Strictly regulated induction system for corynebacterium glutamicum and use thereof

Abstract

The application discloses a stringent induction system for corynebacterium glutamicum and application thereof, and belongs to the fields of genetic engineering and microbiology. The system can be strictly regulated without an inducer and can be expressed after the inducer is added. More specifically, it relates to reducing the leakage expression of Cre recombinase by containing logic gates, repressor regulation, and RNA regulatory elements, and its use for mediating the production of large-scale genomic random rearrangement of SCRaMbLE in the synthetic genome of corynebacterium glutamicum. In particular, it is used for reducing the leakage expression level of Cre recombinase and maintaining a moderate expression level after induction. More importantly, the stringent induction system of the application can be applied to obtain rearranged strains with highly diverse genotypic sequences in genomic rearrangement, so as to achieve the purposes of improving the yield of target products or enhancing the stress resistance of strains.

Description

Technical Field

[0001] This invention pertains to the fields of genetic engineering and microbiology, particularly a method for rigorously regulating the expression of target proteins in Corynebacterium glutamicum. More specifically, it relates to reducing the leakage expression of Cre recombinase by incorporating logic gates, repressive regulation, and RNA regulatory elements, and its use in generating large-scale random genomic rearrangements in Corynebacterium glutamicum through SCRamMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution). 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] Synthetic genomics has deepened our understanding of the fundamental principles of the genome, leading to important applications in the life sciences. To date, humans have only studied mycoplasma (…). Mycoplasma Genome synthesis was achieved in *Escherichia coli* and *Saccharomyces cerevisiae* (Gibson et al.). Science , 2010, 329(5987):52-56; Fredens et al, Nature , 2019, 569(7757):819-822; Zhao et al., Cell, 2023, 186(24):5220-5236), but only in the Sc2.0 project was Cre recombinase and loxPsym site-mediated large-scale random rearrangement of synthetic chromosomes (SCRaMbLE) achieved by inserting thousands of loxPsym sites into synthetic yeast chromosomes (Dymond et al., Nature, 2011, 477(7365):471-476; Richardson et al., 2017, 355(6329), 1040-1044). SCRaMbLE can generate diverse genotypes and phenotypic variants in the synthesized genome, which can be used to study genome evolution or genome miniaturization. However, SCRaMbLE has not yet been applied in prokaryotic cells, both because the workload of writing a large number of loxPsym sites into the genome is huge, and because there are core technical obstacles in the strict expression regulation and leakage expression control of Cre enzymes in prokaryotic cells.

[0004] SCRaMbLE is mediated by Cre recombinase. In eukaryotic cell studies, Cre recombinase fuses with the estrogen-binding domain (EBD) to achieve inducible SCRaMbLE in yeast. However, even in eukaryotic cells, Cre recombinase leaks expression without estradiol induction, leading to the deletion of some essential genes and resulting in growth defects in synthetic yeast or significantly affecting the survival rate of strains after SCRaMbLE. To address this issue, the Sc2.0 research team developed an AND gate switch based on the estrogen effect on daughter cells and nuclear localization control (Wang et al.). Front. Chem. Sci. Eng ., 2018, 12: 806-814), and a light-controlled protein reassembly strategy (Hochrein et al ...). Nat. Commun. (2018, 9(1): 1931) to achieve more precise control over SCRaMbLE. However, Corynebacterium glutamicum, as a prokaryote, lacks a nuclear membrane, and transcription is more likely to occur due to the higher degree of chromatin openness. Furthermore, light-controlled manipulation requires specific equipment, so the existing regulatory strategies cannot be directly applied to the rigorous regulation of SCRaMbLE in Corynebacterium glutamicum. Moreover, there are few existing inducible promoters for Corynebacterium glutamicum, and the extent of leakage expression is unclear. Therefore, it is still necessary to develop a rigorously regulated inducible expression system in Corynebacterium glutamicum.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a rigorous induction system for Corynebacterium glutamicum and its application.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] To meet the requirements of genomic SRaMbLE rearrangement in the *Corynebacterium glutamicum* genome, this invention develops a stringent induction system in *Corynebacterium glutamicum* that allows for precise regulation without an inducing agent and requires the addition of an inducing agent for expression. This induction system significantly reduces the leakage expression of Cre recombinase.

[0009] In a first aspect, the present invention provides a stringent induction system for Corynebacterium glutamicum, comprising multiple promoter-forming logic gates (AND gates) for synergistic regulation, tandem repetition of promoter repression sites to enhance promoter repression, and insertion of self-splicing ribozyme elements into the target gene sequence to further inhibit the post-transcriptional process, significantly reducing the expression of the target gene in the non-inducible state. After the addition of a suitable inducer, the target protein can be expressed normally, and the expression level meets the requirements of practical applications. The stringent induction system is loaded onto a shuttle vector of *Escherichia coli* and *Corynebacterium glutamicum*, enabling the system to stringently regulate the expression of genes of interest in *Corynebacterium* species, such as *Corynebacterium glutamicum*.

[0010] In some implementations, the stringent induction system includes a first inducible promoter element that enables the stringent induction system to induce and regulate gene expression in Corynebacterium glutamicum.

[0011] In some implementations, a second inducible promoter element is also included, which enables the rigorous induction system to induce and regulate gene expression in Corynebacterium glutamicum.

[0012] In some embodiments, the stringent induction system further includes a third constitutive promoter element that enables constitutive gene expression in Corynebacterium glutamicum.

[0013] In some embodiments, the rigorous induction system further includes inserting a ribozyme sequence into the coding sequence of the protein of interest.

[0014] In some embodiments, the rigorous induction system includes a first inducible promoter element, which may be a Cumate induction system, wherein the repressor binding site is CuO Site.

[0015] In some embodiments, the first inducible promoter element Cumate induction system comprises a repressor protein from *Pseudomonas putida* and its binding site, such as CymR and CuO Site.

[0016] In some implementations, the second inducible promoter element is an expression induction system for the protein of interest, such as an anhydrotetracycline (ATc) induction system.

[0017] In some embodiments, the ribozyme sequence is a DNA sequence inserted into the coding sequence of the protein of interest, which undergoes post-transcriptional self-splicing to enable normal mRNA translation, such as the Tetrahymenathermophila self-splicing ribozyme. In some embodiments, the ribozyme sequence needs to be inserted downstream of uracil, with a short 5' fragment and a frameshifted 3' fragment of the coding sequence of the protein of interest. In some embodiments, the ribozyme sequence needs to specifically recognize the six bases at the end of the 5' fragment of the mRNA as an internal guide sequence (IGS), and then ligate the two fragments to form a complete mRNA, enabling the expression of the protein of interest to produce mature mRNA.

[0018] In some embodiments, the third constitutive promoter is a known medium- to high-strength constitutive promoter element from the Corynebacterium glutamicum genome, such as P tuf In some implementations, the third constituent promoter element P tuf Constitutive expression CymR.

