Artificial chromosome for corynebacterium glutamicum

Abstract

The present invention provides an artificial chromosome that can be replicated in a Corynebacterium species, such as Corynebacterium glutamicum, a method for editing a genome of a Corynebacterium species, such as Corynebacterium glutamicum, by using the artificial chromosome, and a system and kit comprising the artificial chromosome.

Description

Artificial chromosomes for Corynebacterium glutamicum Technical Field

[0001] This invention relates to the fields of genetic engineering and microbiology, and in particular to the artificial chromosome (CAC) of Corynebacterium glutamicum and its use in genetic manipulation and genome synthesis in the Corynebacterium glutamicum genome. Background Technology

[0002] Corynebacterium glutamicum is a Gram-positive bacterium with a high GC content in its genome. It is an important industrial microorganism, a GRAS (Generally Recognized As Safe) species, and widely used in the fermentation production of amino acids. Furthermore, Corynebacterium glutamicum shows broad potential in metabolic engineering and synthetic biology, such as in the production of bio-based chemicals, agriculture, and pharmaceuticals.

[0003] Genome synthesis is an emerging technology for expanding genomics knowledge and discovering new functions of genome sequences. By artificially designing, assembling, and synthesizing a genome and replacing the initial genome, it is possible to explore genome-wide codon compression, large-scale genome rearrangement, and genome simplification in organisms with synthetic genomes. This is of great significance for optimizing strain performance and accelerating strain evolution. In the past fifteen years, significant progress has been made in synthesizing complete mycoplasma genomes (1Mb) and recoded Escherichia coli genomes (4Mb) (Gibson et al., Science, 2010, 329(5987):52-56; Fredens et al., Nature, 2019, 569(7757):819-822). Furthermore, as part of the Sc2.0 project, all 16 synthetic chromosomes of *Saccharomyces cerevisiae* have been assembled separately, and all synthetic chromosomes are being integrated into a single yeast strain (12 Mb) (Zhao et al., *Cell*, 2023, 186(24):5220-5236). The rapid progress in genome synthesis for these two microorganisms is largely due to their clear genetic backgrounds, abundant genome editing and recombination tools, and thorough research. More importantly, both microorganisms have available artificial chromosome vectors, which greatly accelerates the time required to synthesize a complete genome.

[0004] Although Corynebacterium glutamicum plays an important role in industrial production, its genome is difficult to manipulate. Furthermore, existing genome editing technologies for large-scale editing and replacement of the Corynebacterium glutamicum genome still suffer from the following major limitations: (a) a lack of technology for introducing large DNA fragments. Traditional plasmids have limited carrying capacity, making it difficult to introduce large DNA fragments into Corynebacterium glutamicum, or existing vector systems lack the ability to shuttle between different hosts; (b) a lack of technology for replacing large DNA fragments. Currently reported examples of Corynebacterium glutamicum genome fragment replacements are all less than 10 kb, limiting the progress of large-scale editing and modification of the Corynebacterium glutamicum genome.

[0005] Therefore, the key to Corynebacterium glutamicum genome synthesis is to develop an artificial chromosome vector that can stably load large DNA fragments, has multi-host shuttle capability, and efficient transfer function, so as to improve the efficiency and flexibility of large-scale genetic manipulation of the Corynebacterium glutamicum genome, thereby promoting its application and development in industrial production. Summary of the Invention

[0006] To meet the needs of genetic manipulation and genome synthesis in Corynebacterium glutamicum, the inventors developed an artificial chromosome vector capable of loading large DNA fragments and stably replicating and passaged in Corynebacterium glutamicum. This vector can also stably replicate and passage in other microorganisms used for molecular biology manipulations, such as Escherichia coli and Saccharomyces cerevisiae.

[0007] In a first aspect, the present invention provides an artificial chromosome vector comprising a first replication distribution element that enables the artificial chromosome vector to replicate in a first host of a Corynebacterium species, such as Corynebacterium glutamicum.

[0008] In some embodiments, the artificial chromosome vector further includes a second replication allocation element that enables the artificial chromosome vector to replicate in a second host. In some embodiments, the artificial chromosome vector further includes a third replication allocation element that enables the artificial chromosome vector to replicate in a third host.

[0009] In some embodiments, the second host is a bacterium used to amplify the artificial chromosome vector, such as *Escherichia coli*. In some embodiments, the third host is yeast, such as *Saccharomyces cerevisiae*.

[0010] In some embodiments, the first replication assignment element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum.

[0011] In some embodiments, the artificial chromosome also includes elements, such as oriT elements, capable of mediating the transfer of the artificial chromosome vector via conjugation.

[0012] In a second aspect, the present invention provides a method for editing the genome of a Corynebacterium species, such as Corynebacterium glutamicum, the method comprising the following steps:

[0013] i) Obtain the nucleic acid fragment or its sub-fragment of interest;

[0014] ii) The nucleic acid fragment of interest is cloned into the artificial chromosome vector of the present invention to obtain an artificial chromosome vector containing the nucleic acid fragment of interest;

[0015] iii) Introducing an artificial chromosome vector containing the nucleic acid fragment of interest into a recipient strain from the Corynebacterium species; and

[0016] iv) Integrate the nucleic acid fragment of interest into the chromosome of the recipient strain.

[0017] In some implementations, step ii) includes assembling a sub-fragment of the nucleosomal fragment of interest into an artificial chromosome vector via transformation-associated recombination (TAR) in a third host.

[0018] In some embodiments, the method further includes amplifying an artificial chromosome vector containing the nucleic acid fragment of interest in the second host between steps ii) and iii).

[0019] In some implementations, step iii) includes engaging the second host with the recipient strain.

[0020] In some implementations, the length of the nucleic acid fragment of interest is up to at least 10 kb.

[0021] In some embodiments, the method further includes introducing recombinases and / or endonucleases into the recipient strain.

[0022] In some implementations, the nucleic acid fragment of interest is derived from the chromosome of a donor strain of the Corynebacterium species.

[0023] In a third aspect, the invention provides the use of the artificial chromosome for delivering nucleic acid fragments of interest to Corynebacterium species, such as Corynebacterium glutamicum.

[0024] In some implementations, the length of the nucleic acid fragment of interest is up to at least 10 kb.

[0025] In a fourth aspect, the present invention provides a system for genome editing in species of the genus Corynebacterium, comprising the artificial chromosome vector of the present invention.

[0026] In some embodiments, the system further comprises a recombinase and / or a nuclease, or a polynucleotide encoding the recombinase and / or the nuclease.

[0027] In a fifth aspect, the present invention provides a kit for genome editing in species of the genus Corynebacterium, comprising the artificial chromosome vector of the present invention.

[0028] In some embodiments, the kit further comprises a recombinase and / or a nuclease, or a polynucleotide encoding the recombinase and / or the nuclease. Attached Figure Description

[0029] Figure 1 shows a schematic diagram of the replicon structure of the artificial chromosome vector of Corynebacterium glutamicum. A: Schematic diagram of the replicon structure required for vector replication in Corynebacterium glutamicum. B: Schematic diagram (left) and verification results (right) of the pCGBAC1-vio plasmid carrying a 9kb purple bacitracin expression gene cluster; C: Schematic diagram (left) and verification results (right) of the pCGBAC1-A1 plasmid carrying a 56kb synthetic genomic DNA fragment.

[0030] Figure 2 illustrates the design strategy for introducing synthetic DNA fragments of Corynebacterium glutamicum in this invention.

[0031] Figure 3 shows the spectra of the recombinant helper plasmids pXMJ19-RecET-ISceI and pXMJ19-RecET-Cas9.

[0032] Figure 4 shows the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmids pCGBAC1-A4a and pCGBAC1-A4, which are loaded with large DNA fragments chunk A4a and chunk A4, into the recipient strain semi-synCG-A3. A: Flowchart of the construction and introduction of Corynebacterium glutamicum artificial chromosome plasmids; B and C: Schematic diagrams of the structure of the Corynebacterium glutamicum artificial chromosome plasmids pCGBAC1-A4a (B) and pCGBAC1-A4 (C) (top), enzyme digestion verification results (bottom left), and transformants obtained by transforming Corynebacterium glutamicum with Corynebacterium glutamicum artificial chromosomes by PCR verification (bottom right); and D: Results of electroconversion efficiency.

[0033] Figure 5 illustrates the strategy and results of large-segment genomic substitution mediated by *Corynebacterium glutamicum* artificial chromosome under RecET and Cas9 or I-SceI mediation. A: Schematic diagram of large-segment genomic substitution mediated by *Corynebacterium glutamicum* artificial chromosome under RecET and Cas9 mediation; B: Schematic diagram of large-segment genomic substitution mediated by *Corynebacterium glutamicum* artificial chromosome under RecET and I-SceI mediation; C: Validation results of large-segment genomic substitution mediated by *Corynebacterium glutamicum* artificial chromosome, the left figure shows the PCR validation results, and the right figure shows the sequencing validation results of the genomic substitution structure.

[0034] Figure 6 shows the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC2-A5 loaded with a large chunk A5 DNA fragment into the recipient strain semi-synCG-A4. A: Flowchart of the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmid; B: Schematic diagram of the structure of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC2-A5 (left) and the enzyme digestion verification results (right); C: Verification of transformants introduced into Corynebacterium glutamicum by electroporation (top left) and conjugation (top right) of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC2-A5, and verification of all synthetic PCR tags of representative transformants (bottom); and D: Transformation efficiency of Corynebacterium glutamicum artificial chromosome plasmid pCGBAC2-A5 by electroporation and conjugation.

[0035] Figure 7 shows the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A6 loaded with a large chunk A6 DNA fragment into the recipient strain semi-synCG-A5. A: Flowchart of the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmid; B: Schematic diagram of the structure of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A6 (left) and enzyme digestion verification results (right); C: Verification of transformants introduced into Corynebacterium glutamicum by electroporation and conjugation of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A6; and D: Transformation efficiency of Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A6 by electroporation and conjugation.

[0036] Figure 8 illustrates the strategy and results of continuous large-segment genomic substitution in Corynebacterium glutamicum artificial chromosome mediated by RecET and I-SceI. A and B: Schematic diagram of chunk A6-A8 substitution strategy; C: Electrophoresis verification of chunk A6-A8 substitution.

[0037] Figure 9 shows the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-80k loaded with the A9b-A10 DNA fragment and introduced into the Corynebacterium glutamicum strain semi-synCG-A9a. A: Flowchart of the construction and introduction of the Corynebacterium glutamicum artificial chromosome plasmid; B: Schematic diagram of the structure of the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-80k (left) and enzyme digestion verification results (right); C: Verification of PCRTag of the transformants of Corynebacterium glutamicum introduced with the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-80k; and D: Transformation efficiency of Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-80k introduced into Corynebacterium glutamicum.

[0038] Invention Details

[0039] This invention is not limited to the specific methods, schemes, reagents, etc., described herein, as these can vary. The terminology used herein is for the purpose of describing specific embodiments only and not for limiting the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Nucleic acid chemistry, molecular biology, and microbiology-related terms and laboratory procedures used herein are widely used terms and routine procedures in their respective fields. Experimental methods not specifically described in this invention are performed according to the specific methods in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th Edition) or according to the relevant product instructions. Unless otherwise specified, the biological reagents used in this invention are commercially available. Those skilled in the art can make various changes, modifications, and substitutions without departing from the spirit of this invention.

