CN116286570A

CN116286570A - Genetically engineered bacterium for high-yield L-isoleucine and application thereof

Info

Publication number: CN116286570A
Application number: CN202211675055.3A
Authority: CN
Inventors: 方海田; 刘慧燕; 魏晓博; 张想军; 张皓杰
Original assignee: Ningxia University
Current assignee: Ningxia University
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-06-23

Abstract

The invention discloses a genetically engineered bacterium for high-yield L-isoleucine and application thereof, and belongs to the technical field of genetic engineering. Firstly, constructing an escherichia coli methionine-deficient strain E.coliNXa1 and a methionine and lysine double-deficient strain E.coliNXa2 by utilizing an ARTP and Ultraviolet (UV) composite mutagenesis method, and then, removing leucine synthesis pathway key enzyme genes LeuA and L-2-amino-3-oxobutyric acid synthesis pathway key enzyme genes tdh, heterologous gene ybgE expression, L-threonine dehydrogenase gene yiaY, tricarboxylic acid cycle local transcription factor iclR and global transcription factor ArcA in escherichia coli to obtain genetically engineered bacteria. The genetically engineered bacterium achieves the aim of improving the yield of the L-isoleucine by cutting off the anabolism pathway of methionine and lysine, cutting off the decomposition pathway of L-threonine, weakening the synthesis pathway of acetic acid and increasing precursors, and the yield of the L-isoleucine reaches 43.22-44.25g/L after the strain is fermented for 40 hours.

Description

Genetically engineered bacterium for high-yield L-isoleucine and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering bacterium for high-yield L-isoleucine and application thereof.

Background

L-isoleucine (L-Ile), also known as "isoleucine", is systematically designated "alphA-Amino-beta-methylpentanoic acid" and is one of the three branched-chain amino acids. The physical properties of the L-isoleucine are mostly white crystal chips or crystalline powder, the taste is odorless but slightly bitter, the sublimation temperature is 168-170 ℃, the L-isoleucine has wide application in the fields of food, medicine, chemical industry and feed, the demand of the L-isoleucine is gradually increased in each field along with the progress of technology, and the application range is gradually enlarged at the same time.

The existing production methods of L-isoleucine include three methods, namely an extraction method, a chemical synthesis method and a fermentation method, wherein the extraction method is used for separating and extracting the L-isoleucine from protein hydrolysate. The product obtained by chemical synthesis is typically a mixture of four optical isomers. To obtain natural L-isoleucine, not only optical resolution is needed, but also L-allo-isoleucine is needed to be separated, the process is complex, and the yield is low. Therefore, it is currently difficult to industrially produce L-isoleucine with high purity by extraction or chemical synthesis. Compared with the method for producing the L-isoleucine by using a protein hydrolysis method and a chemical synthesis method, the microbial fermentation method is environment-friendly, greatly reduces the cost, is easy to control the reaction, can be suitable for large-scale production, and is a main method for producing the L-isoleucine industrially at present.

The microbial fermentation method for synthesizing L-isoleucine mainly depends on the fermentation action of microorganisms, so that the type of microorganisms selected for microbial fermentation is very important. The strain capable of synthesizing the L-isoleucine under natural conditions mainly comes from corynebacterium glutamicum and escherichia coli, has the advantages of clear genetic background, simple technical operation, short generation time and the like, and is widely applied to the production of amino acids such as L-threonine, L-tryptophan, L-phenylalanine and the like. Therefore, it is possible to try to select and breed a strain producing L-isoleucine using E.coli and put it into industrial production.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for high yield of L-isoleucine and application thereof, and the genetically engineered bacterium achieves the aim of improving the yield of the L-isoleucine by cutting off the anabolism pathway of methionine and lysine, cutting off the decomposition pathway of L-threonine, weakening the synthesis pathway of acetic acid and increasing precursors, and the yield of the L-isoleucine reaches 43.22-44.25g/L after the strain is fermented for 40 hours.

In order to achieve the above object, the present invention provides a genetically engineered bacterium for high-yielding L-isoleucine, which is characterized in that leucine synthesis pathway key enzyme genes LeuA, L-2-amino-3-oxobutanoic acid synthesis pathway key enzyme genes tdh, heterologous genes ybgE, L-threonine dehydrogenase genes yiaY, tricarboxylic acid cycle local transcription factors iclR and global transcription factors ArcA are knocked out in genome of methionine and lysine double-defect mutant strains.

