CN115725631A - Construction of controllable high mutation rate corynebacterium glutamicum engineering bacteria and application in stress-resistant breeding - Google Patents
Construction of controllable high mutation rate corynebacterium glutamicum engineering bacteria and application in stress-resistant breeding Download PDFInfo
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Abstract
The invention discloses corynebacterium glutamicum with controllable high mutation rate and application thereof. Knocking out or knocking down corynebacterium glutamicum in bacteria production by using genetic engineering methodnucSOr/andxpbor/andtagIgene, further over-expresseddinPGenes effective in increasing the natural mutation frequency of Corynebacterium glutamicum, there areThe method helps to realize the purpose of mutation and screening under the condition of specific environmental stress, and quickly breeds the genetic engineering bacteria with excellent stress resistance, adaptability and stability. The engineering bacteria have high mutation rate and good controllability, can realize continuous mutation and evolution of in vivo genome, can greatly shorten the adaptive evolution period of a laboratory, and have wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to construction of a controllable high mutation rate corynebacterium glutamicum engineering bacterium and application of the controllable high mutation rate corynebacterium glutamicum engineering bacterium in stress-resistant breeding.
Background
Corynebacterium glutamicum (C)Corynebacterium glutamicum) Is thatAn important food safety grade industrial microbial strain is widely used for industrial fermentation of various amino acids and biosynthesis of organic acids, nucleotides, vitamins and the like, and has important economic value and application prospect. However, under industrial fermentation conditions, such as fermentation production of glutamic acid and its derivative products, corynebacterium glutamicum is often subjected to a variety of physiological or non-physiological stresses, which seriously affect the normal physiological state of the strain and the efficient accumulation of related target products, wherein low-acid stress, high-temperature stress, hyperosmotic stress, and the like are common environmental stresses. High-performance microbial strains are the basis of fermentation engineering and are also important guarantee for improving the yield and quality of biological fermentation products, and engineering microorganisms with excellent stable anti-adversity phenotypes are necessary for realizing higher fermentation yield, yield and production intensity. However, due to the limitation of technical means and theoretical knowledge, traditional metabolic engineering is more focused on the regulation and optimization of local metabolic networks, and the understanding and research on response mechanism analysis and tolerance property modification of the strain external environment stress are always short plates for genetic modification of industrial microorganisms.
Adaptive Laboratory Evolution (ALE) is a common method for achieving breeding of good properties of microbial strains by simulating variation and selection processes in natural Evolution and screening mutants with excellent performance under specific or gradually increased stress. Although the method is an effective strategy for obtaining excellent engineering strains, due to the extremely low natural mutation rate level of the microorganisms, adaptive evolution may require long-time continuous culture to accumulate mutations, and the process has long period and is easy to infect bacteria in passage, so that the evolution fails. In addition, because the nature and frequency of random mutation are difficult to control, the obtained beneficial mutation types and quantity are usually less, and the adaptive laboratory evolution still has great limitation in breeding high-performance microbial chassis bacteria.
The mutant phenotype of a strain is usually caused by the dysfunction of genes encoding DNA repair enzymes and proteins, which ensure the accuracy of DNA replication. In the long-term evolution process of microorganisms, a plurality of strategy methods for repairing DNA damage are developed, including a base excision repair system, a nucleic acid excision repair system, a mismatch repair system, a homologous recombination repair system, an SOS repair system and the like, and theoretically, the replication mutation rate of the genome of the microorganism can be improved by knocking out, knocking down or overexpressing related repair pathway genes, so that the microorganism is accelerated to adapt to physiological environment stress. Microorganisms live in a constantly changing environment and have a survival strategy that regulates the mutation rate according to the degree of adaptation to the environment. When the adaptability is limited by genetic variability, the organism naturally selects for cells with a high mutation rate in the population. Therefore, the controllable high mutation rate is beneficial to the environmental adaptation of the microorganism, and is helpful to increase the generation probability of beneficial mutation, so that the microorganism obtains an evolutionary phenotype with obviously improved robustness in a relatively short continuous stress period.
Disclosure of Invention
The invention aims to provide a construction method and application of a controllable high-mutation-rate corynebacterium glutamicum engineering bacterium.
