Genetically engineered bacteria for producing l-isoleucine and construction method and application thereof

Abstract

The application 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 thereof. rpoB M The genetically engineered bacterium takes E. coli THRD as a host and contains a gene encoding an RNA polymerase beta-subunit mutant The amino acid sequence of the RNA polymerase beta-subunit mutant is sequentially replaced by leucine at the 618th amino acid residue from the N terminal to the C terminal direction compared with the parent RNA polymerase beta-subunit. The genetically engineered bacterium has good industrial application prospects and can tolerate L-isoleucine and accumulate less alpha-ketobutyric acid.

Description

TECHNICAL FIELD

[0001] The present application 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 thereof. BACKGROUND

[0002] L-isoleucine is a branched-chain amino acid essential for human body, and is widely used in the fields of medicine, material, food, and animal husbandry, and has important economic and social values.

[0003] There are three methods for producing L-isoleucine, i.e., microbial fermentation, chemical synthesis, and extraction. The chemical synthesis and extraction methods are difficult to realize industrial production due to the difficulties in separating isomers, the limited source of raw materials, high production cost, and environmental pollution. The microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, and easy realization of large-scale production, and is the main method for producing L-isoleucine at present.

[0004] It is particularly important to breed a strain capable of producing target products in high yield and having strong genetic stability for producing L-isoleucine by microbial fermentation. At present, the strains used for producing L-isoleucine mainly include Escherichia coli and Corynebacterium glutamicum. The E. coli has a clear genetic background, complete genetic modification tools, and easy realization of molecular biology and genetic modification, and has a short fermentation cycle (generally within 48 h) and low equipment requirements, and has a high equipment utilization rate and production intensity.

[0005] The L-isoleucine synthesis pathway in E. coli is relatively long, and involves many branch metabolic pathways, and the flux is regulated by multiple levels including enzyme activity and metabolite concentration. The threonine dehydratase encoded by the gene and the acetyl-CoA synthase encoded by the gene in the pathway are both inhibited by the final product L-isoleucine. ilvA The threonine dehydratase encoded by the gene and the acetyl-CoA synthase encoded by the gene in the pathway are both inhibited by the final product L-isoleucine. ilvIH It is worth noting that the inhibition of L-isoleucine on the activity of acetyl-CoA synthase will cause the intermediate metabolite α-ketobutyric acid to be unable to be further catalyzed to generate α-acetyl-α-hydroxybutyric acid, thereby causing a large amount of accumulation of α-ketobutyric acid in the cell. Existing studies have shown that the excessive accumulation of L-isoleucine and the intermediate metabolite α-ketobutyric acid has obvious toxicity to E. coli cells, and seriously inhibits the growth and survival of the cells, thereby greatly limiting the yield and production efficiency of L-isoleucine in the fermentation process, and becoming the main technical obstacle in the high-efficiency biological manufacturing of the amino acid. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide an E. coli genetically engineered bacterium for stably and efficiently producing L-isoleucine as well as a construction method and application thereof. The engineered bacterium can tolerate L-isoleucine and has less accumulation of α-ketobutyric acid, and has a good industrial application prospect.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a genetically engineered bacterium for producing L-isoleucine, which contains a RNA polymerase β-subunit mutant encoding gene rpoB M ; wherein the amino acid sequence of the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 1, the accession number of the E. coli THRD is CGMCC No. 11074, and it has been published in patent application CN105385702A, and the applicant can obtain this biological material from the deposit institution, China General Microbiological Culture Collection Center (CGMCC).

[0009] The genetically engineered bacterium of the first aspect, further contains a threonine dehydratase mutant encoding gene ilvA M , and / or an acetohydroxy acid synthase mutant encoding gene ilvIH M ; wherein the amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and / or the nucleotide sequence of the acetohydroxy acid synthase mutant encoding gene ilvIH M is shown in SEQ ID NO: 4.

[0010] The genetically engineered bacterium of the first aspect, further contains a threonine dehydratase mutant encoding gene ilvA M and an acetohydroxy acid synthase mutant encoding gene ilvIH M ; and the threonine dehydratase mutant encoding gene ilvA M and the acetohydroxy acid synthase mutant encoding gene ilvIH M are connected by a flexible linker peptide, and the flexible linker peptide is (GGGGS) n , wherein n is any integer from 1 to 4.

[0011] In a second aspect, the present application provides the genetically engineered bacterium of the first aspect for use in fermentative production of L-isoleucine.

