A recombinant microorganism and application of reducing expression of aromatic amino acid transporter in fermentation production
By constructing a recombinant microorganism by deleting the aromatic amino acid transporter aroP from Corynebacterium glutamicum, the problem of low lysine production efficiency was solved, resulting in a significant increase in yield and a reduction in cost, which has broad application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MEIHUA BIOTECH LANGFANG CO LTD
- Filing Date
- 2024-11-28
- Publication Date
- 2026-05-29
AI Technical Summary
Existing methods for producing lysine suffer from limited yield increases, high costs, and complex operations. Furthermore, no studies have been reported on the correlation between aromatic amino acid transporters and bacterial fermentation for amino acid production.
By deleting the aromatic amino acid transporter aroP from Corynebacterium glutamicum using genetic engineering, recombinant microorganisms were constructed, and their expression levels were reduced. Genetic modification was carried out using a secondary recombination strategy with the pK18mobsacB suicide plasmid, resulting in modified strains such as 13032-ΔaroP, 11942-ΔaroP, and 13407-ΔaroP.
It significantly improves the fermentation production efficiency and sugar-acid conversion rate of lysine, is simple to operate and low in cost, and has broad application potential.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to the application of a recombinant microorganism and a method for reducing the expression level of aromatic amino acid transport proteins in fermentation production. Background Technology
[0002] In the field of amino acid production, lysine, as an important essential amino acid, has wide applications in industries such as feed, food, and medicine. Currently, lysine production mainly relies on microbial fermentation, and Corynebacterium glutamicum is a commonly used production strain.
[0003] Aromatic amino acids, including phenylalanine, tyrosine, and tryptophan, play important physiological functions in organisms. Aromatic amino acid transporters are responsible for transporting aromatic amino acids into cells and participating in their metabolic processes. Existing research mainly focuses on the functional characteristics of these transporters and their associations with other physiological processes. Functional characteristic studies include the specific mechanisms of action of the transporters, substrate specificity, and their impact on intracellular aromatic amino acid metabolism. Studies on their associations with other physiological processes include interactions with cell signal transduction and nutrient sensing. Currently, there are no reports of a direct link between aromatic amino acid transporters and bacterial fermentation for amino acid production.
[0004] Furthermore, in lysine production, yields are mainly increased by optimizing fermentation conditions, modifying metabolic pathways, and screening for high-yielding strains. However, these methods often have limitations, such as limited effectiveness, high cost, and complex operation. Therefore, further research on amino acid production methods is necessary. Summary of the Invention
[0005] One of the objectives of this invention is to provide a new recombinant microorganism and method for increasing the yield of lysine fermentation.
[0006] This invention provides a recombinant microorganism, which, compared with the starting strain, has a reduced expression level of the aromatic amino acid transporter aroP; the starting strain is Corynebacterium.
[0007] Preferably, the starting strain is Corynebacterium glutamicum.
[0008] During long-term scientific research, it was unexpectedly discovered that bacteria lacking aromatic amino acid transport proteins have a significantly higher efficiency in fermenting and producing lysine than the control group without the lack of the transport protein, which led to the development of this invention.
[0009] In this invention, the amino acid sequence of the aromatic amino acid transporter aroP is shown in SEQ ID No. 13, and the nucleotide sequence is shown in SEQ ID No. 14 (GenBank number NCgl1062).
[0010] Specifically, this invention utilizes genetic engineering to delete the aromatic amino acid transporter protein of *Corynebacterium glutamicum*, thus creating a *Corynebacterium glutamicum* strain for lysine production. Three modified strains were obtained, numbered 13032-ΔaroP, 11942-ΔaroP, and 13407-ΔaroP. The effects of three control strains (ATCC13032, CGMCCNo.11942, CGMCC No.13407) and three experimental strains (13032-ΔaroP, 11942-ΔaroP, 13407-ΔaroP) were tested in a 1L quadruple fermenter. The experimental groups showed a significant improvement in lysine production efficiency.
[0011] Corynebacterium glutamicum contains a variety of amino acid transport proteins. This invention modifies these amino acid transport proteins by deletion and tests the effects of the modifications. It was found that only the aromatic amino acid transport protein aroP of this invention has a significant positive effect by deletion.