Preferably, the nucleotide sequence of the LeuA gene is shown as SEQ ID No. 1; the nucleotide sequence of the tdh gene is shown as SEQ ID No. 2; the nucleotide sequence of the ybgE gene is shown in SEQ ID No. 3; the nucleotide sequence of the yiaY gene is shown as SEQ ID No. 4; the nucleotide sequence of the iclR gene is shown as SEQ ID No. 5; the nucleotide sequence of the ArcA gene is shown in SEQ ID No. 6.

Preferably, the methionine and lysine double defect mutant strain comprises escherichia coli K12, and an initial strain of the escherichia coli K12 is escherichia coli MG1655.

Preferably, the LeuA, tdh, ybgE, yiaY, iclR and arcA gene knockout nucleotide mutant fragments are obtained by recombinant PCR, and primer sequences for amplifying LeuA, tdh, ybgE, yiaY, iclR and arcA nucleotide mutant fragments are shown in SEQ ID No. 7-42.

Preferably, the gene is knocked out using CRISPR/Cas9 mediated gene editing techniques.

The invention constructs a methionine-deficient strain E.coli NXA1 and a methionine and lysine double-deficient strain E.coli NXA2 of escherichia coli by preferably utilizing an ARTP and Ultraviolet (UV) composite mutagenesis method, and the gene is weakened by utilizing a CRISPR/Cas9 mediated gene editing technology to obtain the genetically engineered bacterium. The genotype of the genetically engineered bacterium is named as E.coli K12 MG1655 delta LeuA delta tdh delta ybgE delta yiaY delta iclR delta ArcA.

The CRISPR/Cas9 mediated gene editing technology is not particularly limited, and a person skilled in the art can knock out the gene conventionally.

In the construction method, a gene site-directed mutagenesis kit is used for point mutation, the gene knockout method is a one-step knockout technology, and the used nucleotide mutation fragment has two homologous arms at the upstream and downstream of the knockout gene and a kanamycin resistance gene box.

The invention also provides a production method for preparing L-isoleucine by using the genetically engineered bacterium, which comprises the following steps:

1) Inoculating genetically engineered bacteria into a seed culture medium for culture to obtain seed liquid;

2) Inoculating the seed solution obtained in the step 1) into a fermentation medium for fermentation to obtain the L-isoleucine.

Preferably, the seed culture medium in the step 1) comprises the following components: 2-5 g/L of ammonium nitrate, 5-10 g/L of glucose, 0.002-0.004 g/L of biotin, 10.5g/L of peptone, 5.5g/L of yeast powder and 10.5g/L of NaCl, wherein the solvent is water; the pH value is 7.0-7.2.

Preferably, the culturing conditions in step 1) are preferably: the temperature is 37 ℃, the rotating speed is 180rpm, and the time is 12 hours.

Preferably, the fermentation medium in the step 2) comprises the following components in percentage by weight: 127.41g/L glucose, 32.97g/L ammonium sulfate, 15.97g/L corn steep liquor, 3.0g/L monopotassium phosphate, 2.5g/L magnesium sulfate, 0.01g/L ferrous sulfate and 0.01g/L manganese sulfate, wherein the solvent is water; the pH value is 7.0-7.2.

Preferably, the fermentation conditions in step 2) include: the temperature is 37 ℃, the time is 40-50 h, and the rotating speed is 200rpm; the seed liquid is preferably inoculated in an amount of 10 to 15%.

The invention also provides application of the genetically engineered bacterium for producing the L-isoleucine at high yield in preparation of the L-isoleucine.

The genetically engineered bacterium for producing high-yield L-isoleucine and the application thereof have the advantages and positive effects that:

1. the genetic engineering bacteria of the invention construct a methionine-deficient strain E.coliNXA1 and a methionine and lysine double-deficient strain E.coliNXA2 by using an ARRTP mutagenesis method, and then the leucine synthesis pathway key enzyme genes LeuA and L-2-amino-3-oxobutyric acid synthesis pathway key enzyme genes tdh, the heterologous gene ybgE expression, the L-threonine dehydrogenase gene yiaY, the local transcription factor iclR and the global transcription factor ArcA in the escherichia coli are knocked out; the purpose of improving the yield of the L-isoleucine is achieved by cutting off the production path of the byproduct methionine and lysine, cutting off the degradation path of the L-threonine, weakening the synthesis path of the acetic acid and further increasing the precursor, and a scientific basis is provided for subsequent more efficient breeding of the L-isoleucine production strain.

