A recombinant strain and method for producing amino acids thereof

Abstract

The present invention belongs to the field of recombinant microbial technology, and specifically relates to a recombinant bacterial strain and a method for producing amino acids therefrom. The present invention provides a waaA mutation, wherein the waaA mutation results in an amino acid change in the catalytic region of the protein encoded by waaA, specifically a mutation of the proline at position 48 of the protein sequence encoded by waaA to a hydroxyl-containing amino acid, or a mutation of the nucleotide sequence of waaA where the nucleotide at position 142 is replaced by cytosine to thymine or adenine. The recombinant strain containing the waaA mutation provided by the present invention has the ability to produce high levels of threonine or isoleucine.

Description

Technical Field

[0001] The present invention relates to the field of recombinant microorganisms, and in particular to constructing a strain containing a mutant waaA or a nucleotide sequence encoding the mutant waaA to produce threonine acid. Background Art

[0002] L-threonine is one of the eight essential amino acids for human and animal growth and is widely used in feed, food additives, and pharmaceutical auxiliary material preparation. Currently, L-threonine is primarily produced through microbial fermentation. A variety of bacteria can be used for L-threonine production, such as mutants induced from wild-type Escherichia coli, Corynebacterium, and Serratia. Specific examples include mutants resistant to amino acid analogs or auxotrophs for methionine, lysine, and isoleucine. However, traditional mutagenesis breeding, due to random mutations that result in slow growth and the production of numerous byproducts, makes it difficult to obtain high-yield strains.

[0003] With the increasing global demand for threonine, the construction and modification of high-threonine-producing strains is particularly important. In Chinese Patent CN03811059.8, filed in 2003 by CJ Corporation of South Korea, a 39-bp deletion from positions 56 to 18 of the threonine operon sequence in Escherichia coli was used to enhance expression of the key thrABC gene for threonine biosynthesis, resulting in a 22% increase in threonine productivity. Kwang Ho Lee et al. (Kwang Ho Lee et al., Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007;3:149) employed a systems metabolic engineering strategy. By mutating the thrA and lysC genes encoding aspartate kinases I and III, they eliminated product feedback inhibition. They also eliminated glycine and isoleucine byproducts by knocking out tdh and weakening ilvA. Furthermore, they inactivated the competing pathway genes metA and lysA to provide more precursors for threonine synthesis. The resulting strain, TH28C (pBRThrABCR3), produced 82.4 g / L of acid in 50 hours of fermentation, with a sugar-to-acid conversion rate of 39.3%. In Chinese Patent No. 201611250306.8 filed by Meihua Group, strain MHZ-0215-2 was obtained by enhancing the pntAB genes and heterologously introducing the pyc gene. This strain achieved a threonine yield of 12.4 g / L with a conversion rate of approximately 16.2% and was free of plasmid burden. Summary of the Invention

[0004] The present invention aims to increase threonine production. Specifically, the present invention provides a mutant waaA that, when expressed in Escherichia coli, can increase the ability of E. coli to synthesize L-threonine. The research process of the present invention is as follows:

[0005] After ultraviolet mutagenesis of W3110, the present invention obtained a mutagenized strain with high threonine production. Whole genome sequencing of the strain revealed a mutation at position 48 in its waaA. To further verify whether the mutation was beneficial to threonine production, the mutation was introduced into the threonine-producing strain MHZ-0215-2, and it was found that the threonine production was greatly improved. Therefore, it was determined that the mutation at this site was beneficial to the improvement of threonine production.

[0006] To explore the underlying mechanism by which mutations at this site are associated with increased threonine production, the function of the enzyme encoded by the mutant waaA was investigated. waaA encodes 3-deoxy-D-mannitol-octanoyltransferase, which participates in the biosynthesis of extracellular membrane lipopolysaccharide. Within its protein sequence, positions 3-23 contain a signal anchor sequence, positions 31-211 comprise the catalytic domain of 3-deoxy-D-mannitol-octanoyltransferase, and positions 268-269, 309-311, and 35-338 comprise the substrate binding region. Proline at position 48 is located within the catalytic domain of the enzyme, so mutations at this position alter the enzyme's catalytic function, further impacting threonine production. Proline, serine, and threonine are all hydroxyl-containing amino acids, with proline being nonpolar and serine and threonine being polar. Mutating amino acid position 48 from proline to serine or threonine does not alter the side chain group, but does alter the polarity of the enzyme, thereby altering its properties.

[0007] Therefore, in a first aspect, the present invention provides a mutant waaA, wherein the mutant waaA refers to a change in the amino acid at position 48 of the amino acid sequence of the protein encoded by the wild-type waaA.

[0008] Specifically, the amino acid sequence of the protein encoded by the mutant waaA of the present invention is as shown in SEQ ID No. 11, wherein the proline at position 48 is mutated to an amino acid containing a hydroxyl group;

[0009] Furthermore, the amino acid sequence of the protein encoded by the mutant waaA is as shown in SEQ ID No.11, in which position 48 of the amino acid sequence is mutated from proline to serine or threonine, or the amino acid sequence of the protein encoded by the mutant waaA is as shown in SEQ ID No.12 or SEQ ID No.13.

