CN116555135A - Construction method of high-yield threonine genetic engineering bacteria - Google Patents

Construction method of high-yield threonine genetic engineering bacteria Download PDF

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CN116555135A
CN116555135A CN202210112898.6A CN202210112898A CN116555135A CN 116555135 A CN116555135 A CN 116555135A CN 202210112898 A CN202210112898 A CN 202210112898A CN 116555135 A CN116555135 A CN 116555135A
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threonine
activity
enzyme
microorganism
iolr
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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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Priority to PCT/CN2022/142931 priority patent/WO2023142853A1/en
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Abstract

The invention provides a construction method of high-yield threonine genetic engineering bacteria. The invention improves the threonine producing capacity of the strain by reducing the activity of the GntR family transcription regulatory factor iolR, and improves the threonine yield by 38.8 percent compared with the unmodified strain. And further strengthens threonine synthesis pathway, and/or knocks out or weakens competing pathway and degradation pathway related to threonine synthesis, so that threonine yield is improved, and L-lysine, L-isoleucine and L-methionine which are byproducts are reduced to different degrees. Provides a new way for large-scale production of threonine and has higher application value.

Description

Construction method of high-yield threonine genetic engineering bacteria
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a construction method of high-yield threonine genetic engineering bacteria.
Background
L-threonine (L-threonine), chemical name of beta-hydroxy-alpha-aminobutyric acid, molecular formula of C 4 H 9 NO 3 The relative molecular mass was 119.12. L-threonine is an essential amino acid, and threonine is mainly used in medicine, 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). Hermann Sahm et al have been working on the development of high threonine-producing cereal strains 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 path, and has few reports on global regulation, byproduct reduction and the like. And the prior report only makes preliminary researches on threonine synthesis pathways, and no system is formed.
Disclosure of Invention
The invention aims to provide a construction method of genetically engineered bacteria with high threonine (L-threonine) yield by reducing or losing the activity of a gntR family regulatory factor iolR and improving the threonine production capacity of a strain.
To achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which has reduced or lost activity of the GntR family regulatory factor iolR as compared to an unmodified microorganism, and which has enhanced threonine-producing ability as compared to an unmodified microorganism. Preferably, the GntR family regulatory factor iolR has the reference sequence number wp_003857140.1 on NCBI, or an amino acid sequence with a similarity of 90%.
Further, the reduction or loss of activity of the GntR family modulator iolR in the microorganism is achieved by reducing expression of a gene encoding the GntR family modulator iolR or knocking out an endogenous gene encoding the GntR family modulator iolR.
Mutagenesis, site-directed mutagenesis, or homologous recombination may be used to reduce expression of the gene encoding GntR family regulator iolR or to knock out the endogenous gene encoding GntR family regulator iolR.
Further, the microorganism has an enhanced activity of an enzyme associated with the threonine synthesis pathway in vivo as compared with an unmodified microorganism; or alternatively, the first and second heat exchangers may be,
the microorganism has reduced or lost enzymatic activity of competing or degrading pathways associated with threonine synthesis in vivo, as compared to the unmodified microorganism; or alternatively, the first and second heat exchangers may be,
the microorganism has an increased activity of an enzyme associated with a threonine synthesis pathway in vivo, while having a decreased or lost activity of an enzyme of a competing or degrading pathway associated with threonine synthesis in vivo, as compared to an unmodified microorganism;
wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase, threonine synthase; preferably, their reference sequence numbers at NCBI are wp_003855724.1, wp_011013506.1, wp_003854900.1, wp_011014183.1, wp_011014964.1, respectively, or amino acid sequences with a similarity of 90% with the above reference sequences.
The enzyme of the competing pathway related to threonine synthesis is at least one selected from diaminopimelate dehydrogenase, threonine dehydratase, 4-hydroxy-tetrahydrodipicolinate synthase and homoserine acetyltransferase; preferably, their reference sequence numbers at NCBI are wp_011015254.1, wp_003862033.1, wp_011014792.1, wp_011013793.1, respectively, or amino acid sequences with a similarity of 90% with the above reference sequences.