[0040] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0041] Unless otherwise stated, nucleic acid sequences are referred to in this article in the 5′ to 3′ direction from left to right.

[0042] 1. Artificial chromosome of Corynebacterium glutamicum and its construction

[0043] To meet the needs of genetic manipulation and genome synthesis in the Corynebacterium glutamicum genome, the inventors developed a bacterial artificial chromosome vector capable of loading large DNA fragments and stably replicating and propagating in Corynebacterium glutamicum, namely Corynebacterium glutamicum artificial chromosome (CAC).

[0044] As used herein, the terms “Corynebacterium glutamicum artificial chromosome,” “CAC,” “CAC vector,” and “CAC plasmid” are used interchangeably to refer to a bacterial artificial chromosome vector that can replicate in Corynebacterium glutamicum cells and contains replication allocation elements required for replication.

[0045] As used in this article, "bacterial artificial chromosome (BAC)" refers to a bacterial chromosome cloning vector constructed based on F plasmids (F-plasmid) or RK2 plasmids (see, for example, Akos et al., Synthetic genomes unveil the effects of synonymous recoding, bioRxiv, 2024). It is commonly used to clone DNA fragments of approximately 150 kb in size and can carry up to 300 k base pairs. BAC plasmids mainly include the following elements: based on F plasmids, oriS, repE (controlling F plasmid replication) and parA, parB (controlling copy number), or based on RK2 plasmids, or oriV, trfA, etc. Vectors cloned based on BACs have a low frequency of chimerism, high transformation efficiency, and exist in a circular structure within bacteria, making them easy to identify, isolate, and purify. They have been widely accepted by the scientific community. They are mainly used for the construction of large-fragment genomic libraries and related research on large gene clusters, and play an important role in various biological genome projects. However, the various BACs known in existing technologies cannot replicate in Corynebacterium glutamicum and are not suitable for genome studies of Corynebacterium glutamicum.

[0046] To address the problems in the prior art, the inventors introduced a new replication allocation element into BAC, thereby endowing the obtained artificial chromosome with the ability to replicate in Corynebacterium glutamicum.

[0047] Therefore, the present invention provides an artificial chromosome vector comprising a first replication distribution element that enables the artificial chromosome vector to replicate in a first host of a Corynebacterium species, such as Corynebacterium glutamicum.

[0048] As used herein, the term "replication allocation element," also known as a "replication allocation sequence," refers to a nucleotide sequence in a vector (e.g., a plasmid) that controls plasmid replication within the host cell and allocation among daughter cells during host cell division. Replication allocation elements typically include a rep gene (e.g., repA II), a nic site and a 22-bp site (repA binding site), parA, parB genes, and / or their recognition sites or parS sites (see Figure 1A). For example, the replication allocation element may include a rep gene (e.g., repA II), a nic site, a 22-bp site, and a parS site.

[0049] The inventors discovered that inserting a suitable parS site into the replication distribution element enables the artificial chromosome to replicate and distribute in Corynebacterium species, particularly Corynebacterium glutamicum.

[0050] In some embodiments, the first replication allocation element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum. Preferably, the parS site comprises the nucleotide sequence of SEQ ID NO:1. In some embodiments, the parS site replaces the parA and parB sequences (parA and parB protein recognition sites) in the replication allocation element.

[0051] For molecular biological manipulation, the CAC vector of this invention can also stably replicate and passage in other microorganisms such as *Escherichia coli* and *Saccharomyces cerevisiae*. In other words, the CAC vector of this invention is a shuttle artificial chromosome.

[0052] In molecular biology research, before transforming a target host with a vector (such as the CAC of the present invention), the vector needs to be amplified in a suitable host in order to obtain a sufficient amount of the vector for transforming the target host (i.e., the first host).

[0053] Therefore, in some embodiments, the artificial chromosome vector further comprises a second replication allocation element that enables the artificial chromosome vector to replicate in a second host. The second host is suitable for amplifying the vector. Suitable hosts for amplifying the vector include, but are not limited to, *Escherichia coli* and yeasts, such as *Saccharomyces cerevisiae*.

[0054] In some implementations, the second host is a bacterium used to amplify the artificial chromosome vector, such as Escherichia coli.

[0055] Furthermore, in order to construct a desired vector capable of carrying large fragments, such as the CAC vector of the present invention, it is necessary to assemble various elements into the desired vector in a suitable host cell through, for example, recombination.

[0056] In some embodiments, the artificial chromosome vector further includes a third replication allocation element that enables the artificial chromosome vector to replicate in a third host. The third host is used to assemble the vector.

[0057] In some implementations, the third host is yeast, such as Saccharomyces cerevisiae.

[0058] In addition to transferring the separated vector into the target host (i.e., the first host), the CAC vector of the present invention can also be transferred from the second host to the first host through conjugation. Therefore, elements capable of mediating vector transfer through conjugation can be incorporated into the CAC vector of the present invention.

[0059] In some embodiments, the artificial chromosome also includes elements capable of mediating the transfer of the artificial chromosome vector through conjugation, such as the oriT element (Fredens et al., Nature, 2019, 569: 514-518).

[0060] To screen host cells containing the vector of the present invention, the vector further comprises selectable markers. These markers can be positive or negative selectable markers.

[0061] As used herein, the term "positive selection marker" refers to a condition under which a transformant containing the positive selection marker can grow, while a transformant without the positive selection marker cannot grow. "Negative selection marker" refers to a condition under which a transformant containing the negative selection marker cannot grow, while a transformant without the negative selection marker can grow.

[0062] For microorganisms, positive selection markers can be antibiotic resistance genes or supplemental auxotrophic genes.

[0063] Examples of screening markers for screening Saccharomyces cerevisiae include, but are not limited to, HIS3 and / or URA3, with HIS3 being preferred. Examples of screening markers for screening Escherichia coli and / or Corynebacterium glutamicum include, but are not limited to, kanR (e.g., SEQ ID NO:2), speR (e.g., SEQ ID NO:3), apmR (e.g., SEQ ID NO:4), and cat (e.g., SEQ ID NO:5).

[0064] Examples of negative selection markers used for genome substitution screening of Corynebacterium glutamicum include, but are not limited to, rpsL, sacB, and pheS*. Specifically, the rpsL negative selection marker indicates that strains possessing a mutant copy of rpsLK43R (as shown in SEQ ID NO:6) in their genome are streptomycin resistant, strains possessing a wild-type copy of rpsL in their genome are sensitive to streptomycin, and strains possessing a wild-type copy of rpsL in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The sacB negative selection marker (as shown in SEQ ID NO:7) indicates that strains possessing the sacB gene in their genome are sensitive to sucrose, and strains possessing the sacB gene in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The pheS* negative selection marker indicates that strains possessing a wild-type pheS copy in their genome, as well as a mutant copy of pheS* (pheS T262A A309G) (as shown in SEQ ID NO:8), are sensitive to 4-chlorophenylalanine. The negative selection markers mentioned above can be expressed using endogenous constitutive promoters of Corynebacterium glutamicum such as Ptuf (as shown in SEQ ID NO:9).

[0065] For the artificial chromosome vector of the present invention, a backbone known in the art for BAC and / or YAC can be used, or a YAC / BAC chimeric artificial chromosome vector backbone can be used (see, for example, Fredens et al., Nature, 2019, 569(7757):514-518).

[0066] The vector's "backbone" refers to the portion of the vector other than the nucleic acid sequence of interest, which may contain replication-assignment elements, selection markers, and / or elements that mediate transfer via conjugation. The vector backbone can be modified by replacing these elements.

[0067] The artificial chromosome vectors of the present invention encompass vectors that do not carry a nucleic acid fragment of interest (also referred to as empty vectors) and vectors that carry a nucleic acid fragment of interest. The nucleic acid fragment of interest may be a DNA fragment to be transferred into a recipient strain, preferably to be integrated into the genome of the recipient strain. The vector can be cleaved (e.g., within the recipient strain) by a nuclease to separate the nucleic acid fragment of interest from the vector backbone. Therefore, in some embodiments, the vector contains nuclease cleavage sites flanking the nucleic acid fragment of interest. The nuclease may be a broad range of nucleases (such as I-SceI), zinc finger nucleases, TALEN, or CRISPR nucleases.

[0068] The nucleic acid fragment of interest, separated from the vector backbone, can be integrated into the genome of the recipient strain via, for example, homologous recombination. In some embodiments, the nucleic acid fragment of interest comprises homologous arms at both ends.

[0069] As used herein, the term "homologous arm" refers to a nucleotide sequence that matches a site of interest in the genome and is capable of mediating homologous recombination, allowing DNA fragments between homologous arms to integrate into the site of interest in the genome. For example, the sequence of a homologous arm can be identical to or complementary to a specific sequence in the genome.

[0070] This invention also provides a method for constructing the artificial chromosome vector of this invention, comprising:

[0071] i) Provide a fragment containing at least the first replication allocation element, a fragment containing a selection marker, and optionally a fragment containing the second replication allocation element, a fragment containing the third replication allocation element, a fragment containing the element mediating the transfer of the artificial chromosome vector via conjugation, and / or a DNA fragment of interest or a sub-fragment thereof, and

[0072] ii) Assemble the fragments from step i) into the artificial chromosome vector.

[0073] In some embodiments, an empty artificial chromosome vector without the nucleic acid sequence of interest is assembled via Gibson Assembly. In some embodiments, the third host is *Saccharomyces cerevisiae*, and an artificial chromosome vector containing the nucleic acid fragment of interest is assembled via yeast TAR.

[0074] 2. Methods for delivering nucleic acid fragments of interest and editing the Corynebacterium genome

[0075] The present invention also provides a method for delivering a nucleic acid fragment of interest to a Corynebacterium species (such as Corynebacterium glutamicum) or editing the genome of a Corynebacterium species (such as Corynebacterium glutamicum), the method comprising the following steps:

[0076] i) Obtain the nucleic acid fragment or its sub-fragment of interest;

[0077] ii) Cloning the nucleic acid fragment of interest into an artificial chromosome vector to obtain an artificial chromosome vector containing the nucleic acid fragment of interest, wherein the artificial chromosome contains a first replication allocation element that enables the artificial chromosome vector to replicate in the Corynebacterium species (first host);

[0078] iii) Introducing an artificial chromosome vector containing the nucleic acid fragment of interest into a recipient strain from the Corynebacterium species; and

[0079] iv) Optionally, the nucleic acid fragment of interest is integrated into the chromosome of the recipient strain.

[0080] As used herein, “editing” the genome of a microorganism (such as a species of Corynebacterium) encompasses introducing nucleotide substitutions, insertions, and / or deletions into the genome of the microorganism, such as inserting a nucleic acid fragment of interest or replacing a DNA fragment in the original genome with a nucleic acid fragment of interest. In some embodiments, the nucleic acid fragment of interest is a naturally occurring DNA fragment, for example, a DNA fragment from a donor strain possessing superior traits (such as higher growth rate, higher carbon source conversion, and higher productivity). The DNA fragment may contain chromosomal segments in the donor strain identified as being associated with the superior trait. The nucleic acid fragment of interest may also be a modified DNA fragment, for example, through synonymous mutations of the stop codon (such as TAG→TAA), insertion of the loxPsym site, decoupling of overlapping genes, deletion of an insertion sequence (IS) of >500 bp, and / or the introduction of a synonymous mutation watermark “PCRTag” onto the genome sequence to distinguish between synthetic and wild-type sequences (see Ye et al., ACS Synthetic Biology, 2022, 11, 1588-1599). In some implementations, the nucleic acid fragment of interest is derived from the chromosome of a donor strain of the Corynebacterium species.