[0019] In some embodiments, the first induced promoter element includes one or more series-connected... CuO Loci, such as 1, 2, or 4.

[0020] In some embodiments, the second inductive promoter element ATC induction system includes tetR-tetA sequence.

[0021] Specifically, in order to better implement the technical solution of the present invention, the following is provided:

[0022] A rigorous induction system for Corynebacterium glutamicum, comprising one or two to four in tandem. CuO The Cumate-ATc induction system at the site, the protein of interest, and the ribozyme sequence elements; specifically including: repressor protein CymR, repressor protein TetR, and P. tetR / tetA Promoter, one or two to four operator genes in tandem CuO Proteins and ribozyme sequence elements of interest;

[0023] Specifically, this includes: CymR-tetR-P tetR / tetA -n CuO- The protein structure of interest, with ribozyme sequence elements inserted downstream of the uracil in the mRNA corresponding to the coding sequence of the protein of interest, and the 5' segment of the coding sequence of the protein of interest being short, with the 3' segment frameshifted; n CuO In this context, n is an integer from 1 to 4.

[0024] The rigorous induction system also includes a promoter that controls CymR gene expression; preferably, the promoter includes the Corynebacterium glutamicum promoter P. hom and the Propionibacterium glutamate promoter P tuf One of them; more preferably, promoter P tuf Its nucleotide sequence is shown as the reverse complementary sequence corresponding to 638-837 bp in SEQ ID NO: 6.

[0025] Preferably, the repressor protein CymR is derived from *Pseudomonas putida* F1, and its amino acid sequence is shown in GenBank: ADI95374.1, specifically as shown in SEQ ID NO: 1; the nucleotide sequence of its encoding gene is shown as the reverse complementary sequence corresponding to the first 621 bp in SEQ ID NO: 6, or the codon GAG of the 17th amino acid in the reverse complementary sequence corresponding to the first 621 bp in SEQ ID NO: 6 is changed to GAA.

[0026] Preferably, the amino acid sequence of the repressor protein TetR is shown in SEQ ID NO: 2; and the nucleotide sequence of its encoding gene is shown in the reverse complementary sequence corresponding to 414-1037 bp in SEQ ID NO: 5.

[0027] Preferably, the P tetR / tetA The nucleotide sequence of the promoter is shown in SEQ ID NO: 5, 1054-1109 bp.

[0028] Preferably, the manipulator gene cuo The nucleotide sequence is shown in SEQ ID NO: 6, 867-898bp;

[0029] The rigorous induction system further includes a ribosome binding site RBS1 for controlling the expression of the protein of interest and a ribosome binding site RBS2 for expressing the repressor protein CymR or the repressor protein TetR; the nucleotide sequence of the ribosome binding site RBS1 for controlling the expression of the protein of interest is shown in SEQ ID NO: 5, 1110-1116 bp; the nucleotide sequence of the ribosome binding site RBS2 for expressing the repressor protein CymR or the repressor protein TetR is shown in the reverse complementary sequence corresponding to SEQ ID NO: 5, 1045-1053 bp.

[0030] Preferably, the protein of interest is an exogenous protein of Corynebacterium glutamicum, which may be sfGFP, Cre recombinase, or more toxic proteins that cause a decrease in the transformation efficiency and growth activity of the strain; further, it is Cre recombinase, specifically Cre recombinase derived from phage P1, whose amino acid sequence is shown in SEQ ID NO: 3, and whose encoding gene nucleotide sequence is shown in SEQ ID NO: 5, 1124-2155bp.

[0031] Preferably, the ribozyme sequence element comprises a Tetrahymena thermophila self-splicing ribozyme, the structure of which is a loop-IGS-Ribozyme structure, and its sequence is shown in SEQ ID NO: 7, loop: 1-20 bp, complementary IGS: 21-26 bp, Ribozyme gene: 27-413 bp.

[0032] Furthermore, the ribozyme sequence element was inserted after the second nucleotide of the 104th amino acid of the Cre recombinase.

[0033] Preferably, plasmid vectors and recombinant bacteria containing the above-mentioned stringent induction system for Corynebacterium glutamicum are also within the scope of protection of this invention. The plasmid vectors provided by this invention can be amplified by Escherichia coli, purified, and then transformed into Corynebacterium glutamicum.

[0034] Preferably, the starting vector of the plasmid vector containing the above-mentioned rigorous induction system for Corynebacterium glutamicum is a vector that can replicate in Escherichia coli and Corynebacterium glutamicum, such as the vector pJYS1Ptac.

[0035] In a second aspect, the present invention provides the use of the above-described rigorous induction system for Corynebacterium glutamicum for rigorous induction of expression of a protein of interest in Corynebacterium species (such as Corynebacterium glutamicum).

[0036] Preferably, the protein of interest may be sfGFP, Cre recombinase, or more toxic proteins that cause a decrease in strain transformation efficiency and growth activity.

[0037] In some implementations, the protein of interest is Cre recombinase.

[0038] In a third aspect, the present invention provides the use of the above-described rigorous induction system for Corynebacterium glutamicum in inducing genomic rearrangement in Corynebacterium glutamicum whose genome contains the loxPsym site, wherein the protein of interest is Cre recombinase.

[0039] Furthermore, the rigorous induction system for Corynebacterium glutamicum is used to generate a loxPsym site-mediated synthetic large-scale random rearrangement of chromosomes (SCRaMbLE) in the Corynebacterium glutamicum genome.

[0040] In a fourth aspect, the present invention provides a system for random genomic rearrangement in Corynebacterium species (such as Corynebacterium glutamicum), comprising the aforementioned rigorous induction system for Corynebacterium glutamicum, wherein the protein of interest is Cre recombinase.

[0041] Furthermore, the system also includes Corynebacterium glutamicum whose genome contains the loxPsym site.

[0042] In a fifth aspect, the present invention provides a method for random genome rearrangement of a Corynebacterium species (such as the SCRaMbLE rearrangement of the synthetic genome of Corynebacterium glutamicum), comprising the following steps:

[0043] i) The plasmid vector containing the above-mentioned rigorous induction system for Corynebacterium glutamicum (the protein of interest is Cre recombinase) was introduced into Corynebacterium glutamicum whose genome contains the loxPsym site to obtain a recombinant strain;

[0044] ii) Induce the recombinant strain obtained in step i) to achieve Cre recombinase expression, so that the genomic fragment containing the loxPsym site undergoes random genomic sequence rearrangement (such as random doubling, deletion, and flipping rearrangement).

[0045] In some implementations, in step i), the Corynebacterium glutamicum containing loxPsym sites in its genome includes more than two loxPsym sites (preferably 2 to 178 loxPsym sites, such as 2, 20, 40, or 178). These sites may contain one or more of the gene coding sequence, or may not contain any gene coding sequence.

[0046] In some implementations, the method further includes Cre recombinase-specific recognition of the loxPsym site, and structural variations such as deletions, flips, duplications, or translocations mediated by Cre / LoxP recombination.