[0010] The mutant waaA provided by the present invention is a nucleotide sequence as shown in SEQ ID No. 14, in which position 142 of the cytosine is mutated to thymine or adenine, or the nucleotide sequence of the mutant waaA is shown in SEQ ID No. 15 or SEQ ID No. 16.

[0011] According to the understanding of those skilled in the art, the present invention seeks to protect the use of the above-mentioned mutant waaA or the above-mentioned DNA molecule in the production of L-threonine or isoleucine.

[0012] In a second aspect, the present invention provides a method for constructing a recombinant strain that produces high L-threonine yield, comprising:

[0013] (1) Using the pTargetF plasmid as a template, the pTF-sgRNA-F / pTF-sgRNA-R primer pair was used to amplify the pTF linear plasmid carrying N20, and the pTF linear plasmid was transformed into Trans1-T1 competent cells to obtain pTargetF-N20(waaA);

[0014] (2) Using the W3110 genome as a template, the upstream homology arm was amplified using the waaA-UF / waaA C142A-UR primer pair or the waaA-UF / waaAC142T-UR primer pair, and the downstream homology arm was amplified using the waaA C142A-DF / waaA-DR primer pair or the waaA C142T-DF / waaA-DR primer pair; using the upstream and downstream homology arms as templates, the waaA-UF / waaA-DR primer pair was used to amplify the full-length waaAC142A fragment or the full-length waaA C142T fragment, also known as Donor DNA;

[0015] (3) The pCas plasmid was electroporated into MHZ-0215-2 competent cells, and the pTargetF-N20 (waaA) plasmid and the Donor DNA constructed in (2) were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells.

[0016] Among them, the nucleotide sequence of the primer pair pTF-sgRNA-F is shown in SEQ ID No.1, the nucleotide sequence of pTF-sgRNA-R is shown in SEQ ID No.2; the nucleotide sequence of waaA-UF is shown in SEQ ID No.5; the nucleotide sequence of waaA-DR is shown in SEQ ID No.6; the nucleotide sequence of waaA C142T-UR is shown in SEQ ID No.7; the nucleotide sequence of waaA C142T-DF is shown in SEQ ID No.8; the nucleotide sequence of waaA C142A-UR is shown in SEQ ID No.9; and the nucleotide sequence of waaA C142A-DF is shown in SEQ ID No.10.

[0017] In a third aspect, the present invention provides a recombinant bacterium containing the aforementioned mutant waaA or constructed using the aforementioned construction method.

[0018] Furthermore, the recombinant bacteria provided by the present invention are Escherichia coli, Corynebacterium glutamicum or Serratia.

[0019] In a fourth aspect, the present invention provides a method for producing threonine or isoleucine, wherein the threonine or isoleucine is obtained by fermentation using the above-mentioned recombinant bacteria.

[0020] According to the understanding of those skilled in the art, the present invention also seeks to protect the use of the aforementioned mutant waaA or the aforementioned recombinant bacteria in increasing threonine production.

[0021] Threonine can be degraded intracellularly to produce glycine and isoleucine. Therefore, those skilled in the art can enhance the synthesis of isoleucine by strengthening genes involved in the threonine-to-isoleucine synthesis pathway. The method provided by the present invention, which facilitates increased threonine production, also facilitates the production of its downstream product, isoleucine.

[0022] Therefore, the present invention also seeks to protect the use of the aforementioned mutant waaA or the aforementioned recombinant bacteria in increasing isoleucine production.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a recombinant strain that produces high levels of threonine or isoleucine. The recombinant strain has a threonine yield of 17.59-18.23 g / L, which is 27.46-32.1% higher than that of the wild-type strain. The present invention demonstrates that amino acid changes in the catalytic region of the waaA-encoded protein or mutation of position 142 of the waaA nucleotide sequence from cytosine to thymine or adenine significantly increase threonine production in the recombinant strain. DETAILED DESCRIPTION

[0025] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0027] The present invention discloses a method for constructing a strain with high threonine production and a method for producing threonine. The present invention is further described below with reference to the following examples. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] The present invention uses MHZ-0215-2 (strain collection number CGMCC No. 13403) as the starting strain and performs relevant modifications on its genome, mainly through waaA mutation.

[0029] 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).

[0030] In the following examples, the final concentration of kanamycin in the culture medium is 50 μg / mL, and the final concentration of spectinomycin in the culture medium is 50 μg / ml.

[0031] In the following examples, all reagents used are commercially available. The parent strain of the high-conversion threonine production strain provided by the present invention is MHZ-0215-2, which belongs to the genus W3110 (Escherichia).

[0032] The primer sequences used in the examples are shown in Table 1 below.

