L-isoleucine high-yield strain and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an L-isoleucine high-yield strain and application thereof. The L-isoleucine high-yield strain is obtained through molecular modification, mutagenesis and resistance screening, and compared with an original strain, the fermentation yield of L-isoleucine and the saccharic acid conversion rate are greatly increased, so that the production cost can be reduced, and the strain has practical value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-yield strain of L-isoleucine and its application, and specifically to a high-yield strain of L-isoleucine obtained based on molecular modification, mutagenesis and resistance screening. Background Technology

[0002] L-Isoleucine, a branched-chain amino acid, is one of the eight essential amino acids for humans. It participates in protein synthesis in humans and animals and regulates many systemic pathways. L-Isoleucine plays a crucial role in maintaining liver function, muscle function, blood circulation, and immune homeostasis. It has broad market and application prospects in the food, livestock feed, and pharmaceutical industries. Adding appropriate amounts of L-Isoleucine to piglet feed can improve their ileal barrier function and effectively prevent diarrhea caused by rotavirus. L-Isoleucine can also promote insulin secretion in the human body in synergy with glucose, achieving a rapid reduction in blood glucose levels. Therefore, the market demand for L-Isoleucine is substantial annually.

[0003] The main methods for producing L-isoleucine include chemical synthesis, hair extraction, and fermentation. Currently, fermentation is the primary method for industrial production, and the strains used are typically *Corynebacterium glutamicum* and *Escherichia coli*. The synthesis of both L-isoleucine and L-valine requires acetolactate synthase, dihydroxy acid reductase, dihydroxy acid dehydratase, and branched-chain amino acid transaminase. Acetolactate synthase is subject to feedback inhibition by L-isoleucine. *E. coli* contains three isoenzymes of acetolactate synthase (AHAS I, AHAS II, and AHAS III, encoded by ilvBN, ilvGM, and ilvIH, respectively). AHAS I has a higher specificity for pyruvate than for α-ketobutyrate, thus favoring the synthesis of L-valine and L-leucine. The ilvGM gene encoding AHASII has a frameshift mutation, therefore this enzyme is not expressed. AHAS III has a higher specificity for α-ketobutyrate than for pyruvate, therefore this enzyme is considered to favor the synthesis of L-isoleucine. Leucine dehydrogenase (LeuDH) is an isoenzyme of branched-chain amino acid transaminase (ilvE) and has similar catalytic functions, both catalyzing the production of L-isoleucine from 2-keto-3-methylvaleric acid. Furthermore, leucine dehydrogenase (LeuDH) is an NAD+ / NADH-dependent oxidoreductase, offering a cost advantage over NADPH in terms of cofactor supply. In the biosynthetic pathway of L-isoleucine, the expression of key enzymes affects the carbon flux of the anabolic flux, thereby influencing the synthesis of the final product.

[0004] The purines and pyrimidines in DNA and RNA have strong ultraviolet absorption capabilities, with the largest absorption peak at 260 nm. Therefore, ultraviolet radiation at a wavelength of 260 nm is the most effective mutagen. Ultraviolet light can cause DNA molecules to form pyrimidine dimers, i.e., two adjacent pyrimidines covalently linked. The appearance of dimers weakens the hydrogen bonding between double bonds and causes distortion of the double-strand structure, hindering normal base pairing and potentially leading to mutations. ARTP is a novel radiofrequency discharge technology that uses inert gas discharge to generate a large number of high-energy active particles under normal pressure and room temperature conditions. Studies have shown that ARTP can efficiently induce the breakage of phosphate groups in DNA mononucleotide triphosphates and the cleavage of bases and ribose bonds in polynucleotides at room temperature and pressure, thereby generating a large number of mutation sites using the cell's own highly fault-tolerant repair mechanism. After mutagenesis, the strains can be screened to obtain superior traits, greatly improving production efficiency.

