Recombinant microorganism for producing threonine and application thereof
By genetically engineering Escherichia coli, reducing thrL expression, and introducing specific amino acid mutations into the threonine/homoserine efflux protein, feedback inhibition was relieved, the threonine synthesis pathway was strengthened, and the problem of low threonine production efficiency in traditional breeding methods was solved, resulting in a significant increase in threonine yield and conversion rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-02
- Publication Date
- 2026-03-10
AI Technical Summary
In existing technologies, traditional mutagenesis breeding results in slow growth of threonine-producing strains and the production of many byproducts, making it difficult to obtain high-yielding strains. Furthermore, there is limited research on the amino acid sequence alterations of the threonine efflux protein RhtA, which affects threonine yield.
By genetically engineering E. coli, the expression level of thrL was reduced, and specific amino acid mutations (such as mutating proline to leucine at position 2) were introduced into the threonine/homoserine efflux protein. At the same time, aspartate kinase/homoserine dehydrogenase I mutant and homoserine kinase were overexpressed to relieve feedback inhibition and enhance the threonine synthesis pathway.
The modified strain significantly improved the yield and conversion rate of threonine, enhancing threonine production efficiency. The threonine yield of the modified strain reached 2.5-2.93 g/L, and the conversion rate increased to 29.33%.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a recombinant microorganism for producing threonine and its applications. Background Technology
[0002] L-Threonine is one of the eight essential amino acids for human and animal growth, and it is widely used in feed, food additives, and pharmaceutical excipients. Currently, L-Threonine is mainly produced through microbial fermentation. Various bacteria can be used for L-Threonine production, such as wild-type mutant strains induced from *Escherichia coli*, *Corynebacterium*, and *Serratia*. However, traditional mutagenesis breeding, due to random mutations, easily leads to slow strain growth and the production of numerous byproducts, making it difficult to obtain high-yielding strains.
[0003] With the increasing demand for threonine, the construction and modification of high-threonine-producing strains are particularly important. Various systems metabolic engineering strategies have been developed for modification. For example, in *E. coli*, proteins that can efflux threonine include RhtA, RhtB, and RhtC, with RhtA and RhtC being the most studied. Research on the L-threonine efflux protein RhtA mainly focuses on its upstream non-coding region, while research on the relationship between changes in its amino acid sequence and threonine production is relatively limited. For instance, existing technology records that replacing the base G at position -1 of the start codon ATG in the RhtA gene with A increases L-threonine production (Livshits VA, Zakataeva NP, Aleshin VV, Vitushkina MV. Identification and characterization of the new gene rhtA involved in threonine and homosine efflux in *Escherichia coli*. *Res Microbiol*. 2003 Mar;154(2):123-35.). Therefore, it is still necessary to provide more new modification methods to improve threonine yield. Summary of the Invention
[0004] One of the objectives of this invention is to provide a new recombinant microorganism and method for improving the yield of threonine fermentation.
[0005] This invention provides a recombinant microorganism for producing threonine, which, compared with the starting strain, has a reduced expression level of thrL and increased expression levels of a threonine / homoserine efflux protein mutant, an aspartate kinase / homoserine dehydrogenase I mutant, homoserine kinase, and threonine synthase. The threonine / homoserine efflux protein mutant is formed by a mutation of proline to leucine at position 2 of the threonine / homoserine efflux protein protein sequence; the aspartate kinase / homoserine dehydrogenase I mutant is formed by a mutation of glycine to arginine at position 433 of the aspartate kinase / homoserine dehydrogenase I protein sequence; the starting strain is *Escherichia coli*.
[0006] The present invention also provides a method for producing L-threonine by fermentation, which includes the step of culturing the above-mentioned recombinant microorganisms.
[0007] This invention also provides a method for constructing recombinant microorganisms, which reduces the expression level of thrL in the recombinant microorganisms compared to the starting strain, and overexpresses a threonine / homoserine efflux protein mutant, an aspartate kinase / homoserine dehydrogenase I mutant, a homoserine kinase, and a threonine synthase; wherein the threonine / homoserine efflux protein mutant is formed by mutating proline to leucine at position 2 of the protein sequence of the threonine / homoserine efflux protein; wherein the aspartate kinase / homoserine dehydrogenase I mutant is formed by mutating glycine to arginine at position 433 of the protein sequence of the aspartate kinase / homoserine dehydrogenase I; and the starting strain is *Escherichia coli*.
