Process for the fermentative production of threonine and use of inactivated or weakened microorganisms for the production of threonine
By reducing or inactivating nucleoside phosphorylase activity and relieving feedback inhibition, the yield of L-threonine from Corynebacterium glutamicum was improved, solving the problem of low yield caused by complex microbial metabolic networks and achieving efficient L-threonine fermentation production.
Patent Information
- Application Number
- CN202411801714.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2026-06-09
AI Technical Summary
In the existing technology for producing L-threonine using Corynebacterium glutamicum, the microbial metabolic network is complex, and there are negative effects caused by unknown mutations, resulting in low yield.
By using genetic engineering methods, the activity of nucleoside phosphorylase in microorganisms was reduced or inactivated, and the feedback inhibition of key genes lysC and hom in threonine synthesis was relieved. The gene thrABC in the threonine terminal synthesis pathway was expressed, and recombinant microorganisms were constructed for the fermentation production of L-threonine.
It significantly improved the yield of L-threonine, enhanced the efficiency of microbial fermentation production, and improved the synthesis and secretion efficiency of threonine.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to a fermentation method for producing threonine and the application of inactivated or weakened nucleoside phosphorylase activity in microorganisms in the production of threonine. Background Technology
[0002] L-Threonine is an essential amino acid with a variety of physiological functions and is widely used in food, feed, and medicine. Its functions include promoting muscle growth, accelerating fat metabolism, and alleviating anxiety symptoms. Market demand is increasing year by year.
[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium glutamicum is a traditional industrial microorganism widely used in the production of various amino acids, nucleotides and other organic acids.
[0004] Various methods of microbial modification can be used to improve amino acid yield, such as removing feedback inhibition of the product, strengthening key enzymes in the terminal synthesis pathway of the product, or enhancing the supply of cofactors for related enzymes, optimizing the carbon flow distribution in central metabolism, and blocking competing pathways.
[0005] However, due to the highly complex metabolic networks of microorganisms, in addition to the established modification strategies mentioned above, there are still other unknown mutations, including point mutations and gene deletions, that can interfere with microbial anabolic and catabolistic metabolism, causing either positive or negative effects. Therefore, further research is necessary. Summary of the Invention
[0006] One of the objectives of this invention is to provide a new method for producing threonine through fermentation.
[0007] This invention provides a fermentation production method for L-threonine, which uses recombinant microorganisms for fermentation production. Compared with the starting strain, the recombinant microorganisms have reduced or inactivated nucleoside phosphorylase activity, and the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30.
[0008] Based on years of research, this invention discovered that the inactivation or weakening of some nucleoside phosphorylases can have a positive effect on the accumulation of L-threonine metabolites in Corynebacterium glutamicum, and further verified this finding, thus completing this invention.
[0009] In the method of the present invention, compared with the starting strain, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the recombinant microorganism is relieved, and the gene thrABC for the threonine terminal synthesis pathway is expressed. Preferably, compared with the starting strain, the recombinant microorganism has a threonine mutation at position 311 of the lysC protein sequence to isoleucine, and a glycine mutation at position 378 of the hom protein to glutamic acid. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.
[0010] This invention specifically uses three Corynebacterium glutamicum model strains, ATCC 13032, ATCC 13869, and ATCC 14067, as test subjects. Through genetic engineering, the L-threonine terminal synthesis pathway was introduced and the feedback inhibition of key genes was relieved, resulting in three L-threonine-producing strains, named SMCT301, SMCT302, and SMCT303, respectively. These genetically engineered mutant strains were then used for shake-flask fermentation to produce L-threonine.
[0011] Further research was conducted using model strains ATCC 13032, ATCC 13869, and ATCC 14067, and three threonine-producing strains SMCT301, SMCT302, and SMCT303 as subjects. Through genetic engineering, the wild-type nucleoside phosphorylases of these strains were inactivated or weakened. Shake-flask fermentation with these modified strains showed an increased L-threonine yield.
