Method for constructing polyploidy corynebacterium glutamicum by utilizing regulatory division gene and application

Abstract

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a method for constructing polyploidy corynebacterium glutamicum by utilizing a regulatory division gene and application of the polyploidy corynebacterium glutamicum. Polyploid corynebacterium glutamicum is successfully constructed by adjusting the expression level of a division gene ftsZ to a proper concentration, and tests prove that compared with original haploid corynebacterium glutamicum, the obtained polyploid corynebacterium glutamicum grows slower, but grows faster when being cultured to a later stage in a CGXII culture medium; according to the present invention, the expression of proteins can be enhanced, and the terpene accumulation rate is high compared with the mode strain ATCC 13032 so as to establish the efficient chassis cell to produce the bio-based product, and the method can be used for the research on the influence of the increase of the chromosome number in the prokaryote on the cell, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and microbial technology, and specifically relates to a method and application of constructing polyploid Corynebacterium glutamicum by regulating fission genes. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Corynebacterium glutamicum has been the main producer of amino acid fermentation in the world for nearly four decades. It was first described by Kinoshita as a glutamate-producing bacterium in 1957. It has the characteristics of being generally recognized as safe for food (GRAS) and has become one of the model microorganisms for industrial production, and is used to build green and efficient microbial cell factories.

[0004] It is reported that Corynebacterium glutamicum produces more than 6 million tons of amino acids per year, and its main products include L-glutamic acid, L-lysine, branched-chain amino acids and L-proline. In recent years, it has been widely used in the production of chemicals, fuels, materials, natural products and recombinant proteins. However, the Corynebacterium glutamicum isolated from nature, its properties are far from meeting the needs of industrial production, so how to improve the microbial chassis to obtain high-yield and efficient industrial microbial cell factories has become the key to industrial applications.

[0005] In the field of synthetic biology, researchers have improved the amino acid production capacity of Corynebacterium glutamicum by metabolic engineering. For example, based on the exploration and optimization of biosynthetic elements and metabolic pathways. The trend of cell evolution is from haploid cells to polyploid cells, and the increase in the number of chromosomes effectively improves its genetic diversity, thereby enhancing the cell's tolerance to environmental stress. In polyploid cells, the presence of gene redundancy can prevent harmful mutations that may escape and persist on other chromosomes, ensuring the stable survival of the cells. In addition, the increase in the number of chromosomes also profoundly affects the overall transcriptional activity, metabolic pathways, and gene expression patterns of the cell. Therefore, the construction of artificial polyploid Corynebacterium glutamicum based on synthetic biology may establish an efficient chassis cell to produce bio-based products, so it is of great significance. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method and application of utilizing a regulated fission gene to construct a polyploid Corynebacterium glutamicum. The present invention has found through experimental studies that ftsZ is the main gene controlling cell division, which recruits other fission proteins to accurately regulate the cell division process in the form of forming a "Z ring". The weak expression of ftsZ below its threshold can prevent cell division, thereby preventing a new round of chromosome replication, while still allowing the ongoing replication process to be completed. This cell grows in a filamentous manner and contains an increased number of incorrectly separated chromosomes, and the chromosomes in the filamentous cells extend in a filamentous manner and are distributed in the cell. However, long-term inhibition of division can cause the filamentous cells to eventually be lysed due to the cell cycle stopping operation. On the contrary, the strong expression of ftsZ allows cells to survive under normal division and chromosome separation. Therefore, by adjusting the expression level of ftsZ to an appropriate concentration, the polyploid Corynebacterium glutamicum is successfully constructed. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a method for constructing a polyploid Corynebacterium glutamicum by regulating a fission gene, the method comprising: during the process of Corynebacterium glutamicum replicating to form a replication fork, inserting a fission gene weak expression control element containing a fission gene encoding an antibiotic resistance gene before the start codon of the fission gene of one of the replication fork chromosomes, thereby controlling the cell to contain multiple chromosomes while dividing normally, thereby forming a polyploid; wherein the fission gene is specifically the ftsZ gene; and the expression intensity of the promoter-RBS expression element in the control element is not more than 200 a.u.