[0008] This invention reveals that by mutating the gene encoding the threonine / homoserine efflux protein in threonine-producing bacteria, the second amino acid of the threonine / homoserine efflux protein is changed from proline to leucine, resulting in an increased threonine yield compared to the unmodified strain. Furthermore, the corresponding recombinant microorganisms are applied to threonine production, further enhancing the threonine production capacity of the threonine-producing bacteria.
[0009] Specifically, this invention uses genetic engineering techniques, with model strains MG1655 and W3110 as starting bacteria, to obtain specific basic strains for threonine production through rational modification (inactivating thrL, relieving feedback inhibition of bifunctional aspartate kinase / homoserine dehydrogenase I, then overexpressing aspartate kinase / homoserine dehydrogenase I to relieve feedback inhibition, and plasmid overexpression of enhanced aspartate kinase / homoserine dehydrogenase I, homoserine kinase, and threonine synthase, thus completing threonine synthesis and transport, obtaining a basic strain for threonine production). The conversion rate is around 25%. Based on this, the second amino acid of the threonine export protein—threonine / homoserine export protein—is replaced with leucine. The resulting modified strain has a significantly higher threonine yield than the unmodified strain, providing a new method for improving threonine production.
[0010] The present invention also provides the application of the above-described recombinant microorganisms or recombinant microorganisms constructed by the above-described method in the fermentation production of L-threonine.
[0011] The present invention also provides the application of the above-mentioned recombinant microorganisms or recombinant microorganisms constructed by the above-mentioned methods in improving the yield of L-threonine fermentation production.
[0012] The present invention also provides the application of the above-described recombinant microorganisms or recombinant microorganisms constructed by the above-described method in microbial genetic breeding for the production of L-threonine.
[0013] This invention also provides the application of a threonine / homoserine export protein mutant in increasing the yield of L-threonine produced by recombinant microbial fermentation; the starting strain of the recombinant microorganism is Escherichia coli; the threonine / homoserine export protein mutant is a protein sequence in which the second position of proline is mutated to leucine.
[0014] In the application of this invention, compared with the original strain, the recombinant microorganism has a reduced expression level of thrL, and an increased expression level of threonine / homoserine efflux protein mutant, aspartate kinase / homoserine dehydrogenase I mutant, homoserine kinase, and threonine synthase; the aspartate kinase / homoserine dehydrogenase I mutant is formed by a mutation of glycine to arginine at position 433 of the aspartate kinase / homoserine dehydrogenase I protein sequence.
[0015] The present invention also provides the application of nucleic acids encoding threonine / homoserine exogenous protein mutants or biological materials containing said nucleic acids in the construction of the above-mentioned recombinant microorganisms; wherein the threonine / homoserine exogenous protein mutant is a protein sequence in which the second position of proline is mutated to leucine.
[0016] In the application of this invention, the biomaterial is an expression cassette or a vector.
[0017] The nucleotide sequence encoding the threonine / homoserine export protein mutant is the rhtA gene nucleotide sequence (NCBI-GeneID: 947045) in which the 5th C is replaced by a T.
[0018] The beneficial effects of this invention are at least as follows: this invention constructs a new threonine-producing bacterium and provides a new method for improving the efficiency of threonine fermentation production. 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 specific sequence information of the gene described in this invention is as follows: Threonine operon leader peptide (encoding gene thrL): UniProt database ID P0AD86; Aspartate kinase / homoserine dehydrogenase I (encoding gene thrA): UniProt database number P00561; ThrA G433R The amino acid sequence of aspartate kinase / homoserine dehydrogenase I is mutated from G to R at position 433. Homoserine kinase (encoding gene thrB): UniProt database number P00547; Threonine synthase (encoding gene thrC): UniProt database ID P00934; Threonine / homoserine efflux protein (encoding gene rhtA): UniProt database ID P0AA67; RhtA P2L The second amino acid position of the threonine / homoserine exogenous protein is mutated from P to L.