[0012] That is, Corynebacterium glutamicum with inactivated nucleoside phosphorylase or carrying weakened nucleoside phosphorylase can be used to produce L-amino acids, especially L-threonine.
[0013] Corynebacterium glutamicum ATCC 13032, ATCC 13869, and ATCC 14067 are model strains of Corynebacterium glutamicum, well-known in the field. These strains can be purchased publicly or obtained from relevant research institutes. Their genome sequences are publicly available and can be found on the NCBI website. Escherichia coli MG1655 is a model strain of Escherichia coli, well-known in the field. This strain can be purchased publicly or obtained from relevant research institutes. Its genome sequence is publicly available and can be found on the NCBI website.
[0014] The present invention also provides a recombinant microorganism, as described above.
[0015] The present invention also provides a method for constructing recombinant microorganisms, wherein the recombinant microorganisms have reduced or inactivated nucleoside phosphorylase activity compared with the starting strain, wherein the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30.
[0016] In the method of the present invention, the activity of the nucleoside phosphorylase can be reduced or inactivated by one or more base mutations or deletions in the coding region, or by replacing the weak promoter. Preferably, compared with the starting strain, the recombinant microorganism has the feedback inhibition of the key genes lysC and hom for threonine synthesis relieved and expresses the thrABC gene for the threonine terminal synthesis pathway. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.
[0017] The present invention also provides a DNA molecule having a nucleotide sequence as shown in any one of SEQ ID No. 25-27.
[0018] The DNA molecules encode nucleoside phosphorylase mutants with amino acid sequences as shown in SEQ ID No. 31-33.
[0019] The present invention also provides biological materials containing the above-mentioned DNA molecules, wherein the biological materials are expression cassettes, vectors or host cells.
[0020] The present invention also provides a nucleoside phosphorylase mutant, the amino acid sequence of which is shown in any one of SEQ ID No. 31-33.
[0021] The mutant of this invention can weaken the activity of nucleoside phosphorylase.
[0022] The present invention also provides the use of recombinant microorganisms, or DNA molecules, or biological materials, or nucleoside phosphorylase mutants in any of the following aspects: (1) L-threonine fermentation production; (2) Genetic breeding of microorganisms for L-threonine fermentation production; (3) Increase the fermentation yield of L-threonine.
[0023] The present invention also provides the application of reducing or inactivating nucleoside phosphorylase activity in microorganisms in increasing the yield of L-threonine produced by microbial fermentation, wherein the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30; Preferably, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the microorganism is further relieved, and the gene thrABC for the threonine terminal synthesis pathway is expressed; And / or, the microorganism can ferment to produce threonine, preferably Corynebacterium glutamicum.
[0024] The beneficial effects of this invention are at least as follows: This invention provides a fermentation method for producing L-threonine, which uses novel recombinant microorganisms (with inactivated or weakened nucleoside phosphorylases) to produce threonine, providing a new and efficient method for producing threonine. Detailed Implementation
[0025] 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.
[0026] 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.
[0027] The key gene for threonine synthesis described in this invention, lysC (NCBI ID NCgl0247 in ATCC13032, BBD29_01500 in ATCC13869, and CEY17_01500 in ATCC14067), encodes aspartate kinase, and hom (NCBI ID NCgl1136 in ATCC13032, BBD29_06325 in ATCC13869, and C in ATCC14067) encodes aspartate kinase. EY17_0645 encodes homoserine dehydrogenase, which is relieved by point mutation of threonine feedback inhibition. The threonine terminal synthesis pathway is achieved by referencing the thrABC operon from E. coli MG1655 (thrA, thrB, and thrC are numbered IEU92_RS00010, IEU92_RS00015, and IEU92_RS00020 in NCBI). Among them, thrA encodes a bifunctional enzyme, namely aspartate kinase and homoserine dehydrogenase, thrB encodes homoserine kinase, and thrC encodes threonine synthase.