[0012] In a third aspect, the present application provides a method for producing L-isoleucine using the genetically engineered bacterium of the first aspect, which comprises: culturing the genetically engineered bacterium in a culture medium to make it produce L-isoleucine; and collecting the L-isoleucine from the genetically engineered bacterium and / or the culture medium.

[0013] In a fourth aspect, the present application provides an Escherichia coli RNA polymerase β-subunit mutant, whose amino acid sequence is shown in SEQ ID NO: 1. According to the present application, the mutant has the effect of improving the host's tolerance to L-isoleucine compared to the parent RNA polymerase β-subunit.

[0014] Beneficial effects:

[0015] The present application is based on the L-threonine production strain E. coli THRD, and a genetically engineered strain with high L-isoleucine tolerance and high production capacity is obtained through chromosomal integration genetic modification, which has the following advantages and beneficial effects:

[0016] 1. By expressing the RNA polymerase β-subunit mutant encoding gene in the high-yield L-threonine strain E. coli THRD rpoB M , the strain's tolerance to L-isoleucine is significantly enhanced. Growth experiments under different concentrations of L-isoleucine stress confirm that the modification effectively improves the strain's survival and growth ability in the end product stress environment.

[0017] 2. Using flexible linker peptide strategy to synergistically regulate key enzyme genes: by expressing threonine dehydratase mutant encoding gene ilvA M and / or acetohydroxy acid synthase mutant encoding gene ilvIH M , not only does it eliminate the feedback inhibition of L-isoleucine on its synthesis pathway, but also it strengthens the biosynthesis flux from L-threonine to L-isoleucine. Further optimization of the number and method of connection effectively reduces the accumulation of toxic intermediate metabolite α-ketobutyric acid, improving the safety and efficiency of the synthesis pathway.

[0018] 3. All genetic modifications of the present application are stably integrated into the chromosome, and the obtained strain does not carry any exogenous plasmid, has high genetic stability, does not need mutagenic treatment, is suitable for industrial continuous fermentation process, and has good prospects for large-scale application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Plasmid map of pREDCas9 and pGRB in the examples.

[0020] Figure 2 : Growth of ILE01 strain and control strain E. coli THRD under different concentrations of L-isoleucine stress in the examples. DETAILED DESCRIPTION

[0021] The present application is described below by specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present application, the essence and scope of the present application are only limited by the claims. For those skilled in the art, various changes or modifications to the composition and amount of the materials in these embodiments without departing from the essence and scope of the present application also fall within the scope of the present application.

[0022] In a first aspect, the present application provides a genetically engineered bacterium for producing L-isoleucine, which contains a RNA polymerase β-subunit mutant encoding gene rpoB M ; wherein the amino acid sequence of the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 1, and the accession number of the E. coli THRD is CGMCC No. 11074.

[0023] According to the first aspect of the present application, compared with the parent RNA polymerase β-subunit, the amino acid sequence of the RNA polymerase β-subunit mutant is sequentially replaced by leucine at the 618th amino acid residue from the N-terminal to the C-terminal direction, and the numbering is based on the sequence shown in SEQ ID NO: 1.

[0024] According to the first aspect of the present application, the accession number of the E. coli THRD is CGMCC No. 11074, and has been published in patent application CN105385702A. The applicant can obtain this biological material from the China General Microbiological Culture Collection Center (CGMCC).

[0025] The genetically engineered bacterium of the first aspect described above, further contains a threonine dehydratase mutant encoding gene ilvA M , and / or an acetyl-CoA carboxylase mutant encoding gene ilvIH M ; wherein the amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and / or the nucleotide sequence of the acetyl-CoA carboxylase mutant encoding gene ilvIH M is shown in SEQ ID NO: 4.

[0026] According to the first aspect of the present application, the threonine dehydratase mutant has an amino acid sequence in which, compared with the parent threonine dehydratase, the amino acid residue at position 447 is replaced by phenylalanine and the amino acid residue at position 451 is replaced by alanine, in order from the N-terminus to the C-terminus, and the numbering is based on the sequence shown in SEQ ID NO: 3.

[0027] According to the first aspect of the present application, the threonine dehydratase mutant has an amino acid sequence in which, compared with the parent threonine dehydratase, the amino acid residue at position 447 is replaced by phenylalanine and the amino acid residue at position 451 is replaced by alanine, in order from the N-terminus to the C-terminus, and the numbering is based on the sequence shown in SEQ ID NO: 3. ilvH M The amino acid sequence is encoded by the gene.