[0005] In L-isoleucine synthesis, excessively high L-isoleucine concentrations can exert feedback inhibition on L-threonine dehydratase and acetolactate synthase, thereby affecting L-isoleucine accumulation. Isoleucine oxime (IleHx) is an L-isoleucine analogue. Using it to cultivate resistance to the target amino acid minimizes the feedback inhibition on the product L-isoleucine, thus promoting L-isoleucine synthesis. Summary of the Invention

[0006] L-Isoleucine shares a similar biosynthetic pathway with L-valine, using multiple catalytic enzymes. This invention addresses the weak L-isoleucine biosynthetic flux in the existing L-valine strain HHVAL-001 (CCTCC NO: M 2020321). Through extensive experiments, pathway modification and mutagenesis were conducted to alter metabolic flux, resulting in a high-yielding L-isoleucine strain. Therefore, the purpose of this invention is to provide a high-yielding L-isoleucine strain obtained through molecular modification, mutagenesis, and resistance screening, and its applications. The strain is named IVL016 and can be applied on a large scale for production.

[0007] The *Escherichia coli* HHVAL-001 was deposited on July 16, 2020, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO:M 2020321, and classified as *Escherichia coli* HHVAL-001.

[0008] And it has already been published in existing technical literature: Vasileios Bampidis, et al. Safety and efficiency of a feed additive consisting of L-valine produced by Escherichiacoli CCTCC M2020321 for allanimal species (Kempex Holland BV). EFSA Journal 2022; 20(2):7163.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides a high-yield L-isoleucine-producing strain of *Escherichia coli* IVL016, which was deposited on October 16, 2023, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), accession number: CCTCC NO: M 20231916, and classified as *Escherichia coli* IVL016. Compared with the original strain, the mutagenic strain can significantly increase the fermentation yield of L-isoleucine and the sugar-acid conversion rate, while reducing production costs.

[0011] The present invention also provides a fermentation method for the aforementioned Escherichia coli IVL016, which is a shake flask fermentation method or a fermenter fermentation method.

[0012] Specifically, the steps of the shake flask fermentation method are as follows: pick a single colony from the plate, inoculate it into the primary seed culture medium, and culture it at 35-37°C until the OD600 reaches 2-4. Then, inoculate it into the secondary shake flask culture medium at a ratio of 4-6% (v / v) for fermentation and culture it at 35-37°C.

[0013] More specifically, the primary seed culture medium is LB liquid medium; the secondary shake flask culture medium comprises the following components: glucose 15-25 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 5-12 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, pH 7.0-7.5.

[0014] In a specific embodiment, the fermentation method in the fermenter is as follows: the seed culture of Escherichia coli IVL016 is inoculated into the fermenter culture medium at a ratio of 2-8% (v / v) for fermentation.

[0015] Preferably, the fermentation medium in the tank comprises the following components: corn steep liquor 13-25 g / L, glucose 25-35 g / L, magnesium sulfate 0.2-0.8 g / L, potassium dihydrogen phosphate 0.6-1.5 g / L, ammonium sulfate 1-5 g / L, diammonium hydrogen phosphate 2-5 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, with water as the solvent and a pH of 6.9-7.1.

[0016] Specifically, the seed culture is obtained from Escherichia coli IVL016 through seed culture, which includes the following steps: inoculating glycerol bacteria into a primary seed culture medium and culturing at 35-37°C until OD600 is between 3 and 5; inoculating at a ratio of 4-6% (v / v) into a secondary seed culture medium and culturing at 35-37°C until OD600 is between 6 and 8.

[0017] More specifically, the primary seed culture medium is LB liquid medium; the secondary seed culture medium comprises the following components: glucose 15-25 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 5-12 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, pH 7.0-7.5.

[0018] The present invention also provides the application of the aforementioned Escherichia coli IVL016 in the preparation of L-isoleucine.

[0019] The present invention further provides a method for preparing L-isoleucine, which uses the fermentation method described above to obtain L-isoleucine.

[0020] This invention is based on the similarity of L-isoleucine and L-valine metabolic pathways. The L-valine strain HHVAL-001 (CCTCC NO: M 2020321) has a weak carbon flux in L-isoleucine synthesis. Through molecular modification, UV and ARTP combined mutagenesis of *E. coli* HHVAL-001, and resistance screening, its metabolic flux was altered to obtain the high-yield L-isoleucine strain *E. coli* IVL016. In a 5L fermentation experiment, *E. coli* IVL016 achieved a fermentation yield of over 39 g / L and a sugar-acid conversion rate of over 30%. Compared with the starting strain *E. coli* HHVAL-001, both L-isoleucine yield and sugar-acid conversion rate were significantly improved, and the strain also exhibited good genetic stability. Attached Figure Description

[0021] Figure 1The mortality curve of Escherichia coli under UV mutagenesis.