[0033] Table 1 Primer sequence list

[0034]

[0035]

[0036] Example 1 Preparation of strain MHZ-0221-13 in which nucleotide 142 of waaA is replaced by cytosine to thymine

[0037] (1) Construction of pTargetF-N20 (waaA C142T) plasmid and Donor DNA

[0038] Step 1: Using the pTargetF plasmid as a template (derived 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 with N20, and the seamless assembly ClonExpress kit was used to assemble this linear plasmid at 37°C. Subsequently, Trans1-T1 competent cells were transformed to obtain pTargetF-N20 (waaA), which was then identified by PCR and verified by sequencing. Step 2: Using the W3110 genome as a template, the waaA-UF / waaA C142T-UR primer pair was selected to amplify the upstream homology arm ①. Step 3: Using the W3110 genome as a template, the waaA The C142T-DF / waaA-DR primer pair amplifies the downstream homology arm ②; Step 4: Using ① and ② as templates, select the waaA-UF / waaA-DR primer pair to amplify the full-length waaA C142T fragment, also known as Donor DNA.

[0039] (2) Preparation of competent cells and electroporation

[0040] Step 1: Electroporate the pCas plasmid (derived 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 referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) into a 5 mL LB tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30°C and 200 rpm until the OD 650After the p-value is 0.4, prepare electrocompetent cells (refer to "Molecular Cloning III" for competent cell preparation). Step 3: Simultaneously electrotransform the pTargetF-N20 (waaA) plasmid and the Donor DNA constructed in (1) into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0041] (3) Recombination verification

[0042] Step 1: Use primer pair waaA-F / waaA-R to perform colony PCR verification on the above single colony; Step 2: Use primer pair waaA-F / waaA-R to amplify the target fragment, and send the amplified product for sequencing to verify the integrity of the sequence.

[0043] (4) Construction-related plasmids are lost

[0044] Step 1: Pick a single colony that has been verified to be correct by sequencing and inoculate it into 5 mL of kanamycin and a final concentration of 0.5 mM Step 2: Pick a single colony and spot it on an LB plate containing kanamycin, spectinomycin and kanamycin alone, and culture it overnight at 30°C. If it cannot grow on the LB plate containing kanamycin and spectinomycin, but grows on the LB plate containing kanamycin, it indicates that the pTargetF-N20 (waaA) plasmid has been lost. Step 3: Pick a positive colony that has lost the pTargetF-N20 (waaA) plasmid, inoculate it into an LB tube without antibiotics, culture it at 42°C for 8 hours, then streak it on an LB plate and culture it overnight at 37°C. Step 4: Pick a single colony and spot it on an LB plate containing kanamycin and an LB plate without antibiotics. If it cannot grow on the LB plate containing kanamycin, but grows on the LB plate without antibiotics, it indicates that the pCas plasmid has been lost, and the MHZ-0221-13 (waaA C142T) strain is obtained.

[0045] Example 2 Preparation of strain MHZ-0221-14 in which nucleotide 142 of waaA is replaced by cytosine to adenine

[0046] (1) Construction of pTargetF-N20 (waaA C142A) plasmid and Donor DNA

[0047] Step 1: Using the pTargetF plasmid as a template (derived 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 with N20, and the seamless assembly ClonExpress kit was used to assemble this linear plasmid at 37°C. Subsequently, Trans1-T1 competent cells were transformed to obtain pTargetF-N20 (waaA), which was then identified by PCR and verified by sequencing. Step 2: Using the W3110 genome as a template, the waaA-UF / waaA C142A-UR primer pair was selected to amplify the upstream homology arm ①. Step 3: Using the W3110 genome as a template, the waaA The C142A-DF / waaA-DR primer pair amplifies the downstream homology arm ②; Step 4: Using ① and ② as templates, select the waaA-UF / waaA-DR primer pair to amplify the full-length waaA C142A fragment, also known as Donor DNA.

[0048] (2) Preparation of competent cells and electroporation

[0049] Step 1: The pCas plasmid (derived 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 competent cell preparation method were both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) into a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30°C 200 r / min until the OD650 is 0.4, and then prepare electroporated competent cells (the competent cell preparation method is referred to "Molecular Cloning III"). Step 3: The pTargetF-N20 (waaA) plasmid and the Donor DNA constructed in (1) were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies were visible.

[0050] (3) Recombination verification

[0051] Step 1: Use primer pair waaA-F / waaA-R to perform colony PCR verification on the above single colony; Step 2: Use primer pair waaA-F / waaA-R to amplify the target fragment, and send the amplified product for sequencing to verify the integrity of the sequence.

[0052] (4) Construction-related plasmids are lost

[0053] Step 1: Pick a single colony that has been verified to be correct by sequencing and inoculate it into 5 mL of kanamycin and a final concentration of 0.5 mM In an LB test tube containing IPTG, culture at 30°C overnight and then streak on an LB plate containing kanamycin; Step 2: Pick a single colony and spot it on an LB plate containing kanamycin, spectinomycin and an LB plate containing only kanamycin, and culture at 30°C overnight. If it cannot grow on the LB plate containing kanamycin and spectinomycin, but grows on the LB plate containing kanamycin, it indicates that the pTargetF-N20 (waaA) plasmid has been lost; Step 3: Pick a positive colony that has lost the pTargetF-N20 (waaA) plasmid, inoculate it into an LB test tube without antibiotics, culture at 42°C for 8 hours, then streak it on an LB plate and culture at 37°C overnight; Step 4: Pick a single colony and spot it on an LB plate containing kanamycin and an LB plate without antibiotics. If it cannot grow on the LB plate containing kanamycin, but grows on the LB plate without antibiotics, it indicates that the pCas plasmid has been lost, and the MHZ-0221-14 (waaAC142A) strain is obtained.

