Genetically engineered bacterium for producing L-isoleucine as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium for stably and efficiently producing L-isoleucine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes E. coliTHRD as a host and contains an RNA (Ribonucleic Acid) polymerase beta-subunit mutant coding gene rpoBM; compared with a parent RNA polymerase beta-subunit, the RNA polymerase beta-subunit mutant has the advantage that the amino acid residue at the 618th site of the amino acid sequence is sequentially replaced with leucine from the N terminal to the C terminal. The genetically engineered bacterium disclosed by the invention can tolerate L-isoleucine and accumulate less alpha-ketobutyric acid, and has a very good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a genetically engineered bacterium that stably and efficiently produces L-isoleucine, its construction method, and its application. Background Technology

[0002] L-Isoleucine is an essential branched-chain amino acid that is widely used in medicine, materials, food, animal husbandry and other fields, and has important economic and social value.

[0003] There are three methods for producing L-isoleucine: microbial fermentation, chemical synthesis, and extraction. Chemical synthesis and extraction methods are difficult to scale up industrially due to drawbacks such as difficulty in separating it from its isomers, limited raw material sources, high production costs, and environmental pollution. Microbial fermentation, on the other hand, has advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production, making it the most important method for L-isoleucine production currently.

[0004] For the production of L-isoleucine using microbial fermentation, selecting a strain that can produce the target product in high quantities and has strong genetic stability is particularly important. Currently, the strains mainly used for L-isoleucine production include *Escherichia coli* and *Corynebacterium glutamicum*. *Escherichia coli* has a clear genetic background, complete gene modification tools, and is easy to implement in terms of molecular biology and genetic modification. Furthermore, it has a short fermentation cycle (generally within 48 hours), low equipment requirements, and high equipment utilization and production intensity.

[0005] The L-isoleucine synthesis pathway in *E. coli* is relatively long and involves multiple branched metabolic pathways, with its flux being regulated at multiple levels, including enzyme activity and metabolite concentration. This pathway... ilvA Gene-encoded threonine dehydratase and ilvIH The gene-encoded acetylhydroxy acid synthases are all subject to feedback inhibition by the end product L-isoleucine. Notably, the inhibition of acetylhydroxy acid synthase activity by L-isoleucine prevents the intermediate metabolite α-ketobutyrate from further catalyzing the formation of α-acetyl-α-hydroxybutyrate, leading to a large accumulation of α-ketobutyrate intracellularly. Existing research indicates that excessive accumulation of L-isoleucine and its intermediate metabolite α-ketobutyrate is significantly toxic to *E. coli* cells, severely inhibiting cell growth and survival, thus greatly limiting the yield and production efficiency of L-isoleucine during fermentation, becoming a major technical obstacle in the efficient biomanufacturing of this amino acid. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a stable and efficient Escherichia coli genetically engineered bacterium for producing L-isoleucine, its construction method, and its applications. This engineered bacterium is tolerant to L-isoleucine and accumulates less α-ketobutyric acid, showing great promise for industrial applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered bacterium for producing L-isoleucine, wherein the genetically engineered bacterium uses E. coliTHRD as a host and contains a gene encoding a mutant β-subunit of RNA polymerase. rpoB M The amino acid sequence of the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 1. The accession number of E. coli THRD is CGMCC No. 11074, and it has been published in patent application CN105385702A. The applicant can obtain the biological material from the China General Microbiological Culture Collection Center (CGMCC).

[0008] As described in the first aspect above, the genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M And / or acetylhydroxyl synthase mutant encoding genes ilvIH M The amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and / or the gene encoding the acetylhydroxyl synthase mutant is as follows: ilvIH M The nucleotide sequence is shown in SEQ ID NO: 4.

[0009] As described in the first aspect above, the genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M Furthermore, the threonine dehydratase mutant gene is encoded by... ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M They are connected by a flexible linker peptide, which is (GGGGS). n , where n is any integer from 1 to 4.

[0010] In a second aspect, the present invention provides the application of genetically engineered bacteria as described in the first aspect in the fermentation production of L-isoleucine.

[0011] Thirdly, the present invention provides a method for producing L-isoleucine using genetically engineered bacteria as described in the first aspect, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-isoleucine; and collecting the L-isoleucine from the genetically engineered bacteria and / or the culture medium.

[0012] Fourthly, the present invention provides a mutant of the β-subunit of Escherichia coli RNA polymerase, the amino acid sequence of which is shown in SEQ ID NO: 1. According to the present invention, the mutant has the effect of improving the host's tolerance to L-isoleucine compared with the parental RNA polymerase β-subunit.

[0013] Beneficial effects: This invention, based on the L-threonine-producing strain E. coliTHRD, utilizes chromosomal integrated genetic modification to obtain a genetically engineered strain that combines high tolerance to and high production capacity of L-isoleucine, offering the following advantages and beneficial effects: 1. By expressing the RNA polymerase β-subunit mutant encoding gene in the high L-threonine-producing strain E. coli THRD rpoB M This significantly enhanced the strain's tolerance to L-isoleucine. Growth experiments under different concentrations of L-isoleucine stress confirmed that this modification effectively improved the strain's survival and growth ability in end-product stress environments.