[0028] The genetically engineered bacterium of the first aspect described above, wherein the genetically engineered bacterium further comprises a threonine dehydratase mutant encoding gene ilvA M and an acetohydroxy acid synthase mutant encoding gene ilvIH M ; and the threonine dehydratase mutant encoding gene ilvA M and the acetohydroxy acid synthase mutant encoding gene ilvIH M are connected by a flexible linker peptide, and the flexible linker peptide is (GGGGS)n, wherein n is any integer from 1 to 4. n

[0029] The present application relates to a flexible linker peptide designed artificially, which comprises a repeat sequence module composed of glycine (G) and serine (S) residues, and the general structure is: (Gly-Gly-Gly-Gly-Ser)n n , i.e. (GGGGS)n. n , wherein n is an integer from 1 to 4. By selecting different n values, the length of the linker peptide can be accurately regulated to adaptively adjust the spatial distance and interaction between the two functional domains on both sides.

[0030] In some embodiments, the flexible linker peptide is preferably (GGGGS)3 or (GGGGS)4.

[0031] In some embodiments, the flexible linker peptide and the gene are preferably 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 ; more preferably ilvA M -(GGGGS)3- ilvIH M or ilvA M -(GGGGS)4- ilvIH M .

[0032] In some embodiments, the RNA polymerase β-subunit mutant-encoding gene rpoB M is also connected with a P rpoB promoter, and the nucleotide sequence of the P rpoB promoter is shown as SEQ ID NO: 5.

[0033] In some embodiments, the threonine dehydratase mutant-encoding gene ilvA M and / or the acetohydroxy acid synthase mutant-encoding gene ilvIH M is also connected with a P ydgI promoter, and the nucleotide sequence of the P ydgI promoter is shown as SEQ ID NO: 6.

[0034] The method for constructing the genetically engineered bacteria according to the first aspect, is based on the directed modification of the starting strain E. coli THRD, mainly including the following three modules:

[0035] (1) Improving the tolerance of the strain to L-isoleucine: RpoB protein is a key β subunit of RNA polymerase, involved in the transcriptional regulation of global genes of microbial cells, and has a wide impact on the phenotype of microbial cells. The mutant-encoding gene rpoB M of RpoB protein is introduced to improve the tolerance of the strain to L-isoleucine;

[0036] (2) Removing feedback inhibition: the mutant-encoding gene ilvIH M of acetohydroxy acid synthase and the mutant-encoding gene ilvA M of threonine dehydratase are introduced, effectively blocking the negative feedback regulation of L-isoleucine on its synthesis pathway, and strengthening the biosynthesis pathway from L-threonine to L-isoleucine;

[0037] (3) Reducing the accumulation of intermediate metabolite α-ketobutyric acid: introducing flexible linker peptide (GGGGS) n acetyl-hydroxy acid synthase mutant encoding gene ilvIH M and threonine dehydratase mutant encoding gene ilvA M , and optimizing the connection mode and the number of connections to promote the ordered assembly of the enzyme complex and the substrate channel effect, thereby reducing the accumulation of intermediate metabolite α-ketobutyric acid, improving the pathway flux and product synthesis efficiency.

[0038] The knockout of the above-mentioned genes can be achieved by any method known in the art for gene knockout or gene silencing, and the integration of the genes can be achieved by any method known in the art, such as homologous recombination, overlap PCR, mutagenesis screening, or gene editing technology. For example, gene knockout can be achieved by removing a specific region of the gene to make it lose the function of expressing the target protein, or by site-specific mutation to introduce one or more nucleotide sequence substitutions, deletions, insertions, etc. in the coding region or promoter region, or by using chemical reagents to reduce or eliminate the transcription of the specific gene.

[0039] In some embodiments, the gene is integrated into the E. coli genome, and the integration site of the gene can be selected according to the routine knowledge of those skilled in the art to select some pseudogene sites that do not have a significant impact on bacterial growth and basic metabolism, such as yeeP 、 ygiP 、 yghX, ygaY 、 yjiT 、 yjiP 、 ycjV 、 ycgH, ygaY, yeeL, ilvG, rph and the like. In other embodiments, the gene is also connected to a promoter, and the promoter can be selected according to the routine knowledge of 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 application preferably uses P rpoB promoter or P ydgI promoter.

[0040] In some embodiments, the construction method comprises using CRISPR / Cas9-mediated gene editing technology for gene integration and / or gene knockout.

[0041] In some embodiments, the construction method comprises constructing a recombinant fragment and a pGRB plasmid.