[0022] Figure 2 Escherichia coli ARTP mutagenesis mortality curve.

[0023] Figure 3 Comparison of yields from shake-flask fermentation of dominant mutagenic strains.

[0024] Figure 4 Genetic stability test of high-yield L-isoleucine strains.

[0025] Figure 5 Biomass and yield curves of Escherichia coli IVL016 fermentation. Detailed Implementation

[0026] The present invention is further illustrated by the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope or embodiments of the invention. The scope of the invention is limited by the appended claims. Based on this specification and general knowledge in the art, those skilled in the art can clearly understand the scope of the claims. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications and alterations to the technical solutions of the invention, and such modifications and alterations are also included within the scope of the invention.

[0027] In this embodiment, the raw materials used to prepare the culture medium are all commonly used in the field and can be purchased on the market.

[0028] The strains and plasmids involved in the following examples are shown in Table 1, and the primers used are shown in Table 2.

[0029] Table 1: Strains and plasmids used in this invention

[0030]

[0031] Table 2: Primers used in this invention

[0032]

[0033] The culture medium formulations used in the following examples are as follows:

[0034] LB liquid medium: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0035] LB solid medium: tryptone 10g / L, sodium chloride 10g / L, yeast extract 5g / L, agar 20g / L.

[0036] Resistance screening solid culture medium: glucose 5 g / L, yeast extract 5 g / L, peptone 10 g / L, sodium chloride 3 g / L, isoleucine oxime 40 g / L, agar 20 g / L.

[0037] In this embodiment of the invention, the method for preparing the seed liquid is as follows:

[0038] The selected bacteria were inoculated into the primary seed culture medium and cultured at a temperature of 35-37℃ and a rotation speed of 200-220 rpm until the OD600 value reached 3-5. Then, the bacteria were inoculated into the secondary seed culture medium at a volume of 4-6% of the secondary seed culture medium volume and cultured at 180-220 rpm and 35-37℃ until the OD600 value reached 6-8 to obtain the secondary seed solution.

[0039] The primary seed culture medium is LB liquid medium.

[0040] The secondary seed culture medium comprises the following components: glucose 10-30 g / L, magnesium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 5-15 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, and yeast extract 0.2-1.5 g / L. The solvent is water, and the pH is adjusted to 7.0-7.2 with sodium hydroxide.

[0041] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0042] Unless otherwise stated, the present invention is tested according to the following method:

[0043] 1. OD value: Measured at a wavelength of 600 nm using a UV-5200(PC) visible spectrophotometer;

[0044] 2. pH value: Monitored online using an InPro3100i / SG / 325 pH sensor;

[0045] 3. Glucose content: determined using an SBA-40D biosensor analyzer;

[0046] 4. L-Isoleucine was detected by high-performance liquid chromatography (HPLC). The specific steps are as follows:

[0047] The fermentation broth was sampled and placed in a centrifuge tube, centrifuged at 5000 rpm for 1 minute, and the supernatant was diluted 100 times. The diluted solution was then filtered through a 0.22 μm microporous membrane before injection for detection. Chromatographic conditions: Column: Yuexu Ultimate HILIC Amphion II 4.6 × 150 mm, 5 μm; Mobile phase: Acetonitrile: 0.05 M potassium dihydrogen phosphate = 3:1; Column temperature: 35℃; Flow rate: 1.0 mL / min; Injection volume: 10 μl; Detector: UV 206 nm;

[0048] The mobile phase (acetonitrile / phosphate mixed solution) was prepared as follows: 0.05M potassium dihydrogen phosphate was weighed and dissolved in ultrapure water, and the pH was adjusted to 3.0 with phosphoric acid. After filtration through a 0.22μm filter membrane, acetonitrile was added in the corresponding volume according to the ratio of acetonitrile:0.05M potassium dihydrogen phosphate = 3:1. The mixture was then sonicated for 20 minutes until no bubbles were present.