[0014] 2. A flexible linker peptide strategy was employed to synergistically regulate key enzyme genes: by expressing the gene encoding a threonine dehydratase mutant. ilvA M and / or acetylhydroxyl synthase mutant encoding genes ilvIH M This not only eliminated the feedback inhibition of L-isoleucine on its synthetic pathway but also enhanced the biosynthetic flux from L-threonine to L-isoleucine. Further optimization of the number and linkage of linker peptides effectively reduced the accumulation of the toxic intermediate metabolite α-ketobutyrate, improving the safety and efficiency of the synthetic pathway.

[0015] 3. All genetic modifications in this invention are stably integrated into the chromosome, and the resulting strain does not carry any exogenous plasmids. It has high genetic stability, requires no mutagenesis treatment, is suitable for industrial continuous fermentation processes, and has good prospects for large-scale application. Attached Figure Description

[0016] Figure 1 The pREDCas9 and pGRB plasmid maps in the examples.

[0017] Figure 2 The examples show the growth of strain ILE01 and control strain E. coli THRD under different concentrations of L-isoleucine stress. Detailed Implementation

[0018] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0019] In a first aspect, the present invention provides a genetically engineered bacterium for producing L-isoleucine, wherein the genetically engineered bacterium uses E. coliTHRD as a host and contains a gene encoding a mutant β-subunit of RNA polymerase. rpoB M The amino acid sequence of the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 1, and the accession number of E. coliTHRD is CGMCC No. 11074.

[0020] According to a first aspect of the present invention, the RNA polymerase β-subunit mutant, compared with the parental RNA polymerase β-subunit, has the amino acid residue at position 618 in the sequence from the N-terminus to the C-terminus replaced by leucine, and the numbering method is based on the sequence shown in SEQ ID NO: 1.

[0021] According to a first aspect of the present invention, the E. coliTHRD has the accession number CGMCC No. 11074 and has been published in patent application CN105385702A. The applicant may obtain the biological material from the China General Microbiological Culture Collection Center (CGMCC).

[0022] As described in the first aspect above, the genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M And / or acetylhydroxyl synthase mutant encoding genes ilvIH M The amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and / or the gene encoding the acetylhydroxyl synthase mutant is as follows: ilvIH M The nucleotide sequence is shown in SEQ ID NO: 4.

[0023] According to a first aspect of the present invention, the threonine dehydratase mutant, compared with the parental threonine dehydratase, has the amino acid residue at position 447 (from the N-terminus to the C-terminus) replaced by phenylalanine, and the amino acid residue at position 451 replaced by alanine, with the numbering method based on the sequence shown in SEQ ID NO: 3.

[0024] According to a first aspect of the present invention, the acetylhydroxyl synthase mutant, compared with the parental acetylhydroxyl synthase, has the following amino acid sequence: the 14th amino acid residue from the N-terminus to the C-terminus is replaced by asparagine, and the 17th amino acid residue is replaced by phenylalanine, with the numbering method based on SEQ ID NO: 7. ilvH M Encoding amino acid sequence.

[0025] As described in the first aspect above, the genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M Furthermore, the threonine dehydratase mutant gene is encoded by... ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M They are connected by a flexible linker peptide, which is (GGGGS). n , where n is any integer from 1 to 4.

[0026] This invention relates to an artificially designed flexible linker peptide comprising a repetitive sequence module composed of glycine (G) and serine (S) residues, with a general structure of (Gly-Gly-Gly-Gly-Ser). n That is, (GGGGS) n Where n is an integer from 1 to 4. By selecting different values ​​of n, the length of the linker peptide can be precisely controlled to adaptively adjust the spatial distance and interaction between its two functional domains.

[0027] In some embodiments, the flexible linker peptide is preferably (GGGGS)3 or (GGGGS)4.

[0028] In some embodiments, the connection method between the flexible linker peptide and the gene is preferably as follows: ilvA M -(GGGGS)3- ilvIH M , ilvA M -(GGGGS)4- ilvIH M, ilvIH M -(GGGGS)3- ilvA M or ilvIH M -(GGGGS)4- ilvA M More preferably ilvA M -(GGGGS)3- ilvIH M or ilvA M -(GGGGS)4- ilvIH M .

[0029] In some embodiments, the RNA polymerase β-subunit mutant encodes a gene rpoB M It also connected to P rpoB The promoter, the P rpoB The nucleotide sequence of the promoter is shown in SEQ ID NO: 5.

[0030] In some embodiments, the threonine dehydratase mutant encodes a gene ilvA M and / or acetylhydroxyl synthase mutant encoding genes ilvIH M It also connected to P ydgI The promoter, the P ydgI The nucleotide sequence of the promoter is shown in SEQ ID NO: 6.

