CN116622598A - Construction method of threonine producing strain - Google Patents

Construction method of threonine producing strain Download PDF

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CN116622598A
CN116622598A CN202210126451.4A CN202210126451A CN116622598A CN 116622598 A CN116622598 A CN 116622598A CN 202210126451 A CN202210126451 A CN 202210126451A CN 116622598 A CN116622598 A CN 116622598A
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gene
threonine
enhanced
microorganism
activity
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康培
吴涛
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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Priority to PCT/CN2022/142844 priority patent/WO2023151407A1/en
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Abstract

The present invention provides a construction method of threonine producing strain. The invention improves the threonine producing capability of coryneform bacteria (such as corynebacterium glutamicum) by superposing and modifying a plurality of enzymes related to threonine synthesis and reducing power supply, including aspartate aminotransferase, aspartokinase, threonine synthase, fructose-1, 6-bisphosphatase, glucose-6-phosphate dehydrogenase 2, 6-phosphogluconate dehydrogenase, NADP dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans, transketolase, NAD kinase and the like, and improves the threonine yield to 6.4g/L. Provides an effective means for producing threonine on a large scale and has wide application prospect.

Description

Construction method of threonine producing strain
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a construction method of threonine production strains.
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 medicine, chemical reagent, food enhancer, feed additive 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 threonine production by corynebacterium glutamicum is mainly focused on the synthesis route, few reports on reducing power supply and the like are provided, and the prior report only makes preliminary researches on the threonine synthesis route and does not form a system.
Disclosure of Invention
The object of the present invention is to provide a method for constructing threonine producing strains.
In order to achieve the object of the present invention, the present invention systematically enhances threonine synthesis pathways by performing additive modification of a plurality of enzymes involved in threonine synthesis, including aspartate aminotransferase, aspartate kinase, threonine synthase, fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans, transketolase, NAD kinase, etc., thereby improving the ability of the strain to produce threonine.
In a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which has an enhanced enzymatic activity associated with threonine synthesis and reducing power supply pathways as compared to an unmodified microorganism, and which has an enhanced threonine-producing ability as compared to an unmodified microorganism.
Wherein the enzyme associated with the threonine synthesis pathway is selected from aspartate aminotransferase, aspartate kinase, threonine synthase; the enzyme related to the supply of reducing power is at least one selected from fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans, 6-phosphogluconate dehydrogenase, transketolase, and NAD kinase.
Preferably, the aspartate aminotransferase, aspartate kinase, threonine synthase, fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase, NAD kinase has reference sequence numbers WP_011013497.1, WP_003855724.1, WP_011014964.1, WP_003856830.1, NP_600790.1, NP_600669.1, NP_600788.1, NP_600631.1, respectively, or amino acid sequences having a similarity of 90% thereto.
The reference sequence number of the gapN gene derived from Streptococcus mutans at NCBI is FOB93_04945, or an amino acid sequence with 90% similarity 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 altering the nucleotide sequence encoding the enzyme.
The microorganism has enhanced in vivo aspartate aminotransferase, aspartate kinase, threonine synthase, fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase and NAD kinase activity as compared to the unmodified microorganism, and overexpresses a Streptococcus mutans-derived glyceraldehyde-3-phosphate dehydrogenase in the microorganism.
Further, the enhancement of aspartate aminotransferase activity was achieved by inserting a sod promoter upstream of the aspB gene start codon.
Further, enhancement of aspartokinase activity was achieved by inserting a sod promoter upstream of the start codon of the lysC gene while mutating the start codon GTG to ATG and mutating the 311 th amino acid encoded by the lysC gene from threonine to isoleucine.
Further, the enhancement of threonine synthase activity is achieved by inserting a sod promoter upstream of the start codon of thrC gene and mutating the start codon GTG to ATG.
Further, the enhancement of fructose-1, 6-bisphosphatase 2 activity is achieved by inserting a tuf promoter upstream of the start codon of the fbp gene.
Further, the glucose-6-phosphate dehydrogenase activity was enhanced by inserting a sod promoter upstream of the start codon of the zwf gene and mutating the 243 st amino acid encoded by the zwf gene from alanine to threonine.
