CN116622599A - Construction method of high-threonine-producing strain - Google Patents

Construction method of high-threonine-producing strain Download PDF

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CN116622599A
CN116622599A CN202210132242.0A CN202210132242A CN116622599A CN 116622599 A CN116622599 A CN 116622599A CN 202210132242 A CN202210132242 A CN 202210132242A CN 116622599 A CN116622599 A CN 116622599A
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threonine
gene
enzyme
microorganism
lost
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康培
宫卫波
何君
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention provides a construction method of a high-yield threonine strain. The invention enhances the expression of one or more of the coryneform bacteria (such as corynebacterium glutamicum) aspartate aminotransferase, aspartokinase and threonine synthase, so that the L-threonine yield of the strain is improved; further, the diaminopimelate dehydrogenase is inactivated, the 4-hydroxy-tetrahydrodipicolinate synthase is weakened, and one or more of threonine dehydratase and homoserine-O-acetyltransferase express the weakened or inactivated strain, so that the L-threonine yield is improved, and finally the L-threonine yield can reach 6.7g/L, thereby providing an effective means for large-scale production of threonine and having wide application prospect.

Description

Construction method of high-threonine-producing strain
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a construction method of a high-yield threonine strain.
Background
L-threonine (L-threonine), chemical name beta-hydroxy-alpha-aminobutyric acid, molecular formula C 4 H 9 NO 3 Relative molecular mass 119.12. L-threonine is an essential amino acid and is mainly used in a plurality of fields such as medicines, chemical reagents, food enhancers, feed additives and the like.
In Corynebacterium glutamicum, five catalytic reactions are required for threonine production from oxaloacetate, respectively aspartokinase (lysC-encoded), aspartyl semialdehyde dehydrogenase (asd-encoded), homoserine dehydrogenase (hom-encoded), homoserine kinase (thrB), and threonine synthase (thrC). Hemann Sahm et al have been working on the development of threonine-producing Corynebacterium glutamicum and have made some breakthroughs to obtain the hom gene against feedback inhibition (Reinscheid D J, eikmanns B J, sahm H.analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase. [ J ]. Journal of Bacteriology,1991,173 (10): 3228-3230), lysC gene (Eikmanns B J, eggeling L, sahm H.molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum. [ J ]. Antonie Van Leeuwenhoek,1993,64 (2): 145-163). Following Hermann Sahm, lothar Eggling increased threonine production from 49mM to 67mM (Simic P, willuhn J, sahm H, et al identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum [ J ]. Applied and Environmental Microbiology,2002,68 (7): 3321-3327) by attenuating the coding gene glyA in the threonine utilization pathway while overexpressing threonine efflux protein ThrE.
The current report of producing L-threonine by using corynebacterium glutamicum is mainly focused on the synthesis path, few reports of branch inactivation, catabolic inactivation, synthesis path combination and the like are provided, and the existing report only carries out preliminary researches on the synthesis path of L-threonine and does not form a system.
Disclosure of Invention
The invention aims to provide a construction method of a high-threonine-producing strain.
To achieve the object of the present invention, the present invention provides an improved ability of a strain to produce L-threonine by enhancing threonine synthesis pathway while reducing competing pathways and/or catabolic pathways associated with threonine synthesis.
In a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which microorganism has an increased enzymatic activity associated with the threonine synthesis pathway and a decreased or lost enzymatic activity associated with competing and/or catabolic pathways associated with threonine synthesis, as compared to an unmodified microorganism, and which microorganism has an increased threonine producing capacity as compared to an unmodified microorganism.
Wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartokinase, threonine synthase;
the enzyme related to the competing pathway and/or catabolic pathway related to threonine synthesis is selected from at least one of diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, homoserine-O-acetyltransferase.
Preferably, the reference sequence numbers of aspartate aminotransferase, aspartokinase, threonine synthase, diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, homoserine-O-acetyltransferase on NCBI are respectively wp_011013497.1, wp_003855724.1, wp_011014964.1, wp_011015254.1, wp_011014792.1, wp_011014022.1, wp_00 011015254.1 033.1, wp_011013793.1, or amino acid sequences with a similarity of 90% thereto.