Preferably, the microorganism is any one of the following (1) to (7):
(1) a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced activity of aspartate aminotransferase and aspartate semialdehyde dehydrogenase;
(2) microorganisms with reduced or lost GntR family regulator iolR activity and enhanced activity of aspartate aminotransferases, aspartate semialdehyde dehydrogenases, homoserine kinases;
(3) microorganisms with reduced or lost activity of GntR family regulatory factor iolR and enhanced activity of aspartate aminotransferase, aspartate semialdehyde dehydrogenase, homoserine kinase, threonine synthase;
(4) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase activities are enhanced, while the diaminopimelate dehydrogenase activity is reduced or lost;
(5) A microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase enzyme activities are enhanced, while the threonine dehydratase activity is reduced or lost;
(6) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase, and threonine synthase enzyme activities are enhanced, while the 4-hydroxy-tetrahydrodipicolinate synthase activity is reduced or lost;
preferably, a decrease in 4-hydroxy-tetrahydrodipicolinate synthase activity refers to a mutation of a in the nucleotide sequence initiation codon ATG encoding 4-hydroxy-tetrahydrodipicolinate synthase to G;
(7) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase enzyme activities are enhanced, while the homoserine acetyltransferase activity is reduced or lost;
preferably, reduced homoserine acetyltransferase activity refers to a mutation of a in the initiation codon ATG of the nucleotide sequence encoding homoserine acetyltransferase to G.
The enhancement of the activity of an enzyme involved in the threonine synthesis pathway in the microorganism is achieved by a compound selected from the following 1) to 5), 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) Enhanced by modification of the amino acid sequence of the enzyme.
Such attenuation includes knocking out or reducing transcription or expression of the gene or altering the amino acid, nucleotide sequence encoding the corresponding enzyme. 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 high-yield threonine-producing genetically engineered bacterium, the method being selected from any one of schemes i to iv:
Scheme i: a strain obtained by reducing or losing the activity of the GntR family regulatory factor iolR encoding in coryneform bacteria having an amino acid-producing ability;
scheme ii:
A. reducing or losing activity of a GntR family regulatory factor iolR encoding in corynebacteria having amino acid production ability to obtain a corresponding strain; and
B. enhancing the enzyme related to threonine synthesis pathway in the strain in the step A to obtain a strain with enhanced enzyme activity;
scheme iii:
a. reducing or losing activity of a GntR family regulatory factor iolR encoding in corynebacteria having amino acid production ability to obtain a corresponding strain; and
b. further weakening the competing pathway and degradation pathway protein activities associated with threonine synthesis in the strain of step a;
the attenuation includes knocking out or reducing gene transcription or expression or altering the amino acid and nucleotide sequences encoding the corresponding enzymes;
scheme iv:
1) Weakening a gene encoding GntR family regulatory factor iolR in coryneform bacteria having amino acid production ability to obtain a gene-weakened strain;
2) Enhancing the enzyme related to threonine synthesis pathway in the gene-attenuated strain of step 1) to obtain an enzyme activity-enhanced strain; and
3) Further attenuating the competing pathway genes associated with threonine synthesis in the enhanced strains of step 2);
The attenuation includes knocking out or reducing transcription or expression of the gene or changing the amino acid, nucleotide sequence encoding the corresponding enzyme;
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) Enhancement by modification of the nucleotide sequence encoding the enzyme;
wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, threonine synthase, homoserine kinase;
the competitive pathway protein related to threonine synthesis is at least one selected from diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, homoserine acetyltransferase, threonine dehydratase.
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 a strain having reduced or lost activity of GntR family regulator iolR in threonine fermentation production or in improving threonine fermentation production.