[0012] In this invention, the preferred method for reducing the expression level of the aromatic amino acid transporter aroP is deletion modification. There are various specific deletion methods, such as deletion modification of the coding region (usually referring to its open reading frame) of the gene encoding the amino acid transporter. This can involve deleting the entire coding region, deleting a portion of the coding region, or introducing nonsense or missense mutations into the coding region. These operations may lead to transcription failure or translation failure, ultimately resulting in the inability to produce a functional protein or a reduction in the expression level of the protein.
[0013] The present invention also provides the application of the above-mentioned recombinant microorganisms in L-lysine fermentation production, in the genetic breeding of microorganisms for L-lysine fermentation production, in increasing L-lysine fermentation yield, or in increasing the sugar-acid conversion rate of L-lysine fermentation production.
[0014] The present invention also provides the application of reducing the expression level of the aromatic amino acid transporter aroP in improving the yield of L-lysine and / or sugar-acid conversion rate in recombinant microbial fermentation.
[0015] The present invention also provides a method for constructing recombinant microorganisms, wherein the expression level of the aromatic amino acid transporter aroP is reduced in the recombinant microorganisms compared with the starting strain; wherein the starting strain is Corynebacterium.
[0016] Preferably, the starting strain is Corynebacterium glutamicum.
[0017] The present invention also provides a fermentation production method for L-lysine, which uses the above-mentioned recombinant microorganisms for fermentation production.
[0018] The beneficial effects of this invention are at least as follows: This invention provides a novel approach and method for lysine production. By preparing novel recombinant microorganisms, this method significantly improves lysine production efficiency. The method boasts advantages such as simple operation, significant results, and low cost, and is expected to be widely applied in lysine production and other amino acid production fields, making a significant contribution to improving the economic efficiency and competitiveness of the amino acid industry. Detailed Implementation
[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.
[0021] The strains used in this invention include one Corynebacterium glutamicum model strain and two lysine-producing strains, with preservation numbers ATCC13032, CGMCC No.11942, and CGMCC No.13407, respectively.
[0022] The genetic information of the Corynebacterium glutamicum type strain ATCC 13032 has been published in GenBank and can be accessed at the following website: https: / / www.ncbi.nlm.nih.gov / nuccore / NC_003450.3.
[0023] CGMCC No. 11942 was deposited on December 25, 2015, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as *Corynebacterium glutamicum*, and this strain has been disclosed in patent CN105734004B.
[0024] CGMCC No. 13407 was deposited on November 30, 2016, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as *Corynebacterium glutamicum*, and has been disclosed in patent application CN106635944A.
[0025] This invention uses the genome of the Corynebacterium glutamicum ATCC 13032 model strain as a PCR amplification template.
[0026] The primer sequence information used in the embodiments of the present invention is shown in Table 1. The embodiments of the present invention are only for illustrating the effects of the present invention and are not intended to limit the scope of the present invention.
[0027] Table 1 Primer sequence information (SEQ ID No. 1-12)
[0028] This invention, taking the deletion of the entire coding region as an example, describes the inactivation modification of the coding regions of amino acid transporter genes in two lysine-producing strains and one Corynebacterium glutamicum model strain. The gene inactivation method employs a classic genetic manipulation technique in Corynebacterium, namely, a secondary recombination strategy based on the pK18mobsacB suicide plasmid. Details are as follows: Example 1: Construction of plasmids modified with transporter protein deletion
[0029] 1.1 Construction of plasmid pK18mobsacB-ΔaroP
[0030] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed using the aroP-1f / aroP-1r primer pair and the aroP-2f / aroP-2r primer pair, respectively, to obtain the upstream and downstream homologous arms of the aroP gene deletion modification. Then, using a mixture of the upstream and downstream homologous arms as a template, amplification was performed using the aroP-1f / aroP-2r primer pair to obtain the full-length fragment after the fusion of the upstream and downstream homologous arms. The full-length fragment was double-digested with restriction endonucleases XbaI and HindIII, and the product was recovered using a gel extraction kit.
[0031] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1). This vector was double-digested with the same restriction endonuclease and then recovered by gel extraction.
[0032] Step 3: The products obtained in Steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. Target transformants were screened using 50 mg / L kanamycin resistance plates, and plasmids were extracted and sequenced from the obtained transformants. The plasmid with correct sequencing was named pK18mobsacB-ΔaroP.
[0033] 1.2 Construction of plasmid pK18mobsacB-ΔbrnQ
[0034] The amino acid and nucleotide sequences of brnQ are shown in SEQ ID No. 15-16, respectively.