2. The genetically engineered bacterium disclosed by the invention has the advantages of simple culture medium, simple fermentation process and low cost, and is suitable for industrial application.

3. The L-isoleucine yield of the genetically engineered bacterium is 116+/-9.17 mg/L of E.coliK12, 499+/-14.04 mg/L of E.coliNX1, 2434+/-70.02 mg/L of E.coliK-12 (delta LeuA), 8.91+/-1.011 g/L of E.coliK-12 (delta LeuA delta tdh), 10.26+/-1.016 g/L of E.coliK-12 (delta LeuA delta tdh), 17.46+/-1.02 g/L of E.coliK-12 (delta LeuA delta tdhGdelta ybgE), 28.64+/-1.07 g/L of E.coliK-12 (delta LeuA delta thddelta GygE delta yY), 30.02+/-1.02+/-1.09 g/L of E.coliK-12 (delta LeuA delta tdh) and 35.37 g/L of E.coliK-12 (delta LeuA delta tdh).

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a schematic diagram showing the L-isoleucine yield of a genetically engineered strain for high-yield L-isoleucine production of the present invention.

Detailed Description

The technical scheme of the invention is further described below through the attached drawings and the embodiments. Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

In the examples described below, materials, reagents, plasmids, kits for exclusive use, strains, etc., were obtained commercially, unless otherwise specified.

Example 1

Construction of methionine and lysine double-defect mutant strain of Escherichia coli

Recombinant E.coli ARTP mutagenesis

The preserved escherichia coli K12 is marked on an LB solid culture medium in a Z shape, and is inversely cultured to a logarithmic phase at 37 ℃; picking single colony on the plate, transferring into LB liquid medium, culturing at 37deg.C and 190rpm for a period of time, and making into OD ₆₀₀ The bacterial suspension with the nm value of 0.6-0.8 is added with 10 percent of glycerol and put into an ultra-clean workbench for standby.

Sterilizing and drying a No.2 bottle of the MMC machine for later use, firstly filling 5mL of special oil, diluting the strain subjected to liquid culture by 10 times, then filling the strain into the No.2 bottle, and taking a growth curve according to operation steps. The temperature of ARTP was maintained at 20℃and the power was adjusted to 100W with a helium flow of 10L/min. Absorbing 10 mu L of bacterial suspension, uniformly coating the bacterial suspension on a metal slide, placing the slide into a mutagenesis chamber by using tweezers, carrying out mutagenesis treatment on bacterial liquid by adopting different times (0 s, 30s, 60s, 90s, 150s, 180s and 240 s), fully eluting the mutagenized bacterial body in 1mL of ultrapure water for 1min, taking 100 mu L of the bacterial suspension, coating the bacterial suspension on an LB culture medium plate, culturing the bacterial suspension overnight at 37 ℃, and determining the optimal mutagenesis time according to the calculated mortality.

1 mol.L of the prepared ^-1 The alpha-AB of (B) is diluted according to a gradient to prepare the final concentration of 0.1 mol.L ^-1 、0.2mol·L ^-1 、0.4mol·L ^-1 、0.6mol·L ^-1 、0.8mol·L ^-1 Resistance plates, on which the cultured strains were spread, respectively.Filling the strain subjected to ARTP mutagenesis into an MMC No.6 bottle, filling bacterial liquid in a No.2 bottle, and filling liquid LB culture medium in a No.4 bottle. The solution in vial 6 was set to 8 concentration gradients of 0%, 9.37%, 19.53%, 29.68%, 39.84%, 50%, 59.37%, 100%, respectively, with 5 parallel concentration gradients each creating a total of 40 droplets, operating according to the instrument protocol. After the liquid drops are cultured for 24 hours, the liquid drops with the highest adaptive concentration and longer than the normal saline are selected for liquid drop collection, and the collected liquid drops are diluted into gradient concentration by the normal saline after being killed and are respectively coated on a resistance screening plate. Then culturing for a period of time under proper conditions, picking single colony with better growth condition on a resistance plate, transferring the single colony into LB liquid culture medium, and culturing at 37 ℃ and 1900rpm overnight to a certain concentration for standby.

