A recombinant microorganism, its preparation method and application in threonine production

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant microorganism, a preparation method thereof and application of the recombinant microorganism in threonine production. Expression of tpx gene and / or yodD gene in the recombinant microorganism is inhibited. The present application finds that inhibition of tpx gene or yodD gene in a strain can effectively improve the ability of the strain to produce amino acids, and the threonine production capacity of Escherichia coli with tpx gene and yodD gene knocked out can reach 27.35 g / L, and the sugar acid conversion rate can reach 27.94%, which has important significance in the field of threonine production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant microorganism, its preparation method, and its application in threonine production. 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* as production strains. Specific examples include amino acid analogue-resistant mutants or auxotrophic strains such as those containing methionine, lysine, and isoleucine (Japanese Patent Application Publication No. 224684 / 83; Korean Patent Application Publication No. 8022 / 87). However, traditional mutagenesis breeding, due to random mutations, results in 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-yield threonine-producing strains are particularly important. In Chinese patent CN03811059.8 filed by CJ Corporation of South Korea in 2003, Escherichia coli was used to enhance the expression of the key gene thrABC for threonine synthesis by deleting a 39bp sequence from position -56 to -18 of the threonine operon sequence, thereby increasing threonine productivity by 22%. Kwang Ho Lee et al. (Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007; 3: 149) used systems metabolic engineering strategies to remove product feedback inhibition by mutating the genes thrA and lysC encoding aspartate kinases I and III, knocking out tdh and weakening ilvA to remove byproducts glycine and isoleucine, and providing more precursors for threonine synthesis by inactivating the competitive pathway genes metA and lysA. The resulting TH28C (pBRThrABCR3) strain produced 82.4 g / L of acid after 50 h of fermentation, with a sugar-acid conversion rate of 39.3%. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a recombinant microorganism, its preparation method, and its application in threonine production.

[0005] In a first aspect, the present invention provides a recombinant microorganism in which the expression of the tpx gene and / or the yodD gene is suppressed.

[0006] Further, the nucleotide sequence of the tpx gene is as shown in SEQ ID NO.1 or has at least 60% sequence identity with SEQ ID NO.1; and / or, the nucleotide sequence of the yodD gene is as shown in SEQ ID NO.3 or has at least 60% sequence identity with SEQ ID NO.3.

[0007] The amino acid sequence encoded by SEQ ID NO.1 is shown in SEQ ID NO.2; the amino acid sequence encoded by SEQ ID NO.3 is shown in SEQ ID NO.4.

[0008] Furthermore, the starting strain of the recombinant microorganism is one or more of Escherichia coli, Bacillus subtilis, or Serratia marcescens.

[0009] Preferably, the starting strain of the recombinant microorganism is MHZ-0215-2 or MHZ-0217-4.

[0010] Among them, strain MHZ-0215-2 is disclosed in Chinese Patent 201611250306.8, and strain MHZ-0217-4 is disclosed in Chinese Patent 202011388854.3.

[0011] Secondly, the present invention provides a method for preparing the recombinant microorganism, comprising:

[0012] Knockout of the tpx gene and / or yodD gene in microorganisms using CRISPR-Cas9 technology.

[0013] The present invention further provides a microbial preparation comprising the recombinant microorganism.

[0014] The present invention further provides the application of the recombinant microorganism or the microbial preparation in the production of amino acids; preferably, its application in the production of threonine.

[0015] The present invention further provides the application of the recombinant microorganism or the microbial preparation in increasing threonine production.

[0016] Thirdly, the present invention provides a method for increasing the amino acid yield of a bacterial strain, comprising inhibiting the expression of the tpx gene and / or the yodD gene in the strain.

[0017] Furthermore, the strain is one or more of Escherichia coli, Bacillus subtilis, or Serratia marcescens.

[0018] Further, the nucleotide sequence of the tpx gene is as shown in SEQ ID NO.1 or has at least 60% sequence identity with SEQ ID NO.1; and / or, the nucleotide sequence of the yodD gene is as shown in SEQ ID NO.3 or has at least 60% sequence identity with SEQ ID NO.3.

[0019] The present invention has the following beneficial effects:

[0020] This invention obtains a recombinant microorganism by knocking out the tpx gene and / or yodD gene in a microorganism. The recombinant microorganism significantly increases the threonine production. After simultaneously knocking out the tpx gene and yodD gene in Escherichia coli, its threonine production capacity can reach 27.35 g / L, and the sugar-acid conversion rate can reach 27.94%, which is of great significance in the field of threonine production. Detailed Implementation

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0022] The genome editing of Escherichia coli 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).