Further, enhancement of 6-phosphogluconate dehydrogenase activity is achieved by inserting a sod promoter upstream of the start codon of the gnd gene.
Further, overexpression of gapN gene was achieved by inserting tuf promoter upstream of the gapN gene start codon.
Further, the enhancement of the transketolase activity was achieved by inserting a sod promoter upstream of the start codon of the tkt gene and performing double copy expression of the tkt gene containing the sod promoter.
Further, enhancement of NAD kinase activity was achieved by inserting a tuf promoter upstream of the start codon of the ppnk 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: genes aspB, lysC, thrC, fbp, zwf, gnd, tkt and ppnk in coryneform bacteria having amino acid productivity are enhanced by genetic engineering means to obtain a gene-enhanced strain, and a gapN gene derived from Streptococcus mutans is overexpressed in the gene-enhanced strain.
The enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having the gene;
2) Enhanced by increasing the copy number of the gene on the chromosome;
3) Enhanced by altering the promoter sequence of said gene on the chromosome;
4) Enhanced by operably linking a strong promoter to the gene;
5) Enhanced by the introduction of enhancers;
6) Enhanced by the use of genes or alleles which encode corresponding enzymes or proteins with high activity.
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).
In a fourth aspect, the present invention provides the use of the modified microorganism of the genus Corynebacterium or the 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 improves the capability of producing threonine of strains by superposing and modifying a plurality of enzymes related to threonine synthesis and reducing power supply of corynebacteria (such as corynebacterium glutamicum), including aspartate aminotransferase, aspartokinase, threonine synthase, fructose-1, 6-bisphosphatase, glucose-6-phosphate dehydrogenase 2, 6-phosphogluconate dehydrogenase, NADP dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans, transketolase, NAD kinase and the like, and improves the threonine yield to 6.4g/L. Provides an effective means for producing threonine on a large scale and has wide application prospect.
Detailed Description
The invention provides a threonine producing strain, which is characterized in that the strain is transformed in the process and the effect:
first, aspartate aminotransferase was expressed intensively in Corynebacterium glutamicum ATCC13032, which was obtained as a strain SMCT286, which produced threonine at 0.8g/L.
On the basis of modifying bacterium SMCT286, aspartokinase is enhanced to obtain strain SMCT289, and the threonine production capacity of the strain is improved from 0.8g/L to 1.8g/L.
On the basis of modifying the strain SMCT289, the threonine synthase is enhanced to obtain the strain SMCT291, and the threonine production capacity of the strain is improved from 1.8g/L to 2.3g/L.
On the basis of modifying bacterium SMCT291, fructose-1, 6-bisphosphatase is expressed in a strengthening way, so that a strain SMCT292 is obtained, and the threonine production capacity of the strain is improved from 2.3g/L to 3.3g/L.
On the basis of modifying bacterium SMCT292, the glucose-6-phosphate dehydrogenase is enhanced to obtain strain SMCT293, and the threonine production capacity of the strain is improved from 3.3g/L to 4.0g/L.
On the basis of modifying bacterium SMCT293, the 6-phosphogluconate dehydrogenase is expressed in an intensified manner to obtain a strain SMCT295, and the threonine production capacity of the strain is improved from 4.0g/L to 4.8g/L.
On the basis of modifying bacterium SMCT295, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans is enhanced to obtain strain SMCT296, and the threonine production capacity of the strain is improved from 4.8g/L to 6.0g/L.
On the basis of modifying bacterium SMCT296, the transketolase is enhanced to obtain a strain SMCT391, and the threonine production capacity of the strain is improved from 6.0g/L to 6.9g/L.
On the basis of modifying bacterium SMCT391, the enhancement expression of transketolase is continued to obtain strain SMCT392, and the threonine production capacity of the strain is improved from 6.9g/L to 8.6g/L.
On the basis of modifying bacterium SMCT392, the ATP-NAD kinase is expressed in a reinforced way, so that a strain SMCT393 is obtained, and the threonine production capacity of the strain is improved from 8.6g/L to 11.0g/L.