[0049] Preparation of standard solution: Prepare a 1 g / L L-isoleucine solution (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., item number I2752-25G), dissolve by sonication, filter through a 0.22 μm membrane, and store at -20℃ for later use.

[0050] Example 1: Construction of L-Isoleucine-producing strain HHVAL005

[0051] In this embodiment, *Escherichia coli* HHVAL-001 was used as the starting strain. Using Red homologous recombination technology, genes causing byproduct accumulation were knocked out of the *E. coli* HHVAL-001 genome, and genes enhancing carbon flux in the main pathway were integrated to construct a genetically engineered bacterium capable of fermenting and producing L-isoleucine. This involved knocking out the lactate dehydrogenase gene *ldhA* in the lactate metabolism pathway and integrating the acetyllactate synthase gene *ilvIH*, resulting in strain HHVAL-003. Similarly, knocking out the threonine dehydrogenase gene *tdh* in the threonine metabolism pathway and integrating the leucine dehydrogenase gene *leuDH*, resulting in strain HHVAL005. The promoters used for the integrated *ilvIH* and *leuDH* genes were both inducible promoters, specifically the *tac* promoter.

[0052] 1. The specific construction steps of strain HHVAL-003 are as follows:

[0053] 1.1 Using pRE112 plasmid as template and ldhA-cs-up and ldhA-cs-down as primers, the DNA fragment was amplified and named ΔldhA-cs, with a length of 2719 bp. DpnI was added for enzyme digestion to eliminate the plasmid template. The PCR product was purified using a kit and used for the first step of homologous recombination.

[0054] Amplification system: PrimeSTAR Max Premix (2×) 25μL, plasmid template 5ng, ldhA-cs-up 1.5μL, ldhA-cs-down 1.5μL, add ddH2O to 50μL;

[0055] Among them, the PrimeSTAR Max Premix (2×) was purchased from Baori Biotechnology (Beijing) Co., Ltd., item number R045A.

[0056] Amplification program: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 45 s, 32 cycles; 72℃ final extension for 5 min.

[0057] 1.2. The PCR fragment ΔldhA-cs was introduced into HHVAL-001 competent cells containing the pKD46 plasmid via electroporation. After incubation at 30°C and 100 rpm for 4 hours, the bacterial culture was centrifuged, and 200 μl of culture medium was reserved to resuspend the cells. The cells were then plated on LB agar plates containing ampicillin and chloramphenicol (both at a final concentration of 100 μg / mL) and incubated overnight at 30°C. The next day, single colonies were selected for PCR verification using the primers yz-ldhA-up / yz-ldhA-down. Products with correct PCR bands were sent for sequencing. Correct single colonies were selected for further culture, and the pKD46 plasmid was eliminated. The strain was named HHVAL-002.

[0058] 1.3. The exogenous inducible promoter tac and the ilvIH gene were synthesized into the pUC19 plasmid to form the pUC19-tac-ilvIH plasmid template. The DNA fragment was amplified using primers ilvIH-up / ilvIH-down and named the PCR fragment p-ilvIH. The PCR product was purified using a kit and used for homologous recombination in the second step. The amplification conditions and system were the same as described in step 1.1. The nucleic acid sequence of the synthesized tac-ilvIH gene is shown in SEQ ID NO.1.

[0059] 1.4. The PCR fragment p-ilvIH was introduced into HHVAL-002 competent cells containing the pKD46 plasmid via electroporation. After culturing at 30°C and 100 rpm for 4 hours, the bacterial culture was transferred to 50 mL of LB liquid medium containing 10% sucrose and free of sodium chloride. After culturing for 24 hours, the bacterial culture was streaked on LB solid medium containing 6% sucrose and free of sodium chloride to verify the primers yz-ldhA-up / yz-ldhA-down. The single-clone PCR amplification products were sent for sequencing. Single clones with correct results were selected for further culture, and the pKD46 plasmid was eliminated. The successfully constructed strain was named HHVAL-003.