[0023] 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.

[0024] In the following examples, all reagents used were commercially available. The parent strains of the high-conversion threonine production strains provided by the present invention are MHZ-0215-2 and MHZ-0217-4, belonging to W3110 (Escherichia).

[0025] The primer sequences used in the examples are shown in the table below.

[0026] Table 1 shows the primers used in the examples.

[0027]

[0028]

[0029] Example 1: Constructing the tpx gene-deleted strain MHZ-0221-6 using MHZ-0215-2 as the substrate bacteria.

[0030] 1. Construction of pTargetF-N20(tpx) plasmid and Donor DNA-1

[0031] 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-sgRNA(tpx)-F / pTF-sgRNA(tpx)-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(tpx), which was then identified by PCR and sequenced for verification.

[0032] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the tpx-UF / tpx-UR primer pair;

[0033] Step 3: Using the W3110 genome as a template, select the tpx-DF / tpx-DR primer pair to amplify the downstream homologous arm ②;

[0034] Step 4: Using ① and ② as templates, select the tpx-UF / tpx-DR primer pair to amplify the up-down full-length fragment, also known as Donor DNA-1.

[0035] 2. Preparation and electroporation of competent cells

[0036] 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 MHZ-0215-2 competent cells (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0037] Step 2: Pick a single colony of MHZ-0215-2 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4 μL of the solution (the method for preparing competent cells is described in Molecular Cloning III).

[0038] Step 3: Electroporate the pTargetF-N20(tpx) plasmid and the Donor DNA-1 constructed in Step 1 into MHZ-0215-2(pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0039] 3. Recombination Verification

[0040] Step 1: Use primer pair tpx-F / tpx-R to perform colony PCR verification on the above single colonies;

[0041] Step 2: Amplify the target fragment using primer pairs tpx-F / tpx-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0042] 4. Loss of constructing related plasmids

[0043] Step 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 the colony onto an LB agar plate containing kanamycin.

[0044] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(tpx) plasmid has been lost.

[0045] Step 3: Pick positive colonies that have lost the pTargetF-N20(tpx) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0046] Step 4: Select a single colony and spot it onto 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 MHZ-0221-6 (tpx deletion) strain is obtained.

[0047] Example 2: Constructing the yodD gene-deleted strain MHZ-0221-7 using MHZ-0215-2 as the substrate bacteria.

[0048] 1. Construction of pTargetF-N20(yodD) plasmid and Donor DNA-2

[0049] 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-sgRNA(yodD)-F / pTF-sgRNA(yodD)-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(yodD), which was then identified by PCR and sequenced for verification.

[0050] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the yodD-UF / yodD-UR primer pair;

[0051] Step 3: Using the W3110 genome as a template, select the yodD-DF / yodD-DR primer pair to amplify the downstream homologous arm ②;

[0052] Step 4: Using ① and ② as templates, select the yodD-UF / yodD-DR primer pair to amplify the up-down full-length fragment, also known as Donor DNA-2.

[0053] 2. Preparation and electroporation of competent cells

[0054] 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 MHZ-0215-2 competent cells (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0055] Step 2: Pick a single colony of MHZ-0215-2 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0056] Step 3: Electroporate the pTargetF-N20 (yodD) plasmid and the Donor DNA-2 constructed in Step 1 into MHZ-0215-2 (pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0057] 3. Recombination Verification

[0058] Step 1: Use primer pair yodD-F / yodD-R to perform colony PCR verification on the above single colonies;

[0059] Step 2: Amplify the target fragment using primer pairs yodD-F / yodD-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0060] 4. Loss of constructing related plasmids

[0061] Step 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 the colony onto an LB agar plate containing kanamycin.

[0062] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(yodD) plasmid has been lost.

[0063] Step 3: Pick positive colonies that have lost the pTargetF-N20(yodD) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0064] Step 4: Select a single colony and spot it onto 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 MHZ-0221-7 (yodD deletion) strain is obtained.

[0065] Example 3: Constructing strain MHZ-0221-8 with simultaneous deletion of the tpx and yodD genes using MHZ-0215-2 as the chassis bacteria.