Preferably, the enhancement of the activity of the enzyme is achieved by a member selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by the use of genes or alleles which encode corresponding enzymes or proteins with high activity.
The reduction or loss of the enzymatic activity associated with competing and/or catabolic pathways associated with threonine synthesis in the microorganism is achieved by a compound selected from the following 1) -5), or an optional combination:
1) Reduced or lost by altering the promoter sequence of the gene encoding the enzyme;
2) Reduced or lost by altering the ribosome binding site of the gene encoding the enzyme;
3) Reduced or lost by altering the amino acid sequence of the enzyme;
4) Reduced or lost by altering the nucleotide sequence encoding the enzyme;
5) Loss by knocking out the coding sequence of the enzyme;
wherein the gene associated with the competing pathway and/or catabolic pathway associated with threonine synthesis is selected from at least one of ddh, dapA, tdcB, ilvA, metX.
Methods of mutagenesis, site-directed mutagenesis or homologous recombination may be employed to reduce expression of or knock-out endogenous genes associated with competing and/or catabolic pathways involved in threonine synthesis.
The microorganism has enhanced in vivo aspartate aminotransferase, aspartate kinase and threonine synthase activity and reduced or lost diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase and homoserine-O-acetyltransferase activity as compared to the unmodified microorganism.
Further, the enhancement of aspartate aminotransferase activity was achieved by inserting a sod promoter upstream of the start codon of the encoding gene aspB.
Further, the enhancement of aspartokinase activity was achieved by inserting a sod promoter upstream of the start codon of lysC gene of the encoding gene, and mutating the start codon of lysC gene from GTG to ATG and mutating amino acid 311 of aspartokinase from T to I.
Further, the threonine synthase activity was enhanced by inserting a sod promoter upstream of the thrC initiation codon of the coding gene and mutating the thrC initiation codon from GTG to ATG.
Further, the reduction or loss of diaminopimelate dehydrogenase activity is achieved by knocking out the ddh gene.
Further, the 4-hydroxy-tetrahydrodipicolinate synthase activity is reduced or lost by mutating the dapA gene start codon from ATG to GTG.
Further, the reduction or loss of threonine dehydratase activity is achieved by knocking out the tdcB gene.
Further, the reduction or loss of threonine dehydratase activity is achieved by knocking out ilvA gene.
Further, the reduction or loss of homoserine-O-acetyltransferase activity is achieved by knocking out metX gene.
Preferably, the corynebacterium of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, tree https:// www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
In a second aspect, the present invention provides a method for constructing a threonine-producing strain, the method comprising: enhancing a gene related to a threonine synthesis pathway in coryneform bacteria having an amino acid production ability by using a genetic engineering means, and inactivating or weakening a gene related to a competing pathway and/or catabolic pathway related to threonine synthesis;
wherein the gene related to threonine synthesis pathway is selected from at least one of aspB, lysC, thrC;
the gene related to the competing pathway or catabolic pathway related to threonine synthesis is selected from at least one of ddh, dapA, tdcB, ilvA, metX;
preferably, the reference sequence numbers of gene aspB, lysC, thrC, ddh, dapA, tdcB, ilvA, metX at NCBI are Cg0294, cg0306, cg2437, cg2900, cg2161, cg1116, cg2334, cg0754, respectively.
The enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by the use of genes or alleles having the corresponding enzymes or proteins encoding high activity;
in the present invention, the weakening is achieved from the following 1) -5), or an optional combination:
1) Reduced or lost by altering the promoter sequence of the gene encoding the enzyme;
2) Reduced or lost by altering the ribosome binding site of the gene encoding the enzyme;
3) Reduced or lost by altering the amino acid sequence of the enzyme;
4) Reduced or lost by altering the nucleotide sequence encoding the enzyme;
5) By knocking out the coding sequence of the enzyme.
The method of attenuation may be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, and the like.
In a third aspect, the present invention provides a method for producing threonine, the method comprising the steps of:
a) Culturing the modified corynebacterium microorganism to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
Preferably, the corynebacterium of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, tree https:// www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
In a fourth aspect, the present invention provides the use of the modified microorganism of the genus Corynebacterium or the high threonine-producing strain constructed as described above for threonine fermentation production or for improving threonine fermentation production.