[0060] 2. The specific construction steps of strain HHVAL005 are as follows:

[0061] 2.1 Using pRE112 plasmid as a template and tdh-cs-up and tdh-cs-down as primers, amplify the DNA fragment named Δtdh-cs, 2715 bp in length. Digest with DpnI to eliminate the plasmid template, purify the PCR product using a kit, and use it for the first step of homologous recombination. The amplification conditions and system are the same as described in step 1.1.

[0062] 2.2 The PCR fragment Δtdh-cs was introduced into HHVAL-003 competent cells containing the pKD46 plasmid via electroporation. After incubation at 30°C and 100 rpm for 4 hours, the bacterial culture was centrifuged, and 200 μl of culture medium was reserved to resuspend the cells. The cells were then plated on LB agar plates containing ampicillin and chloramphenicol (both at a final concentration of 100 μg / mL) and incubated overnight at 30°C. The next day, single colonies were selected for PCR verification using the primers yz-tdh-up / yz-tdh-down. Products with correct PCR bands were sent for sequencing. Correct single colonies were selected for further culture, and the pKD46 plasmid was eliminated. The strain was named HHVAL-004.

[0063] 2.3. The exogenous inducible promoter tac and the leuDH gene were synthesized into the pUC19 plasmid to form the pUC19-tac-leuDH plasmid template. The DNA fragment, named p-leuDH, was amplified using primers leuDH-up / leuDH-down. The amplification system was the same as described in step 1.1, and the amplification program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 20 s, for 32 cycles; and 72℃ final extension for 5 min. The PCR product was purified using a kit and used for homologous recombination in the second step. The nucleic acid sequence of the synthesized tac-leuDH gene is shown in SEQ ID NO.2.

[0064] 2.4. The PCR fragment p-leuDH was introduced into HHVAL-004 competent cells containing the pKD46 plasmid via electroporation. After culturing at 30°C and 100 rpm for 4 hours, the bacterial culture was transferred to 50 mL of LB liquid medium containing 10% sucrose and free of sodium chloride. After culturing for 24 hours, the bacterial culture was streaked onto LB solid medium containing 6% sucrose and free of sodium chloride to verify the primers yz-tdh-up / yz-tdh-down. The single-clone PCR amplification products were sent for sequencing. Single clones with correct results were selected for further culture, and the pKD46 plasmid was eliminated. The successfully constructed strain was named HHVAL005.

[0065] Example 2: Obtaining UV-mutated strains

[0066] Mutagenesis time and intensity have a significant impact on the lethality of the strain; both excessively low and excessively high lethality rates are detrimental to screening. Therefore, it is necessary to investigate the effect of mutagenesis time on the lethality of the original bacteria at different mutagenesis times.

[0067] The starting strain of *Escherichia coli* HHVAL005 was streaked in three zones on LB solid medium and incubated at 37°C inverted mode for 12-16 hours. Single colonies with uniform morphology and saturation were picked and inoculated into 5 mL of LB liquid medium. The culture was continued until the OD600 reached 2-3, and then centrifuged at 5000 rpm for 1 minute. In a clean bench, the supernatant was discarded, and the bacterial cells were resuspended in physiological saline. 3 mL of the bacterial suspension was poured into a sterile agar plate, and a sterile rotor was added. Both the agar plate and the sterile strain were placed on a magnetic stirrer, fixed vertically at a distance of 30 cm from a 30W UV lamp. The magnetic stirrer and the plate lid were opened, and the lights in the clean bench were turned off to ensure that the operation was performed under conditions of no visible light.

[0068] Irradiate the bacterial suspensions under a UV lamp for 0s, 30s, 60s, 90s, 120s, and 150s, respectively, then turn off the UV lamp. Dilute the bacterial suspensions at different irradiation times by 1000 times, and spread 100μL onto LB solid medium. Incubate at 37℃ upside down for 24h. After counting the colonies, calculate the lethality rate using the following formula.

[0069] Lethality = (Number of colonies on control plate - Number of colonies on mutagenized plate) / Number of colonies on control plate × 100%

[0070] In this context, the control plate refers to a plate containing bacterial culture that has not undergone UV irradiation (0 seconds). The optimal mutagenic dose is defined as an irradiation time with a lethality rate of 92%–95%. Figure 1 It is evident that the lethality rate reached 93.6% when UV irradiation lasted 120 seconds during this ultraviolet mutagenesis, thus determining the optimal UV mutagenesis time as 120 seconds.