[0047] The present invention has the following advantages and effects compared with the prior art:

[0048] This invention provides a rigorous induction system and its applications, particularly for reducing the leakage expression level of Cre recombinase and maintaining a moderate expression level after induction. More importantly, the rigorous induction system of this invention can be applied in genome rearrangement to obtain rearranged strains with highly diverse genotype sequences, thereby increasing the yield of the target product or enhancing the strain's stress resistance. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the structure of different Cre recombinase induction systems and their leakage expression. Among them, A, B, C, D, and E are schematic diagrams of the ATc induction system, Cumate induction system, Cumate-ATc induction system, Cumate-ATc induction system with two CuO sites in series, and Cumate-ATc induction system with four CuO sites in series, respectively, as well as the results of the blue-white screening experiment before and after induction. In the horizontal axis, "uninduced" is the proportion of blue-white colonies in the overnight bacterial culture plate, and "induced" is the proportion of blue-white colonies after 8 hours of induction. In the blue-white colony plate, blue colonies are non-retrograded bacteria, and white colonies are retrograded bacteria.

[0050] Figure 2 This diagram illustrates the structure and leakage expression of the Ribozyme-based Cre recombinase induction system. A, B, and C represent Ribozyme inactivation, Ribozyme non-inactivation, and the Cumate-ATc Cre recombinase induction system based on Ribozyme, respectively, along with the blue-white screening results. "Ribo0" indicates an inactivated Ribozyme whose IGS sequence does not pair complementary to the six bases at the 5' end of the mRNA, while "Ribo" indicates a non-inactivated Ribozyme.

[0051] Figure 3 This section presents the expression of the Ribozyme-based Cumate-ATc Cre recombinase induction system in semi-synthetic Corynebacterium glutamicum (semi-synCG-A2). A: Sequencing results of strains rearranged after SCRaMbLE mediated by the Ribozyme-based Cumate-ATc Cre recombinase induction system in semi-synthetic Corynebacterium glutamicum (semi-synCG-A2). B: Plate testing results of strains induced / uninduced for 8 h after SCRaMbLE mediated by the pOri, Cumate-ATc induction system with tandem four CuO sites, and the Ribozyme-based Cumate-ATc Cre recombinase induction system. - represents non-induction conditions, + represents induction conditions; the dilution gradient from top to bottom is 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 .

[0052] Figure 4 These are the fermentation and sequencing results of the violacein-producing strain and the SCRaMbLE rearranged strain; where A: fermentation results of violacein from the rearranged strain; B: sequencing results of the rearranged strain SC13; C: fermentation results of violacein from the multi-copy tryptophan synthesis gene cluster strain.

[0053] Figure 5 This is the sequencing depth map of strain A-S4-2.21. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The present 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 is not intended to limit 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 terms and routine procedures widely used in the respective fields. Experimental methods not specifically described in the present 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 the present invention are commercially available. Those skilled in the art can make various changes, modifications, and substitutions without departing from the spirit of the present invention.

[0055] 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.”

[0056] Unless otherwise stated, nucleic acid sequences are referred to in this article in the 5′ to 3′ direction from left to right.

[0057] 1. The rigorous induction system of Corynebacterium glutamicum and its construction

[0058] To achieve tight regulation of Cre recombinase expression in Corynebacterium glutamicum, this invention develops a Ribozyme-based induction and regulation system that can induce SCRaMbLE in semi-synthetic Corynebacterium glutamicum and maintain Cre recombinase expression at extremely low levels.

[0059] As used in this article, the “Cumate induction system” consists of the repressor protein CymR, the operator gene, and the control gene. cuo The constitutive promoter P that controls CymR gene expression tuf and control cuo An inducible promoter is formed by the promoters of the operator gene and the target gene. Without induction, the repressor protein CymR interacts with the operator gene. cuoThe binding process prevents the transcription of the target gene; after the addition of the inducer cumate, cumate binds to CymR, causing it to separate from the operator gene and induce the transcription of the target gene.

[0060] As used in this article, "ATc inducement system" refers to a system based on tet TetR repressor protein of the operon and promoter P in the regulatory region A P R1 P R2 and manipulating genes tetO1 , tetO2 This constitutes a rigorous induction system. The repressor protein TetR forms a dimer that recognizes and binds to the operator gene. tetO Upstream, it blocks downstream gene transcription. When the inducer ATc (aqueous tetracycline) is added, the two inducer molecules bind to the TetR protein, causing it to dissociate from the promoter. After the promoter is activated, transcription of the target gene begins (see Hillen W et al., Mechanisms underlying expression of Tn10 encoded tetracycline resistance). Annual Review of Microbiology , 1994).

[0061] To address the problems in the prior art, this invention constructs a rigorous induction system composed of two inducible promoter chimeras, thereby endowing the obtained induction system with the ability to be rigorously regulated in Corynebacterium glutamicum.

[0062] This invention discovers that in induced promoter elements, two or four elements connected in series... cuo Manipulating gene sites can further reduce the leakage level of the rigorous induction system, avoiding excessively high background expression levels that could lead to severe rearrangement reactions and death of the strain.

[0063] In some embodiments, the rigorous guidance system is four connected in series. cuo AND-gate induction system for manipulating gene loci.

[0064] Therefore, the present invention provides an AND-gate induction system comprising a first inducible promoter element and a second inducible promoter element, such that the rigorous induction system can reduce the background expression of Cre recombinase protein in Corynebacterium species.

[0065] As used in this article, "self-splicing ribozyme" is derived from the self-splicing intron of Tetrahymena thermophila. It specifically recognizes the six bases at the 5' end of the mRNA through the internal guide sequence (IGS) and cuts the mRNA into 5' and 3' fragments. The two fragments are then joined together to form a complete mRNA, which can be used for targeted splicing of specific RNA.

[0066] This invention has found that the appropriate insertion of the Ribozyme sequence into the Cre recombinase gene sequence can significantly reduce the leakage expression of Cre recombinase protein in Corynebacterium glutamicum using the rigorous induction system, and maintain a moderate protein expression level after the addition of an inducer.

[0067] In some embodiments, the stringent induction system is a stringent induction system comprising a Ribozyme sequence and a second inducible promoter element. In some embodiments, the stringent induction system comprises a Ribozyme sequence and four tandemly connected promoter elements. cuo The AND-gate induction system consists of the Cumate induction system and the ATC induction system, which manipulate gene loci.

[0068] 2. Methods for rearranging the synthetic genome of Corynebacterium

[0069] The present invention also provides a method for rearranging a synthetic genome of a Corynebacterium species (such as Corynebacterium glutamicum), the method comprising the following steps:

[0070] i) Introducing a rigorous induction system into recipient strains from the aforementioned Corynebacterium species;

[0071] ii) Add an appropriate amount of inducer to induce for 8 hours, and then perform serial dilutions of the obtained strains to isolate single colonies on plates;

[0072] iii) This allows us to obtain rearranged strains containing different genome sequences, conduct biological performance testing experiments on them, and screen strains with the desired traits.