In order to better understand and understand the role of the DNA replication repair system of Corynebacterium glutamicum in conferring genomic mutations, the present inventors selected the major components of the base excision repair system, nucleic acid excision repair system, mismatch repair system, homologous recombination repair system and SOS repair system of Corynebacterium glutamicum as test targets (Table 2), respectively, by constructing corresponding gene deletion or overexpression strains, and screened experiments based on rifamycin resistance (rifamycin resistance is usually due to its effect target RNA polymerase beta subunit coding gene)rpoBCaused by mutation), the role of the above gene targets in conferring mutations on the genome of c.glutamicum was evaluated. Through multiple experimental tests, 4 Corynebacterium glutamicum mutants with higher mutation levels are finally obtained, namelynucSGene-deleted StrainnucS，xpbGene-deleted strain∆xpb，tagIGeneLoss and incoordinationdinPOverexpression strains∆tagI+P tuf -dinPChinese medicine and compound mutant fungusnucS ∆xpb ∆tagI+P tuf -dinP。
The gene expression regulation strategy of the invention comprises reducing the expression level of the gene in the genomenucS、xpbAndtagIexpression level, increasedinPThe level of gene expression. The invention provides a method for constructing high mutation rate corynebacterium glutamicum engineering bacteria, which is characterized in that the genetic engineering method is utilized to knock out or knock down the corynebacterium glutamicum engineering bacteria in the developing bacterianucSOr/andxpbor/andtagIa gene. Preferably, further overexpressiondinPGene。
In one embodiment, thedinPThe NCBI accession number of the protein encoded by the gene is CAF20484, but is not limited to this specific protein, and includes proteins encoded by homologous genes in Corynebacterium glutamicum.
In a preferred embodiment, the knockout or knock-down in the developing bacteria is achieved by genetic engineeringnucS、xpbAndtagIgenes and further over-expresseddinPA gene.
Specifically, the genetic engineering method is a gene editing method based on the CRISPR-dCpf1 system or an RNA interference-based method. The CRISPR-dCpf1 system is a widely applied gene editing system at present, and the principle is that sgRNA (small guide ribonucleic acid) generated by CRISPR transcription mediates dCpf1 nuclease without cutting activity to target a specific target sequence, so that the transcription of a target gene is inhibited or activated, and the purpose of regulating and controlling the expression of the specific gene is achieved.
Preferably, the starting vector based on the CRISPR-dCpf1 system is a plasmid pJYS3-dCpf1, and the plasmid based on the temperature-sensitive replicon-containing plasmid cannot replicate under high temperature conditions, so that the mutation of a host cell is stopped, and a controllable high mutation rate is realized. Therefore, the gene expression regulation tool provided by the invention comprises a recombinant plasmid pJYS3-dCpf1-nucS、pJYS3-dCpf1-xpb、pJYS3-dCpf1-tagI+P tuf -dinPAnd pJYS3-dCpf1-nucS- xpb-tagI+P tuf -dinPSaid weight beingA plasmid containing a temperature-sensitive replicon cannot replicate at high temperature, and thus the mutation of a host cell is terminated.
Wherein the sgRNA-mediated cleavage-inactive dCpf1 nuclease produced by CRISPR transcription targets a specific genenucS、xpb、tagIGenes anddinPthe gene can inhibit or activate the gene transcription, so as to achieve the purpose of regulating and controlling the expression of the gene.
The invention further provides the corynebacterium glutamicum engineering bacterium with high mutation rate obtained by the construction method.
In particular to application of the high mutation rate corynebacterium glutamicum engineering bacteria in obtaining mutant bacteria with target characters, phenotypes or characteristics. Preferably, the high mutation rate corynebacterium glutamicum engineering bacteria are cultured under the stress condition, and the obtained mutant strains are screened to obtain mutant bacteria with target characters, phenotypes or characteristics.
More preferably, the method is used for breeding mutant bacteria with improved stress resistance. For example, the acid stress adaptive evolution is adopted, the subculture is carried out under the low acid stress of pH5.8, pH5.5 and pH5.2, and then the domesticated bacteria with better growth characteristics under the low acid stress are screened, so that the corynebacterium glutamicum mutant bacteria with obviously improved acid resistance are obtained. More specifically, the strain serving as a starting strain is applied to a low-acid tolerance evolution experiment, so that the host chassis can obtain an evolved strain with better adaptability to environmental low-acid stress in a relatively short continuous screening period, and the controllable high-mutation-rate strain is prompted to have a wide application prospect in the aspect of stress-resistant breeding.