[0042] In some embodiments, the construction of the pGRB plasmid comprises: designing a target sequence, preparing a DNA fragment comprising the target sequence, and recombining the DNA fragment comprising the target sequence with a linearized vector fragment; in a specific embodiment, the target sequence is 5'-NGG-3'.

[0043] In some embodiments, the constructing method comprises constructing a recombination fragment for gene integration or a recombination fragment for gene knockout. The steps for constructing a recombination fragment for gene integration comprise: using the genome of the starting strain as a template, designing upstream and downstream homology arm primers according to the sequences upstream and downstream of the desired gene insertion site, and designing primers according to the desired gene, amplifying the gene fragment, and then obtaining the recombination fragment by PCR overlap technology. The steps for constructing a recombination fragment for gene knockout comprise: using the upstream and downstream sequences of the gene to be knocked out as a template, designing upstream and downstream homology arm primers; amplifying the upstream and downstream homology arms by PCR, and then preparing the recombination fragment by overlap PCR.

[0044] In some embodiments, the constructing method comprises: simultaneously transforming the pGRB plasmid and the above-mentioned recombination fragment into an electrotransformation competent cell containing pREDCas9, and a step of plasmid elimination, to obtain a recombined genetically engineered strain.

[0045] In a second aspect, the present application provides the use of the genetically engineered bacteria as described in the first aspect in the fermentation production of L-isoleucine.

[0046] In a third aspect, the present application provides a method for producing L-isoleucine using the 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.

[0047] According to the third aspect of the present application, the culturing of the genetically engineered bacteria can be performed using conventional methods in the art. The culture medium for producing L-isoleucine can be a synthetic or natural medium, such as a typical medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic and inorganic components as needed.

[0048] According to the third aspect of the present application, 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 to between 5.0 and 8.0, or between 6.0 and 7.5, or between 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or basic substances, and ammonia gas.

[0049] In some embodiments, the method further comprises maintaining a constant culture temperature of 37°C. In other embodiments, the method further comprises maintaining a pH of about 7.0.

[0050] In other embodiments, the method further comprises supplementing a glucose solution at a concentration during the culturing to maintain a residual glucose concentration in the fermentation medium of less than 5 g / L, for example, 1-5 g / L.

[0051] After the culturing, solids such as cells and cell debris can be removed from the liquid culture medium by conventional techniques (e.g., centrifugation, membrane filtration), and ergothioneine can then be recovered from the fermentation broth by any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0052] The bacteria can also be activated, expanded, etc. prior to inoculation of the fermentation, depending on the storage state of the bacteria. Suitable conditions and media can be selected according to conventional techniques in the art, for example, the seed medium can be the same composition as the fermentation medium, or can be appropriately adjusted based thereon.

[0053] Other molecular biology, genetic engineering, etc. techniques involved can be achieved according to manuals, textbooks, or literature reports readily available to those skilled in the art, and the operation process need not be described in detail herein. In addition, specific strains are selected as hosts in the following examples, and thus specific genetic integration sites, target genes, and primers, etc. are selected according to the hosts, but this does not mean that the purpose of the present application can only be achieved through these specific selections, and the scope of the present application cannot be limited by this. The essence and scope of the present application are only limited by the claims.

[0054] In a fourth aspect, the present application provides an Escherichia coli RNA polymerase β-subunit mutant, the amino acid sequence of which is shown in SEQ ID NO: 1. According to the present application, the mutant has the effect of improving the host's tolerance to L-isoleucine compared to the parent RNA polymerase β-subunit. Preferably, the nucleotide sequence of the gene encoding the RNA polymerase β-subunit mutant is shown in SEQ ID NO: 2.

[0055] The present application is described in more detail below through specific examples.

[0056] 1. Method of gene editing

[0057] The gene editing method used in the present application refers to the literature (Li Y, Lin Z, Huang C, et al .Metabolic engineering of Escherichia coliMetabolic engineering, 2015, 31: 13-21.) using two plasmids shown in the following figures. Figure 1 . In which pREDCas9 carries gRNA expression plasmid pGRB elimination system, λ phage Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), 32℃ culture; pGRB with pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), 37℃ culture.

[0058] The specific steps of the method are as follows:

[0059] 1.1 Construction of pGRB plasmid

[0060] The purpose of constructing plasmid pGRB is to transcribe the corresponding gRNA to form a complex with Cas9 protein, and to achieve the purpose of DNA double-strand break by base pairing and PAM recognition of target gene sites. The method of recombination of DNA fragments containing target sequences with linearized vector fragments is used to construct pGRB plasmid.