[0081] In step i), the nucleic acid fragment or its sub-fragments of interest can be obtained by any method known in the art, such as PCR-based amplification and direct synthesis using a nucleic acid synthesizer.

[0082] If the nucleic acid fragment of interest is too large, a vector carrying the nucleic acid fragment of interest can be obtained by assembling its sub-fragments, for example, via Gibson Assembly.

[0083] In some embodiments, step ii) includes assembling a sub-fragment of the nucleosomal fragment of interest into the artificial chromosome vector via transformation-associated recombination (TAR) in a third host. In step ii), an artificial chromosome vector carrying the nucleosomal fragment of interest is obtained. In some embodiments, the first replication allocation element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum. Preferably, the parS site comprises the nucleotide sequence of SEQ ID NO:1. In some embodiments, the parS site replaces the parA and parB sequences in the replication allocation element.

[0084] In some embodiments, the artificial chromosome vector further includes a third replication allocation element that enables the artificial chromosome vector to replicate in a third host. The third host is used to assemble the vector. Suitable host cells for assembling the vector include, but are not limited to, yeast, such as *Saccharomyces cerevisiae*.

[0085] In some embodiments, the method further includes amplifying an artificial chromosome vector containing the nucleic acid fragment of interest in the second host between steps ii) and iii).

[0086] In some embodiments, the artificial chromosome vector further includes a second replication-distribution element that enables the artificial chromosome vector to replicate in a second host. The second host is suitable for amplifying the vector. Suitable hosts for amplifying the vector include, but are not limited to, *Escherichia coli* and yeasts, such as *Saccharomyces cerevisiae*.

[0087] In some implementations, the second host is a bacterium used to amplify the artificial chromosome vector, such as Escherichia coli.

[0088] In some embodiments, step iii) includes conjugating the second host to the recipient strain. In some embodiments, the artificial chromosome also includes elements capable of mediating the transfer of the artificial chromosome vector via conjugation, such as oriT elements.

[0089] In some implementations, the length of the nucleic acid fragment of interest is up to at least 10 kb.

[0090] In some embodiments, the method further includes introducing a recombinase and / or an endonuclease into the recipient strain. In some embodiments, the vector contains endonuclease cleavage sites flanking the nucleic acid fragment of interest. Examples of recombinases include, but are not limited to, RecE-RecT (RecET), gp61-gp60 (Mycobacterial phage Che9c), and orfC-orfB (Legionella pneumophila) (see, for example, Li et al., Optimizing recombineering in Corynebacterium glutamicum, Biotechnology and Bioengineering, 2021; 118:2255-2264). The endonuclease can be a wide range of nucleases (such as I-SceI), zinc finger nucleases, TALENs, or CRISPR nucleases.

[0091] The isolated fragment can be integrated into the genome of the recipient strain, for example, through homologous recombination. In some embodiments, the nucleic acid fragment of interest includes homologous arms at both ends.

[0092] The present invention also provides the use of the artificial chromosome of the present invention for delivering nucleic acid fragments of interest to Corynebacterium species, such as Corynebacterium glutamicum.

[0093] In some implementations, the length of the nucleic acid fragment of interest is at least 10kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, or more.

[0094] In some implementations, the method further includes a step of removing the carrier skeleton.

[0095] In some implementations, the method further includes iterative steps i)–iv) with different nucleic acid fragments of interest.

[0096] 3. Systems and kits for editing genomes

[0097] The present invention provides a system for genome editing in Corynebacterium species (such as Corynebacterium glutamicum), comprising an artificial chromosome vector including a first replication allocation element that enables the artificial chromosome vector to replicate in a first host of the Corynebacterium species, such as Corynebacterium glutamicum.

[0098] In some embodiments, the first replication allocation element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum. Preferably, the parS site comprises the nucleotide sequence of SEQ ID NO:1. In some embodiments, the parS site replaces the parA and parB sequences in the replication allocation element.

[0099] In some embodiments, the artificial chromosome vector further includes a second replication-distribution element that enables the artificial chromosome vector to replicate in a second host. The second host is suitable for amplifying the vector. Suitable hosts for amplifying the vector include, but are not limited to, *Escherichia coli* and yeasts, such as *Saccharomyces cerevisiae*.

[0100] In some implementations, the second host is a bacterium used to amplify the artificial chromosome vector, such as Escherichia coli.

[0101] In some embodiments, the artificial chromosome vector further includes a third replication allocation element that enables the artificial chromosome vector to replicate in a third host. The third host is used to assemble the vector.

[0102] In some implementations, the third host is yeast, such as Saccharomyces cerevisiae.

[0103] In some embodiments, the artificial chromosome also includes elements, such as oriT elements, capable of mediating the transfer of the artificial chromosome vector via conjugation.

[0104] To screen host cells containing the vector of the present invention, the vector further comprises selectable markers. These markers can be positive or negative selectable markers.

[0105] Examples of screening markers for screening Saccharomyces cerevisiae include, but are not limited to, HIS3 and / or URA3, with HIS3 being preferred. Examples of screening markers for screening Escherichia coli and / or Corynebacterium glutamicum include, but are not limited to, kanR (e.g., SEQ ID NO:2), speR (e.g., SEQ ID NO:3), apmR (e.g., SEQ ID NO:4), and cat (e.g., SEQ ID NO:5).

[0106] Examples of negative selection markers used for genome substitution screening in Corynebacterium glutamicum include, but are not limited to, rpsL, sacB, and pheS*. Specifically, the rpsL negative selection marker indicates that strains possessing a mutant copy of rpsL K43R (as shown in SEQ ID NO:6) in their genome are streptomycin resistant, strains possessing a wild-type copy of rpsL in their genome are sensitive to streptomycin, and strains possessing a wild-type copy of rpsL in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The sacB negative selection marker (as shown in SEQ ID NO:7) indicates that strains possessing the sacB gene in their genome are sensitive to sucrose, and strains possessing the sacB gene in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The pheS* negative selection marker indicates that strains possessing a wild-type pheS copy in their genome, as well as a mutant copy of pheS* (pheS T262A A309G) (as shown in SEQ ID NO:8), are sensitive to 4-chlorophenylalanine. The negative selection markers mentioned above can be expressed using endogenous constitutive promoters of Corynebacterium glutamicum such as Ptuf (as shown in SEQ ID NO:9).

[0107] For the artificial chromosome vector, a backbone known in the art for BAC and / or YAC can be used, or a YAC / BAC chimeric artificial chromosome vector backbone can be used (see, for example, Fredens et al., Nature, 2019, 569(7757):514-518).

[0108] In some embodiments, the system further comprises a recombinase and / or an endonuclease, or a polynucleotide encoding the recombinase and / or endonuclease. Examples of the recombinase include, but are not limited to, RecE-RecT (Escherichia coli Rac phage), gp61-gp60 (Mycobacterium phage Che9c), and orfC-orfB (Legionella pneumophila). The endonuclease can be a broad range of nucleases (such as I-SceI), zinc finger nucleases, TALEN, or CRISPR nucleases.

[0109] In some embodiments, the vector further comprises a site for recognition and / or cleavage by the endonuclease.

[0110] The present invention also provides a kit for genome editing in Corynebacterium species, comprising an artificial chromosome vector or a fragment thereof, the artificial chromosome vector comprising a first replication allocation element that enables the artificial chromosome vector to replicate in a first host of the Corynebacterium species, such as Corynebacterium glutamicum.

[0111] Those skilled in the art will understand that vector fragments can be assembled using conventional techniques to obtain the desired vector. Therefore, they will appreciate that the artificial chromosome vector does not necessarily need to exist as a complete vector, but can be in the form of a fragment. The fragment of the artificial chromosome vector can be a separated polynucleotide fragment, or it can be cloned into a suitable vector.

[0112] In some embodiments, the first replication allocation element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum. Preferably, the parS site comprises the nucleotide sequence of SEQ ID NO:1. In some embodiments, the parS site replaces the parA and parB sequences in the replication allocation element.

[0113] In some embodiments, the artificial chromosome vector further includes a second replication-distribution element that enables the artificial chromosome vector to replicate in a second host. The second host is suitable for amplifying the vector. Suitable hosts for amplifying the vector include, but are not limited to, *Escherichia coli* and yeasts, such as *Saccharomyces cerevisiae*.

[0114] In some implementations, the second host is a bacterium used to amplify the artificial chromosome vector, such as Escherichia coli.

[0115] In some embodiments, the artificial chromosome vector further includes a third replication allocation element that enables the artificial chromosome vector to replicate in a third host. The third host is used to assemble the vector.

[0116] In some implementations, the third host is yeast, such as Saccharomyces cerevisiae.

[0117] In some embodiments, the artificial chromosome also includes elements, such as oriT elements, capable of mediating the transfer of the artificial chromosome vector via conjugation.

[0118] To screen host cells containing the vector of the present invention, the vector further comprises selectable markers. These markers can be positive or negative selectable markers.

[0119] Examples of screening markers for screening Saccharomyces cerevisiae include, but are not limited to, HIS3 and / or URA3, with HIS3 being preferred. Examples of screening markers for screening Escherichia coli and / or Corynebacterium glutamicum include, but are not limited to, kanR (e.g., SEQ ID NO:2), speR (e.g., SEQ ID NO:3), apmR (e.g., SEQ ID NO:4), and cat (e.g., SEQ ID NO:5).

[0120] Examples of negative selection markers used for genome substitution screening in Corynebacterium glutamicum include, but are not limited to, rpsL, sacB, and pheS*. Specifically, the rpsL negative selection marker indicates that strains possessing a mutant copy of rpsL K43R (as shown in SEQ ID NO:6) in their genome are streptomycin resistant, strains possessing a wild-type copy of rpsL in their genome are sensitive to streptomycin, and strains possessing a wild-type copy of rpsL in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The sacB negative selection marker (as shown in SEQ ID NO:7) indicates that strains possessing the sacB gene in their genome are sensitive to sucrose, and strains possessing the sacB gene in the CAC vector tend to lose plasmids without the corresponding positive selection marker. The pheS* negative selection marker indicates that strains possessing a wild-type pheS copy in their genome, as well as a mutant copy of pheS* (pheS T262A A309G) (as shown in SEQ ID NO:8), are sensitive to 4-chlorophenylalanine. The negative selection markers mentioned above can be expressed using endogenous constitutive promoters of Corynebacterium glutamicum such as Ptuf (as shown in SEQ ID NO:9).

[0121] For the artificial chromosome vector, a backbone known in the art for BAC and / or YAC can be used, or a YAC / BAC chimeric artificial chromosome vector backbone can be used (see, for example, Fredens et al., Nature, 2019, 569(7757):514-518).

[0122] In some embodiments, the system further comprises a recombinase and / or an endonuclease, or a polynucleotide encoding the recombinase and / or endonuclease. Examples of the recombinase include, but are not limited to, RecE-RecT, gp61-gp60 (Mycobacterium phage Che9c), and orfC-orfB (Legionella pneumophila). The endonuclease can be a wide range of nucleases (such as I-SceI), zinc finger nucleases, TALEN, or CRISPR nucleases.