[0028] The gene encoding the nucleoside phosphorylase described in this invention is numbered NCgl0094 in ATCC13032, and its corresponding amino acid sequence is shown in SEQ ID No. 28; it is numbered BBD29_00655 in ATCC13869, and its corresponding amino acid sequence is shown in SEQ ID No. 29; it is numbered CEY17_00620 in ATCC14067, and its corresponding amino acid sequence is shown in SEQ ID No. 30.
[0029] The primer sequences used in the examples are shown in Table 1. The plasmid pk18mobsacB-speC used in the specific embodiments of this invention was prepared by metabolically engineering the kanamycin resistance gene of the pK18mobsacB plasmid (GenBank: FJ1287239.1; available for purchase from public sources) to replace it with the spectinomycin resistance gene. The embodiments of this invention are only for illustrating the effects of the invention and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0030] Table 1 Primer sequence information (SEQ ID No. 1-24) Example 1: Construction of an engineered plasmid carrying the thrABC metabolic pathway (threonine terminal pathway). Using the whole genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using the thrABC-f / thrABC-r primer pair. The PCR products were purified by gel electrophoresis and gel extraction. The PCR product contained the thrABC sequence carrying the threonine terminal metabolic pathway.
[0031] The plasmid vector was pVWEx1 (GenBank: MF034723.1; purchased from public sources). This vector was linearized by PCR amplification using the pVWEx1-f / pVWEx1-r primer pair. The PCR product was purified by gel electrophoresis and gel extraction for later use.
[0032] The vectors carrying the thrABC sequence and linearized were then circularized and assembled. The assembly method used was a single-fragment assembly kit from Novizan. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on kanamycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. The correctly sequenced plasmid was named pVWEx1-thrABC.
[0033] Example 2: Carrying the key gene lysC for threonine synthesis T311I Construction of 3 engineered plasmids Using the fully synthesized lysC gene sequence of ATCC13032 as a template, PCR amplification was performed using the lysC-f-1 / lysC-r-1 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a lysC gene carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. T311I(It encodes a mutant of the lysC protein). Using the fully synthesized lysC gene sequence of ATCC13869 as a template, PCR amplification was performed using the lysC-f-2 / lysC-r-2 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was lysC carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. T311I (It encodes a mutant of the lysC protein). Using the fully synthesized lysC gene sequence of ATCC14067 as a template, PCR amplification was performed using the lysC-f-3 / lysC-r-3 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was lysC carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC14067. T311I (It encodes a mutant lysC protein). In all of the above lysC protein mutants, the wild-type lysC protein has a threonine residue at position 311 that is mutated to isoleucine.
[0034] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.
[0035] The lysC carrying the above-mentioned homologous recombination sequences of ATCC13032, ATCC13869 and ATCC14067 T311I The linearized vectors were then circularized and assembled. Assembly was performed using a single-fragment assembly kit from Novizan. Specific instructions were provided in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. LysC vectors correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were identified. T311I The plasmids were named pK18mobsacB-speC-lysC, respectively. T311I -1、pK18mobsacB-speC-lysC T311I -2 and pK18mob sacB-speC-lysC T311I -3.
[0036] Example 3: Carrying the key gene hom for threonine synthesis G378E Construction of 3 engineered plasmids Using the fully synthesized hom gene sequence of ATCC13032 as a template, PCR amplification was performed using the hom-f-1 / hom-r-1 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom gene carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. G378E (It encodes a mutant hom protein). Using the fully synthesized hom gene sequence of ATCC13869 as a template, PCR amplification was performed using the hom-f-2 / hom-r-2 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom protein carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. G378E (It encodes a mutant hom protein). Using the fully synthesized hom gene sequence of ATCC13869 as a template, PCR amplification was performed using the hom-f-3 / hom-r-3 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom protein carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. G378E (It encodes a mutant hom protein). In all of the above hom protein mutants, the wild-type hom protein has a glycine-to-glutamic acid mutation at position 378.
[0037] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.