The expression enhancement in the transformation process comprises means such as promoter replacement, ribosome binding site change, copy number increase, plasmid overexpression and the like, and all the means are known to the 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 aspartate aminotransferase is encoded by the aspB gene, and the invention inserts a sod promoter upstream of the start codon of the aspB gene, thereby realizing the enhancement of the expression of the aspB gene.
The aspartokinase is encoded by the lysC gene, a sod promoter is inserted at the upstream of the start codon of the lysC gene, the GTG of the start codon is mutated into ATG, and the 311 th amino acid is mutated from threonine into isoleucine, so that the overexpression of the lysC gene is realized.
Threonine synthase, encoded by thrC gene, the invention inserts the sod promoter upstream of the thrC gene start codon and mutates the start codon GTG to ATG, thus realizing the overexpression of thrC gene.
The fructose-1, 6-bisphosphatase is encoded by the fbp gene, and the tuf promoter is inserted into the upstream of the initiation codon of the fbp gene, so that the overexpression of the fbp gene is realized.
Glucose-6-phosphate dehydrogenase is encoded by zwf gene, the sod promoter is inserted in upstream of zwf gene start codon, and 243 th amino acid encoded by zwf gene is mutated from alanine to threonine, thus realizing over expression of zwf gene.
The 6-phosphogluconate dehydrogenase is encoded by the gnd gene, and the sod promoter is inserted into the upstream of the start codon of the gnd gene, so that the overexpression of the gnd gene is realized.
The NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans (Streptococcus mutans) is encoded by gapN gene, and the invention introduces the gapN gene derived from Streptococcus mutans and inserts tuf promoter upstream of the initiation codon of the gapN gene, thereby realizing overexpression of the gapN gene.
The invention inserts a sod promoter at the upstream of the start codon of the tkt gene, and carries out double-copy expression on the tkt gene containing the sod promoter, thereby realizing the overexpression of the tkt gene.
NAD kinase, encoded by the ppnk gene, is inserted with tuf promoter at the upstream of the ppnk gene start codon, thus realizing the overexpression of the ppnk gene.
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:
aspartate aminotransferase, coding gene name aspB, NCBI accession number: cg0294, cg0294 and cg0294.
Aspartokinase, coding gene name lysC, NCBI accession number: cg0306, cgl0251, NCgl0247.
Threonine synthase, encoding gene name thrC, NCBI accession No.: cg2437, cgl2220, NCgl2139.
Fructose-1, 6-bisphosphatase 2, coding gene name fbp, NCBI accession number: cg1157, cgl1019, ncgl0976.
Glucose-6-phosphate dehydrogenase, encoding the gene name zwf, NCBI accession No.: cg1778, cgl1576, NCgl1514.
6-phosphogluconate dehydrogenase, coding gene name gnd, NCBI accession number: cg1643, cgl1452, NCgl1396.
An NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans, which codes for the gene name gapN. NCBI numbering: FOB93_04945.
Transketolase, coding gene name tkt, NCBI accession number: cg1774, cgl1574, NCgl1512.
NAD kinase, coding gene name ppnk, NCBI accession number: cg1601, cgl1413, NCgl1358.
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, zwf was derived from Corynebacterium glutamicum, and the nucleotide sequences of wild-type gene lysC, thrC, zwf 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.
The primers referred to in the following examples are shown in Table 1.
TABLE 1
Note that: the primers for introducing the corresponding point mutations are bolded and underlined.