And (3) absorbing 600 mu L of bacterial liquid which is subjected to single-factor multi-level screening culture in 10mL of liquid culture medium, loading the bacterial liquid into an MMC No.2 bottle, loading the No.6 bottle into alpha-AB with the highest concentration suitable for strains, loading the No.4 bottle into the liquid LB culture medium, operating according to an instrument operation flow, setting 8 concentration gradients of 2.78%, 9.95%, 16.67%, 28.05%, 38.00%, 48.01%, 58.00%, 98.83% of the solution in the No.6 bottle, and carrying out three parallel concentration gradients each for 8 hours to create 48 liquid drops. After 48 hours of liquid drop culture, the strain with better growth condition of the highest concentration gradient is collected, diluted by a certain multiple and then coated on a resistance plate for overnight culture at 37 ℃, single colony with better growth condition is selected and transferred into LB liquid culture medium for overnight culture at 37 ℃ and 190rpm for a period of time for standby.

Example 2

Construction of genetically engineered bacterium for high-yield L-isoleucine

(1) LeuA gene of mutant strain E.coliNXA2 is knocked out by CRISPR/Cas9 gene editing technology

1. Coli K-12 competent cells containing pCas plasmid were prepared using E.coli NXA2 obtained in example 1 as an initial strain.

2. The genome DNA of E.coli NXA2 is used as a template, primers LeuA-Up-F (shown as SEQ ID No. 9) and LeuA-Up-R (shown as SEQ ID No. 10) are respectively designed for PCR amplification to obtain an upstream homology arm segment N1, and primers LeuA-Down-F (shown as SEQ ID No. 11) and LeuA-Down-R (shown as SEQ ID No. 12) are respectively used for PCR amplification to obtain a downstream homology arm segment N2.

Overlapping Extension (SOE) PCR was used to ligate the LeuA upstream and downstream homology arm fragments. SOE PCR amplification is carried out by taking upstream and downstream homology arm fragments N1 and N2 as templates, and primers LeuA-Up-F (shown as SEQ ID No. 9) and LeuA-Down-R (shown as SEQ ID No. 12) to synthesize Donor DNA. PCR amplification was performed using pTargetF plasmid as a template, sg20-F-SpeI (shown as SEQ ID No. 7) and sg20-R (shown as SEQ ID No. 8) as primers, and correct transformants were selected to obtain plasmids, thereby obtaining recombinant plasmid pTargetF-LeuA capable of recognizing the LeuA gene.

3. The constructed plasmid pTargetF-LeuA and the knocked-out DNA fragment are added into 40 mu L of E.coli K-12 delta LeuA competent cells containing pCas plasmid, after gentle mixing, the mixture is injected into an electric rotating cup with the diameter of 1mm which is pre-cooled in advance, electric shock is carried out for 5ms under the condition of voltage of 1.8kV, 1mL of LB liquid medium is rapidly added after electric shock is finished, the mixture is cultured for 2h under the condition of 180rpm and 30 ℃ for resuscitation, and then bacterial suspension is uniformly coated on LB resistant solid medium of 50mg/L spectinomycin and 50mg/L kanamycin, and is cultured overnight under the condition of 30 ℃.

4. Clones containing pCas and pTargetF-LeuA identified as correct were inoculated into LB liquid medium containing 0.5mmol/L IPTG and 50mg/L kanamycin and cultured overnight at 37 ℃. Then, the bacterial liquid is coated on an LB solid plate containing 50mg/L kanamycin, cultured overnight, after single colony grows out, randomly picked colonies are inoculated on the LB solid plate containing 50mg/L spectinomycin, cultured overnight to eliminate plasmid pTargetF, then the strain is inoculated into an LB solid culture medium, cultured overnight at 37 ℃ to eliminate pCas plasmid, and the knockout strain E.coliK-12 (delta LeuA) is obtained. Plasmid elimination results were verified using B0871-F, B0871-R as primers.