[0123] In some embodiments, the vector further comprises a site for recognition and / or cleavage by the endonuclease. Beneficial effects

[0124] This invention provides a Corynebacterium glutamicum artificial chromosome (CAC) vector, its construction method, and its uses, particularly for introducing large DNA fragments into Corynebacterium glutamicum. More importantly, the Corynebacterium glutamicum artificial chromosome of this invention can be applied in genome synthesis to achieve efficient replacement of large genome fragments, especially for large-scale genome editing of Corynebacterium glutamicum. Example

[0125] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the embodiments should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0126] Unless otherwise specified, all methods used in the following examples are conventional methods. For specific steps, please refer to, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). All primers used were synthesized by Shanghai Sangon Biotech.

[0127] The Corynebacterium glutamicum strain semi-synCG-An (n=1-10) used in the following examples is a Corynebacterium glutamicum genome synthesis intermediate strain. It is based on the Corynebacterium glutamicum ATCC13032 strain, which undergoes an rpsL K43R mutation and sequentially completes the continuous genome replacement from chunk A1 to chunk An. Specifically, strain semi-synCG-A1 is the strain in which the synthesized DNA fragment chunk A1 (about 55kb) replaces the wild-type sequence in the original genome, as described in Ye et al.'s article (ACS Synthetic Biology, 2022, 11(4):1588-1599); strain semi-synCG-A3 is the strain in which synthetic DNA chunks A2 and A3 (about 110kb in total) are introduced into semi-synCG-A1 according to the method described in Ye et al.'s article, and the wild-type sequence in the genome is replaced. Furthermore, to achieve genomic sequence replacement, a plasmid responsible for recombination was introduced into strain semi-synCG-A3, resulting in strain semi-synCG-A3 / RIS. From strain semi-synCG-A3 / RIS, strain semi-synCG-Am (m = 4-10) was obtained, and chunks A4-A10 (approximately 27-55 kb each) were introduced into semi-synCG-A(m-1) using the vector of this invention. Additionally, for verification purposes, chunk A4a (a fragment of approximately 30 kb from the 5' end of chunk A4) was also constructed. The semi-synCG-A9a strain was obtained by introducing chunk A9a (a fragment of approximately 30 kb from the 5' end of chunk A9) into semi-synCG-A8. The semi-synCG-A9 strain was obtained by introducing chunk A9a (a fragment of approximately 27 kb from the 5' end of chunk A9) and chunk A9b (a fragment of approximately 29 kb from the 3' end of chunk A9) into semi-synCG-A8. Replacing chunk A10 yielded the semi-synCG-A10 strain. Chunk A9b and chunk A10 (approximately 80 kb in total) can also be introduced into semi-synCG-A9a using the vector of this invention. Each synthetic DNA fragment (chunks A1-A10) was designed and synthesized by BGI Genomics into the mini chunks described below (see Figure 2).

[0128] For cloning, each chunk is divided into several mini chunks, with adjacent mini chunks overlapping each other by 60-80 bp as homologous arms, and assembled using yeast TAR. After assembly, interface verification can be performed using PCR tags between adjacent mini chunks, i.e., by performing PCR reactions using i) a forward primer targeting the upstream mini chunk and ii) a reverse primer targeting the downstream mini chunk. If the bands match expectations, it indicates that the adjacent mini chunks have assembled as expected.

[0129] In the following examples, if the plasmid assembly uses the Gibson Assembly method (see https: / / www.neb.cn / applications / cloning-and-synthetic-biology / dna-assembly-and-cloning / gibson-assembly), the assembled plasmid is transformed into *E. coli* DH5α or DH10b strain via chemical transformation and then validated by colony PCR and sequencing. *E. coli* colony PCR validation was performed as follows: a single colony was picked from the solid culture medium and placed in ddH2O, lysed at 98°C for 10 minutes, centrifuged for 5 minutes, and the supernatant was used as a template (added at 10% of the reaction volume) for PCR. The PCR reaction used T5 polymerase from Beijing Qingke Company, with a 15 μL reaction volume. The PCR conditions were: 98°C for 3 min; 98°C for 10 s, 61°C for 10 s, 72°C for 4 kb / min, for a total of 30 cycles; and a final 72°C for 2 min. After the reaction was completed, the PCR amplification products were detected by 1% agarose gel electrophoresis.

[0130] In the following examples, unless otherwise described, the assembly of Corynebacterium glutamicum artificial chromosome (CAC) plasmids loaded with large DNA fragments was performed via transformation-associated recombination (TAR) in *Saccharomyces cerevisiae*: *Saccharomyces cerevisiae* MYA3666 competent cells were prepared using the Zymo Research Frozen-EZ Yeast Transformation II Kit, aliquoted into 100 μL tubes. 200 ng of each fragment was added to each 100 μL competent cell, with homologous arms approximately 60-80 bp between fragments. After incubation at 30°C for 2 hours, cells were plated on SD-His plates containing 2% glucose to screen for positive clones, which were then verified by colony PCR. Colony PCR verification of *Saccharomyces cerevisiae* was performed as follows: single colonies were picked from the solid culture medium and lysed in 0.1% NaOH solution at 95°C for 30 minutes, centrifuged for 5 minutes, and the supernatant was used as a template (added at 10% of the reaction volume) for PCR. PCR reactions were performed using Toyobo's KOD FX polymerase in a 15 μL reaction volume. The PCR conditions were: 94℃ for 3 min; 98℃ for 10 s, 61℃ for 30 s, 68℃ at 1 kb / min for 30 cycles; and a final 68℃ for 5 min. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis. Total DNA was extracted from validated Saccharomyces cerevisiae cultures using the Tiangen yeast genome extraction kit.

[0131] In the following examples, plasmid electroporation of Escherichia coli NEB 10-beta strain was performed as follows: 10 μL of DNA solution was added to 100 μL of competent cells (purchased from New England Biolab (NEB)), and electroporation was performed at 2500 V and 5.0 ms. After recovery at 37°C for 1 hour, the cells were plated on LB plates containing the corresponding antibiotics to screen for positive clones, and verified by colony PCR.

[0132] In the following examples, the restriction enzyme digestion verification of Corynebacterium glutamicum artificial chromosome (CAC) plasmids loaded with large DNA fragments was performed as follows: After extracting plasmids from E. coli NEB 10-beta using the Macherey-Nagel BAC 100kit kit, approximately 4 μL of the extracted DNA solution was mixed with 1 μL CutSmart, 1 μL of DNA endonuclease (ApaI for A4b; I-SceI for pCGBAC1-A4, pCGBAC1-A5, pCGBAC1-A6 and pCGBAC1-80k, purchased from New England Biolab (NEB)), approximately 4 μL ddH2O, or 1 μL CutSmart, 1 μL enzyme 1, 1 μL enzyme 2 (SpeI-HF+NotI-HF for pCGBAC1-A1 and pCGBAC1-A4a, purchased from NEB), approximately 3 μL ddH2O, and incubated at 37°C for 3 hours for restriction enzyme digestion. The enzyme digestion products were separated by 0.6% agarose gel electrophoresis. The gel was imaged and the images were inverted. Grayscale comparison was performed using ImageJ software. The concentration of the extracted plasmid was relatively quantified using 1kb Extended DNA Marker (New England Biolab (NEB)) and DL 15000 DNA Marker (Takara) as references.

[0133] The plasmid electroporation of Corynebacterium glutamicum was performed as follows: The preparation of competent cells for electroporation of Corynebacterium glutamicum was carried out according to the method described by Ye et al., ACS Synthetic Biology, 2022, 11(4):1588-1599. Specifically, the strain to be transformed was inoculated into BHIS liquid medium containing 10 μg / mL chloramphenicol and 150 μg / mL spectinomycin and cultured overnight. The next day, it was transferred to NCM liquid medium and cultured at 30°C and 220 rpm on a shaker until OD600 = 0.3. A final concentration of 1 mM IPTG and 0.5% L-arabinose were added, and the culture was continued at 30°C and 220 rpm until the bacterial concentration reached OD600 = 1.0. Competent cells were prepared and dispensed into 100 μL tubes. Plasmids (minimum 100 ng, maximum 10 μL) were electroporated into 100 μL of *Corynebacterium glutamicum*. After resuscitation in BHIS medium for 1.5 hours, cells were plated on BHIS plates containing the corresponding antibiotics to screen for positive clones, which were then verified by colony PCR. Colony PCR verification of *Corynebacterium glutamicum* was performed as follows: single colonies were picked from the solid medium and lysed in 0.1% NaOH solution at 98°C for 25 minutes, centrifuged for 5 minutes, and the supernatant was used as template (added at 10% of the reaction volume) for PCR. The PCR reaction used Toyobo's KOD FX polymerase in a 15 μL reaction volume. The PCR conditions were: 94°C for 3 min; 98°C for 10 s, 61°C for 30 s, 68°C for 1 kb / min, for a total of 30 cycles; and a final 68°C for 2 min. After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis.

[0134] Example 1: Construction of Corynebacterium glutamicum artificial chromosome (CAC) vector

[0135] The purpose of this embodiment is to construct a bacterial artificial chromosome that can replicate in Corynebacterium glutamicum.

[0136] The BAC-Cat fragment (primers SEQ ID NO:10, SEQ ID NO:11) was amplified using the pBeloBAC11 plasmid (Addgene) as a template. It contains the replication and allocation sequence of the bacterial artificial chromosome (BAC) for Escherichia coli and the Cat fragment.

[0137] The candidate Corynebacterium glutamicum artificial chromosome (CAC) replication allocation sequences were derived from the pCXC100 minimal replicon (GenBank Acc.No. AY 380839; Li et al., FEMS Microbiology Letters, 2004, 236(1):33-39) and pBL90 (GenBank: KU306397.1; Novikov et al., Russian Journal of Genetics, 2016, 1131-1136), respectively. These replication allocation sequences were synthesized by Shanghai Jierui and cloned into the pOK12 backbone (pOK12CXC100, pOK12BL90). The synthetic products were used as templates to amplify the CXC100 fragment (primers: SEQ ID NO:12, SEQ ID NO:13) and BL90 fragment (primers: SEQ ID NO:14, SEQ ID NO:13) of the pCXC100 and pBL90 minimal replicons.

[0138] The selection marker kanR (SEQ ID NO:2) was amplified using pEC-XK99E plasmid (Addgene) as a template; the selection marker speR (SEQ ID NO:3) was amplified using pJYS2_crtYf plasmid (Addgene) as a template; the selection marker apmR (SEQ ID NO:4) was synthesized by Beijing Ruibo; the oriT fragment required for conjugation was amplified using pK18mobsacB plasmid (Addgene) as a template (primers were SEQ ID NO:15 and SEQ ID NO:16).

[0139] pOK12CXC100 and pOK12BL90 were electroporated into Corynebacterium glutamicum, and screened with kanamycin. No transformants were produced after electroporation of Corynebacterium glutamicum with pOK12CX100, indicating that the minimum replicon of pCXC100 cannot serve as a replication element for Corynebacterium glutamicum plasmids on its own. However, transformants were produced after electroporation of Corynebacterium glutamicum with pOK12BL90, suggesting that the replicon of pBL90 has the potential to serve as a replication element for Corynebacterium glutamicum.