[0066] 1. Preparation and electroporation of competent cells

[0067] Step 1: Electrotransfer 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 the MHZ-0221-1 competent cells constructed in Example 1 (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0068] Step 2: Pick a single colony of MHZ-0221-1 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 After 0.4, prepare electroporation competent cells (the method for preparing competent cells is as described in Molecular Cloning III). Step 3: Electroporate the pTargetF-N20 (yodD) plasmid and Donor DNA-2 obtained in Example 2 into MHZ-0221-1 (pCas) competent cells simultaneously (electroplation conditions: 2.5kV, 200Ω, 25μF), plate them on LB agar plates containing spectinomycin and kanamycin, and incubate at 30°C until single colonies are visible.

[0069] 2. Recombination Verification

[0070] Step 1: Use primer pair yodD-F / yodD-R to perform colony PCR verification on the above single colonies;

[0071] Step 2: Amplify the target fragment using primer pairs yodD-F / yodD-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0072] 3. Loss of constructing related plasmids

[0073] Step 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 the colony onto an LB agar plate containing kanamycin.

[0074] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(yodD) plasmid has been lost.

[0075] Step 3: Pick positive colonies that have lost the pTargetF-N20(yodD) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0076] Step 4: Select a single colony and spot it onto 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 MHZ-0221-8 strain (tpx and yodD are simultaneously deleted) is obtained.

[0077] Example 4: Constructing a strain MHZ-0221-9 with the tpx gene deleted using MHZ-0217-4 as the substrate bacteria.

[0078] 1. Construction of pTargetF-N20(tpx) plasmid and Donor DNA-1

[0079] 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-sgRNA(tpx)-F / pTF-sgRNA(tpx)-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(tpx), which was then identified by PCR and sequenced for verification.

[0080] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the tpx-UF / tpx-UR primer pair;

[0081] Step 3: Using the W3110 genome as a template, select the tpx-DF / tpx-DR primer pair to amplify the downstream homologous arm ②;

[0082] Step 4: Using ① and ② as templates, select the tpx-UF / tpx-DR primer pair to amplify the up-down full-length fragment, also known as Donor DNA-1.

[0083] 2. Preparation and electroporation of competent cells

[0084] 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 MHZ-0217-4 competent cells (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0085] Step 2: Pick a single colony of MHZ-0217-4 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0086] Step 3: Electroporate the pTargetF-N20(tpx) plasmid and the Donor DNA-1 constructed in Step 1 into MHZ-0217-4(pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0087] 3. Recombination Verification

[0088] Step 1: Use primer pair tpx-F / tpx-R to perform colony PCR verification on the above single colonies;

[0089] Step 2: Amplify the target fragment using primer pairs tpx-F / tpx-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0090] 4. Loss of constructing related plasmids

[0091] Step 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 the colony onto an LB agar plate containing kanamycin.

[0092] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(tpx) plasmid has been lost.

[0093] Step 3: Pick positive colonies that have lost the pTargetF-N20(tpx) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0094] Step 4: Select a single colony and spot it onto 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 MHZ-0221-9 (tpx deletion) strain is obtained.

[0095] Example 5: Constructing a yodD gene-deleted strain MHZ-0221-10 using MHZ-0217-4 as the substrate bacteria.

[0096] 1. Construction of pTargetF-N20(yodD) plasmid and Donor DNA-2

[0097] 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-sgRNA(yodD)-F / pTF-sgRNA(yodD)-R primer pair was selected to amplify the pTF linear plasmid containing N20. This linear plasmid was assembled at 37℃ using the seamless assembly ClonExpress kit, and then transformed into Trans1-T1 competent cells to obtain pTargetF-N20(yodD), which was then identified by PCR and sequenced for verification.

[0098] Step 2: Using the W3110 genome as a template, the upstream homologous arm ① was amplified by selecting the yodD-UF / yodD-UR primer pair;

[0099] Step 3: Using the W3110 genome as a template, select the yodD-DF / yodD-DR primer pair to amplify the downstream homologous arm ②;

[0100] Step 4: Using ① and ② as templates, select the yodD-UF / yodD-DR primer pair to amplify the up-down full-length fragment, also known as Donor DNA-2.

[0101] 2. Preparation and electroporation of competent cells

[0102] 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 MHZ-0217-4 competent cells (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0103] Step 2: Pick a single colony of MHZ-0217-4 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0104] Step 3: Electroporate the pTargetF-N20 (yodD) plasmid and the Donor DNA-2 constructed in Step 1 into MHZ-0217-4 (pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30℃ until single colonies are visible.

[0105] 3. Recombination Verification

[0106] Step 1: Use primer pair yodD-F / yodD-R to perform colony PCR verification on the above single colonies;

[0107] Step 2: Amplify the target fragment using primer pairs yodD-F / yodD-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0108] 4. Loss of constructing related plasmids

[0109] Step 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 the colony onto an LB agar plate containing kanamycin.