Further, the fermentation yield of threonine is improved by decreasing or losing the activity of GntR family regulatory factor iolR in corynebacteria (Corynebacterium) having an amino acid-producing ability.
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 fifth aspect, the present invention provides the use of the modified corynebacterium microorganism or the high-yield threonine genetic engineering bacterium constructed according to the above method in 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 present invention improves threonine production by a strain (corynebacterium, such as Corynebacterium glutamicum) by inactivating the GntR family regulatory factor iolR. The threonine yield can be improved by 38.8% as compared with that of the unmodified strain. And further strengthening threonine synthesis pathway related genes and/or knocking out or weakening threonine synthesis related competitive pathway genes, so that threonine yield is improved, and L-lysine, L-isoleucine and L-methionine serving as byproducts are reduced to different degrees. The output of SMCT195 threonine in the final constructed strain is highest and reaches 6.5g/L, which is improved by 1.6 times compared with threonine chassis bacteria. Provides a new way for large-scale production of threonine and has higher application value.
Detailed Description
Based on the previous studies, the current emphasis on threonine production by Corynebacterium glutamicum has been focused mainly on the terminal pathways, and no study has been reported on the relationship between the regulatory factor and the production of threonine by the strain. According to the invention, firstly, the influence on threonine is verified by weakening or inactivating the iolR gene on the basis of corynebacterium glutamicum ATCC13032, so that strains SMCT181 and SMCT182 are obtained, and the influence of the iolR on the threonine yield is verified initially, wherein the threonine content of the SMCT181 and the SMCT182 is 0.4g/L and 0.2g/L respectively. Thus, weakening or inactivating the iolR is effective, but is quite different from what is expected. To further verify the effect of iolR on threonine production, threonine-producing chassis bacteria were constructed. In the case of engineering a strain to produce threonine, the synthetic pathway is first opened up, which mainly includes the aspartokinase-aspartate semialdehyde dehydrogenase operon (lysC fbr- asd), homoserine dehydrogenase-homoserine kinase operon (hom) fbr -thrB) is modulated and expression enhanced, threonine synthase (thrC) is enhanced, for which purpose lysC is modified on the basis of the starting strain ATCC13032 fbr Asd obtaining a modified bacterium SMCT183, modified hom on the basis of this fbr thrB is obtained into modified bacterium SMCT184, thrC is reinforced on the basis of SMCT184 to obtain modified bacterium SMCT185, so that the strain has preliminary threonine synthesis capacity, and threonine yield is 4.1g/L.
On this basis, inactivation or attenuation of GntR family regulatory factor iolR resulted in strains SMCT186 and SMCT187 with 26.8% and 17.1% improvement in threonine production capacity, respectively.
To further verify that inactivation of iolR can increase threonine production, at least 1 of diaminopimelate dehydrogenase, threonine dehydratase, 4-hydroxy-tetrahydrodipicolinate synthase, and homoserine was first engineered in the SMCT185 strain to yield a series of strains SMCT188, SMCT189, SMCT190, SMCT192, SMCT191 with further increases in threonine production and various degrees of reduction in the corresponding byproducts. The iolR gene is further inactivated by taking the 5 strains as starting bacteria, so that the strains SMCT193, SMCT194, SMCT195, SMCT196 and SMCT197 are obtained, and the threonine yield is respectively improved by 27.4%, 26.0%, 35.4%, 38.8% and 29.4%.
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.
The invention adopts the following technical scheme:
according to one of the technical scheme, the invention provides a microorganism with reduced or lost activity of a GntR family regulatory factor iolR.
In a second aspect of the present invention, there is provided a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced expression of at least one of aspartic aminotransferase, aspartic semialdehyde dehydrogenase, homoserine kinase, and threonine synthase.
In a third aspect of the present invention, there is provided a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced activity of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase, and reduced or lost activity of diaminopimelate dehydrogenase.