[0140] The YAC-HIS3 fragment (primers SEQ ID NO:17 and SEQ ID NO:18) was amplified using pRSII313 plasmid (Addgene) as a template. It contains the replication and allocation sequence of yeast artificial chromosome (YAC) and the HIS3 marker gene.

[0141] The PCR reaction for fragment amplification was performed as follows: PCR amplification was carried out in a 50 μL system using plasmid DNA as a template. Specifically, Toyobo's KOD One polymerase was used, and amplification was performed under the following conditions: 98℃ for 10 s, 61℃ for 5 s, 68℃ at 20 kb / min, for a total of 30 cycles. After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis, and the amplification products were recovered using a gel recovery kit purchased from Magen.

[0142] The BAC-Cat, oriT, YAC-HIS3, and BL90 fragments were assembled using Gibson Assembly (see https: / / www.neb.cn / applications / cloning-and-synthetic-biology / dna-assembly-and-cloning / gibson-assembly) to obtain the pCGBACYT90 empty vector. The assembly product was transformed into *E. coli* DH10b competent cells (Thermo Fisher) using the CaCl2 heat shock method. Transformed cells were plated on LB agar plates supplemented with 17 μg / mL chloramphenicol for screening positive clones. Plasmids were extracted and sequenced, and the sequencing results showed that the cloned plasmid sequence was correct.

[0143] Using the empty pCGBACYT90 vector as a template, the BAC-oriT fragment (primers SEQ ID NO:19 and SEQ ID NO:20) and the YAC-CAC-Cat fragment (primers SEQ ID NO:21 and SEQ ID NO:22) were amplified. Using the semi-synCG-A1 synthetic genome sequence (Ye et al., ACS Synthetic Biology, 2022, 11, 1588-1599) as a template, seven synthetic DNA sequence fragments were amplified (primers SEQ ID NO:23-SEQ ID NO:36, with lengths of 8152bp, 8116bp, 7820bp, 8219bp, 7910bp, 7713bp, and 7736bp, respectively). These nine fragments were transformed into *Saccharomyces cerevisiae* MYA3666 competent cells (prepared using a kit purchased from Zymo), and assembled using transformation-associated recombination (TAR) to obtain the pCGBACYT90-A1 plasmid. 200 ng of each fragment was added to 100 μL of competent cells and incubated at 30°C for 2 hours. The transformed cells were then plated on SD-His plates containing 2% glucose to screen for positive clones, which were verified by colony PCR. Total DNA was extracted from the validated strain using the Tiangen yeast genome extraction kit. 10 μL of this DNA was transformed into 100 μL of *E. coli* NEB 10-beta competent cells. After thawing at 37°C for 1 hour, the cells were plated on LB plates containing 17 μg / mL chloramphenicol to screen for positive clones, which were verified by colony PCR. Plasmids were extracted from 200 mL of culture of the validated *E. coli* strain using a plasmid extraction kit purchased from Macherey-Nagel, and verified by restriction enzyme digestion and NGS sequencing. The results showed that the obtained plasmids were correctly assembled.

[0144] 10 μL of pCGBACYT90-A1 plasmid was electroporated into 100 μL of *Corynebacterium glutamicum* ATCC13032 competent cells (Bio-rad XCell electroporation instrument, electroporation parameters: 2500 V, 5.0 ms). The cells were then incubated in 900 μL BHIS medium for 1.5 h. Positive clones were screened by plating the transformed cells onto BHIS plates containing 10 μg / mL chloramphenicol. Colony PCR was used to verify the partial synthetic PCR tag (see Ye et al., ACS Synthetic Biology, 2022, 11, 1588-1599) and the interface between the synthetic sequence and the plasmid backbone. The results indicate that the pCGBACYT90-A1 plasmid was correctly introduced into *Corynebacterium glutamicum*.

[0145] Positive clones were transferred to 10 mL of BHIS medium for subculture. After subculture, streaking was performed on single colonies for colony PCR verification. No expected band was found, indicating that the pCGBACYT90 vector, loaded with a large DNA fragment, could not be stably subcultured.

[0146] Based on the pCGBACYT90-A1 plasmid, the pCXC100 minimum replicon was also inserted and tandemly linked with the pBL90 replicon (first constructing the intermediate plasmid pCGBACYT1 using primers SEQ ID NO:37 and SEQ ID NO:13, SEQ ID NO:38 and SEQ ID NO:39, then extracting the plasmid from pCGBACYT1 using primers SEQ ID NO:40 and SEQ ID NO:13). NO:41 Amplified the CXC100BL90 fragment, and then reassembled it with the BAC-oriT fragment, YAC-CAC-Cat fragment, and seven synthetic DNA sequence fragments with lengths of 8152bp, 8116bp, 7820bp, 8219bp, 7910bp, 7713bp, and 7736bp, respectively, as described above. The pCGBACYT1-A1 was then electroporated with Corynebacterium glutamicum. Similarly, the correct band could be verified after electroporation of pCGBACYT1-A1, but the correct band could not be verified after passage. This also indicates that the pCGBACYT1 vector cannot be stably passaged after loading a large DNA fragment.

[0147] Furthermore, using pBL90 as a template, the parA II and parB sequences (recognition sites of parA and parB proteins) were altered to the parS site sequence (SEQ ID NO:1) via PCR to obtain the parSCAC fragment (primers are SEQ ID NO:42 and SEQ ID NO:43). The BAC-Cat fragment, oriT fragment, YAC-HIS3 fragment, and parSCAC fragment were assembled using Gibson Assembly to obtain the pCGBAC1 empty vector. The assembly product was transformed into *E. coli* DH10b competent cells. The transformed cells were plated on LB agar plates supplemented with 17 μg / mL chloramphenicol for screening positive clones, plasmids were extracted, and sequenced. The BAC-oriT and YAC-parSCAC-Cat fragments, amplified using the sequence-verified pCGBAC1 empty vector as a template, and seven synthetic DNA sequence fragments amplified using the synthetic genomic sequence from semi-synCG-A1 as a template, were transformed into *Saccharomyces cerevisiae* MYA3666 competent cells for yeast TAR assembly to obtain the pCGBAC1-A1 plasmid. Using the above method, colony PCR was performed to verify the assembly interface. Total DNA was extracted from 10 mL of the correctly verified strain using the Tiangen yeast genome extraction kit, and 10 μL was transformed into 100 μL of *E. coli* NEB 10-beta competent cells. After thawing at 37°C for 1 hour, the cells were plated on LB agar plates containing 17 μg / mL chloramphenicol for screening positive clones. Colony PCR was then performed to verify the assembly interface. The plasmid was extracted from 200 mL of correctly verified *E. coli* using the Macherey-Nagel kit and verified by enzyme digestion and sequencing.

[0148] 10 μL of pCGBAC1-A1 plasmid was electroporated into 100 μL of *Corynebacterium glutamicum* ATCC13032 competent cells (Bio-rad XCell electroporation instrument, electroporation parameters: 2500 V, 5.0 ms). Cells were then incubated in 900 μL BHIS medium at 37°C for 1.5 h. Positive clones were screened by plating on BHIS plates containing 10 μg / mL chloramphenicol. Colony PCR was used to verify the interface between the synthetic PCR tag and the synthetic sequence and the plasmid backbone. The results showed that the pCGBAC1-A1 plasmid was correctly introduced into *Corynebacterium glutamicum*. Positive clones were transferred to 10 mL of BHIS medium for passage. After passage, single colonies were streaked, and colony PCR and sequencing were used to verify the results. The results indicated that the pCGBAC1 vector can load large DNA fragments into *Corynebacterium glutamicum* and stably replicate and passage.

[0149] Using the pCGBAC1 empty vector as a template, the BAC-oriT fragment (primers SEQ ID NO:44 and SEQ ID NO:45) and the YAC-parSCAC-Cat fragment (primers SEQ ID NO:46 and SEQ ID NO:47) were amplified. Using the violacein expression gene cluster (Sun et al., Microbial Cell Factories, 2016, 15:148) as a template, the ptrc-vioAB fragment (primers SEQ ID NO:48 and SEQ ID NO:49) and the vioCDE fragment (primers SEQ ID NO:50 and SEQ ID NO:51) were amplified. These four fragments were assembled using Gibson Assembly to obtain the pCGBAC1-vio plasmid. The assembled product was transformed into *E. coli* NEB 10-beta competent cells. Transformed cells were plated on LB agar plates supplemented with 17 μg / mL chloramphenicol for screening positive clones. The plasmid was extracted and verified by sequencing. The validated plasmid was electroporated into *Corynebacterium glutamicum* ATCC13032 competent cells (as described above). Positive clones were screened on BHIS plates containing 10 μg / mL chloramphenicol. Colony PCR was used to verify the assembly interface. The results showed that the pCGBAC1-vio plasmid was correctly introduced into *Corynebacterium glutamicum* (see Figure 1B). When the strain was transferred to LB plates containing 0.5 mM IPTG inducer and 17 μg / mL chloramphenicol, the strain turned purple, indicating that the vector could be transferred into *Corynebacterium glutamicum*, replicate stably, and that the purple bacitracin gene cluster on the vector was correctly expressed. This demonstrates that the pCGBAC1 vector has the potential to carry and express large exogenous gene clusters in *Corynebacterium glutamicum*. The results are shown in Table 1 below.

[0150] Table 1: Validation of the ability of the vector constructed in Example 1 to replicate, passage, and load large DNA fragments in Corynebacterium glutamicum.

[0151] Example 2: Construction of recombinant helper plasmid and strain containing said plasmid

[0152] Using the pXMJ19-RecET plasmid (see CN114525234A) as a template, the vector backbone ColE1-BL1-Cat fragment (primers SEQ ID NO:52 and SEQ ID NO:53) and the Ptac-RecET fragment (primers SEQ ID NO:54 and SEQ ID NO:55) were amplified. The PBAD-ISceI fragment (SEQ ID NO:56 and SEQ ID NO:57) was amplified using the I-SceI (Uniprot P03882) fragment synthesized by Shanghai Sangon Biotech as a template. The ColE1-BL1-Cat, Ptac-RecET, and PBAD-ISceI fragments were then assembled using Gibson Assembly to obtain the plasmid pXMJ19-RecET-IsceI. Using the pXMJ19-RecET plasmid as a template, the vector backbone fragments ColE1-BL1-Cat-2 (primers SEQ ID NO:58 and SEQ ID NO:59) and Ptac-RecET-2 (primers SEQ ID NO:60 and SEQ ID NO:61) were amplified, and the PBAD-Cas9 fragment (SEQ ID NO:62 and SEQ ID NO:63) was amplified using the pCas plasmid (Addgene) as a template. The ColE1-BL1-Cat-2, Ptac-RecET-2, and PBAD-Cas9 fragments were assembled using Gibson Assembly to obtain the plasmid pXMJ19-RecET-Cas9 (see Figure 3). The assembled products were transformed into *E. coli* DH5α competent cells. After resuscitation, the transformed cells were plated on LB agar plates supplemented with 34 μg / mL chloramphenicol for screening positive clones, and the plasmid was extracted and sequenced. The sequencing results showed that the obtained plasmid sequence was correct.