[0054] The genotype of the threonine-producing genetically modified strain obtained in Example 1-2 is shown in Table 2.

[0055] Table 2 Genetically engineered bacteria constructed by the present invention

[0056] strain number genotype MHZ-0221-13 MHZ-0215-2,waaA C142T MHZ-0221-14 MHZ-0215-2,waaA C142A

[0057] Example 3: Verification of L-threonine Production by Shake Flask Fermentation of Genetically Engineered Bacteria

[0058] Step 1: Take three strains of bacteria, MHZ-0215-2, MHZ-0221-13, and MHZ-0221-14, from the cryopreserved tubes, streak them on LB plates for activation, and culture at 37°C for 18-24 hours. Step 2: Scrape a loop of bacteria from the plate and inoculate it into a shake flask containing 50 mL of seed culture medium (see Table 3). Culture at 37°C and 90 rpm for about 5 hours until the OD reaches 0. 650 Control within 2; Step 3: Transfer 2 mL of seed liquid to a shake flask containing 20 mL of fermentation medium (see Table 4) and culture on a reciprocating shaker at 37°C and 100 rpm until the residual sugar is exhausted. After the fermentation is completed, measure the sample OD 650The L-threonine content was determined by HPLC, and the residual sugar content was determined by biosensor. To ensure the reliability of the experiment, the shake flask experiment was repeated three times, and the average values ​​of the acid production and conversion rate are shown in Table 5.

[0059] Table 3 Seed culture medium (g / L)

[0060]

[0061]

[0062] Table 4 Fermentation medium (g / L)

[0063] Element concentration glucose 60 corn steep liquor 6 Soybean meal hydrolyzate 7.7 Magnesium sulfate heptahydrate 0.5 <![CDATA[KH2PO4]]> 1.0 Aspartic acid 10 <![CDATA[FeSO4、MnSO4]]> 30mg / L Biotin 50 μg / L Thiamine 500 μg / L pH 7.2

[0064] Table 5 Comparison of productivity of threonine-producing genetically engineered bacteria

[0065]

[0066] Note: * indicates P value < 0.01, indicating that there is a significant difference compared with the control

[0067] As shown in Table 5, the L-threonine production of the novel Escherichia coli of the present invention was higher than that of the respective control strains. The modified strain MHZ-0221-13 produced 17.59 g / L of threonine, with a shake flask conversion rate of 29.31%. This indicates that MHZ-0221-13 produced an average 27.46% more threonine than the starting strain, with a conversion rate of 80.93%. The modified strain MHZ-0221-14 produced 18.23 g / L of threonine, with a shake flask conversion rate of 30.39%. This indicates that MHZ-0221-14 produced an average 32.10% more threonine than the starting strain, with a conversion rate of 87.59%. The shake flask results indicate that the mutation of the waaA nucleotide sequence can significantly improve threonine production.