In a fourth aspect of the present invention, there is provided a microorganism having reduced or lost activity of a GntR family regulatory factor iolR and enhanced enzymatic activity of aspartokinase, aspartyl semialdehyde dehydrogenase, homoserine kinase and threonine synthase, and reduced or lost activity of threonine dehydratase.
In a fifth aspect of the present invention, there is provided a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced enzymatic activity of aspartokinase, aspartyl semialdehyde dehydrogenase, homoserine kinase and threonine synthase, and reduced or lost activity of 4-hydroxy-tetrahydrodipicolinate synthase.
In a sixth aspect of the present invention, there is provided a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced enzymatic activity of aspartokinase, aspartyl semialdehyde dehydrogenase, homoserine kinase and threonine synthase, and reduced or lost activity of homoserine acetyltransferase. The strain is a coryneform bacterium, preferably Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, https:// www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
The protein and the coding gene thereof related to the invention are as follows:
GntR family regulatory factor iolR, coding gene iolR, NCBI accession number: cg0196, cgl0157, NCgl0154.
Aspartokinase, coding gene name lysC, NCBI accession number: cg0306, cgl0251, NCgl0247.
Aspartate semialdehyde dehydrogenase, coding gene name asd, NCBI accession number: cgl0252, cg0307, NCgl0248.
Homoserine dehydrogenase, coding gene name hom, NCBI accession number: cg1337, cgl1183, NCgl1136.
Threonine synthase, encoding gene name thrC, NCBI accession No.: cg2437, cgl2220, NCgl2139.
Homoserine kinase, coding gene thrB, NCBI accession No.: cgl1184, cg1338, NCgl1137.
Diaminopimelate dehydrogenase, coding gene ddh, NCBI accession number: cg2900, cgl2617, NCgl2528.
Threonine dehydratase, coding gene ilvA, NCBI accession No.: cgl2127, cg2334, ncgl2046.
4-hydroxy-tetrahydrodipicolinate synthase, coding gene dapA, NCBI accession number: cgl1971, cg2161, NCgl1896.
Homoserine acetyltransferase (homoserine-O-acetyltransferase), coding gene metX, NCBI accession number: cgl0652, cg0754, 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.
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. Recombinant plasmid pK18mobsacB-Psod-lysC as expression enhancement scheme for aspartokinase-aspartate semialdehyde dehydrogenase operon g1a-T311I Construction of asd
Using ATCC13032 genome as a template, performing PCR amplification by using a P21/P22 primer pair to obtain an upstream homology arm up, performing PCR amplification by using a P23/P24 primer pair to obtain a promoter fragment Psod, and performing PCR amplification by using a P25/P26 primer pair 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-lysCg1a-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 -asd。
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.
2. Recombinant plasmid pK18mobsacB-P of homoserine dehydrogenase-homoserine kinase operon expression enhancement scheme cspB -hom G378E Construction of thrB
PCR amplification was performed using ATCC13032 genome as template and the P29/P30 primer pair to obtain upstream homology arm up, ATPCR amplification is carried out by taking CC14067 genome as a template and using a P31/P32 primer pair to obtain a promoter fragment PcspB, and PCR amplification is carried out by taking ATCC13032 genome as a template and using a P33/P34 primer pair to obtain hom G378E And (3) carrying out PCR amplification by using a P35/P36 primer pair to obtain a downstream homologous arm dn. Fusion PCR is carried out by taking the P29/P32 primer pair and up and PcspB as templates, so as to obtain fragment up-PcspB. With P29/P36 primer pair in the form of up-PcspB, hom G378E Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Psod-hom G378E 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 cspB -hom G378E -thrB。
3. Threonine synthase expression enhancing plasmid pK18mobsacB-P sod -thrC g1a Construction of (3)
The ATCC13032 genome is used as a template, the P37/P38 primer pair is used for PCR amplification to obtain an upstream homology arm up, the P39/P40 primer pair is used for PCR amplification to obtain a promoter segment Psod-thrCg1a, and the P41/P42 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR was performed using the P37/P42 primer pair with up, psod-thrCg1a, dn as templates to obtain full-length fragments up-Psod-thrCg1a-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. Construction of recombinant plasmid pK18 mobsacB-DeltaiolR for GntR family transcription regulatory factor iolR inactivation scheme
The ATCC13032 genome is used as a template, the iolR-1/iolR-2 primer pair is used for PCR amplification to obtain an upstream homology arm up, and the iolR-3/iolR-4 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR is carried out by taking the iolR-1/iolR-4 primer pair and up and dn as templates to obtain the full-length fragment up-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 pK18 mobsacB-delta iolR.
