Recombinant microorganisms for producing threonine, their construction methods, and methods for producing threonine.

Abstract

This invention relates to the field of microbial technology, specifically to recombinant microorganisms for producing threonine, their construction methods, and methods for producing threonine. This invention discovers that reducing the expression of the β-phosphoglucomutase YcjU in microorganisms can significantly increase the yield and conversion rate of threonine and other amino acids. The recombinant microorganisms with reduced β-phosphoglucomutase YcjU expression provided by this invention exhibit significantly higher threonine yield and conversion rate compared to the starting strain, which is beneficial for reducing the fermentation production cost of threonine and provides effective modification targets and strains for the breeding of high-yielding threonine strains.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to recombinant microorganisms for producing threonine, their construction methods, and methods for producing threonine. Background Technology

[0002] L-Threonine is an intermediate metabolite that serves as an important precursor for the synthesis of many amino acids and is an essential amino acid indispensable to the human body. Threonine is widely used in the food, feed, and pharmaceutical industries. The main methods for producing threonine include fermentation, protein hydrolysis, and chemical synthesis. Currently, microbial fermentation has become the mainstream method for threonine production. To produce L-threonine, mutant strains derived from wild-type *Escherichia coli*, *Corynebacteria* sp., *Serratia* sp., or *Providenciasp.* are used. Among methods for producing L-threonine using mutant strains, Japanese Patent No. 10037 / 81 discloses a method using microorganisms belonging to the *Escherichia coli* species, exhibiting a auxotrophic phenotype of diaminopimelic acid and methionine, and whose mutations prevent the biosynthesis of L-threonine from being affected by feedback inhibition from threonine.

[0003] However, traditional mutagenesis breeding, due to random mutations, results in slow bacterial growth and the production of numerous byproducts, making it difficult to obtain high-yielding strains. Screening for high-yielding strains using gene editing methods is more advantageous. For example, Chinese patent CN111019878 A utilizes CRISPR-Cas9 gene editing technology to knock out the key gene dapA in the L-threonine synthesis competitive pathway and the gene tdh encoding threonine dehydrogenase, thereby achieving a threonine yield of 120 g / L.

[0004] With the increasing global demand for threonine, the construction and modification of high-yield threonine-producing strains are particularly important. In 2003, CJ Corporation of South Korea applied for Chinese patent CN03811059.8, which utilized *E. coli* to enhance the expression of the key threonine synthesis gene *thrABC* by deleting a 39bp sequence from positions -56 to -18 of the threonine operon sequence, resulting in a 22% increase in threonine productivity. In 2005, CJ Corporation of South Korea applied for Chinese patent CN1654634A, which increased threonine production by 18%-25% by inactivating the GalR gene.

[0005] The fermentation performance of amino acid production strains is a key factor determining amino acid yield and conversion rate. Therefore, improving the fermentation performance of production strains is of great significance for increasing amino acid yield and conversion rate and further reducing costs. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant microorganism for producing amino acids such as threonine, its construction method, and its method for producing amino acids such as threonine.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] This invention provides any one of the following applications of the β-glucose mutase YcjU or its inhibitor, the β-glucose mutase gene ycjU or its inhibitor, and biological materials containing the gene ycjU or the inhibitor:

[0009] (1) Application in improving the amino acid yield and / or conversion rate of microorganisms;

[0010] (2) Application in the construction of amino acid production strains;

[0011] (3) Application in the fermentation production of amino acids.

[0012] Specifically, the application is achieved by reducing the expression and / or enzyme activity of the β-phosphoglucose mutase YcjU.

[0013] This invention has found that by reducing the expression and / or activity of β-phosphoglucose mutase YcjU in microorganisms, the yield and conversion rate of amino acids can be significantly improved.

[0014] YcjU is the gene encoding β-phosphoglucomutase. The interconversion between glucose-1-phosphate and glucose-6-phosphate is a key step in all cellular metabolism, and this step is accomplished by phosphoglucomutase (EC2.7.5.1) (Ray WJ Jr, Peck EJ Jr (1972) Phosphomutases. In: Boyer PD (ed) The Enzymes, vol. 6. Academic Press, New York, pp 407-458). When the carbon source is galactose, glucose-1-phosphate produced by galactose metabolism is converted to glucose-6-phosphate by phosphoglucomutase. When the carbon source is other than galactose, the main function of phosphoglucomutase is to convert glucose-6-phosphate to glucose-1-phosphate. Glucose-6-phosphate is a common intermediate product of glycolysis, aerobic oxidation, the pentose phosphate pathway, and glycogen synthesis and degradation pathways, and is the intersection of various metabolic pathways.