[0061] 1.1.1 Design of target sequence

[0062] Use CRISPR RGEN Tools to design target sequence (PAM: 5'-NGG-3')

[0063] 1.1.2 Preparation of DNA fragments containing target sequences

[0064] Design primers: 5'-linearized vector end sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (15 bp)-3' and its reverse complementary primer, prepare DNA fragments containing target sequences by annealing of single-stranded DNA. Reaction conditions: pre-denaturation 95℃, 5 min; annealing 30-50℃, 1 min. The annealing system is shown in Table 1.

[0065] Table 1

[0066]

[0067] 1.1.3 Preparation of linear vector

[0068] The linearization of the vector uses the method of reverse PCR amplification.

[0069] 1.1.4 Recombination reaction

[0070] The recombination system is shown in Table 2. The recombination enzyme used is the enzyme of the ClonExpress® II One Step Cloning Kit series, and the recombination conditions are: 37°C, 30 min.

[0071] Table 2

[0072]

[0073] 1.1.5 Transformation of plasmid

[0074] Take 10 μL of the reaction solution and add it to 100 mL of DH5a competent cells. Mix gently, then ice bath for 20 min, 42°C heat shock for 45-90 s, immediately ice bath for 2-3 min, add 900 μL of SOC, and recover at 37°C for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, and resuspend the bacterial cells with about 200 μL of LB medium containing 100 mg / L ampicillin. Then, spread the bacterial cells on a plate containing 100 mg / L ampicillin, invert the plate, and incubate at 37°C overnight. After the plate grows single colonies, identify them by colony PCR, and select positive recombinants.

[0075] 1.1.6 Identification of clones

[0076] After inoculating the PCR-positive colonies into LB medium containing 100 mg / L ampicillin and incubating overnight, the bacteria were preserved, the plasmid was extracted, and identified by enzyme digestion.

[0077] 1.2 Preparation of recombinant DNA fragments

[0078] The recombinant fragment for knockout consists of the upstream and downstream homology arms of the gene to be knocked out (upstream homology arm-downstream homology arm); the recombinant fragment for integration consists of the upstream and downstream homology arms of the integration site and the gene fragment to be integrated (upstream homology arm-target gene-downstream homology arm). Using primer design software primer5, the upstream and downstream homology arm primers (amplification length of about 400-500 bp) are designed with the upstream and downstream sequences of the gene to be knocked out or the integration site as the template; the integration gene amplification primers are designed with the integration gene as the template. After amplifying the upstream and downstream homology arms and the target gene fragment by PCR, the recombinant fragment is prepared by overlap PCR. The PCR amplification system is shown in Table 3:

[0079] Table 3

[0080]

[0081] The overlap PCR system is shown in Table 4:

[0082] Table 4

[0083]

[0084] Note: The template is composed of equimolar amounts of the amplified fragments of the upstream and downstream homology arms and the gene of interest, and the total amount is not more than 10 ng.

[0085] PCR reaction conditions (Bio Biological PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 min; then 30 cycles of denaturation (98°C) for 10 s, annealing ((Tm-3 / 5) °C) for 15 s, 72°C extension (1 min extension per 1 kb of enzyme activity); continue to extend at 72°C for 10 min; maintain (4°C).

[0086] 1.3 Transformation of plasmids and recombinant DNA fragments

[0087] 1.3.1 Transformation of pREDCas9

[0088] The pREDCas9 plasmid was electroporated into the electroporation competent cells of the starting strain by electroporation, and the bacterial cells were recovered and cultured before being plated on LB plates containing spectinomycin and incubated at 32°C overnight. Single colonies growing on the resistant plates were subjected to colony PCR using identification primers to screen positive recombinants.

[0089] 1.3.2 Preparation of electroporation competent cells of the target strain containing pREDCas9

[0090] Cultured at 32°C until OD 600 = 0.1-0.2, 0.1 M IPTG was added (to a final concentration of 0.1 mM), and the culture was continued until OD 600 = 0.6-0.7. The purpose of adding IPTG is to induce the expression of the recombinase on the pREDCas9 plasmid. The medium required for the preparation of the competent cells and the preparation process refer to the conventional standard operation.

[0091] 1.3.3 Transformation of pGRB and recombinant DNA fragments

[0092] pGRB and donor DNA fragments were simultaneously electroporated into electroporation competent cells containing pREDCas9. The recovered and cultured bacterial cells after electroporation were plated on LB plates containing ampicillin and spectinomycin and incubated at 32°C overnight. Colony PCR was performed using specially designed identification primers to screen positive recombinants and preserve them.