[0068] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Meihua (Shanghai) Biotechnology Co., Ltd. <120> A recombinant strain and method for producing amino acids thereof <130> KHP211117787.5 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <400> 1 agttaccgcc atccgctaaa accgttttag agctagaaat agcaa 45 <210> 2 <211> 46 <212> DNA <213> Artificial Sequence <400> 2 ggttttagcg gatggcggta actagtatta tacctaggac tgagct 46 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ccataccggg cggtgtggtg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tttgcatcag gataattctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gcggcgttac agaaatatcg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 gcgactcttt tgtgtgattg 20 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 cggtttttac cgccatccgc taaaatcagg cggcattatg ctgcac 46 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <400> 8 gtgcagcata atgccgcctg attttagcgg atggcggtaa aaaccg 46 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <400> 9 cggtttttac cgccatccgc taaaataagg cggcattatg ctgcac 46 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <400> 10 gtgcagcata atgccgcctt attttagcgg atggcggtaa aaaccg 46 <210> 11 <211> 425 <212> PRT <213> Artificial Sequence <400> 11 Met Leu Glu Leu Leu Tyr Thr Ala Leu Leu Tyr Leu Ile Gln Pro Leu 1 5 10 15 Ile Trp Ile Arg Leu Trp Val Arg Gly Arg Lys Ala Pro Ala Tyr Arg 20 25 30 Lys Arg Trp Gly Glu Arg Tyr Gly Phe Tyr Arg His Pro Leu Lys Pro 35 40 45 Gly Gly Ile Met Leu His Ser Val Ser Val Gly Glu Thr Leu Ala Ala 50 55 60 Ile Pro Leu Val Arg Ala Leu Arg His Arg Tyr Pro Asp Leu Pro Ile 65 70 75 80 Thr Val Thr Thr Met Thr Pro Thr Gly Ser Glu Arg Val Gln Ser Ala 85 90 95 Phe Gly Lys Asp Val Gln His Val Tyr Leu Pro Tyr Asp Leu Pro Asp 100 105 110 Ala Leu Asn Arg Phe Leu Asn Lys Val Asp Pro Lys Leu Val Leu Ile 115 120 125 Met Glu Thr Glu Leu Trp Pro Asn Leu Ile Ala Ala Leu His Lys Arg 130 135 140 Lys Ile Pro Leu Val Ile Ala Asn Ala Arg Leu Ser Ala Arg Ser Ala 145 150 155 160 Ala Gly Tyr Ala Lys Leu Gly Lys Phe Val Arg Arg Leu Leu Arg Arg 165 170 175 Ile Thr Leu Ile Ala Ala Gln Asn Glu Glu Asp Gly Ala Arg Phe Val 180 185 190 Ala Leu Gly Ala Lys Asn Asn Gln Val Thr Val Thr Gly Ser Leu Lys 195 200 205 Phe Asp Ile Ser Val Thr Pro Gln Leu Ala Ala Lys Ala Val Thr Leu 210 215 220 Arg Arg Gln Trp Ala Pro His Arg Pro Val Trp Ile Ala Thr Ser Thr 225 230 235 240 His Glu Gly Glu Glu Ser Val Val Ile Ala Ala His Gln Ala Leu Leu 245 250 255 Gln Gln Phe Pro Asn Leu Leu Leu Ile Leu Val Pro Arg His Pro Glu 260 265 270 Arg Phe Pro Asp Ala Ile Asn Leu Val Arg Gln Ala Gly Leu Ser Tyr 275 280 285 Ile Thr Arg Ser Ser Gly Glu Val Pro Ser Thr Ser Thr Gln Val Val 290 295 300 Val Gly Asp Thr Met Gly Glu Leu Met Leu Leu Tyr Gly Ile Ala Asp 305 310 315 320 Leu Ala Phe Val Gly Gly Ser Leu Val Glu Arg Gly Gly His Asn Pro 325 330 335 Leu Glu Ala Ala Ala His Ala Ile Pro Val Leu Met Gly Pro His Thr 340 345 350 Phe Asn Phe Lys Asp Ile Cys Ala Arg Leu Glu Gln Ala Ser Gly Leu 355 360 365 Ile Thr Val Thr Asp Ala Thr Thr Leu Ala Lys Glu Val Ser Ser Leu 370 375 380 Leu Thr Asp Ala Asp Tyr Arg Ser Phe Tyr Gly Arg His Ala Val Glu 385 390 395 400 Val Leu Tyr Gln Asn Gln Gly Ala Leu Gln Arg Leu Leu Gln Leu Leu 405 410 415 Glu Pro Tyr Leu Pro Pro Lys Thr His 420 425 <210> 12 <211> 425 <212> PRT <213> Artificial Sequence <400> 12 Met Leu Glu Leu Leu Tyr Thr Ala Leu Leu Tyr Leu Ile Gln Pro Leu 1 5 10 15 Ile Trp Ile Arg Leu Trp Val Arg Gly Arg Lys Ala Pro Ala Tyr Arg 20 25 30 Lys Arg Trp Gly Glu Arg Tyr Gly Phe Tyr Arg His Pro Leu Lys Ser 35 40 45 Gly Gly Ile Met Leu His Ser Val Ser Val Gly Glu Thr Leu Ala Ala 50 55 60 Ile Pro Leu Val Arg Ala Leu Arg His Arg Tyr Pro Asp Leu Pro Ile 65 70 75 80 Thr Val Thr Thr Met Thr Pro Thr Gly Ser Glu Arg Val Gln Ser Ala 85 90 95 Phe Gly Lys Asp Val Gln His Val Tyr Leu Pro Tyr Asp Leu Pro Asp 100 105 110 Ala Leu Asn Arg Phe Leu Asn Lys Val Asp Pro Lys Leu Val Leu Ile 115 120 125 Met Glu