[0015] In the above applications, the inhibitory factor is a protein, DNA, or RNA that can inhibit the expression and / or enzyme activity of β-phosphoglucose mutase YcjU.

[0016] In the above applications, the biological materials include recombinant DNA, expression cassettes, vectors, or microorganisms.

[0017] In this invention, the expression and / or reduction of enzyme activity of β-phosphoglucose mutase YcjU can be achieved through a combination of one or more of the following methods (1) and (2):

[0018] (1) Insert, delete or replace one or more bases in the gene encoding the β-glucose mutase YcjU to reduce the expression level, enzyme activity or inactivate the β-glucose mutase YcjU.

[0019] (2) Replace the transcriptional or translational regulatory element of the gene encoding the β-phosphoglucose mutase YcjU with a less active regulatory element to reduce its expression, enzyme activity or inactivation.

[0020] Preferably, the expression and / or activity of the β-phosphoglucose mutase YcjU are reduced by inactivating it.

[0021] In one embodiment of the present invention, the expression and / or activity of the β-phosphoglucose mutase gene ycjU are reduced by deleting it.

[0022] In this invention, the β-phosphoglucose mutase YcjU has any of the following amino acid sequences:

[0023] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0024] (2) An amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acids as shown in SEQ ID NO.1.

[0025] (3) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO.1.

[0026] The amino acid sequence of the β-phosphoglucose mutase YcjU in Escherichia coli is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene ycjU is shown in SEQ ID NO.2.

[0027] The present invention also provides a recombinant microorganism, which, compared with its originating strain, has reduced expression and / or enzyme activity of β-phosphoglucose mutase YcjU or its homologs or functional variants thereof.

[0028] The reduction in expression and / or enzyme activity is achieved through a combination of one or more of the following methods (1) and (2):

[0029] (1) Insert, delete or replace one or more bases in the gene encoding the β-glucose mutase YcjU to reduce the expression level, enzyme activity or inactivate the β-glucose mutase YcjU.

[0030] (2) Replace the transcriptional or translational regulatory element of the gene encoding the β-phosphogluconosome YcjU with a less active regulatory element to reduce its expression level, reduce enzyme activity or inactivate it.

[0031] Preferably, the expression and / or activity of the β-phosphoglucose mutase YcjU are reduced by inactivating it.

[0032] The starting strain described in this invention is a strain used as a starting point before reducing the expression and / or enzyme activity of β-phosphoglucomutase YcjU or its homologs or functional variants. The recombinant microorganism can be obtained by genetically engineering or mutagenesis of the starting strain to reduce the expression and / or enzyme activity of its β-phosphoglucomutase YcjU or its homologs or functional variants.

[0033] Preferably, the starting strain described above is a bacterium capable of accumulating amino acids or their derivatives. The bacterium capable of accumulating amino acids or their derivatives can be a wild-type strain or a strain obtained through genetic engineering or mutagenesis. The starting strain of this invention does not have particular limitations on the yield of the amino acids or their derivatives.

[0034] The amino acids used in this invention are preferably L-amino acids. Decreased expression and / or activity of β-phosphoglucose mutase YcjU can significantly increase the yield of threonine, glycine, or isoleucine. Therefore, the amino acids used in this invention are preferably threonine, glycine, or isoleucine.

[0035] The starting strain of this invention preferably contains one or more of the following mutations:

[0036] (1) Enhanced expression of the pntAB gene;

[0037] (2) Expression of the pyc gene derived from Corynebacterium glutamicum;

[0038] (3) The mutant thrA*(S345P) expressing thrA;

[0039] (4) Knock out the tdh gene;

[0040] (5) Increase the number of copies of thrA*(S345P)BC.

[0041] The starting strains described in this invention are bacteria selected from the genera Escherichia, Corynebacterium, and Serratia.

[0042] The Escherichia spp. bacteria include, but are not limited to, Escherichia coli, and the Corynebacterium spp. bacteria include, but are not limited to, Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, and Corynebacterium aminogenes.

[0043] Preferably, the starting strain is Escherichia coli.