[0073] As used in this paper, a violetin gene cluster expression plasmid and a rigorous induction system were introduced into semi-synthetic Corynebacterium glutamicum to screen rearranged strains with increased violetin production. The violetin gene cluster expression plasmid was based on plasmid pEC-C-vio-1, with its 3111-3905 bp segments modified accordingly. kanR Genes were changed via Gibson Assembly catThe gene (its nucleotide sequence is shown as 4410-5069 bp in GenBank: AJ133195.1) was renamed pVio. The plasmid pEC-C-vio-1 was published in the literature “Sun H , Zhao D , Xiong B , et al.Engineering Corynebacterium glutamicum for violacein hyper production[J].Microbial CellFactories, 2016, 15(1):1-9.DOI:10.1186 / s12934-016-0545-0.”

[0074] In some embodiments, the induction process in step ii) can be carried out under certain stress conditions to obtain rearranged strains that are tolerant to the stress conditions, and the desired strains with enhanced tolerance can be screened out under certain stress conditions.

[0075] 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.

[0076] 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.

[0077] 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. If the plasmid assembly uses the Golden Gate Assembly method, see https: / / goldengate.neb.com / . The assembled plasmid was transformed into *E. coli* DH5α 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 reaction. PCR reactions were performed using T5 polymerase from Beijing Qingke Company, with a reaction volume of 15 μL. The PCR conditions were: 98 ℃ for 3 min; 98 ℃ for 10 s, 61 ℃ for 10 s, 72 ℃ at 4 kb / min, for a total of 30 cycles; and a final induction at 72 ℃ for 2 min. After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis.

[0078] Electroporation of *Corynebacterium glutamicum* plasmids (or linear DNA fragments) was performed as follows: Preparation of electroporation competent cells of *Corynebacterium glutamicum* was carried out according to Ye et al. ACS Synthetic Biology The method described in , 2022, 11(4):1588-1599 is followed. Specifically, the strain to be transformed is inoculated into BHIS liquid medium containing the appropriate antibiotic (25 μg / mL kanamycin, or no antibiotic; if the electroporation fragment is used for RecET-mediated genomic recombination, then 10 μg / mL chloramphenicol) and cultured overnight. The next day, it is transferred to NCM liquid medium and incubated at 30 ℃ with a shaker at 220 rpm to initiate OD. 600 The concentration of bacteria was 0.3, and the bacterial concentration OD was further increased by culturing at 30 °C and 220 rpm. 600=1.0 (If RecET-mediated genomic recombination is performed using electroporated fragments, add 1 mMIPTG). Prepare competent cells and aliquot them into 100 μL tubes. Electroporate plasmids (minimum 100 ng, maximum 10 μL) into 100 μL of Corynebacterium glutamicum. After resuscitation in BHIS medium for 1.5 hours, plate the cells on BHIS plates containing the corresponding antibiotics to screen for positive clones, and verify them by colony PCR. Colony PCR verification of Corynebacterium glutamicum is performed as follows: pick a single colony from the solid medium and lyse it in 0.1% NaOH solution at 98 °C for 25 minutes, centrifuge for 5 minutes, and use the supernatant as a template (added at 10% of the reaction volume) for PCR reaction. PCR was 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 induction at 68 ℃ for 2 min. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis.

[0079] In the following examples, the blue-white screening experiment for Corynebacterium glutamicum was performed as follows: A plasmid containing Cre recombinase expression plasmid and... lacZ Corynebacterium glutamicum was inoculated into 10 mL of BHIS liquid medium containing kanamycin and cultured overnight at 30°C and 220 rpm. A substrate mix was prepared, requiring 80 μL BHIS, 50 μL X-gal, and 13 μL 50 mg / mL IPTG per plate. This mixture was evenly spread onto antibiotic-free BHIS solid plates and air-dried. The overnight bacterial culture was serially diluted with BHIS to the appropriate dilution, and 100 μL was then spread onto antibiotic-free BHIS-X-gal-IPTG plates and incubated at 30°C for 48 hours. The remaining bacterial culture was transferred to 20 mL of BHIS liquid medium containing kanamycin in a 50 mL Erlenmeyer flask, and the initial OD was controlled. 600 The inducing agent was added to a concentration of 0.1 and induced for 8 hours according to the experimental protocol. After the induction was completed, the inducing agent was removed by washing with BHIS. After serial dilution to the appropriate dilution, 100 μL of bacterial culture was spread on BHIS-X-gal-IPTG solid plates and incubated at 30 ℃ for 48 hours.

[0080] In the following examples, the fermentation and measurement experiments of *Corynebacterium glutamicum* containing violetin were conducted as follows: *Corynebacterium glutamicum* strain containing the violetin production plasmid was inoculated into 10 mL of BHIS-Cm15 medium and cultured at 30 °C and 220 rpm for 24 hours; the activated bacterial solution was transferred to 25 mL of BHIS-Cm15 medium at a ratio of 4% and cultured at 30 °C and 220 rpm for 4 hours; IPTG was added to a final concentration of 0.5 mM and cultured at 20 °C and 220 rpm for 48 hours; after fermentation, the OD of the bacterial solution was measured. 600 1 mL of bacterial culture was centrifuged to collect the bacterial cells, 1 mL of anhydrous ethanol was added to resuspend the cells, and the cells were incubated in a 50 °C metal bath until they turned white. The supernatant was collected by centrifugation, and the absorbance of the supernatant at 570 nm was measured to assess the concentration.

[0081] Example 1: Construction and characterization of a strict induction system for Corynebacterium glutamicum

[0082] The purpose of this embodiment is to construct an induction system that can be precisely controlled in Corynebacterium glutamicum.

[0083] To characterize the leakage expression and induction effect of Cre recombinase, a blue-white screening method was first constructed. CG-Δupp:: lacZ Strains. Amplification obtained lacZ - SpeR - Homologous arm fragment, which contains upp Upstream gene fragment (GenBank: NC_003450.3, pp. 698,493-699,292), lacZ promoter, loxPsym. lacZ Gene, speR Gene, rrnB T2 terminator fragment, rrnB T1 terminator fragment, upp Downstream genes (GenBank: NC_003450.3, 699,900-700,699 bp); in SEQ ID NO: 4, lacZ promoter: 1-74 bp, loxPsym: 75-108 bp and 4599-4632 bp, lacZ Gene: 109-3183 bp, T7 terminator: 3208-3255 bp speR The reverse complementary sequence of the rrnBT1 terminator is 3651-4325 bp, and the reverse complementary sequence of the rrnBT1 terminator is 4506-4533 bp. The linear segment ( lacZ - SpeR -Homologous arm fragment) Electroporated into Corynebacterium glutamicum ATCC 13032 (denoted as SCG-pXMJ19-) carrying the pXMJ19-recET plasmidrecET In competent cells, IPTG was added to a final concentration of 1 mM during the preparation process to induce RecET recombinase expression. After resuscitation in 900 μL BHIS medium for 3 hours, the transformed cells were plated on BHIS plates containing 100 μg / mL spectinomycin to screen for positive clones and construct the bacterial strain. CG-Δupp::lacZ Among them, strain SCG-pXMJ19- recET The information was published in the literature “Zhang Ling. Establishment of a strictly regulated promoter system of Corynebacterium glutamicum [D]. South China University of Technology, 2021.”