[0071] The bacterial suspension irradiated for 120 seconds was diluted 1000 times and spread on LB solid medium for culture. Eight single clones (A1-A8) with fast growth and large colonies were picked from the plate and inoculated into 5 mL of primary LB medium. The culture was carried out at 37℃ and 220 rpm for 12-15 h until the OD600 reached 2-4. The primary seed culture was then transferred to shake flask fermentation medium at an inoculation rate of 4% and fermented at 200 rpm and 37℃ for 48 h. The L-isoleucine production was measured to obtain the dominant strain A7, which was then subjected to ARTP mutagenesis.

[0072] Example 3: Obtaining ARTP-mutated strains

[0073] Preparation of mutagenic bacterial culture: The dominant strain A7 obtained by UV mutagenesis was transferred to LB liquid medium and cultured for OD... 600 The concentration was increased to 0.6–0.8, which was used as the culture medium for the bacteria to be mutated.

[0074] The specific steps of the ARTP mutagenesis procedure are as follows:

[0075] (1) Coating: Place the slide in the outer flame of an alcohol lamp and burn for 10 seconds. After cooling, place it in a sterilized petri dish and take 10 μl of the bacterial solution to be induced and evenly coat it on the surface of the slide.

[0076] (2) Collection tube preparation: Prepare several 2mL sterile centrifuge tubes as collection tubes, one for each slide. Add 1mL of sterile physiological saline to the collection tube. Open the cap of the collection tube in the ARTP mutagenesis device, with the cap facing outwards, and clamp it in the corresponding position;

[0077] (3) Mutagenesis: Open the sterile culture dish containing the slide inside the equipment, loosen the sterilized and dried tweezers, and carefully place the slide into the mutagenesis tank with the bacterial solution on top. Set the parameters: temperature 20℃, power 120W, helium flow rate 10SLM, and set the mutagenesis treatment time to 0s, 30s, 60s, 90s, 120s, 150s, 180s, 210s, and 240s respectively. Leave other parameters as default and click Run.

[0078] (4) Incubation: After the run is completed, remove the collection tube, cover it, and mix it in a vortex mixer for 1 min. Collect the mixed bacterial solution, dilute it 100 times, spread it on LB solid medium, and incubate it upside down at 37°C for 24 h. After counting the number of colonies, calculate the lethality rate according to the aforementioned formula.

[0079] The optimal mutagenic dose is determined by the irradiation time with a lethality of 95%–98%. Figure 2 It is evident that the lethality rate reached 97.5% at 180s during this ARTP mutagenesis, thus determining the optimal ARTP mutagenesis time to be 180s.

[0080] The ARTP-mutated bacterial suspension (180s) was diluted 100-fold and spread on LB solid medium for culture. Eight single clones (B1-B8) with fast growth and large colonies were selected from the plate and inoculated into 5 mL of primary LB medium. They were cultured for 12-15 h until the OD600 reached 2-4. The primary seed culture was then transferred to shake-flask fermentation medium at an inoculation rate of 4% and fermented at 200 rpm and 37°C for 48 h. The L-isoleucine production was measured to obtain the dominant strain B5.

[0081] One round of mutagenesis followed by one round of ARTP mutagenesis was performed on the dominant strain B5. Two more rounds of mutagenesis were then performed to obtain the dominant strain F4 (E5 + 1 round of ARTP mutagenesis). The dominant strains in this process were C3 (B5 + 1 round of ARTP mutagenesis), D6 (C3 + 1 round of ARTP mutagenesis), and E5 (D6 + 1 round of ARTP mutagenesis).

[0082] Example 4: Resistance screening of high-yield L-isoleucine mutagen strains

[0083] Isoleucine oxime (IleHx) is a structural analog of L-isoleucine. By breeding isoleucine oxime-resistant mutants, the feedback inhibition of L-isoleucine on L-threonine dehydratase and acetolactate synthase can be genetically overcome, thereby increasing L-isoleucine production. The specific screening steps are as follows:

[0084] (1) Preparation of resistance screening bacterial culture: The dominant strain F4, obtained after three rounds of combined UV and ARTP mutagenesis, was transferred to LB liquid medium and cultured for OD. 600 The solution was prepared at a concentration of 0.6–0.8 as the bacterial culture to be screened.