[0044] In a preferred embodiment of the present invention, the starting strain is MHZ-0215-2, which has been disclosed in Chinese Patent 201611250306.8. Its biodeposit information is as follows: Classification and nomenclature: Escherichia coli, deposited on November 30, 2016 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 13403.

[0045] The present invention also provides a method for constructing the recombinant microorganism, the method comprising: reducing the expression and / or enzyme activity of the β-phosphoglucose mutase YcjU in the starting strain by means of genetic engineering or mutagenesis.

[0046] The genetic engineering or mutagenesis methods described above can employ methods commonly used in the field. Specifically, the genetic engineering methods can utilize conventional gene mutation or deletion techniques. The mutagenesis methods can be physical and / or chemical mutagenesis.

[0047] In one embodiment of the present invention, the genetic engineering method is CRISPR / Cas9 technology. Specifically, a plasmid containing sgRNA targeting the ycjU gene, upstream and downstream homologous arms of the ycjU gene, and Cas9 protein is introduced into the starting strain, and the ycjU gene is knocked out through homologous recombination.

[0048] The present invention provides a significant improvement in the yield and conversion rate of amino acids (especially threonine) or their derivatives from recombinant microorganisms.

[0049] Based on this, the present invention provides the application of the recombinant microorganism in the production of amino acids or their derivatives or in the selection of amino acid-producing strains.

[0050] In the above-described applications, preferably, the amino acid is threonine, glycine, or isoleucine.

[0051] The present invention also provides a method for increasing the amino acid production of microorganisms, comprising: reducing the expression of YcjU and / or enzyme activity of the microorganisms.

[0052] The present invention also provides a method for producing amino acids or their derivatives by fermentation, the method comprising: culturing the recombinant microorganism and recovering the amino acids or their derivatives from the obtained culture medium.

[0053] Preferably, the amino acid is threonine, glycine, or isoleucine. More preferably, it is threonine.

[0054] For threonine, the fermentation medium used to culture the recombinant microorganism preferably comprises the following components: glucose 70-100 g / L, corn steep liquor 5-10 g / L, soybean meal hydrolysate 5-10 g / L, magnesium sulfate heptahydrate 0.2-0.8 g / L, KH2PO4 0.5-1.5 g / L, aspartic acid 5-15 g / L, FeSO4 25-35 mg / L, MnSO4 25-35 mg / L, biotin 40-60 μg / L, thiamine 400-600 μg / L, pH 6.8-7.2.

[0055] For threonine, the seed culture medium for culturing the recombinant microorganism preferably comprises the following components: glucose 20-30 g / L, corn steep liquor 20-30 g / L, soybean meal hydrolysate 6-9 g / L, yeast extract 2-3 g / L, KH2PO4 1-2 g / L, magnesium sulfate heptahydrate 0.2-0.8 g / L, FeSO4 15-25 mg / L, MnSO4 15-25 mg / L, pH 6.8-7.2.

[0056] The above-described method for fermenting to produce amino acids or their derivatives includes: first, culturing activated recombinant microorganisms in a seed culture medium to obtain a mature seed liquid; then, inoculating the seed liquid into a fermentation culture medium for further cultivation; and recovering the amino acids or their derivatives from the obtained culture medium.

[0057] The beneficial effects of this invention are as follows: By reducing the expression of β-phosphoglucoside mutase YcjU in microorganisms, this invention significantly improves the yield and conversion rate of amino acids such as threonine. The recombinant microorganisms with reduced expression of β-phosphoglucoside mutase YcjU provided by this invention exhibit significantly higher threonine yield and conversion rate compared to the starting strain, which is beneficial for reducing the fermentation production cost of threonine and provides an effective modification target and strain for the breeding of high-yield threonine strains. Detailed Implementation

[0058] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0059] The following examples use MHZ-0215-2 as the starting strain (this strain has been disclosed in patent application CN106635945A, and this strain belongs to the genus Escherichia W3110). Related modifications were made to the genome of MHZ-0215-2 to weaken the expression of the ycjU gene, mainly by knocking out the ycjU gene, thereby reducing the conversion of glucose-6-phosphate to glucose-1-phosphate.

[0060] The genome editing of Escherichia coli involved in the following examples mainly draws on the CRISPR-Cas9 gene editing technology reported by Jiang Y et al. (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015).