[0035] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed using primer pairs brnQ-1f / brnQ-1r and brnQ-2f / brnQ-2r to obtain the upstream and downstream homologous arms of the brnQ gene deletion modification. Then, using a mixture of the upstream and downstream homologous arms as a template, amplification was performed using the brnQ-1f / brnQ-2r primer pairs to obtain the full-length fragment after fusion of the upstream and downstream homologous arms. The full-length fragment was double-digested with restriction endonucleases XbaI and HindIII, and the product was recovered using a gel extraction kit.
[0036] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1). This vector was double-digested with the same restriction endonuclease and then recovered by gel extraction.
[0037] Step 3: The products obtained in Steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. Target transformants were screened using 50 mg / L kanamycin resistance plates, and plasmids were extracted and sequenced from the obtained transformants. The sequencing primers were brnQ-1f / brnQ-2r, and the plasmid with correct sequencing was named pK18mobsacB-ΔbrnQ.
[0038] 1.3 Construction of plasmid pK18mobsacB-ΔargV
[0039] The amino acid and nucleotide sequences of argV are shown in SEQ ID No. 17-18, respectively.
[0040] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed using primer pairs argV-1f / argV-1r and argV-2f / argV-2r, respectively, to obtain the upstream and downstream homologous arms of the argV gene deletion modification. Then, using a mixture of the upstream and downstream homologous arms as a template, amplification was performed using the argV-1f / argV-2r primer pairs to obtain the full-length fragment after fusion of the upstream and downstream homologous arms. The full-length fragment was double-digested with restriction endonucleases XbaI and HindIII, and the product was recovered using a gel extraction kit.
[0041] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1). This vector was double-digested with the same restriction endonuclease and then recovered by gel extraction.
[0042] Step 3: The products obtained in Steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. Target transformants were screened using 50 mg / L kanamycin resistance plates, and plasmids were extracted and sequenced from the obtained transformants. The plasmid with correct sequencing was named pK18mobsacB-ΔargV.
[0043] Example 2: Modification of CGMCC No. 11942 by removing transporter proteins
[0044] 2.1 Construction of strain CGMCC No.11942-ΔaroP
[0045] Competent cells of CGMCC No. 11942 were prepared and their genes were recombined according to the methods described in the *C. glutamicum Handbook* (Chapter 23). CGMCC No. 11942 is a high-lysine-producing bacterium, derived from ATCC 13032 through genetic engineering. Specific background information on this modification can be found in patent document CN105734004B.
[0046] Step 1: The recombinant plasmid pK18mobsacB-ΔaroP was transformed into CGMCC No.11942 competent cells by electroporation, and transformants were screened on BHI selective medium containing 15 mg / L kanamycin.
[0047] Step 2: The screened transformants were cultured overnight in ordinary BHI liquid medium at 33°C with shaking on a rotary shaker at 220 rpm. During this culture process, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target deletion mutation.
[0048] Step 3: Perform serial dilutions of the culture (10⁻⁶ ppm). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary BHI solid medium containing 10% sucrose and incubated at 33°C for 48 hours. The resulting transformants should carry the target deletion mutation and not the inserted vector sequence.
[0049] Step 4: Colony PCR amplification of the target sequence was performed using aroP-1f / aroP-2r primers. The PCR band size of the correct transformant was 1 kb. Nucleotide sequencing analysis of the PCR product confirmed that the PCR results met the expected modification, and the target gene deletion was successful. The final modified strain was named 11942-ΔaroP.
[0050] 2.2 Construction of strain CGMCC No.11942-ΔbrnQ
[0051] The modification method is the same as in 2.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔbrnQ, and the PCR and sequencing primers are brnQ-1f / brnQ-2r. The finally obtained modified strain is named 11942-ΔbrnQ.
[0052] 2.3 Construction of strain CGMCC No.11942-ΔargV
[0053] The modification method is the same as in 2.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔargV, and the PCR and sequencing primers are argV-1f / argV-2r. The finally obtained modified strain is named 11942-ΔargV.
[0054] Example 3: Phenotypic testing of CGMCC No. 11942 series modified strains
[0055] Fermentation tests were conducted on the three strains obtained in Example 2 to observe whether their amino acid production capacity was improved. A DasGip quadruple 1L fermenter was used, and fermentation tests were performed simultaneously on both the modified strain and the control strain. Three batches of stability experiments were completed for each strain. The 1L fermentation medium formulation is shown in Table 2, and the fermentation process control parameters are shown in Table 3.