5. GntR family transcription regulatory factor iolR attenuation scheme recombinant plasmid pK18mobsacB-iolR a1g Construction of (3)
PCR amplification was performed using ATCC13032 genome as a template and the iolR-5/iolR-6 primer pair to obtain an upstream homology arm iolR a1g Up, PCR amplification with the pair of iolR-7/iolR-8 primers to obtain the downstream homology arm iolR a1g Dn. With the iolR-5/iolR-8 primer pair with the iolR a1g -up、iolR a1g Fusion PCR is carried out by taking dn as a template to obtain full-length fragment iolR a1g -up-dn. 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-iolR a1g 。
Wherein a1g represents that the 1 st base of the initiation codon of the iolR gene (the iolR wild-type gene sequence is shown in SEQ ID NO: 3) is mutated from a to g.
6. 4-hydroxy-tetrahydrodipicolinate synthase attenuation protocol recombinant plasmid pK18mobsacB-dapA a1g Construction of (3)
PCR amplification was performed using ATCC13032 genome as a template and a P75/P76 primer pair to obtain upstream homology arm dapA a1g Up, PCR amplification with P77/P78 primer pair to obtain downstream homology arm dapA a1g Dn. With the P75/P78 primer pair with dapA a1g -up、dapA a1g Fusion PCR is carried out by taking dn as a template to obtain full-length fragment dapA a1g -up-dn. 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: 4) is mutated from a to g.
7. Threonine dehydratase attenuation protocol recombinant plasmid pK18mobsacB-ilvA a1g Construction of (3)
PCR amplification was performed using ATCC13032 genome as template and the P83/P84 primer pair to obtain upstream homology arm ilvA a1g Up, PCR amplification with P85/P86 primer pair to obtain downstream homology arm ilvA a1g Dn. The primer pair of P83/P86 is ilvA a1g -up、ilvA a1g Fusion PCR is carried out by taking dn as a template to obtain full-length fragment ilvA a1g -up-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-ilvA a1g 。
Wherein a1g represents that the 1 st base of the start codon of ilvA gene (the sequence of ilvA wild type gene is shown in SEQ ID NO: 5) is mutated from a to g.
8. Homoserine acetyltransferase attenuation protocol recombinant plasmid pK18mobsacB-metX a1g Construction of (3)
PCR amplification with ATCC13032 genome as template and P91/P92 primer pair to obtain upstream homology arm metX a1g Up, PCR amplification with P93/P94 primer pair to obtain downstream homology arm metX a1g Dn. With P91/P94 primer pair with metX a1g -up、metX a1g Fusion PCR with dn as template to obtain full length fragment metX a1g -up-dn. 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-metX a1g 。
Wherein a1g represents that the 1 st base of the initiation codon of metX gene (metX wild-type gene sequence is shown in SEQ ID NO: 6) is mutated from a to g.
9. Construction of homoserine acetyltransferase inactivation protocol recombinant plasmid pK18 mobsacB-DeltametX the ATCC13032 genome was used as template, the P95/P96 primer pair was used for PCR amplification to obtain upstream homology arm up, and the P97/P98 primer pair was used for PCR amplification to obtain downstream homology arm dn. Fusion PCR is carried out by taking up and dn as templates by using the P95/P98 primer pair to obtain the full-length fragment up-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 pK18 mobsacB-delta metX.