The invention can effectively improve the natural mutation frequency of the corynebacterium glutamicum, is beneficial to realizing the purposes of mutation and screening under the condition of specific environmental stress, and quickly breeds the genetic engineering bacteria with excellent stress resistance, adaptability and stability. The obtained engineering bacteria have high mutation rate and better controllability, can realize the continuous mutation evolution of in vivo genome, can greatly shorten the adaptive evolution period of a laboratory, and have wide application prospect.
Drawings
FIG. 1 shows the screening test of high mutation rate bacteria of Corynebacterium glutamicum.
FIG. 2 shows pJYS3-dCpf1-nucS-xpb-tagI+P tuf -dinPRecombinant plasmid map.
FIG. 3 shows the application of low acid tolerance fast evolution of high mutation rate engineering bacteria.
Detailed Description
The invention is further illustrated by the following specific examples in order to provide a better understanding of the invention, which are not to be construed as limiting the invention.
The primers used in the examples are shown in Table 1 below.
Primers required in Table 1
| Name of primer | Primer sequence (5 '-3') |
| nucS-up-F | TCCAGCTCTTCATCCCTTATCGCCGGCGTTTCGGATGG |
| nucS-up-R | CCATGTTCTTAGAACAATGTTAAACGCATGCACCCACCATAAC |
| nucS-down-F | GTTATGGTGGGTGCATGCGTTTAACATTGTTCTAAGAACATGG |
| nucS-down-R | TCCAGCTCTTCAAGAGCATCGGCCATCTCAGCATCTGG |
| xpb-up-F | TCCAGCTCTTCATCCCAGGCCTTAATTGATGGCGAAAACCC |
| xpb-up-R | GTTGTGCACGTTATAGCTCTTTAAAAGCCACGAAATGCTTC |
| xpb-down-F | GAAGCATTTCGTGGCTTTTAAAGAGCTATAACGTGCACAAC |
| xpb-down-R | TCCAGCTCTTCAAGAGAGCAGAAGTGGGTGTCAAAGATGTC |
| tagI-up-F | TCCAGCTCTTCATCCGGTTACGGGACTGATTGCCAGCATCG |
| tagI-up-R | CTGATTTCCTAAGCCCACACACTCATGGATTCTCCTTGGGG |
| tagI-down-F | CCCCAAGGAGAATCCATGAGTGTGTGGGCTTAGGAAATCAG |
| tagI-down-R | TCCAGCTCTTCAAGACGGCTTCGATTGCAACGTTTGCCACCATG |
| dinP-up-F | TCCAGCTCTTCATCCCGCGAAACCCGGTGCTTGC |
| dinP-up-R | CTGACACGCTAAAACGCGCTTTTTATGGTAG |
| tuf-F | CTACCATAAAAAGCGCGTTTTAGCGTGTCAG |
| tuf-R | CACCCAGCGTTGCATTGTATGTCCTC |
| dinP-down-F | GAGGACATACAATGCAACGCTGGGTG |
| dinP-down-R | TCCAGCTCTTCAAGATATTCCGATGGTTGCACCGAGG |
| pCRD206-F | CCAGCTCTTCATCTAGAGTCGACCTGCAGGCATGCAAGCTTGG |
| pCRD206-R | CCAGCTCTTCAGGATCCCCGGGTACCGAGCTCGAATTCGTAATC |
| DCpf1-D917A-F | CGTTCGCCGCGAGCGATGGACAGGATGTGCACGTCGTTAGCCTTCTCCTTC |
| DCpf1-D917A-R | CATCCTGTCCATCGCTCGCGGCGAACGCCACCTCGCCTACTACACCCTGGTC |
| DCpf1-E1006A-F | GAAGTTCAGGTCTGCGAAGACCACGATTGCGTTGTAC |
| DCpf1-E1006A-R | CGTGGTCTTCGCAGACCTGAACTTCGGCTTCAAGCG |
| nucS-PAM-F | GTCATCGCCCGTTGCTCAGTTGAATTTCTACTGTTGTAGATATTTAAATAAAACGAAAGGCTC |
| nucS-PAM-R | AACTGAGCAACGGGCGATGACATCTACAACAGTAGAAATTCGGATCCATTATACCTAGGACTGAGCTAG |
| xpb-PAM-F | GATATCGTCCAATCCGATAAGACAGAATTTCTACTGTTGTAGATATTTAAATAAAACGAAAGGCTC |
| xpb-PAM-R | CTGTCTTATCGGATTGGACGATATCTACAACAGTAGAAATTCGGATCCATTATACCTAGGACTG |
| tagI-PAM-F | gatGGCTGCGCAAGATCCCCTGATgaatttctactgttgtagatatttaaataaaacgaaaggctc |
| tagI-PAM-R | cATCAGGGGATCTTGCGCAGCCatctacaacagtagaaattcggatccattatacctaggactg |
| P tuf -dinP-F | CAGTCTAGCTATCGCCAGCGTTTTAGCGTGTCAGTAG |
| P tuf -dinP-R | gtatttcatgggcttacactattcgtcatcccccgtttc |
Functional elements associated with the repair of DNA replication in C.glutamicum are described in Table 2 below.
Example 1: construction and test of high mutation rate engineering strain of corynebacterium glutamicum