[0061] In the following examples, the final concentration of kanamycin in the culture medium was 50 μg / mL, and the final concentration of spectinomycin in the culture medium was 50 μg / mL.

[0062] All reagents used in the following examples are commercially available.

[0063] The primer sequences used in the following examples are shown in Table 1.

[0064] Table 1 Primer sequences used in the examples.

[0065]

[0066] The present invention will be further illustrated below with reference to the embodiments.

[0067] Example 1: Preparation of strain MHZ-0221-12 with inactivated ycjU gene

[0068] 1. Construction of pTargetF-N20(ycjU) plasmid and Donor DNA

[0069] (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-sgRNA-F / pTF-sgRNA-R primer pair was selected to amplify the pTF linear plasmid containing N20. The linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(ycjU), which was then identified by PCR and sequenced for verification.

[0070] (2) Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the ycjU-UF / ycjU-UR primer pair;

[0071] (3) Using the W3110 genome as a template, the downstream homologous arm ② was amplified by selecting the ycjU-DF / ycjU-DR primer pair;

[0072] (4) Using ① and ② as templates, select the ycjU-UF / ycjU-DR primer pair to amplify the up-down full-length fragment, also known as Donor DNA.

[0073] 2. Preparation and electroporation of competent cells

[0074] (1) 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) was electroporated into MHZ-0215-2 competent cells (the transformation method and the preparation method of competent cells are both referred to Molecular Cloning III);

[0075] (2) Pick a single colony of MHZ-0215-2 (pCas) into a 5 mL LB tube containing kanamycin and 10 mM arabinose, and incubate at 30℃ and 200 r / min until OD. 650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0076] (3) The pTargetF-N20(ycjU) plasmid and the Donor DNA constructed in (1) were simultaneously electroporated into MHZ-0215-2(pCas) competent cells (electroplation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30℃ until single colonies were visible.

[0077] 3. Recombination Verification

[0078] (1) Use primer pair ycjU-F / ycjU-R to perform colony PCR verification on the above single colonies;

[0079] (2) The target fragment was amplified using primers ycjU-F / ycjU-R, and the amplified product was sent for sequencing to verify the integrity of the sequence.

[0080] 4. Loss of constructing related plasmids

[0081] (1) Select a single colony that has been correctly sequenced and inoculate it into a 5 mL LB tube containing kanamycin and a final concentration of 0.5 mM IPTG. After incubating overnight at 30°C, streak it onto an LB plate containing kanamycin.

[0082] (2) Pick a single colony and spot it onto LB plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB plates containing kanamycin and spectinomycin, but grows on LB plates containing kanamycin, it indicates that the pTargetF-N20(ycjU) plasmid has been lost.

[0083] (3) Pick positive colonies that have lost pTargetF-N20(ycjU) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them on LB plates and incubate overnight at 37℃.

[0084] (4) Select a single colony and spot it on LB agar plates containing kanamycin and LB agar plates without antibiotics. If it cannot grow on LB agar plates containing kanamycin but grows on LB agar plates without antibiotics, it indicates that the pCas plasmid is lost, and the ycjU knockout strain MHZ-0221-12 is obtained (Table 2).

[0085] Table 2. Genetically engineered bacteria constructed in this embodiment.

[0086] strain number genotype MHZ-0221-12 MHZ-0215-2-ΔycjU

[0087] Example 2: Shake-flask fermentation verification of L-threonine-producing genetically engineered bacteria

[0088] The L-threonine production of strain MHZ-0221-12 constructed in Example 1 and the starting strain MHZ-0215-2 were verified by shake-flask fermentation, as follows:

[0089] 1. Take two strains of bacteria, MHZ-0215-2 and MHZ-0221-12, from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 18-24 hours;

[0090] 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 3). Incubate at 37°C and 90 rpm for approximately 5 hours to allow the OD to reach its maximum. 650 Keep it below 2;

[0091] 3. Transfer 2 mL of seed culture to a shake flask containing 20 mL of fermentation medium (see Table 4), and ferment on a shaker at 37°C and 100 rpm until the residual sugar is exhausted. After fermentation, measure the OD of the sample. 650 The L-threonine content was determined by HPLC, and the residual sugar content was determined by a biosensor method. To ensure the reliability of the experiment, the shake-flask fermentation was repeated three times, and the average values ​​of the acid production and conversion rate are shown in Table 5.