[0085] (2) Spreading: Dilute the bacterial culture to be screened 100 times and spread it on the resistance screening solid medium. Incubate at 37°C with the culture inverted position for 48 hours.

[0086] (3) Screening: 18 single colonies (G1-G18) with fast growth and large colony size were selected from the plate and cultured in shake flask fermentation medium for 48 h for screening. The screening criterion was the yield of L-isoleucine by liquid phase detection. A high-yielding strain of L-isoleucine with a yield of 6.36 g / L was obtained and named G16.

[0087] The results of the shake-flask screening of the key mutagenic strains mentioned above are shown in Table 3 and Figure 3 :

[0088] Table 3: Shake-flask fermentation results of key L-isoleucine mutagenized strains

[0089]

[0090] Example 5: Genetic stability experiment of high-L-isoleucine-producing mutagenic strain

[0091] The high-yielding L-isoleucine strain G16 was inoculated into 50 mL of LB liquid medium and cultured overnight at 37°C for 16 h. 500 μl of the bacterial culture was mixed with 500 μl of 30% glycerol to preserve the strain as the F1 generation. The F1 generation of glycerol bacteria was then inoculated into 50 mL of LB liquid medium and cultured under the same conditions as F1 to preserve the strain as the F2 generation. This process was repeated until the F10 generation.

[0092] The F1-F10 generation strains were cultured in shake flasks, and the yield of L-isoleucine in the fermentation supernatant was determined by liquid chromatography. Figure 4 As shown, the L-isoleucine production remained relatively stable from F1 to F10, with shake-flask fermentation yields generally between 6.0 and 6.4 g / L. The fermentation endpoint results are shown in Table 4, indicating that this mutagenic strain exhibits good genetic stability. The F10 strain was named *Escherichia coli* IVL016 and deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2020321.

[0093] Table 4: Results of shake-flask fermentation of L-isoleucine-producing strain G16

[0094]

[0095]

[0096] Example 6: Fermentation of Escherichia coli IVL016 in a 5L tank

[0097] The starting strain, E. coli HHVAL-001, and the mutagenized high-yield L-isoleucine strain IVL016 were subjected to three consecutive batches of 5L tank fermentation, with a fermentation system of 2L.

[0098] The specific fermentation steps are as follows:

[0099] (1) Primary seed culture: 100 μl of the glycerol bacteria to be tested (HHVAL-001, IVL016) were inoculated into 50 mL of LB liquid medium and cultured overnight at 37℃ and 220 rpm to obtain primary seed culture with an OD600 between 3 and 5. The primary seed culture medium consisted of 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0100] (2) Secondary seed culture: The primary seed cultures of *Escherichia coli* HHVAL-001 and IVL016 were respectively transferred to 100 mL of secondary seed culture medium at a transfer rate of 4% (V / V). The cultures were incubated at 37℃ and 220 rpm until the OD600 reached 6-8 to obtain the secondary seed culture. The secondary seed culture medium consisted of: glucose 25 g / L, magnesium sulfate 1.0 g / L, potassium dihydrogen phosphate 8 g / L, ammonium sulfate 7 g / L, ferrous sulfate 2.5 mg / L, manganese sulfate 0.6 mg / L, zinc acetate 0.7 mg / L, copper chloride 0.03 mg / L, and yeast extract 1 g / L. The solvent was water, and the pH was adjusted to 7.2 with sodium hydroxide.

[0101] (3) 5L tank fermentation: Take 100mL of the above secondary seed liquid and inoculate it into a 5L tank containing 2L of fermentation medium for fermentation.

[0102] The fermentation medium used consisted of the following components: 20 g / L corn steep liquor, 30 g / L glucose, 0.6 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 3 g / L ammonium sulfate, 4.2 g / L diammonium hydrogen phosphate, 3.2 mg / L ferrous sulfate, 0.7 mg / L manganese sulfate, 0.8 mg / L zinc acetate, 0.04 mg / L copper chloride, and 1.2 g / L yeast extract. Water was used as the solvent, and the pH was adjusted to 7.0 with ammonia. Glucose was sterilized separately and then mixed with the other components of the fermentation medium.