Microorganisms have developed many methods or pathways for repairing DNA damage during long-term evolution, such as uracil glycosyl repair enzyme systems and mismatch repair systems to correct replication errors, photoactivation repair systems, excision repair systems, recombination repair systems, SOS repair systems, and the like to repair damage to DNA molecules caused by environmental factors and in vivo chemicals. The C.glutamicum DNA repair system and mechanism differs considerably from that of E.coli, but lacks the MutS/MutL mismatch repair system of important function found in many microorganisms. In order to better understand and understand the role of the DNA replication repair system of Corynebacterium glutamicum in conferring genome mutation, the major components of the base excision repair system, nucleic acid excision repair system, mismatch repair system, homologous recombination repair system and SOS repair system were selected as targets, and the corresponding gene deletion mutant was obtained by gene knockout strategy based on rifamycin resistance experiments (the strain obtained rifamycin resistance is usually due to its action on the coding gene of the beta subunit of the target RNA polymerase)rpoBMutation-induced), the effect of the above gene deletion on the mutation rate at the genome level of Corynebacterium glutamicum was evaluated.
Using a sensor based on temperature sensitivity andSacBthe specific steps of the homologous recombination technology based on the principle of sucrose lethality for traceless knockout of target genes can be seen in the patent publication CN112375726B, CN 103805552B. A fusion fragment containing flanking sequences of upstream and downstream of a target gene-1 kb is obtained by using an overlap extension PCR method, and is subcloned into a common knockout vector of corynebacterium glutamicum such as pCRD206 or pK18mobsacB to construct a target gene knockout plasmid. In one embodiment of the present invention, the substrate is,ΔnucSthe gene deletion mutant can be obtained by the following construction method: the corynebacterium glutamicum is obtained by amplifying nucS-up-F/nucS-up-R and nucS-down-F/nucS-down-R respectively by using primer pairsnucSThe upstream and downstream flanking regions of the site, and the PCR product obtained by the method is fused into a fragment by an overlap extension polymerase chain reaction (OE-PCR) method, and then the fragment is connected with the pCRD206 plasmid skeleton based on a Golden Gate rapid cloning technology to obtain pCRD206-nucSKnocking out the plasmid. Electrotransformation of the knock-out plasmidAfter two cycles of homologous double exchange, the corresponding Corynebacterium glutamicum can be screenedΔnucSA gene deletion mutant. Other deletion mutants of the target gene mentioned in this example were also screened using the same strategy. In another embodiment of the present invention, the substrate is,dinPthe gene in-situ overexpression mutant can be obtained by the following construction method: amplifying by using a primer pair tuf-F/tuf-R to obtain a strong promoter tuf sequence of the corynebacterium glutamicum, and amplifying by using a primer pair dinP-up-F/dinP-up-R and dinP-down-F/dinP-down-R to obtain the corynebacterium glutamicumdinPThe upstream and downstream flanking regions of the site, and the PCR product obtained by the method is fused into a fragment by an overlap extension polymerase chain reaction (OE-PCR) method, and then the fragment is connected with the pCRD206 plasmid skeleton based on the Golden Gate rapid cloning technology to obtain the pCRD206-P tuf -dinPThe plasmid is expressed in situ. After the knockout plasmid is electrically transformed into corresponding corynebacterium glutamicum, P can be obtained by screening after two rounds of homologous double exchange processes tuf -dinPIn situ over-expressing bacteria.