[0056] Table 2 Fermentation medium formulation
[0057] Table 3 Process Control Parameters
[0058] Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and determine the lysine content in the fermentation broth of the recombinant bacteria and control bacteria by HPLC. The average value of the three batches is taken as the final experimental result. Take 1 mL of fermentation broth, dilute it 100 times in a volumetric flask, and detect the OD at a wavelength of 562 nm using a spectrophotometer. The average value of the three batches is taken as the final experimental result. The fermentation test results are shown in Table 4.
[0059] Table 4 Results of L-Lysine fermentation experiment
[0060] In this embodiment, three types of transport protein-deficient modified bacteria were tested simultaneously. Among them, the modified bacteria lacking aromatic amino acid transport proteins (11942-aroP) showed a significantly increased lysine yield. To further demonstrate the effectiveness of this modification method, the three transport protein-deficient modifications were introduced into another lysine-producing bacterium (CGMCC No. 13407) to demonstrate the site effect. The specific introduction method and effect test are shown in Examples 4 and 5 below.
[0061] Example 4: Modification of CGMCC No. 13407 by removing transporter proteins
[0062] 4.1 Construction of strain CGMCC No.13407-ΔaroP
[0063] CGMCC No. 13407 is a high-lysine-producing bacterium, derived from ATCC 13032 through genetic engineering. For details of the modification, please refer to patent document CN106635944A.
[0064] Competent cells of CGMCC No. 13407 were prepared and gene recombination was performed according to the method in the C. glutamicum Handbook (Chapter 23).
[0065] Step 1: The recombinant plasmid pK18mobsacB-ΔaroP was transformed into CGMCC No.13407 competent cells by electroporation, and transformants were screened on BHI selective medium containing 15 mg / L kanamycin.
[0066] Step 2: The screened transformants were cultured overnight in ordinary BHI liquid medium at 33°C with shaking on a rotary shaker at 220 rpm. During this culture process, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target deletion mutation.
[0067] Step 3: Perform serial dilutions of the culture (10⁻⁶ ppm).-2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary BHI solid medium containing 10% sucrose and incubated at 33°C for 48 hours. The resulting transformants should carry the target deletion mutation and not the inserted vector sequence.
[0068] Step 4: Colony PCR amplification of the target sequence was performed using aroP-1f / aroP-2r primers. The PCR band size of the correct transformant was 1 kb. Nucleotide sequencing analysis of the PCR product confirmed that the PCR results met the expected modification, and the target gene deletion was successful. The final modified strain was named 13407-ΔaroP.
[0069] 4.2 Construction of strain CGMCC No.13407-ΔbrnQ
[0070] The modification method is the same as described in 4.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔbrnQ, and the PCR and sequencing primers are brnQ-1f / brnQ-2r. The finally obtained modified strain is named 13407-ΔbrnQ.
[0071] 4.3 Construction of strain CGMCC No.13407-ΔargV
[0072] The modification method is the same as described in 4.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔargV, and the PCR and sequencing primers are argV-1f / argV-2r. The finally obtained modified strain is named 13407-ΔargV.
[0073] Example 5: Phenotypic testing of CGMCC No. 13407 series modified strains
[0074] Fermentation tests were conducted on the three strains obtained in Example 4 to observe whether their amino acid production capacity was improved. A DasGip quadruple 1L fermenter was used, and the modified strains and control strains were validated simultaneously. The fermentation medium formulation is shown in Table 2 of Example 3, and the control process is shown in Table 3 of Example 3.
[0075] Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and determine the lysine content in the fermentation broth of the recombinant bacteria and control bacteria by HPLC. The average value of the three batches is taken as the final experimental result. Dilute 1 mL of fermentation broth 100 times in a volumetric flask and detect the OD at a wavelength of 562 nm using a spectrophotometer. The average value of the three batches is taken as the final experimental result. The fermentation test results are shown in Table 5.
[0076] Table 5 Results of L-Lysine fermentation experiment
[0077] The fermentation results showed that the genetically engineered strain lacking aromatic amino acid transporters had higher lysine production and higher sugar-acid conversion rate compared to the control group. Furthermore, it exhibited a significant increasing effect on different lysine-producing strains.