[0110] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(yodD) plasmid has been lost.

[0111] Step 3: Pick positive colonies that have lost the pTargetF-N20(yodD) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃.

[0112] Step 4: Select a single colony and spot it onto 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 MHZ-0221-10 (yodD deletion) strain is obtained.

[0113] Example 6: Constructing strain MHZ-0221-11 with simultaneous deletion of the tpx and yodD genes using MHZ-0217-4 as the substrate bacteria.

[0114] 1. Preparation and electroporation of competent cells

[0115] Step 1: Electrotransfer 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 the MHZ-0222-1 competent cells constructed in Example 4 (the transformation method and competent cell preparation method are both based on Molecular Cloning III);

[0116] Step 2: Pick a single colony of MHZ-0222-1 (pCas) and place it in a 5 mL LB tube containing kanamycin and 10 mM arabinose. Incubate at 30°C and 200 rpm until OD500. 650 Electrocompetent cells were prepared after 0.4% concentration (the method for preparing competent cells is described in Molecular Cloning III).

[0117] Step 3: Electroporate the pTargetF-N20 (yodD) plasmid and Donor DNA-2 obtained in Example 2 into MHZ-0222-1 (pCas) competent cells simultaneously (electroporation conditions: 2.5kV, 200Ω, 25μF), spread them on LB plates containing spectinomycin and kanamycin, and incubate at 30°C until single colonies are visible.

[0118] 2. Recombination Verification

[0119] Step 1: Use primer pair yodD-F / yodD-R to perform colony PCR verification on the above single colonies;

[0120] Step 2: Amplify the target fragment using primer pairs yodD-F / yodD-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0121] 3. Loss of constructing related plasmids

[0122] Step 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 the colony onto an LB agar plate containing kanamycin.

[0123] Step 2: Pick a single colony and spot it onto LB agar plates containing kanamycin, spectinomycin and kanamycin alone, and incubate overnight at 30°C. If it cannot grow on LB agar plates containing kanamycin or spectinomycin, but grows on LB agar plates containing kanamycin, it indicates that the pTargetF-N20(yodD) plasmid has been lost.

[0124] Step 3: Pick positive colonies with missing pTargetF-N20(yodD) plasmid, inoculate them into antibiotic-free LB tubes, incubate at 42℃ for 8 hours, then streak them onto LB plates and incubate overnight at 37℃; Step 4: Pick single colonies and spot them onto LB plates containing kanamycin and antibiotic-free LB plates. If the colony cannot grow on the LB plate containing kanamycin but grows on the antibiotic-free LB plate, it indicates that the pCas plasmid is missing, and the strain MHZ-0221-11 (tpx and yodD are both missing) is obtained.

[0125] The threonine-producing genetically modified strains obtained in Examples 1-6 are shown in Table 2.

[0126] Table 2 Genetically engineered bacteria constructed in this invention

[0127]

[0128]

[0129] Example 7: Verification of L-threonine-producing genetically engineered bacteria through shake-flask fermentation

[0130] Step 1: Take eight strains of bacteria (MHZ-0215-2, MHZ-0221-6, MHZ-0221-7, MHZ-0221-8, MHZ-0217-4, MHZ-0221-9, MHZ-0221-10, and MHZ-0221-11) from the cryopreservation tubes, streak them on LB plates for activation, and incubate at 37°C for 18-24 hours.

[0131] Step 2: Scrape a loopful of bacterial cells from the plate and inoculate it into a 500 mL 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;

[0132] Step 3: Transfer 2 mL of seed culture to a 500 mL 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 flasks were repeated three times, and the average values ​​of the acid production and conversion rate are shown in Table 5.

[0133] Table 3 Seed culture medium (g / L)

[0134]

[0135]

[0136] Table 4 Fermentation medium (g / L)

[0137] Element concentration glucose 85 Corn syrup 6 Soybean meal hydrolysate 7.7 Magnesium sulfate heptahydrate 0.5 <![CDATA[KH2PO4]]> 1.0 Aspartic acid 10 <![CDATA[FeSO4、MnSO4]]> 30mg / L Biotin 50μg Thiamine 500μg pH 7.2

[0138] Table 5 Comparison of productivity of threonine-producing genetically engineered bacteria

[0139]

[0140] Note: * indicates a p-value < 0.01, meaning there is a significant difference compared to the control.