10. Construction of recombinant plasmid pK18 mobsacB-Deltaddh according to diaminopimelate dehydrogenase inactivation protocol
The ATCC13032 genome is used as a template, the P99/P100 primer pair is used for PCR amplification to obtain an upstream homology arm up, and the P101/P102 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR was performed using the P99/P102 primer pair with up and dn as templates to obtain full-length fragments up-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 pK18 mobsacB-Deltaddh.
The primers used in the construction are shown in Table 1:
TABLE 1
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Note that: the primers for introducing the corresponding point mutations are bolded and underlined.
EXAMPLE 2 construction of genome-engineered Strain
1. Construction of GntR family regulatory factor iolR inactivated or attenuated strains
ATCC13032 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). Recombinant plasmids pK18 mobsacB-DeltaiolR and pK18mobsacB-iolR a1g 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 sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains designated SMCT181 and SMCT182, respectively.
2. Construction of an aspartokinase-aspartate semialdehyde dehydrogenase operon-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 -lysC g1a-T311I Asd 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. 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 SMCT183, respectively.
3. Construction of homoserine dehydrogenase-homoserine kinase operon-enhanced strains
SMCT183 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-P cspB -hom G378E thrB 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. Amplifying the target sequence by PCR, and analyzing the nucleotide sequencing to obtain the targetThe mutant strains of (2) were designated SMCT184, respectively.
4. Construction of threonine synthase expression-enhanced strains
SMCT184 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. 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 SMCT185, respectively.
5. Construction of SMCT185GntR family regulatory factor iolR inactivated or attenuated Strain
SMCT185 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmids pK18 mobsacB-DeltaiolR and pK18mobsacB-iolR a1g The competent cells were transformed separately 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. By passing through The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains designated SMCT186 and SMCT187, respectively.
6. Construction of diaminopimelate dehydrogenase-inactivating Strain
SMCT185 competent cells were prepared according to the classical method of cereal bar (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, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. 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 SMCT188, respectively.
7. Construction of 4-hydroxy-tetrahydrodipicolinate synthase attenuated strains
SMCT185 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-dapA a1g 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. 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 SMCT189, respectively.
8. Construction of threonine dehydratase-attenuated Strain
SMCT185 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-ilvA a1g 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. 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 SMCT190, respectively.
9. Construction of homoserine acetyltransferase weakened Strain
SMCT185 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-metX a1g 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. General purpose medicine The target sequence is amplified by PCR, and the nucleotide sequencing analysis is carried out, so that the target mutant strains are respectively named as SMCT191 strains, the SMCT185 is taken as a starting strain, the metX gene is weakened, and the constructed strains are named as SMCT191.
10. Construction of homoserine acetyltransferase inactivated Strain
SMCT185 competent cells were prepared according to the classical method of cereal bar (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18 mobsacB-. DELTA.metX was used to transform 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, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strains were obtained by the same construction method as described above for SMCT192 strain, with SMCT185 as starting strain, and the metX gene inactivated, and the constructed strain was designated as SMCT192.
11. Construction of iolR inactivated strains
SMCT188, SMCT189, SMCT190, SMCT191 and SMCT192 competent cells were prepared according to the classical method of the valley bar (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18 mobsacB-. DELTA.iolR was transformed into the competent cells by electroporation, respectively, 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 culture was serially diluted (10-2 serial diluted to 10-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Strains grown on sucrose medium do not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strains named SMCT193, SMCT194, SMCT195, SMCT196, and SMCT197, respectively.