EXAMPLE 1 construction of plasmid for genome engineering of Strain
1. Aspartate aminotransferase expression enhancing plasmid pK18mobsacB-P sod Construction of aspB
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P103/P104 primer pair is used for PCR amplification to obtain an upstream homology arm up, and P105/P is usedAnd (3) carrying out PCR amplification on the 106 primer pair to obtain a promoter fragment Psod, and carrying out PCR amplification on the primer pair P107/P108 to obtain a downstream homologous arm dn. And (3) performing fusion PCR by using the P103/P106 primer pair and using up and Psod as templates to obtain fragments up-Psod. Fusion PCR is carried out by taking the P103/P108 primer pair and up-Psod and dn as templates to obtain the full-length fragment up-Psod-dn. 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 -aspB。
2. Aspartokinase expression enhancing plasmid pK18mobsacB-P sod -lysC g1a-T311I Construction of (3)
The corynebacterium glutamicum ATCC13032 genome is used as a template, a P21/P22 primer pair is used for carrying out PCR amplification to obtain an upstream homology arm up, a P23/P24 primer pair is used for carrying out PCR amplification to obtain a promoter fragment Psod, and a P25/P26 primer pair is used for carrying out PCR amplification to obtain lysC g1a-T311I And (3) carrying out PCR amplification by using a P27/P28 primer pair to obtain a downstream homologous arm dn. And carrying out fusion PCR by taking the P21/P24 primer pair and up and Psod as templates to obtain a fragment up-Psod. With the P21/P28 primer pair with up-Psod, lysC g1a-T311I Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Psod-lysC g1a-T311I Dn. 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 corynebacterium glutamicum ATCC13032 genome is used as a template, a P37/P38 primer pair is used for PCR amplification to obtain an upstream homology arm up, and a P39/P40 primer pair is used for PCR amplification to obtain a promoter fragment P sod -thrC g1a And (3) carrying out PCR amplification by using a P41/P42 primer pair to obtain a downstream homologous arm dn. With P37/P40 primer pair up, P sod -thrC g1a Fusion PCR is carried out for the template to obtain a fragment up-P sod -thrC g1a . With P37/P42 primer pair with up-P sod -thrC g1a Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-up-P sod -thrC g1a Dn. 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. Fructose-1, 6-bisphosphatase 2 expression enhancing plasmid pK18mobsacB-P tuf Construction of fbp
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P61/P62 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P63/P64 primer pair is used for PCR amplification to obtain a promoter fragment Ptuf, and a P65/P66 primer pair is used for PCR amplification to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P61/P64 primer pair and using up and Ptuf as templates to obtain fragments up-Ptuf. Fusion PCR was performed using the P61/P66 primer pair and up-Ptuf, dn as templates to obtain full-length fragments up-Ptuf-dn. 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 tuf -fbp。
5. Glucose-6-phosphate dehydrogenase expression enhancing plasmid pK18mobsacB-P sod -zwf A243T Construction of (3)
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P129/P130 primer pair is used for carrying out PCR amplification to obtain an upstream homology arm up, a P131/P132 primer pair is used for carrying out PCR amplification to obtain a promoter fragment Psod, and a P133/P134 primer pair is used for carrying out PCR amplification to obtain zwf A243T And (3) carrying out PCR amplification by using a P135/P136 primer pair to obtain a downstream homologous arm dn. And (3) performing fusion PCR by using the P129/P132 primer pair and using up and Psod as templates to obtain a fragment up-Psod. With the P129/P136 primer pair with up-Psod, zwf A243T Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Psod-zwf A243T Dn. pK18mobsacB was digested with BamHI/HindIII enzymesCutting. 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 -zwf A243T 。
Wherein A243T represents that the 243 rd amino acid of glucose-6-phosphate dehydrogenase encoded by zwf gene is mutated from A to T.
6. 6-phosphogluconate dehydrogenase expression enhancing plasmid pK18mobsacB-P sod Construction of gnd
The Corynebacterium glutamicum ATCC13032 genome is used as a template, the P123/P124 primer pair is used for PCR amplification to obtain an upstream homology arm up, the P125/P126 primer pair is used for PCR amplification to obtain a promoter fragment Psod, and the P127/P128 primer pair is used for PCR amplification to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P123/P126 primer pair and using up and Psod as templates to obtain fragments up-Psod. Fusion PCR was performed using the P123/P128 primer pair and up-Psod, dn as templates to obtain full-length fragments up-Psod-dn. 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 -gnd。
7. Streptococcus mutans-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression enhancing plasmid pK18mobsacB-P tuf Construction of gapN
The corynebacterium glutamicum ATCC13032 genome is used as a template, the P137/P138 primer pair is used for carrying out PCR amplification to obtain an upstream homology arm up, the P139/P140 primer pair is used for carrying out PCR amplification to obtain a promoter fragment Ptuf, the P141/P142 primer pair is used for carrying out PCR amplification to obtain gapN by using the streptococcus mutans genome as a template, and the P143/P144 primer pair is used for carrying out PCR amplification by using the ATCC13032 as a template to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P137/P140 primer pair and using up and Ptuf as templates to obtain fragments up-Ptuf. Fusion PCR was performed using the P137/P144 primer pair and up-Ptuf, gapN, dn as a template to obtain the full-length fragment up-Ptuf-gapN-dn. 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 tuf -gapN。
8. Construction of Protransketogenic potential expression enhancing plasmid pK18mobsacB-Psod-tkt
The Corynebacterium glutamicum ATCC13032 genome is used as a template, a P175/P176 primer pair is used for PCR amplification to obtain an upstream homology arm up, a P177/P178 primer pair is used for PCR amplification to obtain a promoter fragment Psod, and a P179/P180 primer pair is used for PCR amplification to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P175/P178 primer pair and using up and Psod as templates to obtain fragments up-Psod. Fusion PCR was performed using the P175/P180 primer pair and up-Psod, dn as templates to obtain full-length fragments up-Psod-dn. 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-tkt.