[0022] Example 1
[0023] This embodiment describes the modification and fermentation production of the starting strain.
[0024] 1. Using MG1655 and W3110 as starting strains, respectively, threonine production chassis strains were constructed.
[0025] 1.1 Construction of thrL-inactivated strains
[0026] (1) Construction of pTargetF-N20 (ΔthrL) plasmid and Donor DNA
[0027] Step 1: Using pTargetF plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), the pTF linear plasmid containing N20 was amplified using the LWP001 / LWP002 primer pair. This linear plasmid was then assembled at 37℃ using the seamless assembly ClonExpress kit, followed by transformation into Trans1-T1 competent cells to obtain pTargetF-N20 (ΔthrL). After colony growth, colony PCR was performed using LWP003 / pTF-R for verification. Correct transformants showed a band, while the negative control showed no band. The results were verified using pTF-R sequencing. Step 2: Using the MG1655 / W3110 genome as a template, primer pair LWP004 / LWP005 was used to amplify the upstream homologous arm ①. Step 3: Using the MG1655 / W3110 genome as a template, primer pair LWP006 / LWP007 was used to amplify the downstream homologous arm ②. Step 4: Using ① and ② as templates, primer pair LWP004 / LWP007 was used to amplify the full-length ΔthrL fragment, also known as Donor DNA.
[0028] (2) Preparation and transformation of competent cells
[0029] Step 1: Electroporate the pCas plasmid (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MG1655 / W3110 competent cells (the transformation method and competent cell preparation method are the same as those in Molecular Cloning III); Step 2: Pick a single colony of MG1655 (pCas) / W3110 (pCas) into a 5 mL LB tube containing kanamycin and 10 mM arabinose, and culture at 30℃ and 200 r / min until the OD650 reaches 0.4 to prepare electroporated competent cells (the competent cell preparation method is the same as those in Molecular Cloning III). Step 3: Electroporate pTargetF-N20(△thrL) plasmid and Donor DNA simultaneously into MG1655 (pCas) / W3110 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.
[0030] (3) Recombination verification
[0031] Step 1: Use primer pair LWP004 / LWP008 to perform colony PCR verification on the above single colonies. ΔthrL modified strains showed no bands, while unmodified strains showed bands. Step 2: Use primer pair LWP004 / LWP007 to amplify the target fragment. The amplified product is then sequenced to verify the integrity of the sequence.
[0032] (4) Construction of related plasmids lost
[0033] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and 0.5 mM IPTG. Incubate overnight at 30°C and then streak it onto an LB agar plate containing kanamycin. Step 2: Select a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin, and kanamycin only. Incubate overnight at 30°C. If the colony does not grow on the LB agar plates containing kanamycin or spectinomycin, but grows on the LB agar plate containing kanamycin, it indicates that the pTargetF-N20(△thrL) plasmid has been lost. Step 3: Select positive colonies with the lost pTargetF-N20(△thrL) plasmid and inoculate them onto antibiotic-free LB agar plates. After incubating in test tubes at 42℃ for 8 hours, the cultures were streaked onto LB plates and incubated overnight at 37℃. Step 4: Single colonies were picked and spotted onto LB plates containing kanamycin and those without antibiotics. If the colonies could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and strains SMT-MG001 and SMT-W001 were obtained.
[0034] 1.2 Aspartate kinase / homoserine dehydrogenase I desensitization of feedback inhibition (thrA) G433R )
[0035] (1) pTargetF-N20 (thrA) G433R Construction of plasmids and Donor DNA
[0036] Step 1: Using pTargetF plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), the LWP009 / LWP010 primer pair was used to amplify the pTF linear plasmid containing N20. This linear plasmid was then assembled at 37°C using the seamless assembly ClonExpress kit, followed by transformation into Trans1-T1 competent cells to obtain pTargetF-N20(thrA) G433R After colonies grew, colony PCR was performed using LWP011 / pTF-R for verification. Correct transformants showed bands, while negative controls showed no bands. This was then verified using pTF-R sequencing. Step 2: Using the MG1655 / W3110 genome as a template, primers LWP012 / LWP013 were used to amplify the upstream homologous arm ①. Step 3: Using the MG1655 / W3110 genome as a template, primers LWP014 / LWP015 were used to amplify the downstream homologous arm ②. Step 4: Using ① and ② as templates, primers LWP012 / LWP015 were used to amplify thrA. G433R The full-length fragment is also known as Donor DNA.