[0078] Example 6: Modification of ATCC13032 by removing transporter proteins
[0079] 6.1 Construction of strain 13032-ΔaroP
[0080] Competent cells of ATCC13032 were prepared and gene recombination was performed according to the method in the C. glutamicum Handbook (Chapter 23).
[0081] Step 1: The recombinant plasmid pK18mobsacB-ΔaroP was transformed into ATCC13032 competent cells by electroporation, and transformants were screened on BHI selective medium containing 15 mg / L kanamycin.
[0082] Step 2: The screened transformants were cultured overnight in ordinary BHI liquid medium at 33°C with shaking on a rotary shaker at 220 rpm. During this culture process, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target deletion mutation.
[0083] Step 3: Perform serial dilutions of the culture (10⁻⁶ ppm). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary BHI solid medium containing 10% sucrose and incubated at 33°C for 48 hours. The resulting transformants should carry the target deletion mutation and not the inserted vector sequence.
[0084] Step 4: Colony PCR amplification of the target sequence was performed using aroP-1f / aroP-2r primers. The PCR band size of the correct transformant was 1 kb. Nucleotide sequencing analysis of the PCR product confirmed that the PCR results met the expected modification, and the target gene deletion was successful. The final modified strain was named 13032-ΔaroP.
[0085] 6.2 Construction of strain ATCC13032-ΔbrnQ
[0086] The modification method is the same as in 6.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔbrnQ, and the PCR and sequencing primers are brnQ-1f / brnQ-2r. The finally obtained modified strain is named 13032-ΔbrnQ.
[0087] 6.3 Construction of strain ATCC13032-ΔargV
[0088] The modification method is the same as in 6.1 above. The recombinant plasmid used in step 1 is pK18mobsacB-ΔargV, and the PCR and sequencing primers are argV-1f / argV-2r. The finally obtained modified strain is named 13032-ΔargV.
[0089] Example 7 Phenotypic testing of the ATCC13032 series of modified strains
[0090] Fermentation tests were conducted on the three strains obtained in Example 6 to observe whether their amino acid production capacity was improved. A DasGip quadruple 1L fermenter was used, and the modified strains and control strains were validated simultaneously. The fermentation medium formulation is shown in Table 2 of Example 3, and the control process is shown in Table 3 of Example 3.
[0091] Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and determine the lysine content in the fermentation broth of the recombinant bacteria and control bacteria by HPLC. The average value of the three batches is taken as the final experimental result. Dilute 1 mL of fermentation broth 100 times in a volumetric flask and detect the OD at a wavelength of 562 nm using a spectrophotometer. The average value of the three batches is taken as the final experimental result. The fermentation test results are shown in Table 6.
[0092] Table 6 Results of L-Lysine fermentation experiment
[0093] The fermentation results showed that the genetically engineered strain lacking aromatic amino acid transporters had higher lysine production and higher sugar-acid conversion rate compared to the control group. This effect was also significantly increased in the model strain.
[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A recombinant microorganism, characterized in that, Compared with the starting strain, the expression level of the aromatic amino acid transporter aroP was reduced; the starting strain was Corynebacterium.
2. The recombinant microorganism according to claim 1, characterized in that, The starting strain was Corynebacterium glutamicum.
3. The application of the recombinant microorganisms described in claim 1 or 2 in the fermentation production of L-lysine.
4. The application of the recombinant microorganisms described in claim 1 or 2 in the genetic breeding of microorganisms for L-lysine fermentation production.
5. The application of the recombinant microorganism as described in claim 1 or 2 in increasing the yield of L-lysine fermentation.
6. The application of the recombinant microorganism according to claim 1 or 2 in improving the sugar-acid conversion rate of L-lysine fermentation production.
7. Application of reducing the expression level of aromatic amino acid transporter aroP in improving the yield of L-lysine and / or sugar-acid conversion rate in recombinant microbial fermentation.
8. A method for constructing recombinant microorganisms, characterized in that, The recombinant microorganism exhibited a lower expression level of the aromatic amino acid transporter aroP compared to the original strain; the original strain was Corynebacterium.
9. The method according to claim 8, characterized in that, The starting strain was Corynebacterium glutamicum.
10. A fermentation method for producing L-lysine, characterized in that, Fermentation production using the recombinant microorganisms described in claim 1 or 2.