[0141] As shown in Table 5, the L-threonine production of the novel Escherichia coli strain described in this invention is higher than that of the control strain.

[0142] Among them, the threonine yield of the modified strain MHZ-0221-6, which was constructed using MHZ-0215-2 as the substrate bacteria and lacked the tpx gene, was 16.32 g / L, with a shake-flask conversion rate of 19.20%. The yield was 18.26% higher than that of the original strain, and the conversion rate was 18.30% higher.

[0143] The threonine yield of the yodD gene-deleted mutant strain MHZ-0221-7 was 16.64 g / L, with a shake-flask conversion rate of 19.58%, which was 20.58% higher than that of the original strain and 20.64% higher than that of the original strain.

[0144] The modified strain MHZ-0221-8, which simultaneously deletes the tpx and yodD genes, produced 17.83 g / L of threonine, with a shake-flask conversion rate of 20.97%, representing a 29.20% increase in yield and a 29.21% increase in conversion rate compared to the original strain.

[0145] The threonine yield of the modified strain MHZ-0221-9, which was constructed using MHZ-0217-4 as the substrate strain and lacking the tpx gene, was 22.38 g / L, with a shake-flask conversion rate of 26.33%. The yield and conversion rate were both 10.63% higher than those of the original strain.

[0146] The threonine yield of the yodD gene-deleted mutant strain MHZ-0221-10 was 22.36 g / L, with a shake-flask conversion rate of 26.31%, which was 10.53% higher than that of the original strain and 10.55% higher than that of the original strain.

[0147] The modified strain MHZ-0221-11, which simultaneously deletes the tpx and yodD genes, produced 23.30 g / L of threonine, with a shake-flask conversion rate of 27.41%, representing a 15.18% increase in yield and a 15.17% increase in conversion rate compared to the original strain.

[0148] The shake-flask results show that deletion of the tpx and yodD genes can significantly improve the production capacity of recombinant Escherichia coli threonine.