[0009] It should be noted that in the present invention, although Corynebacterium glutamicum is taken as an example to construct and obtain polyploid Corynebacterium glutamicum, based on the inventive concept of the present application, obtaining corresponding polyploids in other haploid organisms is also within the scope of protection of the present invention.

[0010] The second aspect of the present invention provides a polyploid Corynebacterium glutamicum obtained by the above method.

[0011] The third aspect of the present invention provides the use of the polyploid Corynebacterium glutamicum in the preparation of bio-based products.

[0012] Beneficial technical effects of one or more of the above technical solutions:

[0013] The above technical scheme provides a method for constructing a polyploid Corynebacterium glutamicum by regulating a fission gene, and specifically discloses that the polyploid Corynebacterium glutamicum is successfully constructed by regulating the expression level of the fission gene ftsZ to an appropriate concentration. It has been verified by experiments that the obtained polyploid Corynebacterium glutamicum grows slower than the original haploid Corynebacterium glutamicum, but grows faster when cultured in CGXII medium to the later stage; it can enhance protein expression and has higher terpenoid accumulation than the model strain ATCC 13032, thereby establishing an efficient chassis cell to produce bio-based products, and can be applied to the study of the effect of the increase in the number of chromosomes on cells in prokaryotes, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] Figure 1 This is a schematic diagram of the principle of constructing a diploid by suicide plasmid in an embodiment of the present invention.

[0016] Figure 2 This is a schematic diagram of the principle of recET constructing a diploid in an embodiment of the present invention.

[0017] Figure 3 This is the elimination of the recET plasmid and plasmid in the embodiment of the present invention.

[0018] Figure 4 This is the PCR verification and sequencing verification of the polyploid in the embodiment of the present invention.

[0019] Figure 5 This is the morphological analysis of the polyploid Corynebacterium glutamicum in the examples of the present invention.

[0020] Figure 6 This is the protein expression analysis of polyploid Corynebacterium glutamicum in the examples of the present invention.

[0021] Figure 7 It is the color of the polyploid Corynebacterium glutamicum cells in the embodiment of the present invention.

[0022] Figure 8 This is the accumulation of terpenes in the polyploid Corynebacterium glutamicum in the embodiment of the present invention.

[0023] Fig. 9 This is the amino acid accumulation of the polyploid Corynebacterium glutamicum in the embodiment of the present invention. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0026] In a typical embodiment of the present invention, a method for constructing a polyploid Corynebacterium glutamicum by regulating a fission gene is provided, the method comprising: during the process of Corynebacterium glutamicum replicating to form a replication fork, a fission gene weak expression control element containing a fission gene encoding an antibiotic resistance gene is inserted before the start codon of the fission gene of one of the replication fork chromosomes, thereby controlling the cell to contain multiple chromosomes while dividing normally, thereby forming a polyploid; the fission gene is the ftsZ gene; the expression intensity of the promoter-UTR expression element in the control element is not more than 200 a.u.

[0027] The antibiotic resistance gene includes but is not limited to the kanamycin resistance gene nptII, the tetracycline resistance gene tetR and the chloramphenicol acetyltransferase gene CmR. In a specific embodiment of the present invention, the antibiotic resistance gene is the chloramphenicol acetyltransferase gene CmR.

[0028] Specifically, the split gene weak expression control element containing the antibiotic resistance gene encoding is specifically composed of antibiotic resistance gene-terminator-promoter-UTR (Untranslated Regions); the expression intensity of the promoter-UTR expression element in the control element is not more than 200 a.u. (In the present invention, the expression intensity is expressed as a fluorescence intensity value (au), and the fluorescence intensity value is calculated by the ratio of fluorescence intensity to OD600); further not more than 150 a.u.. By controlling the expression intensity of the above-mentioned weak expression control element, that is, controlling the expression intensity of ftsZ, the Corynebacterium glutamicum cell contains both wild-type and engineered chromosomes, thereby forming polyploidy.

[0029] The terminator may be rrnB T1 Terminator.