[0031] As described in the first aspect, the method for constructing genetically engineered bacteria involves starting with a specific strain. E. coli The targeted modification based on THRD mainly includes the following three modules: (1) Improve the strain's tolerance to L-isoleucine: RpoB protein is a key β subunit of RNA polymerase, participates in the transcriptional regulation of global genes in microbial cells, and has a wide range of effects on microbial cell phenotype. Introducing its mutant encoding gene rpoB M Genes were introduced to enhance the strain's tolerance to L-isoleucine; (2) Relief of feedback inhibition: Introduce the gene encoding an acetylhydroxyl synthase mutant that relieves L-isoleucine feedback inhibition. ilvIH M and the gene encoding threonine dehydratase mutant ilvA M It effectively blocked the negative feedback regulation of L-isoleucine on its synthesis pathway and strengthened the biosynthetic pathway from L-threonine to L-isoleucine. (3) Reduce the accumulation of the intermediate metabolite α-ketobutyric acid: Introduce flexible linker peptides (GGGGS) n Linked acetylhydroxy acid synthase mutant encoding gene ilvIH M and the gene encoding threonine dehydratase mutant ilvA M Furthermore, the linkage mode and the number of linking peptides were optimized to promote the orderly assembly of the enzyme complex and the substrate channel effect, thereby reducing the accumulation of the intermediate metabolite α-ketoacetic acid and improving the pathway throughput and product synthesis efficiency.

[0032] The above-mentioned gene knockout can be achieved using any gene knockout or gene silencing method known in the art, and gene integration can also be achieved using any method known in the art, such as homologous recombination, overlap PCR, mutagenesis screening, or gene editing techniques. For example, gene knockout can be achieved by removing a specific region of a gene to prevent it from expressing the target protein, or by introducing one or more nucleotide sequences into the coding region or promoter region through site-specific mutations, such as substitution, deletion, or insertion. Alternatively, chemical reagents can be used to decrease or eliminate the transcription encoded by the specific gene.

[0033] In some embodiments, the gene is introduced or expressed by integrating it into the *E. coli* genome. The integration site is selected based on conventional knowledge among those skilled in the art, choosing pseudogene sites that do not significantly affect bacterial growth and basal metabolism, for example... yeeP , ygiP , yghX、ygaY , yjiT , yjiP , ycjV , ycgH、 ygaY、yeeL、ilvG、rph Gene loci are selectable. In other embodiments, the gene is also linked to a promoter, which can be selected based on conventional understanding among those skilled in the art to regulate gene expression according to production needs, such as P. trc BBa-J23100, T7, etc.; in other embodiments, the present invention preferably uses P. rpoB promoter or P ydgI Promoter.

[0034] In some embodiments, the construction method includes gene integration and / or gene knockout using CRISPR / Cas9-mediated gene editing technology.

[0035] In some embodiments, the construction method includes constructing a recombinant fragment and a pGRB plasmid.

[0036] In some embodiments, the construction of the pGRB plasmid includes: designing a target sequence, preparing a DNA fragment containing the target sequence, and recombining the DNA fragment containing the target sequence with a linearized vector fragment; in one specific embodiment, the target sequence is 5'-NGG-3'.

[0037] In some embodiments, the construction method for recombinant fragments includes constructing either gene-integrated recombinant fragments or gene-knockout recombinant fragments. The steps for constructing gene-integrated recombinant fragments include: using the genome of the starting strain as a template, designing upstream and downstream homologous arm primers based on the upstream and downstream sequences of the intended insertion site of the target gene, designing primers based on the target genome, amplifying the target gene fragment, and then obtaining the recombinant fragment through PCR overlap technology. The steps for constructing gene-knockout recombinant fragments include: using the upstream and downstream sequences of the gene to be knocked out as templates, designing upstream and downstream homologous arm primers; amplifying the upstream and downstream homologous arms separately by PCR, and then preparing the recombinant fragment through overlap PCR.

[0038] In some embodiments, the construction method includes: simultaneously transforming the pGRB plasmid and the above-mentioned recombinant fragment into electrocompetent cells containing pREDCas9, and the plasmid elimination step, to obtain a recombinant genetically engineered strain.

[0039] In a second aspect, the present invention provides the application of genetically engineered bacteria as described in the first aspect in the fermentation production of L-isoleucine.

[0040] Thirdly, the present invention provides a method for producing L-isoleucine using genetically engineered bacteria as described in the first aspect, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-isoleucine; and collecting the L-isoleucine from the genetically engineered bacteria and / or the culture medium.

[0041] According to a third aspect of the invention, the culture of the genetically engineered bacteria can be carried out using methods conventional in the art. The culture medium used for the production of L-isoleucine can be a synthetic or natural culture medium, such as a typical culture medium containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other required organic and inorganic components.

[0042] According to a third aspect of the invention, the genetically engineered bacteria can be cultured under aerobic conditions for 12 to 72 hours, or 16 to 60 hours, or 20 to 48 hours, or 24 to 30 hours; the culture temperature can be controlled at 30 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or 6.0 and 7.5, or 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia.

[0043] In some embodiments, the method further includes maintaining a constant culture temperature of 37°C; in other embodiments, the method further includes maintaining a pH of approximately 7.0.

[0044] In other embodiments, the method further includes adding a certain concentration of glucose solution during the culture process to maintain the residual glucose concentration in the fermentation medium at less than 5 g / L, for example, 1-5 g / L.