5. Single colonies of the activated primordial strain E.collK-12 and the mutant strain E.collK-12 (. DELTA.LeuA) on LB plates were picked up into LB liquid tubes and cultured overnight at 37℃at 180-200 rpm. Bacterial liquid PCR was performed using the identification primers LeuA-S (shown as SEQ ID No. 13) and LeuA-A (shown as SEQ ID No. 14) to verify the knockdown results.

(2) Based on the knock-out strain E.coli K-12 (DeltaLeuA), the gene tdh, ybgE, yiaY, iclR, arcA was knocked out in the same manner as in the above-mentioned step (1). The primer sequences of amplified nucleotide mutant fragments and the identified primer sequences are shown in SEQ ID Nos. 15-42, wherein tdhG-Down-F, tdhG-Down-R is identical to the tdhG-Up-F, tdhG-Up-R sequence, respectively.

Example 3

Shake flask fermentation production of L-isoleucine using E.coli starting strain and knockout strain

The knock-out strains E.coliNXa1, E.coliNXa2, E.coliK-12 (. DELTA.LeuA), E.coliK-12 (. DELTA.LeuA. DELTA.tdh. DELTA.ybgE. DELTA.yiay), E.coliK-12 (. DELTA.LeuA. DELTA.tdh. DELTA.ybgE. DELTA.yiaY. DELTA.iclR) and E.coliK-12 (. DELTA.LeuA. DELTA.tdh. DELTA.ybgE. DELTA.yiaY. DELTA.iclR. DELTA.arcA) prepared in examples 1 and 2 were picked up, respectively, and inoculated on LB slant medium and cultured overnight at 37 ℃. Single colonies stored on the slant medium were spotted with sterile toothpicks, the toothpicks with the colonies attached were placed into 50mL of seed medium, sealed with sterile gauze, and cultured at 37℃for 12 hours at 200 rpm. 5mL of seed fermentation liquid is sucked and mixed in a fermentation culture medium with the volume of 45mL, a sterile gauze is used for sealing, three bacteria are inoculated in parallel, the culture is carried out for 48h under the condition of 200rpm and 37 ℃, ammonia water is added in the fermentation process, the pH value is maintained within the range of 6.5-7, the fermentation liquid is sampled every 2h for measuring the concentration of bacteria and the content of glucose, and the samples are stored in a refrigerator with the temperature of minus 20 ℃. And measuring the L-isoleucine content of the fermented sample by using a high performance liquid chromatograph.

The conditions of the high performance liquid chromatography are as follows: c (C) ₁₈ Chromatographic column, detection wavelength 270nm, flow rate 1mL/min, mobile phase 4% acetonitrile.

Seed culture medium (g/L) with water as solvent: 2-5 g/L of ammonium nitrate, 5-10 g/L of glucose, 0.002-0.004 g/L of biotin, 10.5g/L of peptone, 5.5g/L of yeast powder, 10.5g/L of NaCl and 7.0-7.2.

Shake flask medium (g/L) with water as solvent: 127.41g/L glucose, 32.97g/L ammonium sulfate, 15.97g/L corn steep liquor, 3.0g/L monopotassium phosphate, 2.5g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.01g/L manganese sulfate and pH value of 7.0-7.2.

The fermented sample was subjected to measurement of L-isoleucine content by high performance liquid chromatograph, and the results are shown in Table 1:

TABLE 1 production of wild-type and mutant-containing Gene recombinant Strain L-isoleucine (mg/L)

Fermentation time	E.coliK-12	E.coliNXA1	E.coliNXA2
				40h	116±9.17	490±14.04	2443±70.02

E.coli K-12 (ΔLeuA), E.coli K-12 (ΔLeuA Δtdh ΔybgE ΔyiaY ΔiclR) are hereinafter referred to as knock 1, knock 2, knock 3, knock 4, knock 5, and knock 6. Table 2 shows the L-isoleucine yields after 40h fermentation of the original and knocked-out bacteria. FIG. 1 is a schematic diagram showing L-isoleucine production amounts of the respective strains.