[0030] The promoter is preferably a promoter with lower expression strength, including but not limited to P021, P027, P020, P026, P042, P022, P010 and P gntK , further preferably P020;

[0031] At the same time, since Corynebacterium glutamicum does not have a clear synthetic biology element library, in the present invention, a series of promoter-UTRs of different strengths were obtained by intercepting "promoter-UTR" in the Corynebacterium glutamicum genome according to the transcriptome results (as shown in Table 3), wherein the promoter-UTR includes but is not limited to P021-UTR, P027-UTR, P020-UTR, P026-UTR, P042-UTR, P022-UTR, P010-UTR and P gntK -UTR, more preferably P020-UTR (SEQ ID NO.1).

[0032] In another specific embodiment of the present invention, in the method, the specific method of insertion can be performed by homologous recombination, which will not be described in detail here.

[0033] The method further comprises applying the antibiotic corresponding to the antibiotic resistance gene to form screening pressure in the culture environment of Corynebacterium glutamicum.

[0034] The selected Corynebacterium glutamicum is not specifically limited. In a specific embodiment of the present invention, the selected Corynebacterium glutamicum is specifically Corynebacterium glutamicum ATCC 13032, which is commercially available to the public.

[0035] In addition, it should be noted that in the present invention, although Corynebacterium glutamicum is taken as an example to construct and obtain polyploid Corynebacterium glutamicum, based on the inventive concept of the present application, obtaining corresponding polyploids in other haploid organisms is also within the scope of protection of the present invention.

[0036] In another specific embodiment of the present invention, a polyploid Corynebacterium glutamicum obtained by the above method is provided. It has been verified by experiments that the polyploid Corynebacterium glutamicum obtained in the present application has an increased average cell volume and surface area, a reduced specific surface area, and a slower growth than the original haploid Corynebacterium glutamicum, but when cultured in CGXII medium to the later stage, it grows faster; the chromosomes are very stable and can enhance protein expression.

[0037] In another specific embodiment of the present invention, there is provided the use of the polyploid Corynebacterium glutamicum in the preparation of bio-based products.

[0038] The bio-based products include proteins, terpenes and amino acids.

[0039] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] Example

[0041] 1. Experimental Materials

[0042] 1.1 Strains, plasmids and primers

[0043] The strains, plasmids, primers and sequences used in this example are listed in Tables 1 to 3, respectively.

[0044] Table 1. Strains and plasmids used in this example

[0045]

[0046] Table 2. Primer sequences used in this example

[0047]

[0048]

[0049]

[0050] Table 3. Sequences used in this example

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] 1.2 Culture medium

[0058] The culture medium used in this example is as follows: BHIS culture medium: 38.5 g / L brain heart infusion broth, 91 g / L sorbitol. CGXII culture medium: 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L K2HPO4, 1 g / L KH2PO4, 0.25 g / L MgSO4·7H2O, 42 g / L MOPs, add 1 mL 10 g / L Cacl2 to adjust pH to 7 and then make up to 920 mL; trace element solution: 1 g FeSO4·7H2O, 1 g MnSO4·H2O, 0.1 g ZnSO4·7H2O, 0.02 g CuSO4 and 0.002 g NiSO4·6H2O add 90 mL double distilled water, and adjust pH to 1 with concentrated hydrochloric acid; protocatechuic acid solution: 300 mg protocatechuic acid is added to 8 mL double distilled water, and 1 mL Dissolve in 10M NaOH and filter to sterilize; Biotin solution: 200 mg / L; When using, add 1 mL of trace element solution, 1 mL of biotin solution, 1 mL of protocatechuic acid solution and 80 mL of 50% glucose solution.

[0059] 1.3 Commonly used enzymes and kits in molecular biology

[0060] The enzymes used in the molecular biology techniques in this example are as follows: Phanta Max Super-Fidelity DNA Polymerase for gene cloning was purchased from Novozymes. 2×Es Taq MasterMix (Dye) premixed enzyme for colony PCR verification was purchased from Kangwei Century (Beijing) Company. 2×MultiF Seamless Assembly Mix for constructing recombinant plasmids was purchased from ABclonal. The kits used in this example are as follows: Plasmid mini-extraction kit and agarose gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Company.

[0061] 2. Experimental Methods

[0062] 2.1 Construction of plasmids and strains

[0063] The recombinant plasmid of this example was constructed in E. coli DH5α. tac -UTR fusion was assembled into plasmid pECXK99E by DNA seamless cloning method to obtain the characterization plasmid pECXK99E-P tac -mCherry.