[0084] The 11205-4501bp segments of the plasmid vector pJYS1Ptac (Addgene plasmid number: 85545; total length 12273 bp) remain unchanged, while the 4502-11204bp segments are modified... tetR -P tetR / tetA -The Cre fragment (SEQ ID NO: 5) was used to construct the pT-Cre plasmid; the 4502-11204 bp segments were directly removed to construct the pOri plasmid. Specifically, in SEQ ID NO: 5, the reverse complementary sequence of the rrnBT2 terminator is 33-60 bp, and the reverse complementary sequence of the rrnBT1 terminator is 152-237 bp. tetR The reverse complementary sequence of the gene: 414-1037 bp; the reverse complementary sequence of RBS2: 1045-1053 bp; P tetR / tetA :1054-1109 bp, RBS1: 1110-1116bp, Cre Gene: 1124-2155 bp; rrnB T2 terminator, rrnB T1 terminator tetR The transcription directions of the gene, RBS2, and the Rep101 and pBL ori fragments on the pJYS1Ptac plasmid are the same; P tetR / tetA RBS1 Cre Gene and pJYS1Ptac plasmid KanR The transcription directions of the fragments are the same.

[0085] plasmid pT-Cre tetR -P tetR / tetA (i.e., 243-1109bp in SEQ ID NO: 5) is replaced with cymR -P tuf -P tac - Cuo The fragment (SEQ ID NO: 6) was used to construct the pC-Cre plasmid. Specifically, in SEQ ID NO: 6, cymRThe reverse complementary sequence of the gene: 1-621 bp, the reverse complementary sequence of RBS2: 629-637 bp, P tuf The promoter's inverse complementary sequence: 638-837bp; P tac Promoter: 838-866 bp Cuo : 867-898 bp; This fragment has the same transcriptional direction as the Rep101 and pBLori fragments on the pT-Cre plasmid; P tac promoter, Cuo The transcription direction of the fragment is the same as that of the Cre fragment on the pT-Cre plasmid.

[0086] P in plasmid pC-Cre tac The promoter (i.e., 838-866bp in SEQ ID NO: 6) is replaced with tetR -P tetR / tetA (i.e., 414-1109bp in SEQ ID NO: 5), construct the pT1C-Cre plasmid. Among them, tetR The transcription direction of the fragment is the same as that of the Rep101 fragment and the pBL ori fragment on the pC-Cre plasmid; P tetR / tetA The promoter and the Cre fragment on the pC-Cre plasmid are transcribed in the same direction.

[0087] In pT1C-Cre plasmid Cuo Insert one or three more sites after each site Cuo The fragment (867-898bp in SEQ ID NO: 6) was obtained, and the codon GAG of the 17th amino acid of CymR was changed to GAA to construct the pT2C-Cre plasmid and the pT4C-Cre plasmid.

[0088] Electroporate the plasmid that was correctly sequenced above. CG-Δupp::lacZ Competent cells were revived in 900 μL BHIS medium for 1.5 hours. Transformed cells were then plated on BHIS plates containing 20 μg / mL kanamycin and 100 μg / mL spectinomycin to screen for positive clones and construct the bacterial strain. CGZ / pT-Cre、 CGZ / pC-Cre、 CGZ / pT1C-Cre、 CGZ / pT2C-Cre and CGZ / pT4C-Cre ( CGZ Right now CG-Δupp::lacZ ).

[0089] The strains constructed above were subjected to a blue-white screening experiment, and the results are as follows: Figure 1As shown in Figure AE, the blue-white assay directly determines whether Cre recombinase expression is leaked by observing the different effects exhibited by a single colony. This provides a direct criterion for evaluating the leakage level when this induction system regulates Cre recombinase expression. (Strain) CGZ / pT-Cre、 CGZ / pC-Cre、 CGZ / pT1C-Cre、 CGZ / pT2C-Cre and CGZ The leakage rates of / pT4C-Cre without the addition of an inducer were 99.3%, 97.4%, 89.1%, 71.6%, and 18.6%, respectively; the rearrangement rates with the addition of an inducer were 100%, 99.0%, 95.6%, 98.1%, and 88.0%, respectively (Table 1). The results show that the leakage levels of Cre recombinase expression regulated by using the ATc or Cumate induction systems alone are high, making them unsuitable as tools for inducing Cre recombinase. The use of a chimeric promoter in the Cumate-ATc induction system has some effect on reducing the basal expression of Cre recombinase, but the leakage rate remains high. Increasing the repressor protein binding site can effectively reduce the leakage expression level of Cre recombinase, and the chimeric promoter system with the TC-4×CuO structure is more suitable for mediating Cre recombinase expression.

[0090] Table 1: Plasmids constructed in Example 1 in strains CG-Δupp::lacZ Leakage situation

[0091]

[0092] Example 2: Construction of an induction system for inserting ribozymes

[0093] The Ribozyme-based induction system primarily reduces leakage by splitting the Cre recombinase into two inactive parts without the addition of an inducer. The splitting principles for the Cre recombinase are as follows: to avoid the protein translated from the Cre I or Cre II mRNA fragments after transcription retaining some recombinase function, the design is such that Cre I is short and Cre II is long, with Cre II undergoing a frameshift, deviating from the Cre reading frame; the splicing ribozyme used must be inserted downstream of uracil in the target gene's mRNA to perform its splicing function correctly. Amino acids 18-103 of the Cre protein domain are responsible for recognition and binding, while amino acids 130-338 perform recombination; therefore, the splitting position of the Cre gene is chosen after the second nucleotide of the 104th amino acid. The amino acid sequence of the Cre recombinase is shown in SEQ ID NO: 3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 5, bp 1124-2155: Cre I: 1124-1434bp; Cre II: 1435-2155bp.

[0094] By Gibson assembly, the L1 loop (20 bp, see “S. Hasegawa et al., Imaging Tetrahymenaribozyme splicing activity in single live mammalian cells, Proc. Natl. Acad. Sci. USA 100 (25)14892-14896, https: / / doi.org / 10.1073 / pnas.2036553100(2003).”), the complementary IGS sequence (6 bp), and the Ribozyme gene (loop-IGS-Ribozyme structure, SEQ ID NO: 7) were inserted between CreⅠ and CreⅡ in the pT-Cre plasmid prepared in Example 1 to obtain the plasmid pTRibo-Cre. In SEQ ID NO: 7, L1 loop: 1-20 bp, complementary IGS: 21-26 bp, and Ribozyme gene: 27-413 bp are shown.