The genomic mutation rate was assessed using a rifamycin resistance assay. Inoculating wild type bacteria and corresponding gene mutation bacteria into LBHIS liquid culture medium, culturing overnight at 32 ℃ and 200 r/min, directly coating bacterial suspension with proper concentration on LBHIS plates added with 100 mug/mL rifamycin and LBHIS plates without rifamycin, carrying out static culture at 32 ℃ for 48 h, counting the number of grown colonies, calculating gene mutation rate, and photographing and recording. The genomic mutation rate formula is shown below: v = M/S, where M is expressed as the number of colonies grown on rifamycin-resistant plates and S is expressed as the total number of colonies grown on plates to which rifamycin-resistance was not added. The results are shown in FIG. 1, knock-out genomenucS[NCBI-ProteinID:CAF19919;Endonuclease]、xpb[NCBI-ProteinID:CAF19522；DNA/RNA helicase of superfamily II]、tagI[NCBI-ProteinID:CAF20861；DNA-3-methyladenine glycosylase I]Simultaneous overexpressiondinP[NCBI-ProteinID:CAF20484；Y-family DNA polymerases]The target points can improve the genome mutation frequency of the strain to different degrees. The research result shows that the natural genome mutation frequency of the wild type bacteria is 3.60 +/-1.61 multiplied by 10 -8 ，ΔnucSThe genome mutation frequency of the mutant is 9.01 +/-2.09X 10 -6 ，∆xpbThe genomic mutation frequency of the mutant was 1.17. + -. 0.31X 10 -6 ，∆tagI+P tuf -dinPThe genomic mutation frequency of the mutant was 1.05. + -. 0.12X 10 -6 . Compared with the natural mutation frequency of the wild type bacteria, the genome mutation frequency of the mutant is respectively improved by about 250 times, 32 times and 29 times, and the system is prompted to modify the target site to be conductive to improving the natural mutation frequency of the strain. Single colonies capable of growing on rifamycin-resistant plates were randomly picked and obtained by amplificationrpoBThe coding region of the gene, which was then subjected to Sanger sequencing analysis, was counted for the type and location of the base mutation (where the strain acquired rifamycin resistance based on rifamycin resistance experiments was generally due to its effect on the gene encoding the beta subunit of the target RNA polymeraserpoBCaused by a mutation). Analysis shows that the base mutation of the sequencing region mainly occurs at five positions: namely, A → G (Gln → Arg) at position 426, C → T (Ser → Phe) at position 435, C → T (His → Tyr) at position 439, A → G (His → Arg) at position 439 and C → T (Ser → Leu) at position 444, wherein the genetic mutations include the basic types of pyrimidine substituted pyrimidine and purine substituted purine in SNPs, and the number of C → T base transition mutations is the largest, and the genetic mutations substantially cover all of the high mutation rate Corynebacterium glutamicum engineered bacteria.