[0045] After cultivation, solids, such as cells and cell debris, can be removed from the liquid culture medium using conventional techniques (e.g., centrifugation, membrane filtration). Ergothionein can then be recovered from the fermentation broth using any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0046] Before inoculation and fermentation, bacteria can undergo strain activation, seed culture, etc., depending on their storage state. Appropriate conditions and culture media can be selected according to conventional techniques in this field. For example, the seed culture medium can use the same composition as the fermentation culture medium, or it can be appropriately adjusted based on this.

[0047] Other specific operational methods involving molecular biology and genetic engineering can be implemented using technical manuals, textbooks, or literature reports readily available to those skilled in the art, and need not be described in detail here. Furthermore, specific bacterial strains were selected as hosts in the following examples, and therefore specific gene integration sites, target genes, and primers were chosen based on the host. However, this does not mean that the purpose of the present invention can only be achieved through these specific selections, and should not be construed as limiting the scope of the invention. The essence and scope of the present invention are defined only by the claims.

[0048] Fourthly, the present invention provides a mutant of the β-subunit of Escherichia coli RNA polymerase, the amino acid sequence of which is shown in SEQ ID NO: 1. According to the present invention, the mutant, compared with the parental RNA polymerase β-subunit, has the effect of increasing the host's tolerance to L-isoleucine. Preferably, the nucleotide sequence of the gene encoding the mutant of the β-subunit of RNA polymerase is shown in SEQ ID NO: 2.

[0049] The present invention will be described in more detail below through specific embodiments.

[0050] 1. Methods of gene editing The gene editing method used in this invention is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coliThe procedure was performed using CRISPR–Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21. The two plasmid maps used in this method are attached. Figure 1 The pREDCas9 plasmid carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, and the Cas9 protein expression system. It is resistant to zirconia (working concentration: 100 mg / L) and cultured at 32℃. pGRB uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, and the terminator sequence. It is resistant to ampicillin (working concentration: 100 mg / L) and cultured at 37℃.

[0051] The specific steps of this method are as follows: 1.1 Construction of pGRB plasmid The purpose of constructing plasmid pGRB is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and to achieve a double-strand break in the target DNA through base pairing and PAM recognition of the target gene site. The pGRB plasmid is constructed using a recombination method involving a DNA fragment containing the target sequence and a linearized vector fragment.

[0052] 1.1.1 Target Sequence Design The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools. 1.1.2 Preparation of DNA fragments containing the target sequence Primer design: 5'-linearized vector terminal sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (15 bp)-3' and its reverse complementary primer. DNA fragments containing the target sequence were prepared by annealing single-stranded DNA. Reaction conditions: pre-denaturation 95℃, 5 min; annealing 30-50℃, 1 min. The annealing system is shown in Table 1.

[0053] Table 1

[0054] 1.1.3 Preparation of linear carriers The vector was linearized using reverse PCR amplification.

[0055] 1.1.4 Recombination reaction The recombination system is shown in Table 2. All recombinant enzymes used were from the ClonExpress® II One Step Cloning Kit series. Recombination conditions: 37℃, 30 min.

[0056] Table 2

[0057] 1.1.5 Plasmid Transformation Take 10 μL of reaction solution and add it to 100 mL of DH5α-transformed competent cells. Gently mix and incubate on ice for 20 min. Heat shock at 42℃ for 45-90 s, then immediately incubate on ice for 2-3 min. Add 900 μL of SOC and incubate at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, and resuspend the bacterial cells in about 200 μL. Spread the resuspended cells onto a plate containing 100 mg / L ampicillin. Invert the plate and incubate overnight at 37℃. After single cells have grown on the plate, identify them by colony PCR and select positive recombinants.

[0058] 1.1.6 Cloning Identification PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight for preservation. Plasmids were then extracted and identified by enzyme digestion. 1.2 Preparation of Recombinant DNA Fragments The recombinant fragment used for knockout consists of upstream and downstream homologous arms of the gene to be knocked out (upstream homologous arm - downstream homologous arm); the recombinant fragment used for integration consists of upstream and downstream homologous arms of the integration site and the gene fragment to be integrated (upstream homologous arm - target gene - downstream homologous arm). Primers for upstream and downstream homologous arms (amplification length approximately 400-500 bp) were designed using the primer design software Primer5, with the upstream and downstream sequences of the gene to be knocked out or the integration site as templates; primers for amplifying the integration gene were designed using the gene to be integrated as a template. The upstream and downstream homologous arms and the target gene fragment were amplified separately by PCR, and then recombinant fragments were prepared by overlap PCR. The PCR amplification system is shown in Table 3. Table 3

[0059] The systems for overlap PCR are shown in Table 4: Table 4

[0060] Note: The template consists of equimolar amounts of amplified fragments from upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng.

[0061] PCR reaction conditions (Takara Bio PrimeSTAR HS enzyme): Pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintain (4℃).