Thr Glu Leu Trp Pro Asn Leu Ile Ala Ala Leu His Lys Arg 130 135 140 Lys Ile Pro Leu Val Ile Ala Asn Ala Arg Leu Ser Ala Arg Ser Ala 145 150 155 160 Ala Gly Tyr Ala Lys Leu Gly Lys Phe Val Arg Arg Leu Leu Arg Arg 165 170 175 Ile Thr Leu Ile Ala Ala Gln Asn Glu Glu Asp Gly Ala Arg Phe Val 180 185 190 Ala Leu Gly Ala Lys Asn Asn Gln Val Thr Val Thr Gly Ser Leu Lys 195 200 205 Phe Asp Ile Ser Val Thr Pro Gln Leu Ala Ala Lys Ala Val Thr Leu 210 215 220 Arg Arg Gln Trp Ala Pro His Arg Pro Val Trp Ile Ala Thr Ser Thr 225 230 235 240 His Glu Gly Glu Glu Ser Val Val Ile Ala Ala His Gln Ala Leu Leu 245 250 255 Gln Gln Phe Pro Asn Leu Leu Leu Ile Leu Val Pro Arg His Pro Glu 260 265 270 Arg Phe Pro Asp Ala Ile Asn Leu Val Arg Gln Ala Gly Leu Ser Tyr 275 280 285 Ile Thr Arg Ser Ser Gly Glu Val Pro Ser Thr Ser Thr Gln Val Val 290 295 300 Val Gly Asp Thr Met Gly Glu Leu Met Leu Leu Tyr Gly Ile Ala Asp 305 310 315 320 Leu Ala Phe Val Gly Gly Ser Leu Val Glu Arg Gly Gly His Asn Pro 325 330 335 Leu Glu Ala Ala Ala His Ala Ile Pro Val Leu Met Gly Pro His Thr 340 345 350 Phe Asn Phe Lys Asp Ile Cys Ala Arg Leu Glu Gln Ala Ser Gly Leu 355 360 365 Ile Thr Val Thr Asp Ala Thr Thr Leu Ala Lys Glu Val Ser Ser Leu 370 375 380 Leu Thr Asp Ala Asp Tyr Arg Ser Phe Tyr Gly Arg His Ala Val Glu 385 390 395 400 Val Leu Tyr Gln Asn Gln Gly Ala Leu Gln Arg Leu Leu Gln Leu Leu 405 410 415 Glu Pro Tyr Leu Pro Pro Lys Thr His 420 425 <210> 13 <211> 425 <212> PRT <213> Artificial Sequence <400> 13 Met Leu Glu Leu Leu Tyr Thr Ala Leu Leu Tyr Leu Ile Gln Pro Leu 1 5 10 15 Ile Trp Ile Arg Leu Trp Val Arg Gly Arg Lys Ala Pro Ala Tyr Arg 20 25 30 Lys Arg Trp Gly Glu Arg Tyr Gly Phe Tyr Arg His Pro Leu Lys Thr 35 40 45 Gly Gly Ile Met Leu His Ser Val Ser Val Gly Glu Thr Leu Ala Ala 50 55 60 Ile Pro Leu Val Arg Ala Leu Arg His Arg Tyr Pro Asp Leu Pro Ile 65 70 75 80 Thr Val Thr Thr Met Thr Pro Thr Gly Ser Glu Arg Val Gln Ser Ala 85 90 95 Phe Gly Lys Asp Val Gln His Val Tyr Leu Pro Tyr Asp Leu Pro Asp 100 105 110 Ala Leu Asn Arg Phe Leu Asn Lys Val Asp Pro Lys Leu Val Leu Ile 115 120 125 Met Glu Thr Glu Leu Trp Pro Asn Leu Ile Ala Ala Leu His Lys Arg 130 135 140 Lys Ile Pro Leu Val Ile Ala Asn Ala Arg Leu Ser Ala Arg Ser Ala 145 150 155 160 Ala Gly Tyr Ala Lys Leu Gly Lys Phe Val Arg Arg Leu Leu Arg Arg 165 170 175 Ile Thr Leu Ile Ala Ala Gln Asn Glu Glu Asp Gly Ala Arg Phe Val 180 185 190 Ala Leu Gly Ala Lys Asn Asn Gln Val Thr Val Thr Gly Ser Leu Lys 195 200 205 Phe Asp Ile Ser Val Thr Pro Gln Leu Ala Ala Lys Ala Val Thr Leu 210 215 220 Arg Arg Gln Trp Ala Pro His Arg Pro Val Trp Ile Ala Thr Ser Thr 225 230 235 240 His Glu Gly Glu Glu Ser Val Val Ile Ala Ala His Gln Ala Leu Leu 245 250 255 Gln Gln Phe Pro Asn Leu Leu Leu Ile Leu Val Pro Arg His Pro Glu 260 265 270 Arg Phe Pro Asp Ala Ile Asn Leu Val Arg Gln Ala Gly Leu Ser Tyr 275 280 285 Ile Thr Arg Ser Ser Gly Glu Val Pro Ser Thr Ser Thr Gln Val Val 290 295 300 Val Gly Asp Thr Met Gly Glu Leu Met Leu Leu Tyr Gly Ile Ala Asp 305 310 315 320 Leu Ala Phe Val Gly Gly Ser Leu Val Glu Arg Gly Gly His Asn Pro 325 330 335 Leu Glu Ala Ala Ala His Ala Ile Pro Val Leu Met Gly Pro His Thr 340 345 350 Phe Asn Phe Lys Asp Ile Cys Ala Arg Leu Glu Gln Ala Ser Gly Leu 355 360 365 Ile Thr Val Thr Asp Ala Thr Thr Leu Ala Lys Glu Val Ser Ser Leu 370 375 380 Leu Thr Asp Ala Asp Tyr Arg Ser Phe Tyr Gly Arg His Ala Val Glu 385 390 395 400 Val Leu Tyr Gln Asn Gln Gly Ala Leu Gln Arg Leu Leu Gln Leu Leu 405 410 415 Glu Pro Tyr Leu Pro Pro Lys Thr His 420 425 <210> 14 <211> 1278 <212> DNA <213> Artificial Sequence <400> 14 atgctcgaat tgctttacac cgcccttctc taccttattc agccgctgat ctggatacgg 60 ctctgggtgc gcggacgtaa ggctccggcc tatcgaaaac gctggggtga acgttacggt 120 ttttaccgcc atccgctaaa accaggcggc attatgctgc actccgtctc cgtcggtgaa 180 actctggcgg caatcccgtt ggtgcgcgcg ctgcgtcatc gttatcctga tttaccgatt 240 accgtaacaa ccatgacgcc aaccggttcg gagcgcgtac aatcggcttt cgggaaggat 300 gttcagcacg