Example 2: construction of Corynebacterium glutamicum regulation tool with controllable high mutation rate
This example provides a gene expression regulatory tool based on the CRISPR-dCpf1 system, and targets and effects include reduction of genomic DNAnucS、xpbAndtagIexpression level, increasedinPThe level of gene expression. The CRISPR-dCpf1 system is a widely applied gene editing system at present, and the principle is that sgRNA generated by CRISPR transcription mediates dCpf1 nuclease which loses cutting activity to target a specific target sequence, so that the transcription of a target gene is inhibited or activated, and the purpose of regulating and controlling the expression of the specific gene is achieved. The gene expression regulation tool provided by the invention is constructed by utilizing a commercial plasmid pJYS3_ delta crtYf skeleton, and is obtained by using a primer pair dCpf1-D917A-F/dCpf1-D917A-R and dCpf1-E1006A-F/dCpf1-E1006A-R Point mutations to the Cpf1 proteins D917A and E1006A, inactivating the DNA cleavage activity of the Cpf1 protein, and finally obtaining the plasmid pJYS3-dCpf1. Respectively constructing pJYS3-dCpf1-nucS、pJYS3-dCpf1-xpb、pJYS3-dCpf1-tagIPlasmids each contained a PAM sequence region recognized by Cpf1 protein (shown in Table 3). Using primer pair P tuf -dinP-F/P tuf Amplification of-dinP-R to obtain P tuf The dinP fragment in pJYS3-dCpf1-tagIRespectively constructing and obtaining pJYS3-dCpf1-tagI+P tuf -dinP plasmid. pJYS3-dCpf1 was obtained by combining the above sites-nucS-xpb-tagI+P tuf -dinPThe plasmid sequence of the recombinant plasmid is shown in SEQ ID NO. 1. The recombinant plasmid can express dCpf1 nuclease without cutting activity and sgRNA targeting a specific target sequence, and meanwhile, a plasmid skeleton contains a temperature-sensitive replicon, so that the plasmid cannot replicate at high temperature, and mutation of a host cell is stopped. The recombinant plasmid pJYS3-dCpf1 is prepared-nucS、pJYS3-dCpf1-xpb、pJYS3-dCpf1- tagI+P tuf -dinP andpJYS3-dCpf1-nucS-xpb-tagI+P tuf -dinPcorynebacterium glutamicum ATCC 13032 was transformed electrically and then plated on rifamycin-containing plates, and the mutation rate was calculated.
TABLE 3 Gene elements and gRNA sequences
| Gene eleme nt | 5’- NYTV -3’ | Sequence (5’-3’) |
| nucS | 5’- TTTA -3’ | gaatttctactgttgtagatGTCATCGCCCGTTGCTCAGTTgaatttctactgttgtagat |
| xpb | 5’- TTTA -3’ | gaatttctactgttgtagatATCGTCCAATCCGATAAGACAgaatttctactgttgtagat |
| tagI | 5’- GTTG -3’ | gaatttctactgttgtagatGGCTGCGCAAGATCCCCTGATgaatttctactgttgtagat |
| array | gaatttctactgttgtagatGTCATCGCCCGTTGCTCAGTTgaatttctactgttgtagatATCGTCCA ATCCGATAAGACAgaatttctactgttgtagatGGCTGCGCAAGATCCCCTGATgaatttctactgttg tagat |
The analysis result shows that the recombinant plasmid containing pJYS3-dCpf1-nucSStrain andnucSthe gene deletion bacteria show similar genome mutation frequency which is 270 times that of wild control bacteria; contains recombinant plasmid pJYS3-dCpf1-xpbOr pJYS3-dCpf1-tagI+P tuf -dinPThe genome mutation frequencies of the strains are slightly lower than the corresponding ΔxpbOr∆tagI+ P tuf -dinPThe strains are 12 times and 7 times of wild control strains respectively.
In order to obtain higher genome mutation efficiency, the action targets are subjected to combined operation. In one aspect, the simultaneous knockout is on a chromosomenucS、xpb、tagIAnd overexpressiondinPObtaining combined mutant fungi after genenucS ∆xpb ∆ tagI+P tuf -dinP(ii) a On the other hand, pJYS3-dCpf1 was obtained by construction-nucS-xpb-tagI+P tuf -dinPRecombinant plasmid (shown in FIG. 2), and transferred into Corynebacterium glutamicum for reducingnucS、xpb、tagIGene expression level, simultaneously increasingdinPAt the gene level. Evaluating the genome mutation rate of the two bacteria by a rifamycin resistance experiment, and finding out a combined mutant strain ΔnucS ∆xpb ∆tagI+P tuf -dinP(ii) genome mutation rate (2.42. + -. 0.39X 10) -5 ) Is a wild type control bacterium (3.60 +/-1.61 multiplied by 10) -8 ) 672 times as much as that of the vector containing pJYS3-dCpf1-nucS-xpb-tagI+P tuf -dinPMutant rate of recombinant plasmid strain genome (average mutant rate 1.24 + -0.21X 10) -5 ) Is a wild type control bacterium (3.60 +/-1.61 multiplied by 10) -8 ) 344 times higher.