[0149] 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> Meihua (Shanghai) Biotechnology Co., Ltd. <120> A recombinant microorganism, its preparation method, and its application in threonine production. <130> KHP211117785.3 <160> twenty two <170> SIPOSequenceListing 1.0 <210> 1 <211> 507 <212> DNA <213> Artificial Sequence <400> 1 atgtcacaaa ccgttcattt ccagggcaac ccggttacag tcgccaattc catcccgcag 60 gcgggtagca aagcgcagac ttttactctc gtggcaaaag atctgtctga cgtcaccctc 120 ggtcagtttg cgggtaaacg caaagtgctg aacattttcc cgagtattga taccggtgtt 180 tgcgccgcat cagtacgtaa gtttaaccaa ctggcaactg agatcgacaa caccgttgtg 240 ctgtgtatct ctgccgatct gccgttcgcc cagtctcgtt tctgcggcgc agaaggtctg 300 aacaacgtta tcaccctctc cactttccgt aacgctgaat ttctgcaagc ttacggtgtg 360 gcaattgctg atggcccact gaaaggtctg gcagcgcgtg ccgttgtggt tattgacgaa 420 aatgacaatg tgattttcag ccagctggtg gatgaaatca ccaccgagcc ggattacgaa 480 gcagctctgg ctgtactgaa agcataa 507 <210> 2 <211> 168 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Gln Thr Val His Phe Gln Gly Asn Pro Val Thr Val Ala Asn 1 5 10 15 Ser Ile Pro Gln Ala Gly Ser Lys Ala Gln Thr Phe Thr Leu Val Ala 20 25 30 Lys Asp Leu Ser Asp Val Thr Leu Gly Gln Phe Ala Gly Lys Arg Lys 35 40 45 Val Leu Asn Ile Phe Pro Ser Ile Asp Thr Gly Val Cys Ala Ala Ser 50 55 60 Val Arg Lys Phe Asn Gln Leu Ala Thr Glu Ile Asp Asn Thr Val Val 65 70 75 80 Leu Cys Ile Ser Ala Asp Leu Pro Phe Ala Gln Ser Arg Phe Cys Gly 85 90 95 Ala Glu Gly Leu Asn Asn Val Ile Thr Leu Ser Thr Phe Arg Asn Ala 100 105 110 Glu Phe Leu Gln Ala Tyr Gly Val Ala Ile Ala Asp Gly Pro Leu Lys 115 120 125 Gly Leu Ala Ala Arg Ala Val Val Val Ile Asp Glu Asn Asp Asn Val 130 135 140 Ile Phe Ser Gln Leu Val Asp Glu Ile Thr Thr Glu Pro Asp Tyr Glu 145 150 155 160 Wing Wing Leo Wing Val Leo Lys Wing 165 <210> 3 <211> 228 <212> DNA <213> Artificial Sequence <400> 3 atgaaaaccg caaaagagta cagcgatacc gcaaaacgtg aggtcagcgt cgatgtcgat 60 gccctgctgg cggcgatcaa tgaaattagc gaaagcgaag ttcatcgcag ccagaacgat 120 tctgaacacg ttagcgtcga tggacgtgaa tatcatacat ggcgtgaatt ggcggatgcc 180 ttcgaactgg atattcatga cttcagcgtc tctgaagtga atcgttga 228 <210> 4 <211> 75 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Thr Ala Lys Glu Tyr Ser Asp Thr Ala Lys Arg Glu Val Ser 1 5 10 15 Val Asp Val Asp Ala Leu Leu Ala Ala Ile Asn Glu Ile Ser Glu Ser 20 25 30 Glu Val His Arg Ser Gln Asn Asp Ser Glu His Val Ser Val Asp Gly 35 40 45 Arg Glu Tyr His Thr Trp Arg Glu Leu Ala Asp Ala Phe Glu Leu Asp 50 55 60 Ile His Asp Phe Ser Val Ser Glu Val Asn Arg 65 70 75 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <400> 5 agtcgggttg ccctggaaat gaagttttag agctagaaat agcaa 45 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <400> 6 ttcatttcca gggcaacccg actagtatta tacctaggac tgagct 46 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 cgggttgccc tggaaatgaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 accgatgaca accgtctgtg 20 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <400> 9 atattaaatt atgctgatta tctttcctgt ttaca 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <400> 10 acaggaaaga taatcagcat aatttaatat gcctg 35 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 atcgtctgaa cgtgttgacg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 atccgctcac tgataagatg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 gtgatttgcg ctacgcagaa 20 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <400> 14 agtatggacg tgaatatcat acagttttag agctagaaat agcaa 45 <210> 15 <211> 46 <212> DNA <213> Artificial Sequence <400> 15 tgtatgatat tcacgtccat actagtatta tacctaggac tgagct 46 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 atggacgtga atatcataca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 aagaccgttt catcgcataa 20 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <400> 18 tcagagacgc tgaagcatct ttcctcgcaa ccgtt 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <400> 19 ttgcgaggaa agatgcttca gcgtctctga agtga 35 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 accttgtagc cccaacgttt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <400> 21 gaaccgaatc gagtatgttg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 atgatgcgtg gaaaaccgtc 20

Claims

1. A recombinant microorganism, characterized in that, The tpx and yodD genes were knocked out in the recombinant microorganism; the recombinant microorganism was based on Escherichia coli as the starting strain. The nucleotide sequence of the tpx gene is shown in SEQ ID NO.1; the nucleotide sequence of the yodD gene is shown in SEQ ID NO.

3.

2. The method for preparing the recombinant microorganism according to claim 1, characterized in that, include: The tpx and yodD genes in microorganisms were knocked out using CRISPR-Cas9 technology.

3. A microbial preparation, characterized in that, The microbial preparation contains the recombinant microorganism of claim 1, or the recombinant microorganism prepared by the preparation method of claim 2.

4. Application of recombinant microorganisms or microbial preparations in the production of threonine; wherein the tpx gene and / or yodD gene are knocked out in the recombinant microorganisms; wherein the recombinant microorganisms are derived from Escherichia coli strains; The nucleotide sequence of the tpx gene is shown in SEQ ID NO.1; the nucleotide sequence of the yodD gene is shown in SEQ ID NO.3; The microbial preparation contains the recombinant microorganism.

5. Application of recombinant microorganisms or microbial preparations in increasing threonine production; The tpx gene and / or yodD gene are knocked out in the recombinant microorganism; the recombinant microorganism is based on Escherichia coli as the starting strain. The nucleotide sequence of the tpx gene is shown in SEQ ID NO.1; the nucleotide sequence of the yodD gene is shown in SEQ ID NO.3; The microbial preparation contains the recombinant microorganism.

6. A method for increasing the threonine yield of a bacterial strain, characterized in that, include: Knock out the tpx gene and / or yodD gene in the strain; The strain is Escherichia coli, and the nucleotide sequence of the tpx gene is shown in SEQ ID NO.1; the nucleotide sequence of the yodD gene is shown in SEQ ID NO.3.

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