[0062] 1.3 Transformation of plasmids and recombinant DNA fragments 1.3.1 Conversion of pREDCas9 The pREDCas9 plasmid was electroporated into the electroporation competent cells of the starting strain. After cell resuscitation and culture, the cells were plated on LB agar plates containing zirconia and incubated overnight at 32°C. Single colonies growing on the antibiotic-resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0063] 1.3.2 Preparation of electrotransformation competent cells of the target strain containing pREDCas9 Incubate at 32℃ until OD 600 When the concentration reaches 0.1–0.2, add 0.1 M IPTG (to bring the final concentration to 0.1 mM) and continue culturing until OD reaches 0.2. 600 Competent cells were prepared when the pH was 0.6–0.7. The purpose of adding IPTG was to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process for competent cells followed standard operating procedures.

[0064] 1.3.3 Transformation of pGRB and recombinant DNA fragments pGRB and donor DNA fragments were simultaneously electroporated into electrocompetent cells containing pREDCas9. The revived cells after electroporation were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Colony PCR was performed using specially designed identification primers to verify the colonies, screen for positive recombinants, and maintain the cells.

[0065] 1.4 Plasmid Elimination 1.4.1 Elimination of pGRB Positive recombinants were incubated overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread onto LB plates containing zirconia-resistant bacteria and incubated overnight at 32°C. Single colonies that did not grow on ampicillin-resistant plates but grew on zirconia-resistant plates were selected and preserved.

[0066] 1.4.2 Elimination of pREDCas9 plasmid The positive recombinant was transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, it was spread onto antibiotic-free LB plates and incubated overnight at 37°C. Single colonies that did not grow on bilirubin-resistant plates and grew on antibiotic-free plates were selected for preservation.

[0067] 2. The primer sequences used in the embodiments are shown in Table 5: Table 5

[0068] 3. The culture medium used in the examples can be referred to as follows: The slant culture medium consists of: glucose 1-5 g / L, peptone 5-10 g / L, beef extract 5-10 g / L, yeast extract 1-5 g / L, NaCl 1-5 g / L, agar 15-25 g / L, and the remainder is water, with a pH of 7.0-7.2.

[0069] The seed culture medium consists of: glucose 20-40 g / L, yeast extract 2-5 g / L, peptone 2-5 g / L, citric acid 1-3 g / L, methionine 0.5-1 g / L, monosodium glutamate 1-3 g / L, ammonium sulfate 1-3 g / L, KH₂PO₄ 1-3 g / L, MgSO₄·7H₂O 1-2 g / L, FeSO₄·7H₂O 10-20 mg / L, MnSO₄·7H₂O 10-20 mg / L, V B1 V B3 V B5 V B12 V B6 V H Each component is 0.2-3 mg / L, the remainder is water, pH 7.0-7.2.

[0070] The fermentation medium consists of: glucose 20-40 g / L, yeast extract 2-5 g / L, citric acid 1-3 g / L, ammonium sulfate 1-5 g / L, NaCl 1-5 g / L, KH₂PO₄ 1-3 g / L, MgSO₄·7H₂O 1-2 g / L, FeSO₄·7H₂O 10-20 mg / L, MnSO₄·7H₂O 1-2 mg / L, V B1 V B3 V B5 V B12 V H Each component is 1-3 mg / L, the trace element mixture is 1-2 mg / L, and the remainder is water, pH 7.0-7.2; the trace element mixture consists of: Na2MoO4·2H2O 2.5 g / L, NiCl2·6H2O 1.6 g / L, CaCl2·2H2O 10 g / L, CuSO4·5H2O 0.4 g / L, Al2(SO4)2·18H2O 2.25 g / L, CoCl2·6H2O 1.8 g / L, ZnSO4·2H2O 0.5 g / L, and H3BO4 2.5 g / L.

[0071] The present invention will be described in more detail below through specific embodiments.

[0072] Example 1: This embodiment illustrates the construction of the genetically engineered bacteria for producing L-isoleucine according to the present invention. Specifically, it can be genetically engineered bacteria ILE01, ILE02, ILE03, ILE04, ILE05, ILE06, or ILE07.

[0073] (1) yihF ::P rpoB - rpoB M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its yihF Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948352). yihF -S、UP- yihF -A) and downstream homologous arm primer (DN- yihF -S、DN- yihF -A), PCR amplification of its upstream and downstream homologous arm fragments; using the genome of a laboratory-preserved strain (the genome contains rpoB M () as template, according to rpoB M Primers were designed based on the gene sequence (NCBI GeneID: 948488). rpoB -S、 rpoB -A), PCR amplification rpoB M Fragments. The above fragments were fused using overlap PCR to obtain... rpoB M Integration fragment of gene (upstream homologous arm - P) rpoB - rpoB M -Downstream homologous arm), primer gRNA- yihF -S and gRNA- yihF The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- yihF To prepare competent cells of the engineered strain E. coli THRD, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- yihF and P rpoB - rpoB M The integrated gene fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE01.