The transformation methods of the related strains comprise transformation modes of strengthening and weakening genes and the like which are known to the person skilled in the art, and are referred to the system path engineering of the full-scope high-yield L-arginine corynebacterium crenatum [ D ]. University of Jiangnan, 2016; cui Yi metabolically engineering corynebacterium glutamicum to produce L-leucine [ D ]. Tianjin university of science and technology; xu Guodong construction of L-isoleucine-producing Strain and optimization of fermentation conditions university of Tianjin science and technology 2015.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention enhances the expression of one or more of the coryneform bacteria (such as corynebacterium glutamicum) aspartate aminotransferase, aspartokinase and threonine synthase, so that the L-threonine yield of the strain is improved; further, the diaminopimelate dehydrogenase is inactivated, the 4-hydroxy-tetrahydropyridine formate synthase is weakened, and one or more of threonine dehydratase tdcB, threonine dehydratase ilvA and homoserine-O-acetyltransferase are expressed to be weakened or inactivated, so that the L-threonine yield is improved to 6.7g/L, and an effective means is provided for large-scale production of threonine, and the application prospect is wide.
Detailed Description
The invention provides a threonine producing strain, which adopts a combination scheme of threonine branch inactivation, catabolism inactivation and synthesis pathway enhancement to improve the L-threonine producing capacity of the strain. The strain transformation process and the effect are as follows:
the present invention enhances and reduces the branched expression and the decomposition of products of L-threonine synthesis pathway of an original strain (e.g., corynebacterium glutamicum ATCC 13032), mainly comprising aspartate aminotransferase, aspartokinase, threonine synthase, diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, homoserine-O-acetyltransferase expression enhancement, attenuation, inactivation or demodulation control, to explore the influence thereof on threonine production. The shake flask result shows that the L-threonine production capacity is improved.
The inactivation or weakening in the transformation process comprises means such as replacement of a promoter, change of a ribosome binding site, point mutation, deletion of a sequence and the like, and the expression strengthening in the transformation process comprises means such as replacement of a promoter, change of a ribosome binding site, increase of copy number, over-expression of a plasmid and the like, and all the means are known to a person skilled in the art. The above means are not intended to be exhaustive, and the specific examples are described by way of example only with promoter enhancement.
In particular, the method comprises the steps of,
the invention inserts the sod promoter at the upstream of the start codon of the aspB gene to realize the enhancement of the expression of the aspB gene.
According to the invention, a sod promoter is inserted into the upstream of a lysC gene start codon, the lysC gene start codon is mutated from GTG to ATG, and the 311 th amino acid of a coded protein (aspartokinase) is mutated from T to I, so that the enhancement of the lysC gene is realized.
According to the invention, a sod promoter is inserted into the upstream of the thrC gene start codon, and the thrC gene start codon is mutated from GTG to ATG, so that the thrC gene is enhanced.
The ddh gene is knocked out, so that the ddh gene is inactivated.
The invention changes the initiation codon of the dapA gene from ATG to GTG, thereby realizing attenuation of the dapA gene.
The tdcB gene is knocked out, so that the tdcB gene is inactivated.
The invention knocks out ilvA gene to realize the inactivation of ilvA gene.
The metX gene is knocked out, so that the metX gene is inactivated.
The strain is a coryneform bacterium, preferably Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
Further, the above strain is used for threonine fermentation production.
The protein and the coding gene thereof related to the invention are as follows:
aspartic acid aminotransferase, coding gene aspB, NCBI accession number: cg0294, cgl0240, NCgl0237.
Aspartokinase, coding gene lysC, NCBI accession number: cg0306, cgl0251, NCgl0247.
Threonine synthase, encoding gene thrC, NCBI accession No.: cg2437, cgl2220, NCgl2139.
Diaminopimelate dehydrogenase, coding gene ddh, NCBI accession number: cg2900, cgl2617, NCgl2528.
4-hydroxy-tetrahydrodipicolinate synthase, coding gene dapA, NCBI accession number: cg2161, cgl1971, NCgl1896.
Threonine dehydratase, coding gene tdcB, NCBI accession number: cg1116, cgl0978, NCgl0939.