[0064] To control the expression of ftsZ, promoters P021, P027, and P gntK, P020, P026, P042, P022, and P010 were inserted into the chromosome of the strain C. glutamicum before the start codon of ftsZ by homologous recombination, thus obtaining the engineered strains Cg-21-Z, Cg-27-Z, and Cg-P gntK -Z, Cg-20-Z, Cg-26-Z, Cg-42-Z, Cg-22-Z and Cg-10-Z. tac -mCherry was transferred into C. glutamicum and Cg-20-Z to obtain strains Cg-mCherry and Cg-20-Z-mCherry, respectively, to analyze the protein expression level.

[0065] 2.2ftsZ Characterization of Control Component Strength

[0066] To compare strains Cg-21-Z, Cg-27-Z, Cg-P gntK -Z, Cg-20-Z, Cg-26-Z, Cg-42-Z, Cg-22-Z and Cg-10-Z, and the plasmids pECXK99E-P021-mCherry, pECXK99E-P027-mCherry, pECXK99E-P gntK -mCherry, pECXK99E-P020-mCherry, pECXK99E-P026-mCherry, pECXK99E-P042-mCherry, pECXK99E-P022-mCherry and pECXK99E-P010-mCherry were transformed into strain C. glutamicum for characterization. All strains were cultured in 48-well plates containing 0.5 mL of liquid CGXII medium at 30 ° C for 48 h. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 590 nm and an emission wavelength of 645 nm. The fluorescence intensity value (au) was calculated by comparing the fluorescence intensity with the OD 600 The ratio is calculated.

[0067] 2.8 Protein expression analysis

[0068] In this study, the fluorescent protein mCherry was used for protein expression analysis. In order to compare the protein expression levels between strains C. glutamicum and Cg-20-Z, the plasmid pECXK99E-P tac -mCherry was transferred into C. glutamicum and Cg-20-Z to construct the characterization strains Cg-mCherry and Cg-20-Z-mCherry, respectively. tacThe expression of mCherry was controlled in strains C.glutamicum and Cg-20-Z, and the fluorescence intensity of mCherry in strains C.glutamicum and Cg-20-Z was detected by an enzyme-linked microplate reader. Single colonies of strains Cg-mCherry and Cg-20-Z-mCherry were picked and cultured in 2 mL of BHIS medium at 30°C for 36 h to obtain seeds for characterization. The seeds were then transferred to a 48-well plate containing 500 μL of CGXII medium at a ratio of 2% (v / v) and cultured at 30°C for 48 h. The fluorescence intensity was detected by an enzyme-linked microplate reader at an excitation wavelength of 590 nm and an emission wavelength of 645 nm. The fluorescence intensity value (au) was calculated by comparing the fluorescence intensity with the OD 600 During the characterization process, strain Cg-mCherry required the addition of 25 μg / ml of kanamycin sulfate, while strain Cg-20-Z-mCherry required the addition of 25 μg / ml of kanamycin sulfate and 5 μg / ml of chloramphenicol.

[0069] 3. Experimental results and discussion

[0070] 3.1 Design of polyploid Corynebacterium glutamicum

[0071] In the process of replication of Corynebacterium glutamicum to form replication forks, the ftsZ weak expression control element containing the chloramphenicol acetyltransferase gene CmR is inserted in front of the ftsZ gene start codon of one of the replication fork chromosomes to control the cells to contain multiple chromosomes while dividing normally. Under the selection pressure of adding chloramphenicol and weakly expressing ftsZ, a polyploid Corynebacterium glutamicum containing both wild-type and engineered chromosomes was constructed. The detailed design scheme is as follows ( Figure 1 ). During the replication of Corynebacterium glutamicum, a weak expression element containing "CmR-terminator-promoter-UTR" was inserted in front of the start codon ATG of the ftsZ gene. During chromosome replication, this expression element may be integrated before ftsZ in one or both replication forks. However, only inserting it into one replication fork can ensure that the cell survives by expressing ftsZ normally using another replication fork containing wild-type ftsZ. When chloramphenicol is added, Corynebacterium glutamicum containing only wild-type chromosomes cannot survive because it cannot express CmR ( Figure 1 glutamicum was isolated based on the precise regulation of the divisome formed by FtsZ. FtsZ is a tubulin homologue that forms a "Z ring" to recruit other division proteins and divide the cell into two. The cells use the wild-type chromosome to normally express ftsZ to regulate cell division, and the engineered chromosome to express CmR to resist chloramphenicol stress.