[0093] 1.4 Elimination of plasmids

[0094] 1.4.1 Elimination of pGRB

[0095] The positive recombinants were inoculated into LB medium containing 0.2% arabinose and incubated overnight, then diluted and inoculated onto LB plates containing spectinomycin, and incubated at 32°C overnight. LB plates containing ampicillin and spectinomycin were spotted, and single colonies that did not grow on the ampicillin plates but grew on the spectinomycin plates were selected and preserved.

[0096] 1.4.2 Elimination of pREDCas9 plasmid

[0097] The positive recombinants were inoculated into LB medium containing 0.2% arabinose and incubated overnight, then diluted and inoculated onto LB plates containing spectinomycin, and incubated at 32°C overnight. LB plates containing ampicillin and spectinomycin were spotted, and single colonies that did not grow on the ampicillin plates but grew on the spectinomycin plates were selected and preserved.

[0098] 2 The primer sequences used in the examples are shown in Table 5:

[0099] Table 5

[0100]

[0101] 3 The culture medium used in the examples can be referred to as follows:

[0102] The slope medium consists of glucose 1-5 g / L, proteose peptone 5-10 g / L, beef extract 5-10 g / L, yeast powder 1-5 g / L, NaCl 1-5 g / L, agar 15-25 g / L, and water, pH 7.0-7.2.

[0103] The seed medium consists of glucose 20-40 g / L, yeast extract 2-5 g / L, proteose peptone 2-5 g / L, citric acid 1-3 g / L, methionine 0.5-1 g / L, sodium 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 0.2-3 mg / L each, and water, pH 7.0-7.2.

[0104] 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.

[0105] The present invention will be described in more detail below through specific embodiments.

[0106] Example 1:

[0107] 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.

[0108] (1) yihF ::P rpoB - rpoB M Gene editing

[0109] 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 MPrimers 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.

[0110] (2) ilvA ::P ydgI - ilvA M Gene editing

[0111] 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 MPrimers 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.

[0112] (3) yjiP ::P ydgI - ilvIH M Gene editing

[0113] 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 ( ilvINCBI 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.

[0114] (4) ilvA ::P ydgI - ilvA M -(GGGGS)3- ilvIH M Gene editing

[0115] 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...) ilvAM () 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 genes 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.

[0116] (5) ilvA ::P ydgI - ilvIH M-(GGGGS)4- ilvA M Gene editing

[0117] 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 ( ilvA - ilvA -DN-S、DN- ydgI -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ilvA Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ydgI - ydgI -S、 ilvIH - ydgI -A), PCR amplification ilvIH Promoter fragment; genome of a laboratory-preserved strain (containing...) ilvIH M () as template, according to ilvI M Primers were designed based on the gene sequence (NCBI GeneID: 948287). ilvH - ydgI -S、 ​ -GS4-A), PCR amplification ​ M Fragments; genomes of laboratory-preserved strains (containing...) ​ M () as template, according to ​ M Gene sequence ( ​ NCBI GeneID: 948793 ​ Design primers (GS4-) using NCBI GeneID: 947267. ​ -S、 ​ -A), PCR amplification ​ M Fragment; flexible linker peptide (GGGGS)4 is designed in ​ M Downstream primers of the gene and ​ M In the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain P. ydgI - ​ M-(GGGGS)4- ​ M The integrated fragment (upstream homologous arm - P) ydgI - ​ M -(GGGGS)4- ​ M -Downstream homologous arm), primer gRNA- ​ S and gRNA- ​ The DNA fragment obtained by -A annealing was ligated to plasmid pGRB to construct pGRB- ​ To prepare competent cells of the engineered strain ILE01, follow the procedures shown in 1.3 and 1.4, and insert plasmid pGRB- ​ and P ydgI - ​ M -(GGGGS)4- ​ M The integrated fragment was simultaneously electroporated into competent cells, ultimately yielding strain ILE05.

[0118] (6) ​ ::P ydgI - ​ M -(GGGGS)3- ​ M Gene editing

[0119] Using the E. coli MG1655 (ATCC700926) genome as a template, based on its ​ Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI GeneID: 948287). ​ -S、UP- ​ -A) and downstream homologous arm primer (DN- ​ -S、DN- ​ -A), PCR amplification of its upstream and downstream homologous arm fragments; using the E. coli MG1655 (ATCC700926) genome as a template, according to ​ Primers were designed based on the gene promoter sequence (NCBI GeneID: 945159). ​ - ​ -S、 ​ - ​ -A), PCR amplification ​ Promoter fragment; genome of a laboratory-preserved strain (containing...) ​ M () as template, according to ​ M Gene sequence ( ​ NCBI GeneID: 948793​ Primers were designed using NCBI GeneID: 947267. ​ - 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.