Threonine dehydratase, coding gene ilvA, NCBI accession No.: cg2334, cgl2127, ncgl2046.
homoserine-O-acetyltransferase, coding gene metX, NCBI accession No.: cg0754, cgl0652, NCgl0624.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
In the following examples, gene lysC, thrC, dapA was derived from Corynebacterium glutamicum, and the nucleotide sequences of wild-type gene lysC, thrC, dapA are shown in SEQ ID NOS: 1-3, respectively.
The experimental materials used in the following examples are as follows:
the experimental methods involved in the following examples are as follows:
the PCR amplification system was as follows:
composition of the components Volume (microliter)
Sterilized deionized water 29
5×pfu buffer 10
2.5mM dNTP 5
10 mu M upstream primer 2
10 mu M downstream primer 2
Pfu 1
Template 1 (fusion PCR template added to 2. Mu.l maximum)
Sum up 50
The PCR amplification procedure was as follows:
the strain transformation method comprises the following steps:
1. seamless assembly reaction procedure: reference is made to ClonExpress MultiS One Step Cloning Kit.
2. The transformation method comprises the following steps: refer to the description of Trans1-T1 Phage Resistant Chemically Competent Cell.
3. Preparation of competent cells: reference c.glutamicum Handbook, charter 23.
EXAMPLE 1 construction of plasmid for genome engineering of Strain
1. Aspartic acid aminotransferase protocol recombinant plasmid pK18mobsacB-P sod Construction of aspB
The Corynebacterium glutamicum ATCC13032 genome is used as a template, the P103/P104 primer pair is used for PCR amplification to obtain an upstream homology arm up, the P105/P106 primer pair is used for PCR amplification to obtain a Psod, the P107/P108 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and the P103/P108 primer pair is used for fusion PCR with up, psod, dn as a template to obtain a full-length fragment Psod-aspB. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain a recombinant plasmid pK18mobsacB-Psod-aspB.
2. Aspartokinase expression enhancement scheme recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I Construction of (3)
The Corynebacterium glutamicum ATCC13032 genome is used as a template, and the P21/P22 primer pair is used for PCR amplification to obtain upstream homologyArm up, PCR amplification with P23/P24 primer pair to obtain Psod, and PCR amplification with P25/P26 primer pair to obtain lysC g1a The sequence mid, the downstream homologous arm dn is obtained by PCR amplification with the P27/P28 primer pair, and the full-length fragment P is obtained by fusion PCR with the P21/P28 primer pair as a template and up, psod, mid, dn sod -lysC g1a-T311I . pK18mobsacB was digested with BamHI/HindIII. Assembling the two by using a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I 。
Wherein g1a represents that the 1 st base of the start codon of lysC gene (the wild-type gene sequence of lysC is shown in SEQ ID NO: 1) is mutated from g to a, and T311I represents that the 311 st amino acid of aspartokinase encoded by lysC gene is mutated from T to I.
3. Threonine synthase expression enhancing plasmid pK18mobsacB-P sod -thrC g1a Construction of (3)
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P37/P38 primer pair is used for carrying out PCR amplification to obtain an upstream homology arm up, a P39/P40 primer pair is used for carrying out PCR amplification to obtain a Psod, a P41/P42 primer pair is used for carrying out PCR amplification to obtain a downstream homology arm dn, and a P37/P42 primer pair is used for carrying out fusion PCR with up, psod, dn as a template to obtain a full-length fragment P sod -thrC g1a . pK18mobsacB was digested with BamHI/HindIII. Assembling the two by using a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-P sod -thrC g1a 。
Wherein g1a represents that the 1 st base of the initiation codon of thrC gene (thrC wild-type gene sequence is shown in SEQ ID NO: 2) is mutated from g to a.
4. Construction of diaminopimelate dehydrogenase-inactivating plasmid pK18 mobsacB-Deltaddh
The construction method of the plasmid is the same as that of the above, and the primers are P99, P100, P101 and P102.
The Corynebacterium glutamicum ATCC13032 genome is used as a template, a P99/P100 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P101/P102 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and a P99/P102 primer pair is used for fusion PCR with up and dn as templates to obtain a full-length fragment delta ddh. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain a recombinant plasmid pK18 mobsacB-Deltaddh.