tttatctgcc gtatgatctg cccgatgcac tcaaccgttt cctgaataaa 360 gtcgacccta aactggtgtt gattatggaa accgaactat ggcctaacct gattgcggcg 420 ctacataaac gtaaaattcc gctggtgatc gctaacgcgc gactctctgc ccgctcggcc 480 gcaggttatg ccaaactggg taaattcgtc cgtcgcttgc tgcgtcgtat tacgctgatt 540 gctgcgcaaa atgaagaaga tggtgcacgt tttgtggcgc tgggcgcaaa aaataatcag 600 gtgaccgtta ccggtagcct gaaattcgat atttctgtaa cgccgcagtt ggctgctaaa 660 gccgtgacgc tgcgccgcca gtgggcacca caccgcccgg tatggattgc caccagcact 720 cacgaaggcg aagagagtgt ggtgatcgcc gcacatcagg cattgttaca gcaattcccg 780 aatttattgc tcatcctggt accccgtcat ccggaacgct tcccggatgc gattaacctt 840 gtccgccagg ctggactaag ctatatcaca cgctcttcag gggaagtccc ctccaccagc 900 acgcaggttg tggttggcga tacgatgggc gagttgatgt tactgtatgg cattgccgat 960 ctcgcctttg ttggcggttc actggttgaa cgtggtgggc ataatccgct ggaagctgcc 1020 gcacacgcta ttccggtatt gatggggccg catactttta actttaaaga catttgcgcg 1080 cggctggagc aggcaagcgg gctgattacc gttaccgatg ccactacgct tgcaaaagag 1140 gtttcctctt tactcaccga cgccgattac cgtagtttct atggccgtca tgccgttgaa 1200 gtactgtatc aaaaccaggg cgcgctacag cgtctgcttc aactgctgga accttacctg 1260 ccaccgaaaa cgcattga 1278 <210> 15 <211> 1278 <212> DNA <213> Artificial Sequence <400> 15 atgctcgaat tgctttacac cgcccttctc taccttattc agccgctgat ctggatacgg 60 ctctgggtgc gcggacgtaa ggctccggcc tatcgaaaac gctggggtga acgttacggt 120 ttttaccgcc atccgctaaa atcaggcggc attatgctgc actccgtctc cgtcggtgaa 180 actctggcgg caatcccgtt ggtgcgcgcg ctgcgtcatc gttatcctga tttaccgatt 240 accgtaacaa ccatgacgcc aaccggttcg gagcgcgtac aatcggcttt cgggaaggat 300 gttcagcacg tttatctgcc gtatgatctg cccgatgcac tcaaccgttt cctgaataaa 360 gtcgacccta aactggtgtt gattatggaa accgaactat ggcctaacct gattgcggcg 420 ctacataaac gtaaaattcc gctggtgatc gctaacgcgc gactctctgc ccgctcggcc 480 gcaggttatg ccaaactggg taaattcgtc cgtcgcttgc tgcgtcgtat tacgctgatt 540 gctgcgcaaa atgaagaaga tggtgcacgt tttgtggcgc tgggcgcaaa aaataatcag 600 gtgaccgtta ccggtagcct gaaattcgat atttctgtaa cgccgcagtt ggctgctaaa 660 gccgtgacgc tgcgccgcca gtgggcacca caccgcccgg tatggattgc caccagcact 720 cacgaaggcg aagagagtgt ggtgatcgcc gcacatcagg cattgttaca gcaattcccg 780 aatttattgc tcatcctggt accccgtcat ccggaacgct tcccggatgc gattaacctt 840 gtccgccagg ctggactaag ctatatcaca cgctcttcag gggaagtccc ctccaccagc 900 acgcaggttg tggttggcga tacgatgggc gagttgatgt tactgtatgg cattgccgat 960 ctcgcctttg ttggcggttc actggttgaa cgtggtgggc ataatccgct ggaagctgcc 1020 gcacacgcta ttccggtatt gatggggccg catactttta actttaaaga catttgcgcg 1080 cggctggagc aggcaagcgg gctgattacc gttaccgatg ccactacgct tgcaaaagag 1140 gtttcctctt tactcaccga cgccgattac cgtagtttct atggccgtca tgccgttgaa 1200 gtactgtatc aaaaccaggg cgcgctacag cgtctgcttc aactgctgga accttacctg 1260 ccaccgaaaa cgcattga 1278 <210> 16 <211> 1278 <212> DNA <213> Artificial Sequence <400> 16 atgctcgaat tgctttacac cgcccttctc taccttattc agccgctgat ctggatacgg 60 ctctgggtgc gcggacgtaa ggctccggcc tatcgaaaac gctggggtga acgttacggt 120 ttttaccgcc atccgctaaa aacaggcggc attatgctgc actccgtctc cgtcggtgaa 180 actctggcgg caatcccgtt ggtgcgcgcg ctgcgtcatc gttatcctga tttaccgatt 240​​​​​​​​​​​gctgcgcaaa atgaagaaga tggtgcacgt tttgtggcgc tgggcgcaaa aaataatcag 600 gtgaccgtta ccggtagcct gaaattcgat atttctgtaa cgccgcagtt ggctgctaaa 660 gccgtgacgc tgcgccgcca gtgggcacca caccgcccgg tatggattgc caccagcact 720 cacgaaggcg aagagagtgt ggtgatcgcc gcacatcagg cattgttaca gcaattcccg 780 aatttattgc tcatcctggt accccgtcat ccggaacgct tcccggatgc gattaacctt 840 gtccgccagg ctggactaag ctatatcaca cgctcttcag gggaagtccc ctccaccagc 900 acgcaggttg tggttggcga tacgatgggc gagttgatgt tactgtatgg cattgccgat 960 ctcgcctttg ttggcggttc actggttgaa cgtggtgggc ataatccgct ggaagctgcc 1020 gcacacgcta ttccggtatt gatggggccg catactttta actttaaaga catttgcgcg 1080 cggctggagc aggcaagcgg gctgattacc gttaccgatg ccactacgct tgcaaaagag 1140 gtttcctctt tactcaccga cgccgattac cgtagtttct atggccgtca tgccgttgaa 1200 gtactgtatc aaaaccaggg cgcgctacag cgtctgcttc aactgctgga accttacctg 1260 ccaccgaaaa cgcattga 1278