[0072] Because the suicide plasmid pK18mobsacB relies on sucrose selection after passage for homologous recombination, the ftsZ promoter cannot be replaced by homologous recombination with the suicide plasmid pK18mobsacB. Figure 1 In order to replace the ftsZ promoter, the RecET-mediated homologous recombination system was used to insert the "CmR-terminator-promoter-UTR" expression element before the ATG start codon of ftsZ in one step ( Figure 2 ).

[0073] Construction of pECXK99E-Δper-P brnFE -recET plasmid, the removal of the replicon per in the plasmid can be achieved by removing pECXK99E-Δper-P overnight in antibiotic-free medium brnFE -recET plasmid ( Figure 3 ). Promoter P brnFE The induction intensity of branched-chain amino acids was the strongest among them, and pECXK99E-Δper-P brnFE -recET plasmid was transferred into C. glutamicum, and after overnight culture in 2 mL BHIS medium, 10% was transferred to BHIS medium containing 13.1 g / L L-Leu and induced for 5 h until OD 600 1, to make competent cells. Transfer 4 μg of the phosphorylated and purified "CmR-terminator-promoter-UTR" expression element fragment, resume culture for 5 hours, and apply BHIS plates containing Cm ( Figure 2 ).

[0074] P021-UTR,P027-UTR,P gntK -UTR, P020-UTR, P026-UTR, P042-UTR, P022-UTR, P010-UTR expression elements, the above expression elements control strains Cg-21-Z, Cg-27-Z, Cg-P gntK The intensity of ftsZ in Cg-Z, Cg-20-Z, Cg-26-Z, Cg-42-Z, Cg-22-Z and Cg-10-Z was determined by isolating single colonies under the condition of adding chloramphenicol and amplifying by PCR with primers 1.9-ftsZ-F / 1.9-ftsZ-R designed upstream and downstream of the expression element "CmR-terminator-promoter-UTR" ( Figure 4 ). Two DNA fragments were amplified from strain Cg-20-Z: 2474 bp of the wild-type chromosome and 3825 bp of the engineered chromosome. However, the other strains only had the 2474 bp DNA fragment from the engineered chromosome ( Figure 4). Therefore, strain Cg-20-Z may contain both wild-type and engineered chromosomes. Therefore, regulating ftsZ to an appropriate expression level can make cells contain both wild-type and engineered chromosomes.

[0075] 3.2 Verification of polyploidy

[0076] In order to further confirm that polyploid Corynebacterium glutamicum can be constructed by regulating cell division, we used Cg-20-Z as the research object and the model strain C. glutamicum as the control strain, and verified it through the following multiple methods.

[0077] We first verified the polyploidy of C. glutamicum Cg-20-Z by PCR amplification of the expression element. Twelve isolated single colonies were randomly selected from C. glutamicum Cg-20-Z for verification. The results are shown in Figure 4 As shown, Corynebacterium glutamicum Cg-20-Z contains both a 2474 bp wild-type chromosome and a 3825 bp engineered chromosome, which indicates that strain Cg-20-Z is a polyploid cell.

[0078] 3.3 Morphological analysis of polyploid Corynebacterium glutamicum

[0079] The increase in chromosome number in C. glutamicum may affect cell morphology. We compared the morphological changes of C. glutamicum and Cg-20-Z strains, such as Figure 5 As shown in the figure, after C. glutamicum changed from haploid cell C. glutamicum to polyploid cell Cg-20-Z, the cell morphology changed. The decrease in ftsZ intensity prevented FtsZ from forming a septum to constrict the two daughter cells after the genome replication was completed, causing the morphology of C. glutamicum to be hypertrophic and filamentous. The abnormal division of C. glutamicum diploid may cause abnormal assembly of Z rings, forming branch-like cells.