[0037] (2) Preparation and transformation of competent cells
[0038] Step 1: Electroporate the pCas plasmid (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into SMT-MG001 / SMT-W001 competent cells (transformation and competent cell preparation methods are as described in Molecular Cloning III); Step 2: Pick single colonies of SMT-MG001 (pCas) / SMT-W001 (pCas) in 5 mL LB tubes containing kanamycin and 10 mM arabinose, and incubate at 30°C and 200 rpm until OD650 reaches 0.4 to prepare electroporated competent cells (competent cell preparation method is as described in Molecular Cloning III). Step 3: Electroporate pTargetF-N20(thrA G433RThe plasmid and Donor DNA were simultaneously electroporated into SMT-MG001 (pCas) / SMT-W001 (pCas) competent cells (electroplation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30°C until single colonies were visible.
[0039] (3) Recombination verification
[0040] The target fragment was amplified using primers LWP012 / LWP015, and the amplified product was sent for sequencing to verify the integrity of the sequence.
[0041] (4) Construction of related plasmids lost
[0042] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and 0.5 mM IPTG. Incubate overnight at 30°C and then streak it onto an LB agar plate containing kanamycin. Step 2: Select a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin, and kanamycin only. Incubate overnight at 30°C. If the colony does not grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that pTargetF-N20(thrA) is functioning correctly. G433R The plasmid has been lost; Step 3: Pick pTargetF-N20(thrA) G433R Positive colonies with plasmid loss were inoculated into antibiotic-free LB tubes, incubated at 42°C for 8 hours, and then streaked onto LB plates and incubated overnight at 37°C. Step 4: Single colonies were picked and dotted onto LB plates containing kanamycin and LB plates without antibiotics. If the colonies could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and strains SMT-MG002 and SMT-W002 were obtained.
[0043] 1.3 Expression plasmid pTrc99A-thrA G433R BC build
[0044] Using the SMT-MG002 genome as a template, thrA was amplified using primer pair LWP016 / LWP017. G433R BC fragment. pTrc99A was digested with EcoRI / SmaI, and thrA was then assembled using the ClonExpress Seamless Assembly Kit. G433R BC was assembled with enzyme-digested pTrc99A, and then transformed into Trans1-T1 competent cells to obtain pTrc99A-thrA. G433RBC, use 99A-F / 99A-R primers to perform colony PCR verification, select test tubes to extract plasmids from the verified colonies, perform enzyme digestion verification with EcoRI / SmaI, and send for sequencing.
[0045] 2. Construct a system containing pTrc99A-thrA G433R BC strains
[0046] pTrc99A-thrA G433R BC was electroporated into SMT-MG002 / SMT-W002 competent cells (transformation and competent cell preparation methods were both referred to in Molecular Cloning III) to obtain cells containing pTrc99A-thrA. G433R The strain with BC plasmid is SMT-MG002(ABC) / SMT-W002(ABC).
[0047] 3. Verification of threonine production by the modified strain
[0048] Step 1: Take 8 strains of bacteria from cryopreservation tubes, namely MG1655, SMT-MG001, SMT-MG002, SMT-MG002 (ABC), W3110, SMT-W001, SMT-W002, and SMT-W002 (ABC), and activate them by streaking on LB plates and incubating at 37°C for 18-24 hours; Step 2: Scrape a loopful of bacterial cells from the plate and inoculate it into a shake flask containing 50 mL of seed culture medium (see Table 1). Incubate at 37°C and 90 rpm for about 5 hours to keep the OD650 below 2. Step 3: Transfer 2 mL of seed culture to a shake flask containing 20 mL of fermentation medium (see Table 2). Fermentation was carried out on a shaker at 37°C and 100 rpm until the residual sugar was exhausted. After fermentation, the OD650 of the sample was measured, and the L-threonine content was determined by HPLC. The residual sugar content was determined by a biosensor method. To ensure the reliability of the experiment, the shake flask was tested three times at a fermentation temperature of 37°C. The acid production and conversion rate results are shown in Table 3.