[0074] (2) ilvA ::P ydgI - ilvA M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ilvA Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ilvA -S、UP- ilvA -A) and downstream homologous arm primer (DN- ilvA -S、DN- ilvA -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ilvA - ydgi -S、 ydgi - ilvA -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvA M () as template, according to ilvA M Primers were designed based on the gene sequence (NCBI GeneID: 948287). ydgi - ilvA -S、 ilvA -A), PCR amplification ilvA M Fragments. The above fragments were fused using overlap PCR to obtain... ilvA M Integration fragment of gene (upstream homologous arm - P) ydgI - ilvA M -Downstream homologous arm), primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ilvA To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ilvA and P ydgI - ilvA M The integrated gene fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE02.

[0075] (3) yjiP ::P ydgI - ilvIH M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its yjiP Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 38094982). yjiP -S、UP- yjiP -A) and downstream homologous arm primer (DN- yjiP -S、DN- yjiP -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). yjiP - ydgi -S、 ydgi - ilvIH -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvIH M () as template, according to ilvIH M Gene sequence ( ilvI NCBI GeneID: 948793 ilvH Primers were designed using NCBI GeneID: 947267. ydgi - ilvIH -S、 ilvIH -A), PCR amplification ilvIH M Fragments. The above fragments were fused using overlap PCR to obtain... ilvIH M Integration fragment of gene (upstream homologous arm - P) ydgI - ilvIH M -Downstream homologous arm), primer gRNA- yjiP -S and gRNA- yjiP The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- yjiP To prepare competent cells of the engineered strain ILE02, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- yjiP and ilvIH M The integrated gene fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE03.

[0076] (4) ilvA ::P ydgI - ilvA M -(GGGGS)3- ilvIH M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ilvA Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ilvA -S、UP- ilvA -A) and downstream homologous arm primers ( ilvIH - ilvA -DN-S、DN- ilvA -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ilvA - ydgi -S、 ydgi - ilvA -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvA M () as template, according to ilvA M Primers were designed based on the gene sequence (NCBI GeneID: 948287). ydgi - ilvA -S、 ilvA -GS3-A), PCR amplification ilvA M Fragments; genomes of laboratory-preserved strains (containing...) ilvIH M () as template, according to ilvIH M Gene sequence ( ilvI NCBI GeneID: 948793 ilvH Design primers (GS3-) using NCBI GeneID: 947267. ilvIH -S、 ilvIH -A), PCR amplification ilvIH M Fragments; flexible linker peptide (GGGGS)3 is designed in ilvA M Downstream primers of the gene and ilvIH M In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain P. ydgI - ilvA M -(GGGGS)3- ilvIH M The integrated fragment (upstream homologous arm - P) ydgI - ilvA M -(GGGGS)3- ilvIH M -Downstream homologous arm), primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ilvA To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ilvA and P ydgI - ilvA M -(GGGGS)3- ilvIH M The integrated fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE04.

[0077] (5) ilvA ::P ydgI - ilvA M -(GGGGS)4- ilvIH M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ilvA Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ilvA -S、UP- ilvA -A) and downstream homologous arm primers ( ilvIH - ilvA -DN-S、DN- ilvA -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ilvA - ydgi -S、 ydgi - ilvA -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvA M () as template, according to ilvA M Primers were designed based on the gene sequence (NCBI GeneID: 948287). ydgi - ilvA -S、 ilvA -GS4-A), PCR amplification ilvAM Fragments; genomes of laboratory-preserved strains (containing...) ilvIH M () as template, according to ilvIH M Gene sequence ( ilvI NCBI GeneID: 948793 ilvH Design primers (GS4-) using NCBI GeneID: 947267. ilvIH -S、 ilvIH -A), PCR amplification ilvIH M Fragment; flexible linker peptide (GGGGS)4 is designed in ilvA M Downstream primers of the gene and ilvIH M In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain P. ydgI - ilvA M -(GGGGS)4- ilvIH M The integrated fragment (upstream homologous arm - P) ydgI - ilvA M -(GGGGS)4- ilvIH M -Downstream homologous arm), primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ilvA To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ilvA and P ydgI - ilvA M -(GGGGS)4- ilvIH M The integrated fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE05.

[0078] (6) ilvA ::P ydgI - ilvIH M -(GGGGS)3- ilvA M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ilvA Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ilvA -S、UP- ilvA -A) and downstream homologous arm primer (DN- ilvA -S、DN- ilvA -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ilvA - ydgi -S、 ydgi - ilvIH -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvIH M () as template, according to ilvIH M Gene sequence ( ilvI NCBI GeneID: 948793 ilvH Primers were designed using NCBI GeneID: 947267. ydgi - ilvIH -S、 ilvIH -GS3-A), PCR amplification ilvIH M Fragments; genomes of laboratory-preserved strains (containing...) ilvA M () as template, according to ilvA M Primers (GS3-) were designed based on the gene sequence (NCBI GeneID: 948287). ilvA -S、 ilvA -A), PCR amplification ilvA M Fragments; flexible linker peptide (GGGGS)3 is designed in ilvIH M Downstream primers of the gene and ilvA M In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain P. ydgI - ilvIH M -(GGGGS)3- ilvA M The integrated fragment (upstream homologous arm - P) ydgI - ilvIH M -(GGGGS)3- ilvA M - ilvIH M -Downstream homologous arm), primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ilvA To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ilvA and P ydgI - ilvIH M -(GGGGS)3- ilvA M The integrated fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE06.