TABLE 2 yield of original and knocked out Strain L-isoleucine (g/L)

Fermentation time	Yield of products
		Original bacteria	0.116±0.091
Knock out 1	8.91±0.41
		Knock-out 2	10.26±0.56
Knock-out 3	17.46±1.02
		Knock out 4	28.64±1.07
Knock-out 5	30.02±1.09
		Knock-out 6	43.22±1.032

As can be seen from the results in Table 2 and FIG. 1, the yield of L-isoleucine was greatly increased, up to 44.25g/L, in the knockout 6 strain, compared with the original strain.

Therefore, the invention adopts a genetically engineered bacterium with high L-isoleucine yield, and achieves the purpose of improving the L-isoleucine yield by cutting off the anabolism pathway of methionine and lysine, cutting off the decomposition pathway of L-threonine, weakening the synthesis pathway of acetic acid and increasing precursors, and the L-isoleucine yield reaches 43.22-44.25g/L after fermentation for 40 hours.

Finally, it should be noted that: the above examples are only for illustrating the technical solution of the present invention and not for limiting it, and it should be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Claims

1. A genetically engineered bacterium for high yield of L-isoleucine is characterized in that: the gene is characterized in that leucine synthesis pathway key enzyme genes LeuA, L-2-amino-3-oxobutyric acid synthesis pathway key enzyme genes tdh, heterologous genes ybgE, L-threonine dehydrogenase genes yiaY, tricarboxylic acid cycle local transcription factors iclR and global transcription factors ArcA are knocked out in genome of methionine and lysine double-defect mutant strains.

2. The genetically engineered bacterium for high yield of L-isoleucine of claim 1, wherein: the nucleotide sequence of the LeuA gene is shown as SEQ ID No. 1; the nucleotide sequence of the tdh gene is shown as SEQ ID No. 2; the nucleotide sequence of the ybgE gene is shown in SEQ ID No. 3; the nucleotide sequence of the yiaY gene is shown as SEQ ID No. 4; the nucleotide sequence of the iclR gene is shown as SEQ ID No. 5; the nucleotide sequence of the ArcA gene is shown as SEQ ID No. 6.

3. The genetically engineered bacterium for high yield of L-isoleucine of claim 1, wherein: the methionine and lysine double-defect mutant strain comprises escherichia coli K12, and an initial strain of the escherichia coli K12 is escherichia coli MG1655.

4. The genetically engineered bacterium for high yield of L-isoleucine of claim 1, wherein: the LeuA, tdh, ybgE, yiaY, iclR and ArcA gene knockout nucleotide mutant fragments are obtained through recombinant PCR, and primer sequences for amplifying LeuA, tdh, ybgE, yiaY, iclR and ArcA nucleotide mutant fragments are shown in SEQ ID No. 7-42.

5. The method for producing L-isoleucine by using the genetically engineered bacterium as claimed in any one of claims 1 to 4, comprising the steps of:

6. The method for producing L-isoleucine by using the genetically engineered bacterium of claim 5, wherein the method comprises the steps of: the seed culture medium in the step 1) comprises the following components: 2-5 g/L of ammonium nitrate, 5-10 g/L of glucose, 0.002-0.004 g/L of biotin, 10.5g/L of peptone, 5.5g/L of yeast powder and 10.5g/L of NaCl, and the solvent is water; the pH value is 7.0-7.2; the culture conditions were 37℃at a rotational speed of 180rpm for 12 hours.

7. The method for producing L-isoleucine by using the genetically engineered bacterium of claim 5, wherein the method comprises the steps of: the fermentation medium in the step 2) comprises the following components in percentage by weight: 127.41g/L glucose, 32.97g/L ammonium sulfate, 15.97g/L corn steep liquor, 3.0g/L monopotassium phosphate, 2.5g/L magnesium sulfate, 0.01g/L ferrous sulfate and 0.01g/L manganese sulfate, wherein the solvent is water; the pH value is 7.0-7.2.

8. The method for producing L-isoleucine by using the genetically engineered bacterium of claim 5, wherein the method comprises the steps of: the fermentation conditions in step 2) include: the temperature is 37 ℃, the time is 40-50 h, and the rotating speed is 200rpm; the seed liquid is preferably inoculated in an amount of 10 to 15%.

9. An application of genetically engineered bacterium for high-yield L-isoleucine in preparing L-isoleucine.