[0095] Using Gibson assembly and plasmid pTRibo-Cre as a template, a mutation was made to change the complementary IGS sequence to a non-complementary IGS sequence (GGGTCA), resulting in Ribozyme inactivation and plasmid pTRibo0-Cre.

[0096] Electroporate the plasmid that was correctly sequenced above. CG-Δupp::lacZIn competent cells, transformed cells were plated on BHIS plates containing 20 μg / mL kanamycin and 100 μg / mL spectinomycin to screen for positive clones and construct the bacterial strain. CGZ / pTRibo-Cre and CGZ / pTRibo0-Cre.

[0097] The strains constructed above were subjected to a blue-white screening experiment, such as... Figure 2 As shown in A and B, strains CGZ The / pTRibo-Cre strain exhibited a leakage rate of 7.4% without an inducer and a rearrangement rate of 62.0% with an inducer. CGZ Under both non-induced and induced conditions, all colonies on the / pTRibo0-Cre plate were blue, indicating that Cre / loxP recombination did not occur after the addition of the inducer. Cre recombinase could not be expressed when Ribozyme ribozyme was inactivated, proving that the strict regulation of this system is due to the splicing of Ribozyme.

[0098] The above results demonstrate that Ribozyme can function normally in Corynebacterium glutamicum for splicing, and that the Ribozyme induction system can further reduce leakage levels.

[0099] Using Gibson assembly, with pT4C-Cre plasmid as a template, Cre was replaced with CreⅠ-Ribozyme-CreⅡ in pTRibo-Cre plasmid to obtain plasmid pT4CRibo-Cre.

[0100] Electroporate the correctly sequenced pT4CRibo-Cre plasmid into CG-Δupp::lacZ Competent cells were revived in 900 μL BHIS medium for 1.5 hours. Transformed cells were then plated on BHIS plates containing 20 μg / mL kanamycin and 100 μg / mL spectinomycin to screen for positive clones and construct the bacterial strain. CGZ / pT4CRibo-Cre.

[0101] strains CGZ The blue-white screening experiment was performed using / pT4CRibo-Cre, and the results are as follows: Figure 2 As shown in C, the leakage rate was 0% without the inducer, while the rearrangement rate under cumate and ATc was 29.0% with the inducer. This demonstrates that the Ribozyme-based splicease-induced system, combined with the Cumate-induced chimeric promoter, can reduce the leakage level to 0%.

[0102] Example 3: Synthetic strain containing 40 loxPsym sites and application of SCRaMbLE

[0103] The *Corynebacterium glutamicum* strain semi-synCG-An (n=1-10) is a mid-genome synthesis strain of *Corynebacterium glutamicum*. It is derived from the *Corynebacterium glutamicum* ATCC13032 strain by undergoing an rpsL K43R mutation, and sequentially completing consecutive genome substitutions from chunk A1 to chunk An. Specifically, strain semi-synCG-A1 is the strain described in the article by Ye et al. (…). ACS Synthetic Biology In the study “Zhang Z, Hong P, Li Z, et al. Expediting genome synthesis of Corynebacterium glutamicum with an artificial chromosome vector[J]. Trends in Biotechnology.DOI:10.1016 / j.tibtech.2025.02.019.”, strain semi-synCG-A1 was developed by replacing the wild-type sequence in the original genome with a synthetic DNA fragment chunk A1 (approximately 55 kb) and inserting 20 loxPsym sites. In the study “Zhang Z, Hong P, Li Z, et al. Expediting genome synthesis of Corynebacterium glutamicum with an artificial chromosome vector[J]. Trends in Biotechnology.DOI:10.1016 / j.tibtech.2025.02.019.”, strains semi-synCG-A1 and semi-synCG-A2 were published in the literature ...

[0104] The pT4CRibo-Cre plasmid prepared in Example 2, along with the pT4C-Cre plasmid and pOri plasmid constructed in Example 1, were electroporated into semi-synCG-A2 competent cells. The transformed cells were plated on BHIS plates containing 25 μg / mL kanamycin to screen for positive clones and construct strains CGA2 / pT4CRibo-Cre, CGA2 / pT4C-Cre, and CGA2 / pOri.

[0105] SCRaMbLE induction was performed on strain CGA2 / pT4CRibo-Cre, and the induction steps are as follows:

[0106] (1) Take strain CGA2 / pT4CRibo-Cre and add it to 5 mL of BHIS-Kan25 medium (BHIS + 25 μg / mL Kan; the same below) and shake overnight at 30 ℃;

[0107] (2) Take the overnight bacterial culture and serially dilute it by 10.5 The coating was then applied onto a BHIS-Kan25 solid plate.

[0108] (3) Measure the OD of overnight bacterial culture 600 Transfer to 20 mL BHIS-Kan25 medium to initiate OD. 600 The concentration was 0.1, and SCRaMbLE was induced by adding a final concentration of 25 μM cumate and a final concentration of 50 ng / mL ATc. The induction was carried out at 30 ℃ and 220 rpm for 4 h, 8 h, 12 h and 24 h, respectively.

[0109] (4) Take the induced bacterial culture, wash away the inducing agent to shut down SCRaMbLE, and finally serially dilute it by 10. 5 After dilution, the sample was spread onto a BHIS-Kan25 solid plate and incubated at 30 ℃ for 48 hours.

[0110] Two strains were randomly selected at each of the four induction times, and their whole genomes were sequenced by Shanghai Sangon Biotech Co., Ltd. The sequencing results showed that the strains underwent structural variations such as deletions, flips, translocations, and duplications at different frequencies after induction. The results are as follows: Figure 3 As shown in A, the results indicate that deletion events accounted for the largest proportion among the eight strains, with 30 events, representing 49% of the total rearrangement events; replication events accounted for 21 events, representing 34% of the total rearrangement events; while flipping events were fewer, with only 10 events, representing 16% of the total rearrangement events; demonstrating that this system can mediate the occurrence of SCRaMbLE after induction.

[0111] TLC experiments were performed on strains CGA2 / pOri, CGA2 / pT4CRibo-Cre, and CGA2 / pT4C-Cre, and the results are as follows: Figure 3 As shown in Figure B, with the addition of an inducer (+) (8 hours of induction), the activity of strain CGA2 / pT4C-Cre decreased by three orders of magnitude compared to the case without an inducer (-); the activity of strain CGA2 / pT4CRibo-Cre before and after induction differed by one order of magnitude. This demonstrates that excessively high SCRaMbLE levels lead to decreased strain fitness, and strain CGA2 / pT4CRibo-Cre also exhibited a certain level of SCRaMbLE after induction.

[0112] The above results demonstrate that the Ribozyme-induced system pT4CRibo-Cre can rigorously regulate Cre recombinase expression and mediate SCRaMbLE in semi-synthetic Corynebacterium glutamicum, making it suitable for subsequent work on regulating genome rearrangement in semi-synthetic Corynebacterium glutamicum.