[0079] (7) ilvA ::P ydgI - ilvIH M -(GGGGS)4- ilvA M Gene editing Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ilvA Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ilvA -S、UP- ilvA -A) and downstream homologous arm primer (DN- ilvA -S、DN- ilvA -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ydgi Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ilvA - ydgi -S、 ydgi - ilvIH -A), PCR amplification ydgi Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvIH M () as template, according to ilvIH M Gene sequence ( ilvI NCBI GeneID: 948793 ilvH Primers were designed using NCBI GeneID: 947267. ydgi - ilvIH -S、 ilvIH -GS4-A), PCR amplification ilvIH M Fragments; genomes of laboratory-preserved strains (containing...) ilvAM () as template, according to ilvA M Primers (GS4-) were designed based on the gene sequence (NCBI GeneID: 948287). ilvA -S、 ilvA -A), PCR amplification ilvA M Fragment; flexible linker peptide (GGGGS)4 is designed in ilvIH M Downstream primers of the gene and ilvA M In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain P. ydgI - ilvIH M -(GGGGS)4- ilvA M The integrated fragment (upstream homologous arm - P) ydgI - ilvIH M -(GGGGS)4- ilvA M - ilvIH M -Downstream homologous arm), primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ilvA To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ilvA and P ydgI - ilvIH M -(GGGGS)4- ilvA M The integrated fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE07.

[0080] Example 2: This example illustrates the effect of different concentrations of L-isoleucine on the growth of a bacterial strain, which may be ILE01 or E. coli THRD.

[0081] (1) Slant culture: Take the -80℃ preserved strain and streak it onto the activated slant, incubate at 37℃ for 12 h, and subculture once; (2) Shake flask culture: A loopful of seed culture was scraped from the slant and inoculated into a series of 500 mL Erlenmeyer flasks containing 30 mL of basic culture medium. The concentrations of L-isoleucine in the culture medium were 20 g / L and 40 g / L, respectively. 1 mL of bottom sugar was added as a carbon source. The flasks were sealed with nine layers of gauze and cultured at 37℃ and 220 rpm for 14 h. After 4 h of culture, samples were taken every 2 h to determine the OD of the strain. 600 The values ​​were used to plot growth curves for analysis and comparison.

[0082] The preferred composition of the slant culture medium in this embodiment is: 1 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L NaCl, 25 g / L agar, and the remainder is water, pH 7.0.

[0083] The preferred basic culture medium composition in this embodiment is: Na₂HPO₄·12H₂O 15 g / L, KH₂PO₄ 3 g / L, (NH₄)₄SO₄ 5 g / L, NaNO₃ 1 g / L, V B1 V B3 V B5 V B12 V H Each component was 1 mg / L, the trace element mixture was 1 mg / L, and the remainder was water, pH 7.0; the trace element mixture consisted of: Na2MoO4·2H2O 2.5 g / L, NiCl2·6H2O 1.6 g / L, CaCl2·2H2O 10 g / L, CuSO4·5H2O 0.4 g / L, Al2(SO4)2·18H2O 2.25 g / L, CoCl2·6H2O 1.8 g / L, ZnSO4·2H2O 0.5 g / L, and H3BO4 2.5 g / L.

[0084] The base sugar composition is: 60% glucose and 3% MgSO4·7H2O.

[0085] Experimental results are as follows Figure 2 As shown, in a basic culture medium containing 20 g / L or 40 g / L L-isoleucine, the growth of the engineered strain ILE01 of this invention after 14 h of culture (expressed as OD) was [data missing]. 600 The RNA polymerase β-subunit mutant (as described in this invention) showed significant improvement compared to the control strain E. coli THRD. This result confirms that the RNA polymerase β-subunit mutant or its encoding gene, as described in this invention, can be effectively introduced. rpoB M This can effectively enhance the strain's tolerance to L-isoleucine, thus making it more conducive to the strain's growth and production of L-isoleucine.

[0086] Example 3: This embodiment illustrates a method for producing L-isoleucine using the genetically engineered bacteria described in this invention. Specifically, the genetically engineered bacteria can be ILE01, ILE02, ILE03, ILE04, ILE05, ILE06, or ILE07.

[0087] (1) Slant culture: Take the -80℃ preserved strain and streak it onto the activated slant, incubate at 37℃ for 12 h, and subculture once; (2) Shake flask seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Seal the flask with nine layers of gauze and culture at 37℃ and 220 rpm for 7-10 h. (3) Shake-flask fermentation: Inoculate 10% of the seed culture volume into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), seal with nine layers of gauze, and incubate at 37℃ with shaking at 220 r / min. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia; add 60% ( m / f Fermentation was maintained using glucose solution; the fermentation cycle was 24 hours.

[0088] The preferred composition of the slant culture medium in this embodiment is: 1 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L NaCl, 25 g / L agar, and the remainder is water, pH 7.0.