5. 4-hydroxy-tetrahydrodipicolinate synthase attenuation protocol recombinant plasmid pK18mobsacB-dapA a1g Construction of (3)
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P75/P76 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P77/P78 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and a P75/P78 primer pair is used for fusion PCR with up and dn as templates to obtain a full-length fragment dapA a1g . pK18mobsacB was digested with BamHI/HindIII. Assembling the two with a seamless cloning kit, and transforming the Trans 1T 1 competent cells to obtain a recombinant plasmid pK18mobsacB-dapA a1g 。
Wherein a1g represents that the 1 st base of the initiation codon of dapA gene (the wild-type gene sequence of dapA is shown in SEQ ID NO: 3) is mutated from a to g.
6. Construction of recombinant plasmid pK18 mobsacB-DeltatdcB in inactivation protocol of threonine dehydratase tdcB
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P145/P146 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P147/P148 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and the P145/P148 primer pair is used for fusion PCR with up and dn as templates to obtain a full-length fragment delta tdcB. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain recombinant plasmid pK18 mobsacB-delta tdcB.
7. Construction of recombinant plasmid pK18 mobsacB-DeltailvA for threonine dehydratase ilvA inactivation protocol
The Corynebacterium glutamicum ATCC13032 genome is used as a template, a P87/P88 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P89/P90 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and a P87/P90 primer pair is used for fusion PCR with up and dn as templates to obtain a full-length fragment delta ilvA. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain recombinant plasmid pK18 mobsacB-delta ilvA.
8. Construction of homoserine-O-acetyltransferase inactivation protocol recombinant plasmid pK18 mobsacB-DeltametX
The Corynebacterium glutamicum ATCC13032 genome is used as a template, a P95/P96 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P97/P98 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and a P95/P98 primer pair is used for fusion PCR with up and dn as templates to obtain a full-length fragment delta metX. pK18mobsacB was digested with BamHI/HindIII. The two are assembled by a seamless cloning kit, and Trans 1T 1 competent cells are transformed to obtain a recombinant plasmid pK18 mobsacB-delta metX.
The primers used in the plasmid construction procedure are shown in Table 1:
TABLE 1
Note that: the primers for introducing the corresponding point mutations are bolded and underlined.
EXAMPLE 2 construction of genome-engineered Strain
1. Construction of an aspartate aminotransferase enhanced Strain
ATCC13032 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod aspB transformation of the competent cells by electroporation and selection of transformants on selection medium containing 15mg/L kanamycin, in which the gene of interest is inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequences were amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strains were obtained and designated SMCT241, respectively.
2. Construction of aspartokinase enhanced expression and demodulation control strain
SMCT241 competent cells were prepared according to the classical method of cereal bars (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequences were amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains designated SMCT242, respectively.
3. Construction of threonine synthase expression-enhanced strains
SMCT242 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod -thrC g1a The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During the culture, the transformant undergoes a second recombination, and the vector sequence is transferred from the gene by gene exchangeRemoved from the group. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequences were amplified by PCR and analyzed by nucleotide sequencing, and the obtained target mutant strains were designated SMCT256, respectively.
4. Construction of diaminopimelate dehydrogenase-inactivating Strain
SMCT256 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod -thrC g1a The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequences were amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains designated SMCT257, respectively.
5. Construction of 4-hydroxy-tetrahydrodipicolinate synthase expression attenuated Strain
SMCT257 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18 mobsacB-. DELTA.ddh was transformed into the competent cells by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, the transformantA second recombination event occurs and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strains were obtained by PCR amplification of the target sequences and nucleotide sequencing analysis and were designated SMCT258, respectively.
6. Construction of threonine dehydratase tdcB-inactivated Strain
SMCT258 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18 mobsacB-DeltatdcB. The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequences were amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains designated SMCT259, respectively.
7. Construction of threonine dehydratase ilvA-inactivated Strain
SMCT259 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18 mobsacB-DeltailvA. The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. This cultureDuring the process, the transformant undergoes a second recombination, and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strains were obtained by PCR amplification of the target sequences and nucleotide sequencing analysis and were designated SMCT260, respectively.
