The seed culture was then inoculated at a 5% (v / v) inoculation rate into 500 mL Erlenmeyer flasks containing 100 mL of fermentation medium and fermented at 30 °C and 300 rpm for 72-96 h. After fermentation, twice the volume of 95% ethanol was added to the fermentation broth, and the mixture was vigorously shaken to mix. After filtration through gauze, the residue was washed twice with anhydrous ethanol and dried in a 65 °C oven to constant weight. The product was then weighed. The results are as follows: Figure 4 As shown, the fermentation products contain virtually no mycotoxin, and their yield is not significantly different from that of the wild-type strain.

[0057] Comparative Example 1: Comparison of toxicity and editing efficiency of different Cas proteins in Xcc

[0058] Editing plasmids including Cas9, codon-optimized Cas9 (opt-Cas9), Cpf1, and codon-optimized Cpf1 (opt-Cpf1) were constructed and compared. The growth and editing (gene knockout) efficiency of strains transformed into Xcc were compared. All strains transformed into Xcc with Cas9 and opt-Cas9 died, resulting in no correct transformants. Both Cpf1 and opt-Cpf1 transformed into Xcc produced correct transformants, while the growth of strains transformed with opt-Cpf1 was slightly lower than that of strains transformed with Cpf1. Figure 5 However, editing efficiency is 20% higher than Cpf1. Figure 6 ).

[0059] Comparative Example 2: Effects of different promoters on Cas protein expression on editing efficiency

[0060] To further improve the editing (gene knockout) efficiency, the Cas protein promoter was optimized. Different endogenous promoters, including P1-1, P1-3, P1-4, P1-6, P1-23, P2-1, P2-4, P2-18, and the ARA inducible promoter, were selected. The effects of different promoters on editing efficiency were compared, and the results are as follows: Figure 7 As shown, P1-23 showed the highest efficiency in initiating Cas protein expression editing.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A gene editing system for Xanthomonas brasiliensis, characterized in that, The gene editing system includes a Cpf1 protein expression vector and a crRNA expression vector. The Cpf1 protein expression vector contains an Fncpf1 expression cassette and a λRed expression cassette, wherein: The Fncpf1 expression cassette consists of a temporal promoter, an RBS sequence, and a Cpf1 protein-coding gene, wherein the temporal promoter initiates expression during the host's logarithmic growth phase. The λRed expression cassette consists of the repressor protein AraC and the promoter P. ara It consists of the λRed encoding gene.

2. The gene editing system according to claim 1, characterized in that: The Cpf1 protein expression vector uses pBBR1MCS-2 plasmid as its backbone.

3. The gene editing system according to claim 1, characterized in that: The nucleotide sequence of the timing promoter is shown in SEQ ID NO.

1.

4. The gene editing system according to claim 1, characterized in that: The RBS sequence is shown in SEQ ID NO.

2.

5. The gene editing system according to claim 1, characterized in that: The Cpf1 protein encoding gene was optimized using codons from Xanthomonas campestris.

6. The gene editing system according to claim 1, characterized in that: The crRNA expression vector consists of a promoter, a spacer sequence, a crRNA insertion region, and a terminator.

7. The gene editing system according to claim 6, characterized in that: The promoter is P. j23119 .

8. The application of the system according to any one of claims 1-7 in gene editing of Xanthomonas brasiliensis.

9. The application according to claim 8, characterized in that: The gene editing refers to gene knockout or gene knock-in.

10. A gene editing method for Xanthomonas brasiliensis, characterized in that: The step involves converting the system described in any one of claims 1-7 into the host bacteria to be modified.

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