[0080] 3.4 Analysis of protein expression levels in polyploid Corynebacterium glutamicum

[0081] In order to analyze the effect of polyploid Corynebacterium glutamicum on protein synthesis, we used the reporter gene mCherry for characterization analysis. Figure 7 As shown, with promoter P tac The expression of the reporter gene mCherry was controlled and transferred into the haploid C. glutamicum and the polyploid C. glutamicum Cg-20-Z. The C. glutamicum and Cg-20-Z strains were cultured in CGXII medium. Figure 7 As shown, compared with strain C. glutamicum, the red fluorescent protein expression level in strain Cg-20-Z cells increased significantly after 96h of culture ( Figure 6 ). The above results indicate that polyploid Corynebacterium glutamicum can enhance protein expression, which is beneficial to the synthesis of metabolites and protein production.

[0082] 3.5 Analysis of terpenoid accumulation levels in polyploid C. glutamicum

[0083] During the culture process, it was found that the color of the Cg-20-Z strain was darker than that of C. glutamicum ( Figure 8 ), C. glutamicum synthesizes C50 carotenoids through the native and complete MEP pathway. The yellow phenotype of C. glutamicum comes from C50 carotenoids, glycosylated decaprenyl xanthin ( Figure 7 The accumulation of decaprenylflavin in the polyploid C. glutamicum Cg-20-Z was 4.92 times that of the wild-type C. glutamicum ( Figure 8 ).

[0084] 3.6 Analysis of amino acid accumulation levels in polyploid C. glutamicum

[0085] After culturing haploid C. glutamicum and polyploid C. glutamicum Cg-20-Z with ion culture medium CGXII for 48 h, the amino acid analyzer detected the ability of C. glutamicum polyploid Cg-20-Z to produce various amino acids compared with haploid C. glutamicum. Among the 18 amino acids, only Thr, Glu, Gly, Ala, Val, Phe and Lys were detected. The titers of Val and Lys produced by diploid C. glutamicum were increased by 169% and 109%, respectively, compared with the model strain of C. glutamicum. Fig. 9 ).

[0086] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for constructing polyploid Corynebacterium glutamicum by regulating fission genes, characterized in that: The method comprises: during the process of Corynebacterium glutamicum replicating and forming a replication fork, inserting a weak expression control element of a fission gene containing a gene encoding an antibiotic resistance before the start codon of the fission gene of one of the replication fork chromosomes, controlling the cell to contain multiple chromosomes while dividing normally, thereby forming a polyploid; the fission gene is the ftsZ gene; and the expression intensity of the promoter-UTR expression element in the control element is no more than 200 a.u.

2. The method according to claim 1, characterized in that The antibiotic resistance genes include kanamycin resistance gene nptII, tetracycline resistance gene tetR and chloramphenicol acetyltransferase gene CmR.

3. The method according to claim 1, characterized in that The split gene weak expression control element containing the antibiotic resistance gene is specifically composed of antibiotic resistance gene-terminator-promoter-UTR; the expression intensity of the promoter-UTR expression element in the control element is no more than 200 a.u.; further no more than 150 a.u.

4. The method according to claim 1, characterized in that The terminator is rrnB T1 Terminator.

5. The method according to claim 1, characterized in that The promoters are promoters with lower expression strength, including P021, P027, P020, P026, P042, P022, P010 and P gntK , further preferably P020; the promoter-UTR includes P021-UTR, P027-UTR, P020-UTR, P026-UTR, P042-UTR, P022-UTR, P010-UTR and P gntK -UTR, more preferably P020-UTR (SEQ ID NO.1).

6. The method according to claim 3, characterized in that The method further comprises applying an antibiotic corresponding to the antibiotic resistance gene to form a screening pressure in a culture environment of Corynebacterium glutamicum.

7. The method according to claim 1, characterized in that The selected Corynebacterium glutamicum was Corynebacterium glutamicum ATCC 13032.

8. The polyploid Corynebacterium glutamicum obtained by the method according to any one of claims 1 to 7.

9. Use of the polyploid Corynebacterium glutamicum according to claim 8 in the preparation of bio-based products.

10. The use according to claim 9, characterized in that: The bio-based products include proteins, terpenes and amino acids.

Images