The strains obtained are shown in table 2:
TABLE 2
Strain Genotype of the type
SMCT181 ATCC13032,ΔiolR
SMCT182 ATCC13032,iolR a1g
SMCT183 ATCC13032,P sod -lysC g1a-T311I -asd
SMCT184 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB
SMCT185 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a
SMCT187 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,iolR a1g
SMCT186 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,ΔiolR
SMCT188 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,Δddh
SMCT189 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,dapA a1g
SMCT190 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,ilvA a1g
SMCT191 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,metX a1g
SMCT192 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,ΔmetX
SMCT193 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,Δddh,ΔiolR
SMCT194 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,dapA a1g ,ΔiolR
SMCT195 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,ilvA a1g ,ΔiolR
SMCT196 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,metX a1g ,ΔiolR
SMCT197 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB,P sod -thrC g1a ,ΔmetX,ΔiolR
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: pick the slant seeds 1 of smtt 181, smtt 182, smtt 183, smtt 184, smtt 185, smtt 186, smtt 187, smtt 188, smtt 189, smtt 190, smtt 191, smtt 192, smtt 193, smtt 194, smtt 195, smtt 196 and smtt 197 were looped into 500mL flasks with 20mL seed medium and shake-cultured at 30 ℃ for 16h at 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 the L-threonine in the fermentation broths of the engineering bacteria and the control bacteria was detected by HPLC, the concentrations of which are shown below.
Threonine shake flask fermentation results are shown in table 3:
TABLE 3 Table 3
As can be seen from table 3, the threonine producing chassis bacteria SMCT185 is knocked out or inactivated, and the threonine yield is improved by 26.8% and 17.1%, respectively, which indicates that the modified iolR has a positive effect on improving the threonine yield, and the inactivated iolR has a better effect. A series of strains of the chassis (SMCT 188, SMCT189, SMCT190, SMCT191 and SMCT 192) are constructed by inactivating or weakening the by-product through key genes on the basis of the SMCT185, and the threonine yield is further improved. Based on this inactivation of the iolR, a series of iolR inactivated strains (SMCT 193, SMCT194, SMCT195, SMCT196 and SMCT 197) were constructed with different levels of improvement in threonine production compared to the control strain, by 27.4%, 26.0%, 35.4%, 38.8% and 29.4%, respectively. Again, engineering iolR was shown to be an effective site for increasing threonine production. In addition, the threonine amino acid yield is improved when the iolR modification is combined with other sites, and the combination of the iolR modification and the independent modification of the iolR shows that the iolR and other sites (such as the enhancement and deregulation of the expression of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase, homoserine dehydrogenase and threonine synthase and the down regulation of the expression of diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase and homoserine acetyltransferase) can effectively improve 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
Construction method of <120> high-yield threonine genetic engineering bacteria