9. Transketolase two-copy expression enhancing plasmid pK18mobsacB-Psod-tkt 2nd Construction of (3)
The genome of corynebacterium glutamicum ATCC13032 is used as a template, a P301/P302 primer pair is used for carrying out PCR amplification to obtain an upstream homology arm up, a P303/P304 primer pair is used for carrying out PCR amplification to obtain a promoter fragment Psod, a P305/P306 primer pair is used for carrying out PCR amplification to obtain a tkt, and a P307/P308 primer pair is used for carrying out PCR amplification to obtain a downstream homology arm dn by using ATCC13032 as a template. And carrying out fusion PCR by taking the P301/P304 primer pair and up and Psod as templates to obtain a fragment up-Psod. Fusion PCR was performed using the P301/P308 primer pair and up-Psod, tkt, dn as a template to obtain the full-length fragment up-Psod-tkt-dn. 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 pK18mobsacB-Psod-tkt 2nd 。
10. Construction of NAD kinase expression enhancing plasmid pK18mobsacB-Ptuf-ppnk
The Corynebacterium glutamicum ATCC13032 genome is used as a template, the P109/P110 primer pair is used for PCR amplification to obtain an upstream homology arm up, the P111/P112 primer pair is used for PCR amplification to obtain a promoter fragment Ptuf, and the P113/P114 primer pair is used for PCR amplification to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P109/P112 primer pair and using up and Ptuf as templates to obtain fragments up-Ptuf. Fusion PCR is carried out by taking the P109/P114 primer pair and up-Ptuf and dn as templates to obtain the full-length fragment up-Ptuf-dn. 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-Ptuf-ppnk.
EXAMPLE 2 construction of genome-engineered Strain
1. Construction of aspartate aminotransferase-enhanced expression 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 mutant strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and designated SMCT286.
2. Construction of aspartokinase expression-enhancing Strain
SMCT286 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 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% sucroseAnd (3) standing and culturing at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and designated SMCT289.
3. Construction of threonine synthase expression-enhanced strains
SMCT289 competent cells were prepared according to the classical method of cereal bars (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 mutant strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and was designated SMCT291.
4. Construction of fructose-1, 6-bisphosphatase 2 expression-enhancing Strain
SMCT291 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P tuf The competent cells were transformed by electroporation with fbp 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 the substrate containingAnd (3) standing and culturing for 48h at 33 ℃ on a common solid brain heart infusion culture medium containing 10% of sucrose. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target mutant strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and designated SMCT292.
5. Construction of glucose-6-phosphate dehydrogenase expression-enhancing Strain
SMCT292 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod -zwf A243T 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 strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and was designated SMCT293.
6. Construction of 6-phosphogluconate dehydrogenase expression-enhancing Strain
SMCT293 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P sod Gnd transformed the competent cells by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, wherein 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 strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and designated SMCT295.
7. Construction of Streptococcus mutans-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression-enhancing Strain
SMCT295 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P tuf gapN 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 mutant strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and was designated SMCT296.
8. Construction of a Proketogenic potential expression-enhancing Strain
SMCT296 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18mobsacB-Psod-tkt 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 the culture process, the transformant undergoes a second recombination, and the vector sequence is changed through gene exchangeColumns are removed from the genome. 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 strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and designated SMCT391.