Claims

1. Use of a mutant waaA protein or a gene encoding the protein in producing threonine, characterized in that: The amino acid sequence of the mutant waaA protein is shown in SEQ ID No. 12 or SEQ ID No.

13.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the mutant waaA protein is shown in SEQ ID No. 15 or SEQ ID No.

16.

3. Use of a mutant waaA protein or a gene encoding the protein in producing isoleucine, characterized in that: The amino acid sequence of the mutant waaA protein is as shown in SEQ ID No. 11, in which the proline at position 48 is mutated to serine or threonine.

4. A method for constructing a recombinant strain that produces high L-threonine yield, characterized in that: include; (1) Using the pTargetF plasmid as a template, the primer pair with the nucleotide sequence shown in SEQ ID No. 1-2 was used to amplify the pTF linear plasmid containing N20, and the pTF linear plasmid was transformed into Trans1-T1 competent cells to obtain pTargetF-N20; (2) Using the W3110 genome as a template, using the primer pair with nucleotide sequences as shown in SEQ ID No. 5 and 9, or the primer pair with nucleotide sequences as shown in SEQ ID No. 5 and 7, amplify the upstream homology arm, and using the primer pair with nucleotide sequences as shown in SEQ ID No. 6 and 8, or the primer pair with nucleotide sequences as shown in SEQ ID No. 6 and 10, amplify the downstream homology arm; using the upstream homology arm and the downstream homology arm as templates, using the primer pair with nucleotide sequences as shown in SEQ ID No. 5 and 6, amplify the DNA fragment as shown in SEQ ID No. 15 or SEQ ID No. 16, which is called Donor DNA; (3) The pCas plasmid was electroporated into MHZ-0215-2 competent cells, and the pTargetF-N20 plasmid and the Donor DNA constructed in (2) were simultaneously electroporated into MHZ-0215-2 competent cells.

5. A recombinant strain that produces high amounts of L-threonine, characterized in that: The mutant waaA gene contains a nucleotide sequence as shown in SEQ ID No. 15 or SEQ ID No. 16 or is constructed using the construction method according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria are Escherichia coli, Corynebacterium glutamicum or Serratia marcescens.

7. A method for producing threonine or isoleucine, characterized in that: Threonine or isoleucine is obtained by fermentation using the recombinant bacteria according to any one of claims 5 to 6.

8. Use of a mutant waaA protein in which the proline at position 48 of the amino acid sequence shown in SEQ ID No. 11 is mutated to serine or threonine, or a gene encoding the protein, or the recombinant bacterium according to any one of claims 5 to 6, in increasing threonine production.

9. Use of a mutant waaA protein in which the proline at position 48 of the amino acid sequence shown in SEQ ID No. 11 is mutated to serine or threonine, or a gene encoding the protein, or the recombinant bacterium according to any one of claims 5 to 6, in increasing isoleucine production.

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