[0153] Plasmids pXMJ19-RecET-ISceI and pXMJ19-RecET-Cas9 were transformed into *Corynebacterium glutamicum* semi-synCG-A3 competent cells. Transformed cells were plated on BHIS plates containing 10 μg / mL chloramphenicol for selection of positive clones, and colony PCR was used for verification. The verification results showed that plasmids pXMJ19-RecET-ISceI and pXMJ19-RecET-Cas9 were successfully transformed into *Corynebacterium glutamicum* semi-synCG-A3 cells.

[0154] Strains infused with pXMJ19-RecET-ISceI or pXMJ19-RecET-Cas9 plasmids are named semi-synCG-A3 / RIS or semi-synCG-A3 / RCas.

[0155] Example 3: Constructing a CAC vector loaded with a large DNA fragment, chunk A4 or chunk A4a, and transforming it into Corynebacterium glutamicum strain semi-synCG-A3 / RIS or semi-synCG-A3 / RCas.

[0156] Using pCGBAC1 prepared in Example 1 as a template, the parSCAC-HA3ISceI fragment (primers SEQ ID NO:64 and SEQ ID NO:21) and the MHArsL-BAC-oriT-YAC fragment (primers SEQ ID NO:65 and SEQ ID NO:20) were amplified by overlap PCR. The first to sixth minichunk fragments (lengths of 4967bp, 4913bp, 4959bp, 4991bp, 4981bp, and 4737bp, respectively) were amplified using chunk A4 as a template. The A4a-sS-DHA fragment (primers SEQ ID NO:66-SEQ ID NO:79) was constructed to connect the last minichunk of the synthetic sequence to the pCGBAC1 vector backbone and to contain a bidirectional selection marker (sacB-speR) for screening for antibiotic substitution in Corynebacterium glutamicum genome. The MHAArpsL-BAC-oriT-YAC fragment, parSCAC-HA3ISceI fragment, six mini-chunk fragments of chunk A4a, and the A4aD fragment were transformed into *Saccharomyces cerevisiae*. 200 ng of each fragment was added to 100 μL of competent *Saccharomyces cerevisiae* cells to assemble the pCGBAC1-A4a plasmid via *Saccharomyces cerevisiae* TAR. Positive clones were screened on SD-His plates containing 2% glucose and verified by colony PCR.

[0157] Using the pCGBAC1 empty vector prepared in Example 1 as a template, the MHAArpsL-BAC-oriT fragment and the parSCAC-HA3ISceI fragment were amplified respectively; using chunk A4 as a template, 11 minichunk fragments (with lengths of 4967bp, 4913bp, 4959bp, 4991bp, 4981bp, 4737bp, 4984bp, 4961bp, 4883bp, 4982bp, and 4960bp respectively) were amplified respectively; the A4-sS-DHA fragment (primers are SEQ ID NO: 64-89) was constructed to connect the last minichunk of the synthetic sequence to the vector backbone and contain a bidirectional selection marker (sacB-speR) for screening for antibiotic substitution in Corynebacterium glutamicum genome. The MHAArpsL-BAC-oriT-YAC fragment, parSCAC-HA3ISceI fragment, 11 mini-chunk fragments of chunk A4, and the A4D fragment were transformed into *Saccharomyces cerevisiae*. 200 ng of each fragment was added to 100 μL of competent *Saccharomyces cerevisiae* cells, and the pCGBAC1-A4 plasmid was obtained by TAR assembly. Cells were plated on SD-His plates containing 2% glucose for screening of positive clones, and validation was performed by colony PCR.

[0158] Total DNA was extracted from 10 mL of a validated *Saccharomyces cerevisiae* strain culture using the Tiangen Yeast Genome Extraction Kit. 10 μL of the DNA solution was transformed into 100 μL of *E. coli* NEB 10-beta competent cells. After 1 hour of recovery, the cells were plated on LB agar plates containing 40 μg / mL spectinomycin to screen for positive clones, which were then validated by colony PCR. Plasmids were extracted from 200 mL of a validated *E. coli* culture using the Macherey-Nagel BAC 100kit kit and verified by enzyme digestion (see Figures 4B and C), indicating that plasmids pCGBAC1-A4a and pCGBAC1-A4 were successfully constructed. 10 μL of pCGBAC1-A4a and pCGBAC1-A4 plasmids were electroporated into 100 μL of *Corynebacterium glutamicum* semi-synCG-A3 / RIS competent cells, respectively. Cells were plated on BHIS plates containing 150 μg / mL spectinomycin for screening positive clones, and verification was performed by colony PCR (amplification of synthetic PCR tags and plasmid backbones on the plasmids). The results showed that pCGBAC1-A4a and pCGBAC1-A4 plasmids were correctly introduced into *Corynebacterium glutamicum* (see Figures 4B and C), with an electroporation efficiency of 10-1. 2 -10 3CFU / μg DNA (see Figure 4D). In addition, pCGBAC1-A4a was transformed into strain semi-synCG-A3 / RCas using the method described above.

[0159] Example 4: Large fragment substitution in the genome of Corynebacterium glutamicum

[0160] 4.1 Large-fragment genome replacement was performed using the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A4a mediated by RecET and Cas9.

[0161] The Corynebacterium glutamicum strain semi-synCG-A3 / RCas, inoculated with the pCGBAC3-A4a plasmid in Example 3, was inoculated into BHIS liquid medium containing 10 μg / mL chloramphenicol and 150 μg / mL spectinomycin and cultured overnight. The next day, it was transferred to NCM liquid medium and cultured at 30°C and 220 rpm in a shaker until OD600 = 0.3. Then, 1 mM IPTG and 0.5% L-arabinose were added to the final concentration, and the bacterial concentration was induced to OD600 = 1.0 at 30°C and 220 rpm. Competent cells were prepared and dispensed into 100 μL tubes. 2–3 μg of pEC-sgRNA plasmid expressing sgRNA was added to each 100 μL of competent cells for electroporation. sgRNA expression was driven by the constitutive promoter PH36 (SEQ ID NO: 117, Li, Ning et al., Biotechnology Journal, 2021, 16, e2100093). The four sgRNA sequences were SEQ ID NO: 118-121 (Figure 5A). After 3 hours of recovery, the bacterial culture was plated on BHIS solid medium containing 10 μg / mL chloramphenicol, 20 μg / mL streptomycin, and 150 μg / mL spectinomycin. After approximately 3 days of incubation, single colonies were transferred to fresh BHIS solid medium containing 10 μg / mL chloramphenicol, 20 μg / mL streptomycin, 150 μg / mL spectinomycin, and 20 μg / mL kanamycin, respectively, and cultured overnight for phenotypic screening. Strains that grew on chloramphenicol, streptomycin, and spectinomycin solid media but not on kanamycin BHIS solid medium were selected. Colony PCR was used to verify the replacement of upstream and downstream interfaces and to verify whether the plasmid backbone remained within the cell. Single colonies were used to verify all PCR tags to determine if the replacement of chunk A4a was complete. As shown in Figure 5C, the selected strains underwent genomic replacement, and the replacement was complete.

[0162] 4.2. Large-fragment genomic substitution was performed using the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-A4a mediated by RecET and I-SceI.

[0163] The *Corynebacterium glutamicum* strain semi-synCG-A3 / RIS, inoculated with the pCGBAC3-A4a plasmid as described in Example 3, was inoculated overnight into BHIS liquid medium containing 10 μg / mL chloramphenicol and 150 μg / mL spectinomycin. The next day, it was transferred to 100 mL of fresh BHIS medium and cultured at 30°C and 220 rpm on a shaker until OD600 = 0.3. A final concentration of 1 mM IPTG was added, and the cells were induced for 4 hours at 30°C and 220 rpm. The bacterial concentration OD600 was then measured (hereinafter, 1 mL of cells with an OD600 of 1 is referred to as 1 OD). The cells were collected by centrifugation at 4000 rpm for 10 minutes, resuspended in sterile water, and spread onto BHIS solid medium containing 10 μg / mL chloramphenicol, 20 μg / mL streptomycin, and 150 μg / mL spectinomycin, with an average of approximately 3 OD of cells spread per solid medium (Figure 5B). After approximately 3 days of incubation, single bacteria were transferred to fresh BHIS solid medium containing 10 μg / mL chloramphenicol, 20 μg / mL streptomycin, 150 μg / mL spectinomycin, and 20 μg / mL kanamycin, respectively, and cultured overnight for phenotypic screening. Strains that grew on chloramphenicol, streptomycin, and spectinomycin solid media but not on kanamycin BHIS solid medium were selected for colony PCR verification of the upstream and downstream interfaces of the replacement and to verify whether the plasmid backbone remained within the cell. Stranded isolates of correctly verified strains were then used to verify all PCR tags to determine the completeness of chunk A4a replacement. The results were consistent with those in Figure 5C, indicating that the selected strains underwent genome replacement, and that the replacement was complete.

[0164] The same method was used on the Corynebacterium glutamicum strain semi-synCG-A3 / RIS that had the pCGBAC3-A4 plasmid introduced in Example 3. The results showed that chunk A4 completely replaced the genome sequence, and the resulting strain was named semi-synCG-A4.

[0165] Example 5: Constructing the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC2-A5 loaded with a large chunk A5 DNA fragment and transforming it into the Corynebacterium glutamicum strain semi-synCG-A4.

[0166] Using the pCGBAC1 empty vector from Example 1 as a template, the MHAsacB-BAC-oriT fragment (primers SEQ ID NO: 90 and SEQ ID NO: 91) and the YAC-parSCAC-HA2ISceI fragment (SEQ ID NO: 92 and 93) were obtained by overlap PCR; using chunk Using A5 as a template, 12 minichunk fragments (4512bp, 4554bp, 4539bp, 4542bp, 4520bp, 4609bp, 4522bp, 4533bp, 4536bp, 4543bp, 4525bp, and 4902bp in length) were obtained by digestion with enzymes (XhoI, XbaI, BglII, BstBI, XbaI, NdeI, HindIII, and EcoRI, purchased from NEB). The fragment A5-rK-DHA was constructed and digested to ligate the synthetic sequence A5 to the vector backbone (pCGBAC2, which, compared to pCGBAC1, has the oriT sequence and some downstream repetitive or redundant sequences deleted, totaling 757bp) and contains a bidirectional selection marker (rpsL-kanR) for screening Corynebacterium glutamicum genome replacement antibiotics. The MHAsacB-BAC-oriT fragment, YAC-parSCAC-HA2ISceI fragment, 12-chunk A5 minichunk fragment, and A5D fragment were transformed into competent *Saccharomyces cerevisiae* cells, and the pCGBAC2-A5 plasmid was assembled using TAR. Positive clones were screened by plating cells on SD-His plates containing 2% glucose and validated by colony PCR. Plasmids were extracted from 10 mL of culture of the validated *Saccharomyces cerevisiae* strain using the Tiangen Yeast Genome Extraction Kit.

[0167] The pCGBAC2-A5 plasmid was introduced into Corynebacterium glutamicum semi-synCG-A4 competent cells via conjugation and electroporation, respectively.