[0120] (7) ilvA ::P ydgI - ilvIH M -(GGGGS)4- ilvA M Gene editing

[0121] The upstream homology arm primer (UP- S, UP- A) and the downstream homology arm primer (DN- S, DN- A) were designed according to the upstream and downstream sequences of the gene (NCBI GeneID: 948287), and the upstream and downstream homology arm fragments were amplified by PCR. The primer (P- S, P- A) was designed according to the promoter sequence of the gene (NCBI GeneID: 945159), and the promoter fragment was amplified by PCR. The primer (GS4- S, GS4- A) was designed according to the gene sequence (NCBI GeneID: 948793, NCBI GeneID: 947267), and the gene fragment was amplified by PCR. The primer (GS4- S, GS4- A) was designed according to the gene sequence (NCBI GeneID: 948287), and the gene fragment was amplified by PCR. The flexible connection peptide (GGGGS)4was designed in the downstream primer of the gene and the upstream primer of the gene. The above fragments were fused by overlapping PCR to obtain the integration fragment (upstream homology arm-P- (GGGGS)4- downstream homology arm) of the P- (GGGGS)4- gene. ilvA ilvA ilvA ilvA ilvA ydgI ilvA ydgI ydgI ilvIH ydgI ilvIH M ilvIH M ilvI NCBI GeneID: 948793, ilvH NCBI GeneID: 947267 ydgI ilvIH ilvIH ilvIH M ilvA M ilvA M NCBI GeneID: 948287 ilvA ilvA ilvA M ilvIH M ilvA M ydgI ilvIH M ilvA M ydgI ilvIH ​​​​​​​​​​​​​​​​​​​​​​​​​​​M -(GGGGS)4- ilvA M - ilvIH M - downstream homology arm), the primer gRNA- ilvA- S and gRNA- ilvA The DNA fragment obtained by annealing A was ligated with plasmid pGRB to construct pGRB- ilvA The competent cells of engineered strain ILE01 were prepared, and the plasmid pGRB- ilvA and P ydgI - ilvIH M -(GGGGS)4- ilvA M The integration fragment was simultaneously electroporated into the competent cells, and finally the strain ILE07 was obtained.

[0122] Example 2:

[0123] This example is used to illustrate the influence of different concentrations of L-isoleucine on the growth of the strain, which can be ILE01 or E. coli THRD.

[0124] (1) Inclined surface culture: the preserved strain at -80℃ was streaked and inoculated on the activated inclined surface, and cultured at 37℃ for 12 h, and subcultured once;

[0125] (2) Shake flask culture: a loop of the inclined surface seed was scraped with an inoculation loop and inoculated into a series of 500 mL triangular flasks containing 30 mL of basic medium, the concentration of L-isoleucine in the medium was 20 g / L and 40 g / L respectively, and 1 mL of bottom sugar was added as a carbon source, nine layers of gauze were sealed, and cultured at 37℃, 220 rpm for 14 h, after 4 h of culture, the OD 600 value of the strain was measured every 2 h, and the growth curve was drawn for analysis and comparison.

[0126] The preferred inclined surface culture medium of this example is composed of glucose 1 g / L, proteose peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, NaCl 5 g / L, agar 25 g / L, and the rest is water, pH 7.0.

[0127] The preferred basic medium of this example is composed of Na2HPO4·12H2O 15 g / L, KH2PO4 3 g / L, (NH)4SO4 5 g / L, NaNO3 1 g / L, V B1 , V B3 , V B5 , V B12 , V H1 mg / L, trace element mixture 1 mg / L, the rest is water, pH 7.0; wherein, 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, H3BO4 2.5 g / L.

[0128] The basal sugar consists of: 60% glucose, 3% MgSO4·7H2O.

[0129] The experimental results are shown in Table 1. Figure 2 As shown in Table 1, the growth amount (calculated by OD value) of the engineered bacteria ILE01 of the present application in the basic medium containing 20 g / L or 40 g / L L-isoleucine after 14 h of culture was significantly improved compared with the control strain E. coli THRD. The results confirmed that the introduction of the RNA polymerase β-subunit mutant or the coding gene thereof of the present application can effectively enhance the tolerance of the strain to L-isoleucine, thereby more conducive to the growth of the strain and the production of L-isoleucine. 600 rpoB M

[0130] Example 3:

[0131] This example is used to illustrate the method for producing L-isoleucine by using the genetically engineered bacteria of the present application, which can be the genetically engineered bacteria ILE01, ILE02, ILE03, ILE04, ILE05, ILE06 or ILE07.