[0038] The above-mentioned homogeneous recombination sequences carrying ATCC13032, ATCC13869 and ATCC14067 were used. G378E The linearized vectors were then circularized and assembled. Assembly was performed using Novizan's single-fragment assembly kit. Specific instructions were found in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB broth. Plasmids were extracted the following day and sequenced. Transformants correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were identified. G378E The plasmids were named pK18mobsacB-speC-hom respectively. G378E -1、pK18mobsacB-speC-hom G378E -2、pK18mobsacB-speC-hom G378E -3.
[0039] Example 4: Construction of three engineered plasmids inactivated by nucleoside phosphorylase Using the genomic sequences of Corynebacterium glutamicum ATCC13032, ATCC13869, and ATCC14067 as templates, PCR amplification was performed using primers P1 / P2 to obtain upper homologous arm fragments UP-1, UP-2, and UP-3. PCR amplification was then performed using primers P3 / P4 to obtain lower homologous arm fragments DN-1, DN-2, and DN-3. The PCR products were purified by gel electrophoresis and gel extraction. The PCR products were NCgl0094-UP-1, BBD29_00655-UP-2, and CEY17_00620-UP-3 and NCgl0094-DN-1, BBD29_00655-DN-2, and CEY17_00620-DN-3, respectively, each carrying a homologous recombination sequence.
[0040] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.
[0041] The aforementioned NCgl0094-UP-1 and NCgl0094-DN-1 / BBD29_00655-UP-2 and BBD29_00655-DN-2 / CEY17_00620-UP-3 and CEY17_00620-DN-3, carrying homologous recombination sequences, were circularized and assembled with linearized vectors, respectively. Assembly was performed using Novizan's multi-fragment assembly kit. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day, and sequencing analysis was performed. The plasmids that were correctly sequenced and carried homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were named pK18mobsacB-speC-ΔNCgl0094-1, pK18mobsacB-speC-ΔBBD29_00655-2, and pK18mobsacB-speC-ΔCEY17_00620-3, respectively.
[0042] Example 5: Construction of three engineered plasmids with weakened nucleoside phosphorylase Using the genomic sequences of Corynebacterium glutamicum ATCC13032, ATCC13869, and ATCC14067 as templates, PCR amplification was performed using primers P5 / P6 to obtain upper homologous arm fragments UP-1', UP-2', and UP-3'. PCR amplification was then performed using primers P7 / P8 to obtain lower homologous arm fragments DN-1', DN-2', and DN-3'. The PCR products were purified by gel electrophoresis and gel extraction. The PCR products were NCgl0094r-UP-1, BBD29_00655r-UP-2, CEY17_00620r-UP-3 and NCgl0094r-DN-1, BBD29_00655r-DN-2, CEY17_00620r-DN-3, respectively, each carrying a homologous recombination sequence.
[0043] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.
[0044] The aforementioned NCgl0094r-UP-1 and NCgl0094r-DN-1 / BBD29_00655r-UP-2 and BBD29_00655r-DN-2 / CEY17_00620r-UP-3 and CEY17_00620r-DN-3, carrying homologous recombination sequences, were circularized and assembled with linearized vectors, respectively. Assembly was performed using Novizan's multi-fragment assembly kit. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day, and sequencing analysis was performed. The plasmids that were correctly sequenced and carried homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were named pK18mobsacB-speC-NCgl0094r-1, pK18mobsacB-speC-BBD29_00655r-2, and pK18mobsacB-speC-CEY17_00620r-3, respectively.
[0045] The nucleotide sequence of the gene encoding the nucleoside phosphorylase of the attenuated ATCC13032 is shown in SEQ ID No. 25, and the corresponding amino acid sequence is shown in SEQ ID No. 31. The nucleotide sequence of the gene encoding the nucleoside phosphorylase of the attenuated ATCC13869 is shown in SEQ ID No. 26, and the corresponding amino acid sequence is shown in SEQ ID No. 32. The nucleotide sequence of the gene encoding the nucleoside phosphorylase of the attenuated ATCC14067 is shown in SEQ ID No. 27, and the corresponding amino acid sequence is shown in SEQ ID No. 33.