[0089] The preferred seed culture medium composition in this embodiment is: glucose 20 g / L, yeast extract 5 g / L, peptone 4 g / L, citric acid 3 g / L, methionine 0.5 g / L, monosodium glutamate 2 g / L, ammonium sulfate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 10 mg / L, MnSO4·7H2O 10 mg / L, V B1 V B3 V B5 V B12 V B6 V H Each 2 mg / L, the remainder being water, pH 7.0.

[0090] The preferred fermentation medium composition in this embodiment is: glucose 20 g / L, yeast extract 3 g / L, citric acid 2 g / L, ammonium sulfate 4 g / L, NaCl 1 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 1.2 mg / L, V B1 VB3 V B5 V B12 V H Each component was 2 mg / L, the trace element mixture was 1 mg / L, and the remainder was water, pH 7.0; the trace element mixture consisted of: Na2MoO4·2H2O 2.5 g / L, NiCl2·6H2O 1.6 g / L, CaCl2·2H2O 10 g / L, CuSO4·5H2O 0.4 g / L, Al2(SO4)2·18H2O 2.25 g / L, CoCl2·6H2O 1.8 g / L, ZnSO4·2H2O 0.5 g / L, and H3BO4 2.5 g / L.

[0091] Using *E. coli* THRD as a control strain, strains ILE01-ILE07 were subjected to 24-hour shake-flask fermentation, and the L-isoleucine content in the fermentation broth was detected by high-performance liquid chromatography (HPLC). The results of the shake-flask fermentation are shown in Table 6. The fermentation data indicate that L-isoleucine was introduced into the *E. coli* THRD genome. rpoB M It had no significant effect on the growth of the strain. The L-isoleucine production of strain ILE03 was lower than that of ILE04-ILE07, but the accumulation of α-ketobutyric acid was significantly higher than that of the aforementioned strains. Therefore, overexpression alone... ilvA M and ilvIH M This can easily lead to the accumulation of the toxic intermediate metabolite α-ketobutyric acid; however, using flexible linker peptides can... ilvA M and ilvIH M Fusion expression after ligation can effectively reduce the accumulation of the toxic intermediate metabolite α-ketobutyrate and increase the yield of L-isoleucine. Specifically, the method employed... ilvA M -(GGGGS)3- ilvIH M The fusion expression strain ILE04 showed the best results, achieving an L-isoleucine yield of 10.8 g / L, which is sufficient for most applications. ilvA M -(GGGGS)3- ilvIH M The enzyme fusion expression is more suitable for the current strain to produce L-isoleucine.

[0092] Table 6

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing L-isoleucine, characterized in that: The genetically engineered bacteria used E. coli THRD as a host and contained a gene encoding a mutant β-subunit of RNA polymerase. rpoB M The amino acid sequence of the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 1, and the accession number of E. coliTHRD is CGMCC No. 11074.

2. The genetically engineered bacteria as described in claim 1, characterized in that: The genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M And / or acetylhydroxyl synthase mutant encoding genes ilvIH M The amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and / or the gene encoding the acetylhydroxyl synthase mutant is as follows: ilvIH M The nucleotide sequence is shown in SEQ ID NO:

4.

3. The genetically engineered bacteria as described in claim 2, characterized in that: The genetically engineered bacteria also contain a gene encoding a threonine dehydratase mutant. ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M ; Furthermore, the threonine dehydratase mutant gene is encoded by... ilvA M and the gene encoding acetylhydroxy acid synthase mutant ilvIH M They are connected by a flexible linker peptide, which is (GGGGS). n , where n is any integer from 1 to 4.

4. The genetically engineered bacteria as described in claim 3, characterized in that: The flexible linker peptide is (GGGGS)3 or (GGGGS)4.

5. The genetically engineered bacteria as described in claim 4, characterized in that: The flexible linker peptide and the gene are connected in the following manner: ilvA M -(GGGGS)3- ilvIH M , ilvA M -(GGGGS)4- ilvIH M , ilvIH M -(GGGGS)3- ilvA M or ilvIH M -(GGGGS)4- ilvA M .

6. The genetically engineered bacteria according to any one of claims 1-5, characterized in that: The RNA polymerase β-subunit mutant encodes the gene rpoB M It also connected to P rpoB The promoter, the P rpoB The nucleotide sequence of the promoter is shown in SEQ ID NO:

5.

7. The genetically engineered bacteria according to any one of claims 2-5, characterized in that: The gene encoding the threonine dehydratase mutant ilvA M and / or acetylhydroxyl synthase mutant encoding genes ilvIH M It also connected to P ydgI The promoter, the P ydgI The nucleotide sequence of the promoter is shown in SEQ ID NO:

6.

8. The use of the genetically engineered bacteria according to any one of claims 1-7 in the fermentation production of L-isoleucine.

9. A method for producing L-isoleucine, comprising: The genetically engineered bacteria according to any one of claims 1-7 are cultured in a culture medium to produce L-isoleucine; In addition, the L-isoleucine is collected from the genetically engineered bacteria and / or the culture medium.

10. A mutant of the β-subunit of Escherichia coli RNA polymerase, the amino acid sequence of which is shown in SEQ ID NO: 1.

Images