The results further suggest that a universal recombinant plasmid system can be constructed and obtained based on CRISPR-dCpf1, and effective regulation and control can be realizednucS、xpb、tagI、dinPAnd the gene expression level of the target point is equal, so that the natural mutation frequency of the corynebacterium glutamicum can be effectively improved. The method is simple and convenient to operate, has high efficiency and universality, and can control the mutation rate level required by the engineering strain by selecting different regulating tools.
Example 3: stress-resistant breeding application of corynebacterium glutamicum with controllable high mutation rate
In order to verify the applicability of the high mutation rate engineering bacteria in high-performance strain breeding, a low-acid tolerance experiment is randomly selected for testing. As shown in a-c in FIG. 3, wild type WT and high mutation engineering bacteria are selectednucS、∆ xpb、∆tagI+P tuf -dinPAnnucS ∆xpb ∆tagI+P tuf -dinPFor the initial strain to carry out acid stress adaptive evolution, subculturing under the conditions of low acid stress of pH5.8, pH5.5 and pH5.2, carrying out three rounds of passage for 9 days (as shown in a-c in figure 3), rapidly screening to obtain domesticated bacteria with good growth characteristics under low acid stress, and carrying out subsequent separation and purification to obtain an acid-resistant strain from 100 single coloniesThe Mutant strain Mutant-isolated of the corynebacterium glutamicum can be improved most obviously. As shown in d in FIG. 3, the mutant strain has obviously better growth performance than the wild control strain under the different low-acid pH stress conditions. The results suggest that the high mutation rate engineering bacteria can greatly improve the spontaneous mutation frequency of the bacterial strain, shorten the adaptive evolution period and be beneficial to subsequent optimization and breeding of engineering chassis bacteria with excellent stress resistance.
Claims (10)
1. A method for constructing high mutation rate corynebacterium glutamicum engineering bacteria is characterized in that the genetic engineering method is used for knocking out or knocking down the corynebacterium glutamicum in the developing bacterianucSOr/andxpbor/andtagIa gene.
2. The method of construction of claim 1, further overexpressingdinPGene。
3. The method of construction of claim 2, wherein the method comprisesdinPThe NCBI accession number for the gene encoded protein is CAF20484.
4. The method of claim 2, wherein the knockout or knock-down in the developing bacteria is achieved by genetic engineeringnucS、xpbAndtagIgenes and further over-expresseddinPA gene.
5. The method of any one of claims 1 to 4, wherein the genetic engineering method is a CRISPR-dCpf1 system-based gene editing method or an RNA interference-based method.
6. The construction method according to claim 5, wherein the starting vector based on the CRISPR-dCpf1 system is plasmid pJYS3-dCpf1, and the plasmid containing the temperature-sensitive replicon cannot replicate under high temperature conditions, so that the mutation of the host cell is stopped, and a controllable high mutation rate is realized.
7. The method of claim 5, wherein the sgRNA-mediated loss of cleavage activity of dCpf1 nuclease produced by CRISPR transcription targets a particular saidnucS、xpb、tagIGenes anddinPthe gene can inhibit or activate the gene transcription, so as to achieve the purpose of regulating and controlling the expression of the gene.
8. The engineered Corynebacterium glutamicum with high mutation rate obtained by the construction method according to any one of claims 1 to 7.
9. Use of the engineered bacterium of corynebacterium glutamicum of claim 8, to obtain a mutant of a desired trait, phenotype or characteristic.
10. The use of claim 9, wherein the high mutation rate corynebacterium glutamicum engineered bacteria is cultured under stress conditions, and the obtained mutant strain is screened to obtain mutant bacteria with target traits, phenotypes or characteristics; preferably, the method is used for breeding mutant bacteria with improved stress resistance.
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| CN113774077A (en) * | 2021-09-17 | 2021-12-10 | 中国医学科学院病原生物学研究所 | Single-base gene editing system and method applied to mycobacterium tuberculosis |
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