9. Construction of a transketolase two-copy expression-enhanced Strain
SMCT391 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-Psod-tkt 2nd 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 strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and was designated SMCT392.
10. Construction of NAD kinase expression-enhanced Strain
SMCT392 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18mobsacB-Ptuf-ppnk 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 the culture, the transformant undergoes a second recombination by gene crossingThe vector sequence is removed from the genome instead. 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 strain was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis and was designated SMCT393.
The strains obtained are shown in table 2:
TABLE 2
Strain name Genotype of the type
SMCT286 ATCC13032,P sod -aspB
SMCT289 SMCT286,P sod -lysC g1a-T311I
SMCT291 SMCT289,P sod -thrC g1a
SMCT292 SMCT291,P tuf -fbp
SMCT293 SMCT292,P sod -zwf A243T
SMCT295 SMCT293,P sod -gnd
SMCT296 SMCT295,P tuf -gapN
SMCT391 SMCT296,P sod -tkt
SMCT392 SMCT391,P sod -tkt 2nd
SMCT393 SMCT392,P tuf -ppnk
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: the engineering bacteria slant seeds 1 are picked up and are looped into a 500mL triangular flask filled with 20mL of seed culture medium, and are subjected to shaking culture for 16h at 30 ℃ and 220 r/min.
(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 threonine producing ability of Corynebacterium glutamicum
As can be seen from Table 3, the superposition modification of aspartate aminotransferase, aspartokinase, threonine synthase, fructose-1, 6-bisphosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans, transketolase, NAD kinase and the like has a positive effect on the increase of threonine yield, and the threonine yield is increased to 11.0g/L, indicating that the superposition combination of the invention is effective, and can significantly increase the threonine yield.
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> KHP211124943.7
<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
<210> 2
<211> 1446
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 2
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
<210> 3
<211> 1455
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 3
atggtgatct tcggtgtcac tggcgacttg gctcgaaaga agctgctccc cgccatttat 60
gatctagcaa accgcggatt gctgccccca ggattctcgt tggtaggtta cggccgccgc 120
gaatggtcca aagaagactt tgaaaaatac gtacgcgatg ccgcaagtgc tggtgctcgt 180
acggaattcc gtgaaaatgt ttgggagcgc ctcgccgagg gtatggaatt tgttcgcggc 240
aactttgatg atgatgcagc tttcgacaac ctcgctgcaa cactcaagcg catcgacaaa 300
acccgcggca ccgccggcaa ctgggcttac tacctgtcca ttccaccaga ttccttcaca 360
gcggtctgcc accagctgga gcgttccggc atggctgaat ccaccgaaga agcatggcgc 420
cgcgtgatca tcgagaagcc tttcggccac aacctcgaat ccgcacacga gctcaaccag 480
ctggtcaacg cagtcttccc agaatcttct gtgttccgca tcgaccacta tttgggcaag 540
gaaacagttc aaaacatcct ggctctgcgt tttgctaacc agctgtttga gccactgtgg 600
aactccaact acgttgacca cgtccagatc accatggctg aagatattgg cttgggtgga 660
cgtgctggtt actacgacgg catcggcgca gcccgcgacg tcatccagaa ccacctgatc 720
cagctcttgg ctctggttgc catggaagaa ccaatttctt tcgtgccagc gcagctgcag 780
gcagaaaaga tcaaggtgct ctctgcgaca aagccgtgct acccattgga taaaacctcc 840
gctcgtggtc agtacgctgc cggttggcag ggctctgagt tagtcaaggg acttcgcgaa 900
gaagatggct tcaaccctga gtccaccact gagacttttg cggcttgtac cttagagatc 960
acgtctcgtc gctgggctgg tgtgccgttc tacctgcgca ccggtaagcg tcttggtcgc 1020
cgtgttactg agattgccgt ggtgtttaaa gacgcaccac accagccttt cgacggcgac 1080
atgactgtat cccttggcca aaacgccatc gtgattcgcg tgcagcctga tgaaggtgtg 1140
ctcatccgct tcggttccaa ggttccaggt tctgccatgg aagtccgtga cgtcaacatg 1200
gacttctcct actcagaatc cttcactgaa gaatcacctg aagcatacga gcgcctcatt 1260
ttggatgcgc tgttagatga atccagcctc ttccctacca acgaggaagt ggaactgagc 1320
tggaagattc tggatccaat tcttgaagca tgggatgccg atggagaacc agaggattac 1380
ccagcgggta cgtggggtcc aaagagcgct gatgaaatgc tttcccgcaa cggtcacacc 1440
tggcgcaggc cataa 1455

Claims (7)

1. A modified coryneform microorganism, characterized in that the microorganism has an enhanced activity of an enzyme related to threonine synthesis pathway and an enhanced activity of an enzyme related to supply of reducing power as compared to an unmodified microorganism, and the microorganism has an enhanced threonine-producing ability as compared to an unmodified microorganism;
wherein the enzyme associated with the threonine synthesis pathway is selected from aspartate aminotransferase, aspartate kinase, threonine synthase; the enzyme related to the supply of reducing power is at least one selected from fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans, 6-phosphogluconate dehydrogenase, transketolase, and NAD kinase.