[0103] Fermentation conditions: initial temperature 37℃, stirring speed 300 rpm, initial aeration ratio 0.5 vvm. After the cell OD600 reaches 3, lactose with a final concentration of 4 g / L and threonine with a final concentration of 8 g / L are added for induction, and the stirring speed and dissolved oxygen cascade are controlled to maintain dissolved oxygen at 25%. Simultaneously, the glucose flow rate is controlled to maintain the glucose content in the fermentation broth at 2-5 g / L, and 25% ammonia is added throughout the process to maintain the pH of the fermentation broth at 7.0. Fermentation is terminated when the production rate of L-isoleucine in the fermentation broth slows down or stops increasing.

[0104] (4) Liquid phase detection: The fermentation endpoint detection results of Escherichia coli HHVAL-001 and IVL016 are shown in Table 5. The original strain HHVAL-001 produced about 2 g / L of L-isoleucine and had a sugar-acid conversion rate of about 1.6%. After modification, mutagenesis and resistance screening, the strain IVL016 produced about 39 g / L and had a sugar-acid conversion rate of more than 30%. Compared with the original strain HHVAL-001, both the yield and the sugar-acid conversion rate were significantly improved, and it can be applied to industrial production.

[0105] Table 5: Results of fermentation endpoint (45h) of Escherichia coli HHVAL-001 and IVL016

[0106]

[0107] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A high-yield L-isoleucine-producing strain of Escherichia coli IVL016 ( Escherichia coli ), characterized in that, Its accession number is CCTCC NO:M 20231916.

2. The fermentation method for Escherichia coli IVL016 as described in claim 1, characterized in that, It is either the shake flask fermentation method or the fermentation tank fermentation method.

3. The fermentation method as described in claim 2, characterized in that, The steps of the shake flask fermentation method are as follows: pick a single colony from the plate, inoculate it into the primary seed culture medium, and culture it at 35~37℃ until the OD600 is between 2~4. Then, inoculate it into the secondary shake flask culture medium at a ratio of 4~6% (v / v) and culture it at 35~37℃.

4. The fermentation method as described in claim 3, characterized in that, The primary seed culture medium is LB liquid medium; the secondary shake flask culture medium comprises the following components: glucose 15-25 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 5-12 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, pH 7.0-7.

5.

5. The fermentation method as described in claim 2, characterized in that, The fermentation method in the fermenter is as follows: the seed culture of Escherichia coli IVL016 is inoculated into the fermenter culture medium at a ratio of 2-8% (v / v) for fermentation.

6. The fermentation method as described in claim 5, characterized in that, The fermentation medium in the tank comprises the following components: corn steep liquor 13-25 g / L, glucose 25-35 g / L, magnesium sulfate 0.2-0.8 g / L, potassium dihydrogen phosphate 0.6-1.5 g / L, ammonium sulfate 1-5 g / L, diammonium hydrogen phosphate 2-5 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, with water as the solvent and a pH of 6.9-7.

1.

7. The fermentation method as described in claim 5, characterized in that, The seed culture is obtained from Escherichia coli IVL016 through seed culture, which includes the following steps: inoculating glycerol bacteria into a primary seed culture medium and culturing at 35-37°C until the OD600 reaches 3-5; inoculating 4-6% into a secondary seed culture medium and culturing at 35-37°C until the OD600 reaches 6-8.

8. The fermentation method as described in claim 7, characterized in that, The primary seed culture medium is LB liquid medium; the secondary seed culture medium comprises the following components: glucose 15-25 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 5-12 g / L, ammonium sulfate 3-10 g / L, ferrous sulfate 2-5 mg / L, manganese sulfate 0.3-0.8 mg / L, zinc acetate 0.4-1.0 mg / L, copper chloride 0.02-0.05 mg / L, yeast extract 0.5-2 g / L, pH 7.0-7.

5.

9. The use of Escherichia coli IVL016 as described in claim 1 in the preparation of L-isoleucine.

10. A method for preparing L-isoleucine, characterized in that, Obtained by the fermentation method as described in any one of claims 2 to 8.

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