[0049] Table 1 Seed Culture Medium
[0050] Table 2 Fermentation Culture Media
[0051] Table 3 Comparison of productivity of threonine-producing genetically engineered bacteria
[0052] The fermentation results show that, for both MG1655 and W3110, after removing transcriptional repression and feedback inhibition in the threonine synthesis pathway, and enhancing the expression of aspartate kinase / homoserine dehydrogenase I, homoserine kinase, and threonine synthase in the threonine synthesis pathway, the yield and conversion rate of threonine significantly improved. Threonine production increased from almost none to approximately 2.5 g / L, with a conversion rate of about 25%. Therefore, the modified strains possess the ability to produce threonine and have the potential for further improvement.
[0053] 4. SMT-MG002, SMT-W002 genome rhtA P2L Transformation
[0054] (1) pTargetF-N20 (rhtA) P2L Construction of plasmids and Donor DNA
[0055] Step 1: Using pTargetF plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), the LWP044 / LWP045 primer pair was used to amplify the pTF linear plasmid containing N20. This linear plasmid was then assembled at 37℃ using the seamless assembly ClonExpress kit, followed by transformation into Trans1-T1 competent cells to obtain pTargetF-N20(rhtA) P2L After colonies grow, colony PCR is performed using LWP046 / pTF-R for verification. Correct transformants show a band, while negative controls show no band. This is then verified using pTF-R sequencing. Step 2: Using the MG1655 / W3110 genome as a template, primers LWP047 / LWP048 are used to amplify the upstream homologous arm up. Step 3: Using the MG1655 / W3110 genome as a template, primers LWP049 / LWP050 are used to amplify the downstream homologous arm dn. Step 4: Using up and dn as templates, primers LWP047 / LWP050 are used to amplify up-dn, also known as Donor DNA.
[0056] (2) Preparation and transformation of competent cells
[0057] Step 1: Electroporate the pCas plasmid (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into SMT-MG002 / SMT-W002 competent cells (transformation method and competent cell preparation method are both referred to "Molecular Cloning III"); Step 2: Pick single colonies of SMT-MG002 (pCas) / SMT-W002 (pCas) into 5 mL LB tubes containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30℃ and 200 r / min until the OD650 reaches 0.4 to prepare electroporated competent cells (competent cell preparation method is referred to "Molecular Cloning III"). Step 3: Electroporate pTargetF-N20(rhtA) into SMT-MG002 / SMT-W002 competent cells into SMT-MG002 / SMT-W002 competent cells (competent cell preparation method is referred to "Molecular Cloning III"). P2L The plasmid and Donor DNA were simultaneously electroporated into SMT-MG002 (pCas) / SMT-W002 (pCas) competent cells (electroplation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30°C until single colonies were visible.
[0058] (3) Recombination verification
[0059] The target fragment was amplified using primers LWP047 / LWP050, and the amplified product was sent for sequencing to verify the integrity of the sequence.
[0060] (4) Construction of related plasmids lost
[0061] Step 1: Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and 0.5 mM IPTG. Incubate overnight at 30°C and then streak it onto an LB agar plate containing kanamycin. Step 2: Select a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin, and kanamycin only. Incubate overnight at 30°C. If the colony does not grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that pTargetF-N20(rhtA) is functioning correctly. P2L The plasmid has been lost; Step 3: Pick pTargetF-N20(rhtA) P2LPositive colonies with plasmid loss were inoculated into antibiotic-free LB tubes, incubated at 42°C for 8 hours, and then streaked onto LB plates and incubated overnight at 37°C. Step 4: Single colonies were picked and dotted onto LB plates containing kanamycin and LB plates without antibiotics. If the colonies could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and strains SMT-MG005 and SMT-W005 were obtained.
[0062] 5. Construction of plasmid-containing strains
[0063] pTrc99A-thrA respectively G433R BC was electroporated into SMT-MG005 / SMT-W005 competent cells (transformation and competent cell preparation methods were both based on Molecular Cloning III) to obtain cells containing pTrc99A-thrA. G433R The strain with BC plasmid is SMT-MG005(ABC) / SMT-W005(ABC).