[0046] Example 6: The threonine terminal pathway thrABC was introduced into the model bacteria ATCC 13032, ATCC 13869, and ATCC 14067, respectively. Competent cells of C. glutamicum model bacteria ATCC13032, ATCC13869, and ATCC14067 were prepared according to the method described in the C. glutamicum Handbook (Charpter 23) and exogenous genes were expressed.
[0047] The expression plasmid pVWEx1-thrABC was transformed into ATCC13032, ATCC13869, and ATCC14067 competent cells using electroporation, and transformants were screened on BHI selective medium containing 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed as the final results. The resulting modified strains were named 13032-thrABC, 13869-thrABC, and 14067-thrABC, respectively.
[0048] Example 7: lysC was introduced into 13032-thrABC, 13869-thrABC, and 14067-thrABC, respectively. T311I and hom G378E Competent cells of 13032-thrABC, 13869-thrABC, and 14067-thrABC were prepared and their genes were recombined according to the method in the C. glutamicum Handbook (Charpter 23).
[0049] The recombinant plasmid pK18mobsacB-speC-lysC was electroporated. T311I -1、pK18mobsacB-speC-lysCT311I -2 and pK18mobsacB-speC-lysC T311I Transformations were performed on 13032-thrABC, 13869-thrABC, and 14067-thrABC competent cells, respectively, and transformants were screened on BHI selective media containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The selected transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The cultures were serially diluted (to 10⁻⁶ ppm). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated statically at 30°C for 48 h. The resulting transformants should carry the target mutation and not the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed as the final result. The resulting modified strains were named 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C, respectively.
[0050] Following the methods described in the C. glutamicum Handbook (Charpter 23), competent cells of 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C were prepared and gene recombination was performed.
[0051] The recombinant plasmid pK18mobsacB-speC-hom was electroporated. G378E -1、pK18mobsacB-speC-hom G378E -2、pK18mobsacB-speC-hom G378E Transformations were performed on 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C competent cells, and transformants were screened on BHI selective media containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The selected transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The cultures were serially diluted (to 10⁻⁶ ppm). -2The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated statically at 30°C for 48 h. The resulting transformants should carry the target mutation and not the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the final results. The resulting modified strains were named SMCT301, SMCT302, and SMCT303, respectively.
[0052] Example 8: Shake-flask test of fermentation performance of three model strains after introduction of the threonine terminal pathway and removal of threonine feedback inhibition. The culture medium used in the shake flask test is as follows: Plate activation medium: BHI 37 g / L, 20 g / L agar powder.
[0053] Seed culture medium: peptone 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ammonium sulfate 16 g / L, urea 8 g / L, potassium dihydrogen phosphate 10.4 g / L, dipotassium hydrogen phosphate 21.4 g / L, biotin 5 mg / L, magnesium sulfate 3 g / L, glucose 50 g / L, pH 7.2.
[0054] Fermentation medium: corn steep liquor 50 mL / L, glucose 30 g / L, ammonium sulfate 4 g / L, MOPS 30 g / L, potassium dihydrogen phosphate 10 g / L, urea 20 g / L, biotin 10 mg / L, magnesium sulfate 6 g / L, ferrous sulfate 1 g / L, vitamin B1•HCl 40 mg / L, calcium pantothenate 50 mg / L, nicotinamide 40 mg / L, manganese sulfate 1 g / L, zinc sulfate 20 mg / L, copper sulfate 20 mg / L, pH 7.2.
[0055] Fermentation method: 1. Seed activation: Take the strain to be verified from the cryopreservation tube, streak it on seed activation medium, and incubate at 30℃ for 24h; 2. Seed culture: Pick 1 plate of activated seeds and transfer it to a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Incubate at 30 °C and 230 r / min for 6 h with shaking. 3. Fermentation culture: Inoculate 6 mL of seed culture into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 30 °C and 150 r / min for 24 h with shaking. Perform 3 replicates for each strain.