[0113] The violacein production plasmid pVio and the pT4CRibo-Cre plasmid prepared in Example 2 were sequentially electroporated into semi-synthetic Corynebacterium glutamicum semi-synCG-A2 to construct strains CGA2 / vio and CGA2 / vio / pT4CRibo-Cre.

[0114] SCRaMbLE induction was performed on strain CGA2 / vio / pT4CRibo-Cre, and the induction steps are as follows:

[0115] (1) Take strain CGA2 / vio / pT4CRibo-Cre and add it to 5 mL of BHIS-Cm10-Kan20 medium and shake overnight at 30 °C;

[0116] (2) Take the overnight bacterial culture and serially dilute it by 10. 5 The coating was then applied to a BHIS-Cm10-Kan20-IPTG0.5 solid plate.

[0117] (3) Measure the OD of overnight bacterial culture 600 Transfer to 20 mL BHIS-Cm10-Kan20 medium to initiate OD. 600 The concentration was 0.1, and 25 μM cumate and 50 ng / mL ATc were added to induce SCRaMbLE. 0.5 mM MIPTG was added to induce violacein production. SCRaMbLE was induced in a shaker at 30 ℃ and 220 rpm for 8 h.

[0118] (4) Take the induced bacterial culture, wash away the inducing agent to shut down SCRaMbLE, and finally serially dilute it by 10. 3 It was then coated onto a BHIS-Cm10-Kan20-IPTG0.5 solid plate.

[0119] Plates were incubated at 30 °C for approximately 2 days until colonies of suitable size appeared, and at 25 °C for approximately 4 days until the colonies turned purple. It was observed that the difference in purple intensity was not significant on plates without SCRaMbLE induction, while the difference in purple intensity was significant on plates with SCRaMbLE induction. Strains with deeper purple colonies were selected for violetin fermentation experiments. Figure 4 Based on the A in the sequence and combined with the sequencing results, high-yielding strains SC13 and SC18 were screened out.

[0120] Whole genome sequencing was performed on strains SC13 and SC18. The sequencing results for SC13 are as follows: Figure 4 As shown in B, a copy of the loxP fragment at positions 48935-58208 was found. This fragment contains the NCgl2927-2932 gene cluster (hereinafter referred to as...). TrpThe gene cluster is associated with tryptophan (Trp) biosynthesis, a key precursor to violacein. Therefore, it is particularly important to investigate whether SC13 is related to... Trp The increased production of violacein due to gene cluster duplication was verified.

[0121] Amplification Trp - rk -1、 Trp - rk -2、 Trp - rk -3、 Trp - rk -4 segment, the structure of which is the upstream homologous arm. Trp Gene cluster-P tuf - rpsL - KanR Fragment - Downstream homologous arm. The nucleotide sequences of the upstream homologous arms of the four fragments mentioned above are, in GenBank: NC_003450.3, 1,011,264-1,011,793 bp, 3,086,566-3,087,052 bp, 1,662,029-1,662,555 bp, and 2,368,580-2,369,087 bp; Trp The nucleotide sequence of the gene cluster is shown in GenBank: NC_003450.3, pp. 3,233,113-3,240,178; P tuf The reverse complementary sequence of the promoter is shown in SEQ ID NO: 6, 638-837 bp; rpsL - KanR The nucleotide sequence of the fragment is shown in SEQ ID NO: 8, wherein rpsL : 1-369 bp, KanR The nucleotide sequences of the downstream homologous arms of the above four fragments are as follows: GenBank: NC_003450.3, 1,011,794-1,012,323 bp, 3,087,099-3,087,626 bp, 1,662,627-1,663,088 bp, and 2,369,114-2,369,640 bp.

[0122] Amplification rk -del 1、 rk -del 2、 rk -del 3、 rkThe -del 4 fragment has an upstream homologous arm-downstream homologous arm structure. The nucleotide sequences of the upstream homologous arms of the four fragments are shown in GenBank: NC_003450.3, 3,239,179-3,240,178 bp. The nucleotide sequences of the downstream homologous arms are, in GenBank: NC_003450.3, 1,011,794-1,012,323 bp, 3,087,099-3,087,626 bp, 1,662,627-1,663,088 bp, and 2,369,114-2,369,640 bp.

[0123] Will Trp - rk -1 Transferred to SCG-pXMJ19- recET In competent cells, IPTG at a final concentration of 1 mM was added during the preparation process to induce RecET recombinase expression, thus constructing the strain CG / Trp1rk. Then... rk -del1 was electroporated into CG / Trp1rk competent cells, which were induced to express RecET recombinase by adding IPTG to a final concentration of 1 mM during the preparation of these competent cells, and the selection marker P was removed. tuf - rpsL - KanR Construct strain CG / Trp1. Trp - rk -2 was electroporated into CG / Trp1 competent cells, which had been pre-treated with 1 mM IPTG to induce RecET recombinase expression, thus constructing strain CG / Trp12rk. Then... rk -del 2 was electroporated into CG / Trp12rk competent cells, which had been induced to express RecET recombinase with a final concentration of 1 mM IPTG during the preparation process, and the selection marker P was removed. tuf - rpsL - KanR Strains CG / Trp12 were constructed. Strains CG / Trp123 and CG / Trp1234 were then constructed sequentially using the same method.

[0124] The violacein production plasmid pVio was electroporated into CG / Trp1, CG / Trp12, CG / Trp123, and CG / Trp1234 competent cells to construct strains CG / Trp1 / vio, CG / Trp12 / vio, CG / Trp123 / vio, and CG / Trp1234 / vio.

[0125] The above-constructed strain and strain CGA2 / vio were subjected to violetin fermentation experiments, and the results are as follows: Figure 并将其替换为Figure 4 As shown in Figure C, there was no significant difference in fermentation biomass between the strain and the wild type after the copy number of the tryptophan synthesis gene cluster increased; however, the yield of violacein produced by the strain increased significantly with the increase in the copy number of the tryptophan synthesis gene cluster. This indicates that the increase in the copy number of gene clusters related to tryptophan biosynthesis promotes tryptophan synthesis and thus promotes violacein production. This result verifies that SCRaMbLE-induced genomic rearrangement is the main reason for the increased pigment production in high-yielding strains.

[0126] Example 4: SCRaMbLE of Corynebacterium glutamicum at 178 sites and its application

[0127] The strain semi-synCG-A was developed according to the methods described in the articles by Ye et al. and Zhang et al. (Trends in Biotechnology, 2025, 43(6):1425-1445). The strain involved sequentially replacing the wild-type sequence in the original genome with synthetic DNA fragments across approximately 524 kb of genome sequence, and inserting 178 loxPsym sites. The strain semi-synCG-A was disclosed in the literature “Zhang Z , Hong P , Li Z , et al. Expediting genomesynthesis of Corynebacterium glutamicum with an artificial chromosome vector[J].Trends in Biotechnology.DOI:10.1016 / j.tibtech.2025.02.019.”