8. Construction of homoserine-O-acetyltransferase inactivated Strain
SMCT260 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB- ΔmetX. The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strains were obtained by PCR amplification of the target sequences and nucleotide sequencing analysis and were designated SMCT261, respectively.
The strains obtained are shown in table 2:
TABLE 2
EXAMPLE 3 construction of strains shake flask verification
1. Culture medium
Seed activation medium: BHI 3.7%, agar 2%, pH7.
Seed culture medium: 5g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 16g/L ammonium sulfate, 8g/L urea, 10.4g/L potassium dihydrogen phosphate, 21.4g/L dipotassium hydrogen phosphate, 5mg/L biotin and 3g/L magnesium sulfate. Glucose 50g/L, pH 7.2.
Fermentation medium: corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, monopotassium phosphate 10g/L, urea 20g/L, biotin 10mg/L, magnesium sulfate 6g/L, ferrous sulfate 1g/L, VB1 & HCl 40mg/L, calcium pantothenate 50mg/L, nicotinamide 40mg/L, manganese sulfate 1g/L, zinc sulfate 20mg/L, copper sulfate 20mg/L, and pH 7.2.
2. Engineering bacterium shake flask fermentation production of L-threonine
(1) Seed culture: SMCT241, SMCT242, SMCT256, SMCT257, SMCT258, SMCT259, SMCT260, SMCT261 slant seed 1 was inoculated into a 500mL Erlenmeyer flask containing 20mL of seed medium, and cultured at 30deg.C with 220r/min shaking for 16h.
(2) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 20mL of the fermentation medium, and cultured at 33℃under 220r/min with shaking for 24 hours.
(3) 1mL of the fermentation broth was centrifuged (12000 rpm,2 min), and the supernatant was collected and tested for L-threonine in the fermentation broth of the engineering bacteria and the control bacteria by HPLC (Table 3).
TABLE 3 shaking flask fermentation results of L-threonine
As can be seen from Table 3, the L-threonine production by one or more of the strains whose expression is enhanced by aspartate aminotransferase, aspartokinase and threonine synthase is improved; deactivation of diaminopimelate dehydrogenase; weakening of 4-hydroxy-tetrahydrodipicolinate synthase; the threonine dehydratase tdcB, threonine dehydratase ilvA, homoserine-O-acetyltransferase, or the like, expresses a strain or strains that are attenuated or inactivated with an increased L-threonine production, and the highest L-threonine production is 6.7g/L.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> gallery plum blossom biotechnology development Co., ltd
<120> construction method of threonine-producing Strain
<130> KHP211124941.5
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1266
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 1
gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60
aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120
tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180
ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240
gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300
ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360
gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420
aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480
ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540
accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600
atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660
cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720
attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780
gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840
aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900
tcttctgtag aagacggcac caccgacatc accttcacct gccctcgttc cgacggccgc 960
cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020
gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080
accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140
tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200
ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260
cgctaa 1266
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<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
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gtggactaca tttcgacgcg tgatgccagc cgtacccctg cccgcttcag tgatattttg 60
ctgggcggtc tagcaccaga cggcggcctg tacctgcctg caacctaccc tcaactagat 120
gatgcccagc tgagtaaatg gcgtgaggta ttagccaacg aaggatacgc agctttggct 180
gctgaagtta tctccctgtt tgttgatgac atcccagtag aagacatcaa ggcgatcacc 240
gcacgcgcct acacctaccc gaagttcaac agcgaagaca tcgttcctgt caccgaactc 300
gaggacaaca tttacctggg ccacctttcc gaaggcccaa ccgctgcatt caaagacatg 360
gccatgcagc tgctcggcga acttttcgaa tacgagcttc gccgccgcaa cgaaaccatc 420
aacatcctgg gcgctacctc tggcgatacc ggctcctctg cggaatacgc catgcgcggc 480
cgcgagggaa tccgcgtatt catgctgacc ccagctggcc gcatgacccc attccagcaa 540
gcacagatgt ttggccttga cgatccaaac atcttcaaca tcgccctcga cggcgttttc 600
gacgattgcc aagacgtagt caaggctgtc tccgccgacg cagaattcaa aaaagacaac 660
cgcatcggtg ccgtgaactc catcaactgg gcacgcctta tggcacaggt tgtgtactac 720
gtttcctcat ggatccgcac cacaaccagc aatgaccaaa aggtcagctt ctccgtacca 780
accggcaact tcggtgacat ttgcgcaggc cacatcgccc gccaaatggg acttcccatc 840
gatcgcctca tcgtggccac caacgaaaac gatgtgctcg acgagttctt ccgtaccggc 900
gactaccgag tccgcagctc cgcagacacc cacgagacct cctcaccttc gatggatatc 960
tcccgcgcct ccaacttcga gcgtttcatc ttcgacctgc tcggccgcga cgccacccgc 1020
gtcaacgatc tatttggtac ccaggttcgc caaggcggat tctcactggc tgatgacgcc 1080
aactttgaga aggctgcagc agaatacggt ttcgcctccg gacgatccac ccatgctgac 1140
cgtgtggcaa ccatcgctga cgtgcattcc cgcctcgacg tactaatcga tccccacacc 1200
gccgacggcg ttcacgtggc acgccagtgg agggacgagg tcaacacccc aatcatcgtc 1260
ctagaaactg cactcccagt gaaatttgcc gacaccatcg tcgaagcaat tggtgaagca 1320
cctcaaactc cagagcgttt cgccgcgatc atggatgctc cattcaaggt ttccgaccta 1380
ccaaacgaca ccgatgcagt taagcagtac atagtcgatg cgattgcaaa cacttccgtg 1440
aagtaa 1446
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<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 3
atgagcacag gtttaacagc taagaccgga gtagagcact tcggcaccgt tggagtagca 60
atggttactc cattcacgga atccggagac atcgatatcg ctgctggccg cgaagtcgcg 120
gcttatttgg ttgataaggg cttggattct ttggttctcg cgggcaccac tggtgaatcc 180
ccaacgacaa ccgccgctga aaaactagaa ctgctcaagg ccgttcgtga ggaagttggg 240
gatcgggcga agctcatcgc cggtgtcgga accaacaaca cgcggacatc tgtggaactt 300
gcggaagctg ctgcttctgc tggcgcagac ggccttttag ttgtaactcc ttattactcc 360
aagccgagcc aagagggatt gctggcgcac ttcggtgcaa ttgctgcagc aacagaggtt 420
ccaatttgtc tctatgacat tcctggtcgg tcaggtattc caattgagtc tgataccatg 480
agacgcctga gtgaattacc tacgattttg gcggtcaagg acgccaaggg tgacctcgtt 540
gcagccacgt cattgatcaa agaaacggga cttgcctggt attcaggcga tgacccacta 600
aaccttgttt ggcttgcttt gggcggatca ggtttcattt ccgtaattgg acatgcagcc 660
cccacagcat tacgtgagtt gtacacaagc ttcgaggaag gcgacctcgt ccgtgcgcgg 720
gaaatcaacg ccaaactatc accgctggta gctgcccaag gtcgcttggg tggagtcagc 780
ttggcaaaag ctgctctgcg tctgcagggc atcaacgtag gagatcctcg acttccaatt 840
atggctccaa atgagcagga acttgaggct ctccgagaag acatgaaaaa agctggagtt 900
ctataa 906

Claims (9)

1. A modified coryneform microorganism, characterized in that the microorganism has an enhanced activity of an enzyme related to threonine synthesis pathway and a decreased or lost activity of an enzyme related to competing and/or catabolic pathways related to threonine synthesis as compared to an unmodified microorganism, and the microorganism has an enhanced threonine producing ability as compared to an unmodified microorganism;
wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartokinase, threonine synthase;
the enzyme related to the competing pathway and/or catabolic pathway related to threonine synthesis is selected from at least one of diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, homoserine-O-acetyltransferase.