[0168] Specifically, for the introduction of plasmids into *Corynebacterium glutamicum* via electroporation: 10 μL of yeast-derived DNA was used to transform 100 μL of *E. coli* NEB 10-beta competent cells. After 1 hour of recovery, the cells were plated on LB agar plates containing 40 μg / mL kanamycin to screen for positive clones. Colony PCR was used to verify the assembly interface. The plasmid was extracted from 200 mL of correctly validated *E. coli* using the Macherey-Nagel BAC100 kit and verified by enzyme digestion (I-SceI, purchased from NEB) (see Figure 6B). The results showed that the pCGBAC2-A5 plasmid was correctly assembled. 100 ng of the pCGBAC2-A5 plasmid was electroporated into 100 μL of *Corynebacterium glutamicum* semi-synCG-A4 competent cells. After 1.5 hours of recovery, the cells were plated on BHIS agar plates containing 150 μg / mL spectinomycin to screen for positive clones, which were then verified by colony PCR. The results showed that the pCGBAC2-A5 plasmid could be introduced into Corynebacterium glutamicum via electroporation (see Figure 6C).

[0169] For the introduction of Corynebacterium glutamicum via bacterial conjugation: 10 μL of DNA extracted from yeast was used to transform 100 μL of competent E. coli NEB 10-beta (denoted as NEB 10-beta / mob) cells containing the conjugation helper plasmid pTA-mob (Trine Aakvik Strand et al., PLoS One, 2014, 9(3):e90372). After 1 hour of recovery, the cells were plated on LB plates containing 20 μg / mL gentamicin and 40 μg / mL kanamycin to screen for positive clones, which were then verified by colony PCR. Then, validated positive *E. coli* clones and *Corynebacterium glutamicum* semi-synCG-A4 competent cells were added at a ratio of 1:3 (v / v, same OD600) onto 0.45 μm cellulose acetate filters (Millipore Corp., 0.45-μm-pore-size cellulose acetate filter) and incubated for 20 hours. The cells were washed off with liquid LB medium, diluted, and plated onto BHIS plates containing 50 μg / mL nalidixic acid, 10 μg / mL chloramphenicol, and 150 μg / mL spectinomycin to screen for positive clones, which were then verified by colony PCR (see Figure 6C). The results showed that the pCGBAC2-A5 plasmid could be introduced into *Corynebacterium glutamicum* via conjugation.

[0170] The strain prepared above was replaced and verified with chunk A5 in the manner described in Example 4.2, and the resulting strain was named semi-synCG-A5.

[0171] Example 6: Constructing the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC3-A6 loaded with a large fragment of A6 DNA and introducing it into the Corynebacterium glutamicum strain semi-synCG-A5

[0172] Using the pCGBAC1 empty vector and apmR fragment from Example 1 as templates, the fragments MHAArpsL-YAC-oriT-apmR (SEQ ID NO: 93 and SEQ ID NO: 94) and BAC-CAC-HA3ISceI (SEQ ID NO: 95 and SEQ ID NO: 96) were obtained by overlap PCR; using chunk Eleven minichunk fragments (4928bp, 4962bp, 4774bp, 4993bp, 4988bp, 4730bp, 4950bp, 4742bp, 4961bp, 4840bp, and 4885bp in length) were obtained by digestion with HindIII, SpeI, BstBI, XbaI, XhoI, AflII, and BamHI, respectively. An A6-sS-DHA fragment was constructed to connect the synthetic sequence A6 to the vector backbone and to contain a bidirectional selection marker (sacB-speR) for screening for antibiotic substitution in Corynebacterium glutamicum genome.

[0173] The MHAArpsL-BAC-oriT-YAC fragment, BAC-parSCAC-HA3ISceI fragment, 11 A6 minichunks, and A6D fragment were transformed into competent *Saccharomyces cerevisiae* cells, and the pCGBAC3-A6 plasmid was assembled using TAR. Positive clones were screened on SD-His plates containing 2% glucose, and the assembly interface was verified by colony PCR. Total DNA was extracted from the verified strains and transformed into *E. coli* NEB 10-beta competent cells. Positive clones were screened on LB plates containing 40 μg / mL spectinomycin, and the plasmid was extracted and verified by I-SceI restriction enzyme digestion. The results showed that the pCGBAC3-A6 plasmid was correctly assembled (see Figure 7B).

[0174] Total DNA was extracted from the validated *Saccharomyces cerevisiae* strain and transformed into *E. coli* NEB 10-beta competent cells. Positive clones were screened by plating the cells onto LB agar plates containing 40 μg / mL kanamycin. Plasmids were extracted from validated *E. coli* cells and verified by I-SceI digestion. Using the method described in Example 4, the pCGBAC3-A6 plasmid was introduced into *Corynebacterium glutamicum* semi-synCG-A5 competent cells via conjugation or electroporation. Colony PCR was used to verify the synthetic PCR tag and plasmid backbone on the plasmid (see Figure 7C). The results showed that the pCGBAC3-A6 plasmid could be introduced into *Corynebacterium glutamicum* semi-synCG-A5 cells via conjugation and electroporation.

[0175] The strains prepared above were used to replace and verify chunk A6 according to the method described in Example 4.2, and the resulting strain was named semi-synCG-A6.

[0176] Example 7: Large-fragment iterative replacement of genome in Corynebacterium glutamicum artificial chromosome mediated by RecET and I-SceI

[0177] Following the methods described above, an artificial chromosome plasmid pCGBAC1-A7 loaded with chunk A7 was constructed and transformed into *Corynebacterium glutamicum* semi-synCG-A6 obtained in Example 5. I-SceI and RecET expression was induced to allow the synthetic fragment chunk A7 to replace the wild-type genomic fragment (see Figures 8A and 8B). Resistance phenotype screening was performed under chloramphenicol, 20% sucrose, and kanamycin conditions, and genotype verification was conducted via colony PCR. The verified strain underwent pCGBAC1-A7 plasmid removal and re-stripe purification, followed by colony PCR. Single colonies were used to verify all PCR tags, confirming that the synthetic sequence had been completely replaced on the genome and that the plasmid backbone had been removed (see Figure 8C). The *Corynebacterium glutamicum* strain confirming that the synthetic chunk A7 had completely replaced the wild-type sequence was named semi-synCG-A7, which can be used as a starting strain for introducing new plasmids for further replacement.

[0178] Examples 5 and 6 can be iteratively performed with this example. That is, by designing two sets of positive and negative selection markers (see Figures 8A and 8B), the first set is rpsL-kanR (correspondingly, rpsL on the genome has undergone a K43R mutation), and the second set is sacB-speR. After the first set of markers is replaced on the genome, it can provide a negative selection marker for the replacement of the second set of markers. When the second set of markers is replaced on the genome with the synthetic fragment, the selection conditions are streptomycin and spectinomycin. After the second set of markers is replaced on the genome, it provides a negative selection marker for the first set of replacements. When the second set of markers is replaced on the genome with the synthetic fragment, the selection conditions are sucrose and kanamycin.

[0179] Furthermore, to improve the efficiency of negative screening, the negative screening conditions can be changed or superimposed. For example, the second set of markers can be changed to pheS*-sacB-speR.

[0180] As described above, the Corynebacterium glutamicum strains that can sequentially integrate chunks A8 and A9a and confirm that the synthetic chunks completely replace the wild-type sequences are named semi-synCG-A8 and semi-synCG-A9a, respectively, starting with semi-synCG-A7.

[0181] Example 8: Constructing the Corynebacterium glutamicum artificial chromosome plasmid pCGBAC1-80k loaded with a large chunk A9b-A10 DNA fragment and introducing it into the Corynebacterium glutamicum strain semi-synCG-A9a / RIS

[0182] The purpose of this embodiment is to verify whether the CAC of the present invention can load and deliver larger-sized fragments.

[0183] Therefore, as described above, the CAC plasmid pCGBAC1-80k loaded with a large chunk A9b-A10 DNA fragment (approximately 80kb) was constructed. The vector backbone was amplified using pCGBAC1 as a template; nine minichunk fragments (5651bp, 9045bp, 9366bp, 7893bp, 9200bp, 8476bp, 8834bp, 6845bp, and 6642bp in length) were amplified using chunks A9 and A10 as templates, respectively; the A10-sS-DHA fragment (primers were SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:93, SEQ ID NO:97-SEQ ID NO:116) was constructed to connect the last minichunk of the synthetic sequence to the vector backbone and to contain a bidirectional selection marker (sacB-speR) for screening for antibiotic substitution in Corynebacterium glutamicum genome; the above fragments were transformed into competent Saccharomyces cerevisiae cells and assembled using TAR to obtain the pCGBAC1-80k plasmid, which carries an 80kb DNA fragment of interest. Cells were plated on SD-His plates containing 2% glucose to screen for positive clones, and colony PCR was used to verify the assembly interface. Total DNA was extracted from the validated strains and transformed into *E. coli* NEB 10-beta competent cells. Cells were plated on LB plates containing 40 μg / mL spectinomycin to screen for positive clones, and plasmids were extracted and verified by I-SceI in vitro digestion. The results showed that the pCGBAC1-80k plasmid was correctly assembled (see Figure 9B). Using the method described in Example 4, the pCGBAC1-80k plasmid was introduced into *Corynebacterium glutamicum* strain semi-synCG-A9a competent cells by electroporation. Colony PCR was used to verify the synthetic PCR tags and plasmid backbone on the plasmid (see Figure 9C). The results showed that the pCGBAC1-80k plasmid could be introduced into *Corynebacterium glutamicum* semi-synCG-A9a by electroporation, demonstrating that the CAC of the present invention can be used to deliver DNA fragments up to 80 kb.

[0184] sequence

[0185] SEQ ID NO:1 Corynebacterium glutamicum replication allocation element, 16 bp in length.

[0186] The SEQ ID NO:2kanR fragment is 795bp in length.

[0187] The SEQ ID NO:3speR fragment is 1134bp in length.

[0188] The SEQ ID NO:4apmR fragment is 1103bp in length.

[0189] The SEQ ID NO:5cat fragment is 660bp in length.

[0190] The SEQ ID NO:6rpsLK43R fragment is 369bp in length.

[0191] The SEQ ID NO:7sacB fragment is 1590bp in length.

[0192] The SEQ ID NO:8pheS* fragment is 1038bp in length.

[0193] The SEQ ID NO:9Ptuf fragment is 200bp in length.

[0194] SEQ ID NO: 10 Primer BAC-F, 41 nt in length

[0195] SEQ ID NO:11 Primer BAC-R, 34 nt in length

[0196] SEQ ID NO: 12 Primer CXC100-F, 20nt in length

[0197] SEQ ID NO: 13 Primer CAC-R, 20 nt in length

[0198] SEQ ID NO: 14 Primer BL90-F, 24nt in length

[0199] SEQ ID NO:15 Primer oriT-F, 35nt in length

[0200] SEQ ID NO:16 Primer oriT-R, 31 nt in length

[0201] SEQ ID NO:17 Primer YAC-F, 34nt in length

[0202] SEQ ID NO:18 Primer YAC-R, 55nt in length

[0203] SEQ ID NO:19 Primer V2F2, 54nt in length

[0204] SEQ ID NO: 20 Primer V2R, 30 nt in length

[0205] SEQ ID NO:21 Primer V1F, 22nt in length

[0206] Primer V1R, SEQ ID NO:22, is 47 nt in length.