[0056] 4. OD 562 Measurement: The fermentation broth was diluted 100 times, and the absorbance was measured at a wavelength of 562 nm using a spectrophotometer. Each strain was tested in triplicate, and the average value was calculated. The results are shown in Table 2, where the data represents the average of the three parallel tests.
[0057] 5. Amino acid concentration determination: Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and detect it using Agilent high-performance liquid chromatography (HPLC). Three replicates were performed for both the recombinant strain and the control strain, and the average value was calculated. The results are shown in Table 2.
[0058] Table 2 Comparison of amino acid yield detection results of recombinant strains The amino acid content in the fermentation broth was analyzed, and it was found that the threonine concentration of Corynebacterium glutamicum, which carried the threonine terminal pathway and removed the threonine terminal restriction, was significantly increased. Table 2 shows that compared to the wild-type strain ATCC13032, SMCT301 increased threonine production from 2.5 g / L to 7.6 g / L, an increase of 240.8%; compared to the wild-type strain ATCC13869, SMCT302 increased threonine production from 2.3 g / L to 7.1 g / L, an increase of 246.2%; and compared to the wild-type strain ATCC14067, SMCT303 increased threonine production from 2.2 g / L to 7.0 g / L, an increase of 262.3%. This indicates that opening the threonine terminal synthesis pathway significantly improves the strain's threonine production capacity. Therefore, it can be proven that after metabolic engineering modification, SMCT301, SMCT302, and SMCT303 are better threonine-producing strains.
[0059] Example 9: Inactivation or weakening of nucleoside phosphorylases in the model strain ATCC13032 of Corynebacterium glutamicum and the threonine-producing strain SMCT301. Competent cells of the C. glutamicum model strain ATCC13032 and the threonine-producing strain SMCT301 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0060] The recombination method was the same as in Example 7, i.e., inactivated or weakened recombinant plasmids (pK18mobsacB-speC-ΔNCgl0094-1 and pK18mobsacB-speC-NCgl0094r-1) were transformed into ATCC13032 and SMCT301 competent cells, respectively, using electroporation. Transformants were screened on BHI selective medium containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ oz). -2The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The transformed strains were amplified by PCR and analyzed by nucleotide sequencing to obtain the final results. The resulting modified strains with inactivated nucleoside phosphorylase were named SMCT346 (originating strain ATCC13032) and SMCT352 (originating strain SMCT301), respectively, while the modified strains with weakened nucleoside phosphorylase were named SMCT347 (originating strain ATCC13032) and SMCT353 (originating strain SMCT301), respectively.
[0061] Example 10: Inactivation or weakening of nucleoside phosphorylases in the model strain ATCC13869 of Corynebacterium glutamicum and the threonine-producing strain SMCT302. Competent cells of the C. glutamicum model strain ATCC13869 and the threonine-producing strain SMCT302 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0062] The recombination method was the same as in Example 7, i.e., inactivated and weakened recombinant plasmids (pK18mobsacB-speC-ΔBBD29_00655-2 and pK18mobsacB-speC-BBD29_00655r-2) were transformed into ATCC13869 and SMCT302 competent cells, respectively, using electroporation. Transformants were screened on BHI selective medium containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ oz). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The transformed strains were amplified by PCR and analyzed by nucleotide sequencing to obtain the final results. The resulting modified strains with inactivated nucleoside phosphorylase were named SMCT348 (originating strain ATCC13869) and SMCT354 (originating strain SMCT302), respectively, while the modified strains with weakened nucleoside phosphorylase were named SMCT349 (originating strain ATCC13869) and SMCT355 (originating strain SMCT302), respectively.