2. The microorganism according to claim 1, characterized in that the enhancement of the enzymatic activity associated with the threonine synthesis pathway is achieved by a compound selected from the following 1) to 6), or optionally:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by the use of genes or alleles having the corresponding enzymes or proteins encoding high activity;
the reduction or loss of the enzymatic activity associated with competing and/or catabolic pathways associated with threonine synthesis in the microorganism is effected by a compound selected from the following groups 1) -5), or an optional combination:
1) Reduced or lost by altering the promoter sequence of the gene encoding the enzyme;
2) Reduced or lost by altering the ribosome binding site of the gene encoding the enzyme;
3) Reduced or lost by altering the amino acid sequence of the enzyme;
4) Reduced or lost by altering the nucleotide sequence encoding the enzyme;
5) Loss by knocking out the coding sequence of the enzyme;
wherein the gene associated with the competing pathway and/or catabolic pathway associated with threonine synthesis is selected from at least one of ddh, dapA, tdcB, ilvA, metX.
3. The microorganism according to claim 2, wherein the expression of genes involved in the competing and/or catabolic pathways involved in threonine synthesis is reduced or endogenous genes involved in the competing and/or catabolic pathways involved in threonine synthesis are knocked out by means of mutagenesis, site-directed mutagenesis or homologous recombination.
4. The microorganism of claim 1, wherein the microorganism has increased in vivo aspartate aminotransferase, aspartate kinase and threonine synthase activity and reduced or lost diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase and homoserine-O-acetyltransferase activity as compared to an unmodified microorganism.
5. The microorganism according to claim 4, wherein the enhancement of the aspartate aminotransferase activity is achieved by inserting a sod promoter upstream of the start codon of the encoding gene aspB; and/or
The enhancement of aspartokinase activity is achieved by inserting a sod promoter upstream of the start codon of the coding gene lysC, and mutating the start codon of the lysC gene from GTG to ATG and the 311 th amino acid of aspartokinase from T to I; and/or
The threonine synthase activity is enhanced by inserting a sod promoter upstream of the thrC initiation codon of the coding gene and mutating the thrC initiation codon from GTG to ATG; and/or
The reduced or lost diaminopimelate dehydrogenase activity is achieved by knocking out the ddh gene; and/or
The 4-hydroxy-tetrahydrodipicolinate synthase activity is reduced or lost by mutating the dapA gene start codon from ATG to GTG; and/or
The reduction or loss of threonine dehydratase activity is achieved by knocking out the tdcB gene; and/or
The reduced or lost threonine dehydratase activity is achieved by knocking out the ilvA gene; and/or
The reduced or lost homoserine-O-acetyltransferase activity is achieved by knocking out the metX gene.
6. A microorganism according to any of claims 1 to 5, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
7. A method for constructing a threonine-producing strain, comprising: enhancing a gene related to a threonine synthesis pathway in coryneform bacteria having an amino acid production ability by using a genetic engineering means, and inactivating or weakening a gene related to a competing pathway and/or catabolic pathway related to threonine synthesis;
wherein the gene related to threonine synthesis pathway is selected from at least one of aspB, lysC, thrC;
the gene related to the competing pathway or catabolic pathway related to threonine synthesis is selected from at least one of ddh, dapA, tdcB, ilvA, metX;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by the use of genes or alleles having the corresponding enzymes or proteins encoding high activity;
the weakening is selected from the following 1) -5), or optionally in combination:
1) Reduced or lost by altering the promoter sequence of the gene encoding the enzyme;
2) Reduced or lost by altering the ribosome binding site of the gene encoding the enzyme;
3) Reduced or lost by altering the amino acid sequence of the enzyme;
4) Reduced or lost by altering the nucleotide sequence encoding the enzyme;
5) By knocking out the coding sequence of the enzyme.
8. The method of claim 7, wherein the method of attenuation is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination.
9. A method for producing threonine, characterized in that the method comprises the steps of:
a) Culturing the microorganism of any one of claims 1-6 to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
CN202210132242.0A 2022-02-14 2022-02-14 Construction method of high-threonine-producing strain Pending CN116622599A (en)

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DE102010003419B4 (en) * 2010-03-30 2019-09-12 Evonik Degussa Gmbh Process for the fermentative production of L-ornithine
CN109554322B (en) * 2018-12-03 2020-08-04 江南大学 Recombinant Escherichia coli with high L-threonine yield and construction method thereof
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