[0207] SEQ ID NO:23 Primer 0409A1-F, 58 nt in length

[0208] SEQ ID NO:24 Primer 0409A1-R, 21nt in length

[0209] SEQ ID NO: 25 Primer 0409A2-F, 21nt in length

[0210] SEQ ID NO:26 Primer 0409A2-R, 23nt in length

[0211] SEQ ID NO:27 Primer 0409A3-F, 29nt in length

[0212] SEQ ID NO: 28 Primer 0409A3-R, 28nt in length

[0213] SEQ ID NO:29 Primer 0409A4-F, 26nt in length

[0214] SEQ ID NO: 30 Primer 0409A4-R, 22nt in length

[0215] SEQ ID NO:31 Primer 0409A5-F, 22nt in length

[0216] SEQ ID NO:32 Primer 0409A5-R, 24nt in length

[0217] SEQ ID NO:33 Primer 0409A6-F, 24nt in length

[0218] SEQ ID NO:34 Primer 0409A6-R, 23nt in length

[0219] SEQ ID NO:35 Primer 0409A7-F, 21nt in length

[0220] SEQ ID NO:36 Primer A72R, 52nt in length

[0221] SEQ ID NO:37 Primer 210301-F, 51 nt in length

[0222] SEQ ID NO:38 Primer 210302-F, 44nt in length

[0223] SEQ ID NO:39 Primer 210302-R, 29nt in length

[0224] SEQ ID NO:40 Primer 210407-F, 50 nt in length

[0225] SEQ ID NO:41 Primer 210407-R, 56 nt in length

[0226] SEQ ID NO:42 Primer parS-F, 56nt in length

[0227] SEQ ID NO:43 Primer parS-R, 56 nt in length

[0228] SEQ ID NO:44 Primer Z220527-3-F, 46nt in length

[0229] SEQ ID NO:45 Primer Z220527-3-R, 22nt in length

[0230] SEQ ID NO:46 Primer Z220527-2-F, 44nt in length

[0231] SEQ ID NO:47 Primer Z220527-2-R, 39nt in length

[0232] SEQ ID NO:48Z220527-1-F, length 21nt

[0233] SEQ ID NO:49 Primer Z210629-1-R, 21nt in length

[0234] SEQ ID NO: 50 Primer Z210629-2-F, 22nt in length

[0235] SEQ ID NO:51 Primer Z220527-1-R, 21nt in length

[0236] SEQ ID NO:52 Primer B220617-1-F, 21nt in length

[0237] SEQ ID NO:53 Primer B220617-1-R, 22nt in length

[0238] SEQ ID NO:54 Primer B220617-2-F, 21nt in length

[0239] SEQ ID NO:55 Primer B220617-2-R, 22nt in length

[0240] SEQ ID NO:56 Primer B220617-3-F, 42nt in length

[0241] SEQ ID NO:57 Primer B220617-3-R, 50 nt in length

[0242] SEQ ID NO:58 Primer Z220311-1-F, 31nt in length

[0243] SEQ ID NO:59 Primer Z220316-2-R, 21nt in length

[0244] SEQ ID NO: 60 Primer Z220316-3-F, 36nt in length

[0245] SEQ ID NO:61 Primer Z220316-3-R, 59nt in length

[0246] SEQ ID NO:62 Primer Z220316-1-F, 21nt in length

[0247] SEQ ID NO:63 Primer Z220316-1-R, 45 nt in length

[0248] SEQ ID NO:64 Primer Z220612-TAR-R, 50 nt in length

[0249] SEQ ID NO:65 Primer Z221104-6-F, 59nt in length

[0250] SEQ ID NO:66 Primer Z220612-11-F, 59nt in length

[0251] SEQ ID NO:67 Primer Z220716-A1-R, 23 nt in length

[0252] SEQ ID NO:68 Primer Z220716-A2-F, 22nt in length

[0253] SEQ ID NO:69 Primer Z220716-A2-R, 18nt in length

[0254] SEQ ID NO:70 Primer Z220716-A3-F, 22nt in length

[0255] SEQ ID NO:71 Primer Z220716-A3-R, 19nt in length

[0256] SEQ ID NO:72 Primer Z220721-A4-F, 20nt in length

[0257] SEQ ID NO:73 Primer Z220721-A4-R, 23nt in length

[0258] SEQ ID NO:74 Primer Z220721-A5-F, 26nt in length

[0259] SEQ ID NO:75 Primer Z220721-A5-R, 30 nt in length

[0260] SEQ ID NO:76 Primer Z220721-A6-F, 26nt in length

[0261] SEQ ID NO:77 Primer Z220716-A6-R, 21 nt in length

[0262] SEQ ID NO:78 Primer Z220716-A7-F, 20nt in length

[0263] SEQ ID NO:79 Primer Z220612-13-R, 50 nt in length

[0264] SEQ ID NO: 80 Primer Z220716-A7-R, 20 nt in length

[0265] SEQ ID NO:81 Primer Z220716-A8-F, 29nt in length

[0266] SEQ ID NO:82 Primer Z220716-A8-R, 20nt in length

[0267] SEQ ID NO: 83 Primer Z220716-A9-F, 22nt in length

[0268] SEQ ID NO: 84 Primer Z220716-A9-R, 19nt in length

[0269] SEQ ID NO: 85 Primer Z220716-A10-F, 24nt in length

[0270] SEQ ID NO: 86 Primer Z220716-A10-R, 20 nt in length

[0271] SEQ ID NO: 87 Primer Z220716-A11-F, 22nt in length

[0272] SEQ ID NO: 88 Primer Z230723-1-R, 43nt in length

[0273] SEQ ID NO: 89 Primer Z221104-11-F, 23nt in length

[0274] SEQ ID NO:90 Primer Z230306-1-F, 39nt in length

[0275] SEQ ID NO:91 Primer Z231029-1-R, 19nt in length

[0276] SEQ ID NO:92 Primer Z220527-2-F, 44nt in length

[0277] SEQ ID NO:93 Primer Z230514-3-F, 26nt in length

[0278] SEQ ID NO:94 Primer Z220413-1-R, 24nt in length

[0279] SEQ ID NO:95 Primer Z230516-IF, 25nt in length

[0280] SEQ ID NO:96 Primer Z230307-BAC-F, 23nt in length

[0281] SEQ ID NO:97 Primer A9-4-F, 19nt in length

[0282] SEQ ID NO:98 Primer A9-3-R, 19nt in length

[0283] SEQ ID NO:99 Primer A9-5-F, 19nt in length

[0284] SEQ ID NO: 100 Primer A9-4-R, 21 nt in length

[0285] SEQ ID NO: 101 Primer A9-6-F, 24nt in length

[0286] SEQ ID NO:102 Primer A9-5-R, 20 nt in length

[0287] SEQ ID NO: 103 Primer A10-1-F, 21 nt in length

[0288] SEQ ID NO:104 Primer A9-6-R-TAR, 20 nt in length

[0289] SEQ ID NO: 105 Primer A10-2-F, 21 nt in length

[0290] SEQ ID NO: 106 Primer A10-1-R, 24 nt in length

[0291] SEQ ID NO: 107 Primer A10-3-F, 22nt in length

[0292] SEQ ID NO: 108 Primer A10-2-R, 23 nt in length

[0293] SEQ ID NO: 109 Primer A10-4-F, 28 nt in length

[0294] SEQ ID NO:110 Primer A10-3-R, 30 nt in length

[0295] SEQ ID NO:111 Primer A10-5-F, 29nt in length

[0296] SEQ ID NO:112 Primer A10-4-R, 25 nt in length

[0297] SEQ ID NO: 113 Primer A10-6-F, 26nt in length

[0298] SEQ ID NO:114 Primer A10-5-R, 27 nt in length

[0299] SEQ ID NO: 115 Primer A10D-F(7.19), 20 nt in length.

[0300] SEQ ID NO: 116 Primer A10-6D-R, 21 nt in length

[0301] The fragment SEQ ID NO:117PH36 is 95bp in length.

[0302] SEQ ID NO:118sgRNA fragment 1, 507bp in length

[0303] SEQ ID NO:119sgRNA fragment 2, 507bp in length

[0304] SEQ ID NO:120sgRNA fragment 3, 507bp in length

[0305] SEQ ID NO:121sgRNA fragment 4, 507bp in length

Claims

1. An artificial chromosome vector comprising a first replication distribution element that enables the artificial chromosome vector to replicate in a first host of a Corynebacterium species, such as Corynebacterium glutamicum.

2. The artificial chromosome vector of claim 1, further comprising a second replication distribution element, the second replication distribution element enabling the artificial chromosome vector to replicate in a second host.

3. The artificial chromosome vector of claim 1 or 2, further comprising a third replication distribution element that enables the artificial chromosome vector to replicate in a third host.

4. The artificial chromosome vector of claim 2, wherein the second host is a bacterium used to amplify the artificial chromosome vector, such as Escherichia coli.

5. The artificial chromosome of claim 3, wherein the third host is yeast, such as Saccharomyces cerevisiae.

6. The artificial chromosome of any one of claims 1-5, wherein the first replication allocation element comprises a parS site from a Corynebacterium species, such as Corynebacterium glutamicum, preferably the parS site of SEQ ID NO:

1.

7. The artificial chromosome of any one of claims 1-6, further comprising an element capable of mediating the transfer of the artificial chromosome vector via conjugation, such as an oriT element.

8. A method for editing the genome of a Corynebacterium species, such as Corynebacterium glutamicum, said method comprising the following steps: i) Obtain the nucleic acid fragment or its sub-fragment of interest; ii) Cloning the nucleic acid fragment of interest into the artificial chromosome vector of any one of claims 1-7 to obtain an artificial chromosome vector containing the nucleic acid fragment of interest; iii) Introducing an artificial chromosome vector containing the nucleic acid fragment of interest into a recipient strain from the Corynebacterium species; and iv) Integrate the nucleic acid fragment of interest into the chromosome of the recipient strain.

9. The method of claim 8, wherein step ii) comprises assembling a sub-fragment of the nucleosomal fragment of interest into an artificial chromosome vector via transformation-associated recombination (TAR) in a third host.

10. The method of claim 8 or 9, further comprising amplifying an artificial chromosome vector containing the nucleic acid fragment of interest in the second host between steps ii) and iii).

11. The method of claim 10, wherein step iii) comprises engaging the second host with the recipient strain.

12. The method of any one of claims 8-11, wherein the length of the nucleic acid fragment of interest is at least 10 kb.

13. The method of any one of claims 8-12, further comprising introducing a recombinase and / or an endonuclease into the recipient strain.

14. The method of any one of claims 8-13, wherein the nucleic acid fragment of interest is derived from the chromosome of a donor strain of the Corynebacterium species.

15. Use of the artificial chromosome of any one of claims 1-7 for delivering a fragment of nucleic acid of interest to a species of Corynebacterium, such as Corynebacterium glutamicum.

16. The use of claim 15, wherein the length of said nucleic acid fragment of interest is at least 10 kb.

17. A system for genome editing in species of the genus Corynebacterium, comprising an artificial chromosome vector according to any one of claims 1-7.

18. The system of claim 17, further comprising a recombinase and / or a nuclease, or a polynucleotide encoding said recombinase and / or nuclease.

19. A kit for genome editing in Corynebacterium species, comprising an artificial chromosome vector of any one of claims 1-7.

20. The kit of claim 19, further comprising a recombinase and / or a nuclease, or a polynucleotide encoding said recombinase and / or nuclease.

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