[0132] (1) Inclination culture: take the -80℃ preserved bacteria to inoculate on the activated slope, cultivate at 37℃ for 12 h, and subculture once;

[0133] (2) Seed culture in a flask: use the inoculation ring to scrape a ring of the slope seed to inoculate in the 500 mL triangular flask containing 30 mL of seed culture medium, nine layers of gauze are sealed, cultivate at 37℃, 220 rpm for 7-10 h;

[0134] (3) Fermentation culture in a flask: inoculate into the 500 mL triangular flask containing the fermentation culture medium according to the inoculation amount of 10% of the seed culture liquid (the final volume is 30 mL), nine layers of gauze are sealed, cultivate at 37℃, 220 r / min, maintain the pH at 7.0-7.2 by supplementing ammonia water during the fermentation process; supplement 60% (w / v) glucose solution to maintain the fermentation process; the fermentation period is 24 h. m / v ​​​

[0135] The preferred slant medium of the embodiment is composed of glucose 1 g / L, proteose peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, agar 25 g / L, and the rest is water, pH 7.0.

[0136] The preferred seed medium of the embodiment is composed of glucose 20 g / L, yeast extract 5 g / L, proteose peptone 4 g / L, citric acid 3 g / L, methionine 0.5 g / L, sodium 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 2 mg / L, and the rest is water, pH 7.0.

[0137] The preferred fermentation medium of the embodiment is composed of 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 , V B3 , V B5 , V B12 , V H 2 mg / L, trace element mixture 1 mg / L, and the rest is water, pH 7.0; wherein the trace element mixture is composed 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.

[0138] E. coli THRD as a control strain, strains ILE01-ILE07 were subjected to 24 h shake flask fermentation, and the L-isoleucine content in the fermentation broth was detected by high performance liquid chromatography. The shake flask fermentation results are shown in Table 6. The fermentation data show that the introduction of the L-isoleucine biosynthetic gene cluster into the genome of E. coli THRD can improve the L-isoleucine production of the strain. rpoB MStrains ILE03, ILE04, ILE05, ILE06 and ILE07 were constructed by inserting the genes encoding the enzymes into the pET-28a(+) vector, respectively. ilvA M However, the overexpression of the enzymes alone led to the accumulation of toxic intermediate metabolite α-ketobutyric acid, and the production of L-isoleucine was low. ilvIH M The accumulation of toxic intermediate metabolite α-ketobutyric acid was reduced, and the production of L-isoleucine was improved. ilvA M However, the overexpression of the enzymes alone led to the accumulation of toxic intermediate metabolite α-ketobutyric acid, and the production of L-isoleucine was low. ilvIH M The accumulation of toxic intermediate metabolite α-ketobutyric acid was reduced, and the production of L-isoleucine was improved. ilvA M -(GGGGS)3- ilvIH M The strain ILE04 had the best effect, and the production of L-isoleucine reached 10.8 g / L. ilvA M -(GGGGS)3- ilvIH M The enzyme fusion expression of (GGGGS)3- is more suitable for the production of L-isoleucine by the current strains.

[0139] Table 6

[0140]

[0141] Although the present application has been disclosed in the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various changes, modifications, replacements and variations in the form and details of the embodiments without departing from the spirit and principles of the present application, and the scope of the present application 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. 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 The amino acid sequence of the threonine dehydratase mutant is shown in SEQ ID NO: 3, and the gene encoding the acetylhydroxy acid synthase mutant is... ilvIH M The nucleotide sequence is shown in SEQ ID NO: 4; and the threonine dehydratase mutant encodes the gene. 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.

2. The genetically engineered bacteria as described in claim 1, characterized in that: The flexible linker peptide is (GGGGS)3 or (GGGGS)4.

3. The genetically engineered bacteria as described in claim 2, 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 .

4. The genetically engineered bacteria according to any one of claims 1-3, 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.

5. The genetically engineered bacteria according to any one of claims 1-3, 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.

6. The use of the genetically engineered bacteria according to any one of claims 1-3 in the fermentation production of L-isoleucine.

7. A method for producing L-isoleucine, comprising: The genetically engineered bacteria according to any one of claims 1-3 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.

Images