[0064] 6. Verification of Threonine Production by Modified Bacteria
[0065] The seed culture medium fermentation process was the same as described above. The validation strains were SMT-MG002(ABC), SMT-MG005(ABC), SMT-W002(ABC), and SMT-W005(ABC). The fermentation results are shown in Table 4 below.
[0066] Table 4
[0067] As shown in the table above, the threonine yield and conversion rate were improved after the second amino acid of rhtA in different strains was mutated to leucine. The threonine yield of SMT-MG005(ABC) was approximately 2.93 g / L, with a conversion rate of 29.33%, which was 14.57% higher than the control SMT-MG002(ABC). The threonine yield of SMT-W005(ABC) was approximately 2.76 g / L, with a conversion rate of 27.63%, which was 12.32% higher than the control SMT-W002(ABC). The threonine / homoserine transporter mutant facilitates the efflux of threonine, thereby reducing the intracellular threonine concentration and further increasing threonine yield.
[0068] The strain information obtained by constructing this invention is shown in Table 5 below.
[0069] Table 5
[0070] The primer sequences are shown in Table 6 below.
[0071] Table 6 (SEQ ID NO.1-27)
[0072] 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 for the production of threonine, characterized in that, The expression of thrL is reduced, and the expression of threonine / high-sine export protein mutant, aspartate kinase / high-sine dehydrogenase I mutant, high-sine kinase and threonine synthase is increased compared with the starting strain; The threonine / high-sine export protein mutant is a mutation of proline to leucine at the 2nd position in the protein sequence of threonine / high-sine export protein; the aspartate kinase / high-sine dehydrogenase I mutant is a mutation of glycine to arginine at the 433rd position in the protein sequence of aspartate kinase / high-sine dehydrogenase I; and the starting strain is Escherichia coli.
2. A method for the fermentative production of L-threonine, characterized in that The step of culturing the recombinant microorganism of claim 1.
3. A method of constructing a recombinant microorganism, comprising, The expression of thrL is reduced, and the expression of threonine / high-sine export protein mutant, aspartate kinase / high-sine dehydrogenase I mutant, high-sine kinase and threonine synthase is increased compared with the starting strain; The threonine / high-sine export protein mutant is a mutation of proline to leucine at the 2nd position in the protein sequence of threonine / high-sine export protein; the aspartate kinase / high-sine dehydrogenase I mutant is a mutation of glycine to arginine at the 433rd position in the protein sequence of aspartate kinase / high-sine dehydrogenase I; and the starting strain is Escherichia coli.
4. Use of the recombinant microorganism of claim 1 or the recombinant microorganism constructed by the method of claim 3 in the fermentation production of L-threonine.
5. Use of the recombinant microorganism of claim 1 or the recombinant microorganism constructed by the method of claim 3 in improving the yield of L-threonine in the fermentation production.
6. Use of the recombinant microorganism of claim 1 or the recombinant microorganism constructed by the method of claim 3 in the genetic breeding of microorganisms for producing L-threonine.
7. Use of threonine / high-sine export protein mutant in improving the yield of L-threonine in the fermentation production of recombinant microorganism; the starting strain of the recombinant microorganism is Escherichia coli; and the threonine / high-sine export protein mutant is a mutation of proline to leucine at the 2nd position in the protein sequence of threonine / high-sine export protein.
8. Use according to claim 7, characterized in that, The expression of thrL is reduced, and the expression of threonine / high-sine export protein mutant, aspartate kinase / high-sine dehydrogenase I mutant, high-sine kinase and threonine synthase is increased compared with the starting strain; and the aspartate kinase / high-sine dehydrogenase I mutant is a mutation of glycine to arginine at the 433rd position in the protein sequence of aspartate kinase / high-sine dehydrogenase I.
9. Use of nucleic acid encoding threonine / high-sine export protein mutant or biological material containing the nucleic acid in constructing the recombinant microorganism of claim 1; the threonine / high-sine export protein mutant is a mutation of proline to leucine at the 2nd position in the protein sequence of threonine / high-sine export protein.
10. Use according to claim 9, characterized in that, The biological material is an expression cassette or a vector.