[0063] Example 11: Inactivation or weakening of nucleoside phosphorylases in the model strain ATCC14067 of Corynebacterium glutamicum and the threonine-producing strain SMCT303. Competent cells of the C. glutamicum model strain ATCC14067 and the threonine-producing strain SMCT303 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0064] The recombination method was the same as in Example 7, i.e., inactivated and weakened recombinant plasmids (pK18mobsacB-speC-ΔCEY17_00620-3 and pK18mobsacB-speC-CEY17_00620r-3) were transformed into ATCC14067 and SMCT303 competent cells, respectively, using electroporation. Transformants were screened on BHI selective medium containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ oz). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The transformed strains were amplified by PCR and analyzed by nucleotide sequencing to obtain the final results. The resulting modified strains with inactivated nucleoside phosphorylase were named SMCT350 (originating strain ATCC14067) and SMCT356 (originating strain SMCT303), respectively, while the modified strains with weakened nucleoside phosphorylase were named SMCT351 (originating strain ATCC14067) and SMCT357 (originating strain SMCT303), respectively.
[0065] Example 12: Shake-flask test of the fermentation performance of the above-mentioned *Corynebacterium glutamicum* model strain and threonine-producing strain after nucleoside phosphorylase gene inactivation or attenuation modification. The culture medium and fermentation method used in the shake flask test were the same as in Example 8, and the test results are shown in Table 3.
[0066] Table 3 Comparison of amino acid yield detection results of recombinant strains The amino acid content in the fermentation broth was tested, and it was found that the threonine concentration in the culture broth of strains modified by nucleoside phosphorylase inactivation or weakening was increased to varying degrees, suggesting that the efficiency of bacterial synthesis or secretion of threonine was improved.
[0067] The results indicate that the inactivation or weakening of the nucleoside phosphorylase provided in this invention promotes the production and yield of threonine in Corynebacterium. Nucleoside phosphorylase inactivation is more conducive to the synthesis of threonine or promotes its secretion into the extracellular space. Furthermore, the related modifications are universal and can improve the synthesis efficiency of various amino acids.
[0068] 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 fermentation method for producing L-threonine, characterized in that, The fermentation production is carried out using recombinant microorganisms, wherein the recombinant microorganisms have reduced or inactivated nucleoside phosphorylase activity compared to the starting strain, and the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30.
2. The method according to claim 1, characterized in that, Compared with the original strain, the recombinant microorganism showed that the feedback inhibition of the key genes lysC and hom for threonine synthesis was relieved, and the gene thrABC for the threonine terminal synthesis pathway was expressed. Preferably, compared with the starting strain, the recombinant microorganism has a threonine mutation at position 311 of the lysC protein sequence to isoleucine, and a glycine mutation at position 378 of the hom protein to glutamic acid. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.
3. A recombinant microorganism, characterized in that, As described in any one of claims 1-2.
4. A method for constructing recombinant microorganisms, characterized in that, The recombinant microorganism has reduced or inactivated nucleoside phosphorylase activity compared to the starting strain, wherein the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30.
5. The method according to claim 4, characterized in that, The activity of the nucleoside phosphorylase is reduced or inactivated by one or more base mutations or deletions in the coding region, or by replacing the weak promoter. Preferably, compared with the starting strain, the recombinant microorganism has the feedback inhibition of the key genes lysC and hom for threonine synthesis relieved and expresses the thrABC gene for the threonine terminal synthesis pathway. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.
6. A DNA molecule, characterized in that, The nucleotide sequence is shown in any one of SEQ ID No. 25-27.
7. A biomaterial containing the DNA molecule of claim 6, wherein the biomaterial is an expression cassette, a vector, or a host cell.
8. A nucleoside phosphorylase mutant, characterized in that, The amino acid sequence is shown in any one of SEQ ID No. 31-33.
9. The use of the recombinant microorganism of claim 3, or the DNA molecule of claim 6, or the biological material of claim 7, or the nucleoside phosphorylase mutant of claim 8, in any of the following aspects: (1) L-threonine fermentation production; (2) Genetic breeding of microorganisms for L-threonine fermentation production; (3) Increase the fermentation yield of L-threonine.
10. The application of reducing or inactivating nucleoside phosphorylase activity in microorganisms in increasing the yield of L-threonine produced by microbial fermentation, wherein the amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30; Preferably, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the microorganism is further relieved, and the gene thrABC for the threonine terminal synthesis pathway is expressed; And / or, the microorganism can ferment to produce threonine, preferably Corynebacterium glutamicum.