[0128] The pT4CRibo-Cre constructed in Example 2 was electroporated into semi-synCG-A competent cells. The transformed cells were plated on BHIS plates containing 25 μg / mL kanamycin to screen for positive clones and construct strain CGA / pT4CRibo-Cre.

[0129] The strain CGA / pT4CRibo-Cre was passaged overnight, and after subculturing, it was shaken for 8 h without an inducer (as a blank control). The strain sequence remained unchanged, indicating that the strict induction system can still strictly repress the expression of Cre enzyme without leakage.

[0130] The strain CGA / pT4CRibo-Cre was induced to express Cre recombinase-mediated SCRaMbLE for four consecutive rounds under pH ≤ 6.0 conditions. The induction steps are as follows:

[0131] (1) Take strain CGA / pT4CRibo-Cre and add it to 10 mL of BHIS-Kan20 medium and shake overnight at 30 ℃;

[0132] (2) Measure the OD of overnight bacterial culture 600 Transfer to 10 mL of pH 6.0 BHIS-Kan20 medium to initiate OD. 600 The concentration was 0.3, and the mixture was incubated at 30 °C and 220 rpm for 24 h.

[0133] (3) Determine the OD of overnight bacterial culture 600 Transfer to 50 mL of BHIS-Kan25 medium at the appropriate pH value to initiate OD. 600 The concentration was 0.1, and 25 μM cumate and 50 ng / mL ATc were added to induce SCRaMbLE. SCRaMbLE was induced in a shaker at 30 °C and 220 rpm for 8 h.

[0134] (4) After induction, the OD of the bacterial culture at the end of induction was measured. 600 Transfer to 10 mL of BHIS medium at the appropriate pH value to allow the initial OD to develop. 600 Incubate at 0.3°C, 30°C, and 220 rpm until the bacterial culture OD reaches its maximum. 600 The value is 0.8–1, and the cells are continuously passaged for a total of 4 generations;

[0135] (5) After passage, wash away the inducing agent to shut down SCRaMbLE, and dilute to OD. 600 0.3×10 -4 It was coated onto a BHIS-Kan25 solid plate with the corresponding pH value.

[0136] After each round of induction, dominant bacteria were enriched through subculturing. After plating, strains with larger colonies were visually selected from the solid plates. Growth curves and final OD values ​​were obtained using a fully automated Bioscreen C growth curve analyzer. 600 Data were selected based on final OD values ​​at pH 6.0 and pH 5.5. 600 The strain with the best numerical value was used for the next round of evolution. Through SCRaMbLE evolution at pH 6.0, 5.8, 5.6 and 5.4 in sequence, a strain A-S4-2.21 was finally obtained.

[0137] Whole genome sequencing was performed on strain A-S4-2.21, and the results are as follows: Figure 5As shown, sequences between multiple loxPsym sites in the genome of strain A-S4-2.21 underwent replication and flipping, generating a novel SCRaMbLE interface. This demonstrates that the Ribozyme-induced system pT4CRibo-Cre can regulate Cre recombinase expression and mediate SCRaMbLE in semi-synthetic Corynebacterium glutamicum-A.

[0138] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A stringent induction system for Corynebacterium glutamicum, characterized in that: The strict inducible system comprises repressor CymR, repressor TetR, P tetR / tetA Promoter, 1 or 2-4 in series of operon CuO , protein of interest, ribozyme sequence element; structure is CymR-tetR-P tetR / tetA -n CuO -structure of protein of interest, n CuO n in the above is an integer from 1 to 4; the protein of interest is Cre recombinase, and the ribozyme sequence element is inserted after the second nucleotide of the 104th amino acid of the Cre recombinase.

2. The stringent induction system for Corynebacterium glutamicum according to claim 1, wherein: the repressor protein CymR is from Pseudomonas putida F1, and has an amino acid sequence as shown in GenBank: ADI95374.1, specifically as shown in SEQ ID NO: 1; the repressor protein TetR has an amino acid sequence as shown in SEQ ID NO: 2; and the Cre recombinase has an amino acid sequence as shown in SEQ ID NO:

3.

3. The stringent induction system for Corynebacterium glutamicum according to claim 1, wherein: the nucleic acid sequence of the ribosome binding site RBS1 is as shown in SEQ ID NO: 5, and the nucleic acid sequence of the ribosome binding site RBS2 is as shown in the reverse complement sequence of SEQ ID NO:

5.

4. The stringent induction system for Corynebacterium glutamicum according to claim 1, wherein: the stringent induction system further comprises a ribosome binding site RBS1 for controlling expression of a protein of interest and a ribosome binding site RBS2 for expressing the repressor protein CymR or the repressor protein TetR; the nucleotide sequence of the ribosome binding site RBS1 for controlling expression of a protein of interest is as shown in SEQ ID NO: 5, 1110-1116 bp; and the nucleotide sequence of the ribosome binding site RBS2 for expressing the repressor protein CymR or the repressor protein TetR is as shown in the reverse complement sequence of SEQ ID NO: 5, 1045-1053 bp.

5. The stringent induction system for Corynebacterium glutamicum according to claim 1, wherein: the ribozyme sequence element comprises a Tetrahymena self-splicing ribozyme.

6. The stringent induction system for Corynebacterium glutamicum according to claim 5, wherein: the ribozyme sequence element has a structure of loop-IGS-Ribozyme, and has a sequence as shown in SEQ ID NO:

7. The stringent induction system also includes a promoter controlling expression of the CymR gene, the promoter including one of a Corynebacterium glutamicum promoter P hom and a Corynebacterium glutamicum promoter P tuf glutamicum. The P tetR / tetA The nucleotide sequence of the promoter is shown in SEQ ID NO: 5 at 1054-1109 bp; The nucleotide sequence of the operator 7. A plasmid vector or a recombinant bacterium comprising the stringent induction system for Corynebacterium glutamicum according to any one of claims 1-6. is shown in SEQ ID NO: 6 at positions 867-898 bp. for one of the following purposes: (1) expressing a protein of interest in Corynebacterium glutamicum by stringent induction; and (2) inducing genome rearrangement in Corynebacterium glutamicum having a loxPsym site in the genome. comprising the following steps: i) introducing the plasmid vector comprising the stringent induction system for Corynebacterium glutamicum according to any one of claims 1-6 into Corynebacterium glutamicum having two or more loxPsym sites in the genome, to obtain a recombinant strain; and ii) inducing the recombinant strain obtained in step i) to express the Cre recombinase, so that the genome sequence of the genome fragment containing the loxPsym site is randomly rearranged. ​ 8. Use of the stringent induction system for C. glutamicum as claimed in any one of claims 1 to 6, the plasmid vector or the recombinant bacteria as claimed in claim 7, characterized in that ​ ​ ​ 9. A method of synthetic genome SCRaMbLE rearrangement of Corynebacterium glutamicum, characterized in that, ​ ​ ​

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