[0092] Table 3 Seed Culture Medium

[0093]

[0094]

[0095] Table 4 Fermentation Culture Media

[0096] Element concentration glucose 85g / L Corn syrup 6g / L Soybean meal hydrolysate 7.7g / L Magnesium sulfate heptahydrate 0.5g / L <![CDATA[KH2PO4]]> 1.0g / L Aspartic acid 10g / L <![CDATA[FeSO4、MnSO4]]> 30mg / L Biotin 50μg Thiamine 500μg pH 7.2

[0097] Table 5 Comparison of productivity of threonine-producing genetically engineered bacteria

[0098]

[0099] Table 5 shows that the L-threonine production of strain MHZ-0221-12 was significantly higher than that of the starting strain MHZ-0215-2. The threonine production of MHZ-0215-12 was 13.83 g / L, with a sugar-acid conversion rate of 16.4%, representing a 14.3% increase in yield and a 15.5% increase in conversion rate compared to the starting strain. The shake-flask fermentation experiment results indicate that knocking out the ycjU gene in E. coli strains can significantly improve the threonine production capacity of the strains.

[0100] Example 3: Fermentation verification of L-threonine-producing genetically engineered bacteria in a fermenter

[0101] The strain MHZ-0221-12 constructed in Example 1 and the starting strain MHZ-0215-2 were used to verify the production of L-threonine in a 1L fermenter, as follows:

[0102] 1. Take two strains of bacteria, MHZ-0215-2 and MHZ-0221-12, from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 18-24 hours;

[0103] 2. Scrape a loopful of bacterial cells from the plate and inoculate it into a shake flask containing 100 mL of seed culture medium (see Table 3). Incubate at 37°C and 90 rpm for approximately 5 hours to allow the OD to reach its maximum. 650 Keep it below 2;

[0104] 3. Preparation for fermentation in a 1L tank: pH calibration, pump calibration, in-situ cleaning, dissolved oxygen calibration.

[0105] 4. Transfer 60 mL of seed culture to a 1 L fermenter containing 360 mL of fermentation medium (see Table 4). Ferment at 37 °C and 500 rpm-1200 rpm until the residual sugar is depleted. After fermentation, measure the OD of the sample. 650 The L-threonine content was determined by HPLC, and the residual sugar content was determined by a biosensor method. To ensure the reliability of the experiment, the fermentation in the fermenter was repeated three times, and the average values ​​of the acid production and conversion rate are shown in Table 6.

[0106] Table 6 Comparison of productivity of threonine-producing genetically engineered bacteria

[0107]

[0108] Table 6 shows that the L-threonine production of strain MHZ-0221-12 was significantly higher than that of the starting strain. The L-threonine production of strain MHZ-0215-12 was 19 g / L, with a sugar-acid conversion rate of 22.3%, representing a 42.8% increase in yield and a 42.9% increase in conversion rate compared to the starting strain. The fermentation experiment results in the fermenter indicate that knocking out the ycjU gene in E. coli can significantly improve the strain's threonine production capacity.