2. The microorganism of claim 1, wherein the enhancement of enzymatic activity is achieved by a member selected from the group consisting of 1) to 6), or an optional combination of:
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 altering the nucleotide sequence encoding the enzyme.
3. The microorganism of claim 1, wherein the microorganism has enhanced in vivo aspartate aminotransferase, aspartate kinase, threonine synthase, fructose-1, 6-bisphosphatase 2, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase, and NAD kinase activity as compared to the unmodified microorganism, and overexpresses a streptococcus mutans-derived glyceraldehyde-3-phosphate dehydrogenase in the microorganism.
4. A microorganism according to claim 3, characterized in that the enhancement of aspartate aminotransferase activity is achieved by inserting a sod promoter upstream of the aspB gene start codon; and/or
Enhancement of aspartokinase activity was achieved by inserting a sod promoter upstream of the start codon of the lysC gene while mutating the start codon GTG to ATG and mutating the 311 th amino acid encoded by the lysC gene from threonine to isoleucine; and/or
Enhancement of threonine synthase activity is achieved by inserting a sod promoter upstream of the start codon of thrC gene and mutating the start codon GTG to ATG; and/or
The enhancement of fructose-1, 6-bisphosphatase 2 activity is achieved by inserting a tuf promoter upstream of the start codon of the fbp gene; and/or
The enhancement of glucose-6-phosphate dehydrogenase activity is achieved by inserting a sod promoter upstream of the start codon of the zwf gene and mutating the 243 th amino acid encoded by the zwf gene from alanine to threonine; and/or
Enhancement of 6-phosphogluconate dehydrogenase activity is achieved by inserting a sod promoter upstream of the start codon of the gnd gene; and/or
Through inserting tuf promoter at upstream of gapN gene start codon, it realizes the over expression of gapN gene; and/or
Enhancement of transketolase activity was achieved by inserting a sod promoter upstream of the start codon of the tkt gene and performing two-copy expression of the tkt gene containing the sod promoter; and/or
Enhancement of NAD kinase activity was achieved by inserting a tuf promoter upstream of the start codon of the ppnk gene.
5. A microorganism according to any of claims 1 to 4, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
6. A method of constructing a threonine-producing strain, the method comprising: enhancing genes aspB, lysC, thrC, fbp, zwf, gnd, tkt and ppnk in coryneform bacteria having amino acid production ability by using genetic engineering means to obtain a gene-enhanced strain, and overexpressing a gapN gene derived from Streptococcus mutans in the gene-enhanced strain;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having the gene;
2) Enhanced by increasing the copy number of the gene on the chromosome;
3) Enhanced by altering the promoter sequence of said gene on the chromosome;
4) Enhanced by operably linking a strong promoter to the gene;
5) Enhanced by the introduction of enhancers;
6) Enhanced by the use of genes or alleles which encode corresponding enzymes or proteins with high activity.
7. A method for producing threonine, characterized in that the method comprises the steps of:
a) Culturing the microorganism of any one of claims 1-5 to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
CN202210126451.4A 2022-02-10 2022-02-10 Construction method of threonine producing strain Pending CN116622598A (en)

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