[0109] 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. sequence list <110> Li Yan <120> Recombinant microorganisms for producing threonine, their construction methods, and methods for producing threonine. <130> KHP211117779.6 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 219 <212> PRT <213> Artificial Sequence <400> 1 Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp 1 5 10 15 Thr Ala His Leu His Phe Gln Ala Trp Gln Gln Ile Ala Ala Glu Ile 20 25 30 Gly Ile Ser Ile Asp Ala Gln Phe Asn Glu Ser Leu Lys Gly Ile Ser 35 40 45 Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly 50 55 60 Asp Phe Asn Ser Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu 65 70 75 80 Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro 85 90 95 Gly Ile Arg Ser Leu Leu Ala Asp Leu Arg Ala Gln Gln Ile Ser Val 100 105 110 Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu 115 120 125 Glu Leu Arg Glu Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys 130 135 140 Asn Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu 145 150 155 160 Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly 165 170 175 Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Ala 180 185 190 Gly Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Glu Ser Leu Thr 195 200 205 Trp Pro Arg Leu Ser Ala Phe Trp Gln Asn Val 210 215 <210> 2 <211> 660 <212> DNA <213> Artificial Sequence <400> 2 atgaaactgc aaggggtaat tttcgatctg gatggtgtaa tcaccgatac cgcgcatctg 60 catttccagg cgtggcagca gattgccgct gaaattggca tcagcattga tgcgcagttt 120 aacgaatccc taaaagggat cagccgcgat gagtctctgc ggcgcattct gcaacacggg 180 ggcaaagagg gcgactttaa ctcgcaggag agggcgcaac tggcgtatcg caaaaatctg 240 ctctatgtcc actcactacg cgagttgacg gtcaacgctg ttctacccgg cattcgctct 300 ttgctggcag atctccgtgc acagcagatc tcggttgggc tggcttctgt ctccctgaat 360 gcgccgacga ttttagcggc gctggagctg cgcgagtttt tcaccttctg cgcggatgct 420 tcccaactta aaaactcgaa accggacccg gaaatctttc tcgccgcctg tgcagggctg 480 ggcgtgccgc cgcaggcatg tatcggcatt gaagatgcgc aggcgggcat tgacgccatt 540 aacgccagcg gtatgcgctc ggtggggatc ggcgcgggct taaccggggc gcaattactg 600 ttgccttcaa cggaatcact cacctggccg cggttatcgg ccttctggca aaacgtatag 660 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 agtatctttc tcgccgcctg tgcgttttag agctagaaat agcaa 45 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <400> 4 gcacaggcgg cgagaaagat actagtatta tacctaggac tgagct 46 <210> 5 <211> 52 <212> DNA <213> Artificial Sequence <400> 5 accgctacca aacatcagga ggatgacaaa ggaatcaaca tggctcagct tt 52 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <400> 6 tgcacgcgca gcttggcatc aaggttag 28 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 gtgatgctca attacatgct gccgga 26 <210> 8 <211> 52 <212> DNA <213> Artificial Sequence <400> 8 aaagctgagc catgttgatt cctttgtcat cctcctgatg tttggtagcg gt 52 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <400> 9 ggatgattcg tttatggcta agccgg 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <400> 10 tcggcgcacc tacttgctga acaatc 26

Claims

1. Any of the following applications that reduce the expression and / or activity of β-phosphoglucose mutase YcjU: (1) Application in increasing the yield and / or conversion rate of threonine in Escherichia coli; (2) Application in constructing Escherichia coli for threonine production; The amino acid sequence of the β-phosphoglucose mutase YcjU is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The reduction in expression and / or enzyme activity is achieved through a combination of one or more of the following methods (1) and (2): (1) Insert, delete or replace one or more bases in the gene encoding the β-glucose mutase YcjU to reduce the expression level, enzyme activity or inactivation of the β-glucose mutase YcjU. (2) Replace the transcriptional or translational regulatory element of the gene encoding the β-phosphoglucose mutase YcjU with a regulatory element with lower activity so as to reduce its expression level and enzyme activity.

3. The application according to claim 1 or 2, characterized in that, The expression and / or activity of the β-phosphoglucose mutase YcjU are reduced by knocking down or knocking out the enzyme.

4. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli, compared with its originating strain, has reduced expression and / or enzyme activity of β-phosphoglucose mutase YcjU. The amino acid sequence of the β-phosphoglucose mutase YcjU is shown in SEQ ID NO.

1.

5. The recombinant Escherichia coli according to claim 4, characterized in that, The reduction in expression and / or enzyme activity is achieved through a combination of one or more of the following methods (1) and (2): (1) Insert, delete or replace one or more bases in the gene encoding the β-glucose mutase YcjU to reduce the expression level, enzyme activity or inactivation of the β-glucose mutase YcjU. (2) Replace the transcriptional or translational regulatory element of the gene encoding the β-phosphoglucose mutase YcjU with a regulatory element with lower activity so as to reduce its expression level and enzyme activity.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The expression and / or activity of the β-phosphoglucose mutase YcjU are reduced by knocking down or knocking out the enzyme.

7. The method for constructing recombinant Escherichia coli according to any one of claims 4 to 6, characterized in that, include: The expression and / or enzyme activity of the β-phosphoglucose mutase YcjU in the starting strain were reduced by genetic engineering methods.

8. The use of the recombinant Escherichia coli according to any one of claims 4 to 6 in the production of threonine or the selection of threonine-producing strains.

9. A method for producing threonine by fermentation, characterized in that, Cultivate the recombinant Escherichia coli according to any one of claims 4 to 6, and recover the threonine from the obtained culture medium.

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