<130> KHP211124125.9
<160> 6
<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> 762
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 3
atgaccaccg aagctcccat ttggccagcc gaactcttcg aagacctcga ccgcaacgga 60
ccaatccccc tctacttcca agtagcccaa cgcctcgaag acggcatccg cagcggagtc 120
ctcccacccg gagcacgcct agaaaacgag atctccgtgg cgaaacacct caacgtatcc 180
cgccccaccg tccgacgcgc catccaagaa gtcgtagaca aaggcctctt agttcgccgc 240
cgcggtgttg gcacccaggt cgtccaaagc cacgtcaccc gcccagtcga actgaccagt 300
ttcttcaacg acctcaaaaa cgccaacctg gaccccaaaa cccgagtcct cgagcaccgc 360
ctccttgcag caagttccgc catcgcagaa aaactcggag tttccgcagg tgacgaagtc 420
ctcctcatcc gccgcctccg ctccaccgga gacatccccg tagcgatcct ggaaaactac 480
ctccccccag cgttcaacga cgtctccctc gacgaactag aaaagggtgg actctacgat 540
gcgctgcgca gccgaggtgt tgtcttaaaa atcgccaacc agaaaatcgg tgcgcgccga 600
gcagtcggtg aagaaagcac cctcctcgac atcgaagacg gcggaccact tctcaccgtc 660
gaacgcgttg cattggataa ttccggccaa gtaatcgagt tgggaagcca ctgctaccgc 720
ccagatatgt acaactttga aaccactctg gtggccaggt aa 762
<210> 4
<211> 906
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 4
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
<210> 5
<211> 1311
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 5
atgagtgaaa catacgtgtc tgagaaaagt ccaggagtga tggctagcgg agcggagctg 60
attcgtgccg ccgacattca aacggcgcag gcacgaattt cctccgtcat tgcaccaact 120
ccattgcagt attgccctcg tctttctgag gaaaccggag cggaaatcta ccttaagcgt 180
gaggatctgc aggatgttcg ttcctacaag atccgcggtg cgctgaactc tggagcgcag 240
ctcacccaag agcagcgcga tgcaggtatc gttgccgcat ctgcaggtaa ccatgcccag 300
ggcgtggcct atgtgtgcaa gtccttgggc gttcagggac gcatctatgt tcctgtgcag 360
actccaaagc aaaagcgtga ccgcatcatg gttcacggcg gagagtttgt ctccttggtg 420
gtcactggca ataacttcga cgaagcatcg gctgcagcgc atgaagatgc agagcgcacc 480
ggcgcaacgc tgatcgagcc tttcgatgct cgcaacaccg tcatcggtca gggcaccgtg 540
gctgctgaga tcttgtcgca gctgacttcc atgggcaaga gtgcagatca cgtgatggtt 600
ccagtcggcg gtggcggact tcttgcaggt gtggtcagct acatggctga tatggcacct 660
cgcactgcga tcgttggtat cgaaccagcg ggagcagcat ccatgcaggc tgcattgcac 720
aatggtggac caatcacttt ggagactgtt gatccctttg tggacggcgc agcagtcaaa 780
cgtgtcggag atctcaacta caccatcgtg gagaagaacc agggtcgcgt gcacatgatg 840
agcgcgaccg agggcgctgt gtgtactgag atgctcgatc tttaccaaaa cgaaggcatc 900
atcgcggagc ctgctggcgc gctgtctatc gctgggttga aggaaatgtc ctttgcacct 960
ggttctgtcg tggtgtgcat catctctggt ggcaacaacg atgtgctgcg ttatgcggaa 1020
atcgctgagc gctccttggt gcaccgcggt ttgaagcact acttcttggt gaacttcccg 1080
caaaagcctg gtcagttgcg tcacttcctg gaagatatcc tgggaccgga tgatgacatc 1140
acgctgtttg agtacctcaa gcgcaacaac cgtgagaccg gtactgcgtt ggtgggtatt 1200
cacttgagtg aagcatcagg attggattct ttgctggaac gtatggagga atcggcaatt 1260
gattcccgtc gcctcgagcc gggcacccct gagtacgaat acttgaccta a 1311
<210> 6
<211> 1134
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 6
atgcccaccc tcgcgccttc aggtcaactt gaaatccaag cgatcggtga tgtctccacc 60
gaagccggag caatcattac aaacgctgaa atcgcctatc accgctgggg tgaataccgc 120
gtagataaag aaggacgcag caatgtcgtt ctcatcgaac acgccctcac tggagattcc 180
aacgcagccg attggtgggc tgacttgctc ggtcccggca aagccatcaa cactgatatt 240
tactgcgtga tctgtaccaa cgtcatcggt ggttgcaacg gttccaccgg acctggctcc 300
atgcatccag atggaaattt ctggggtaat cgcttccccg ccacgtccat tcgtgatcag 360
gtaaacgccg aaaaacaatt cctcgacgca ctcggcatca ccacggtcgc cgcagtactt 420
ggtggttcca tgggtggtgc ccgcacccta gagtgggccg caatgtaccc agaaactgtt 480
ggcgcagctg ctgttcttgc agtttctgca cgcgccagcg cctggcaaat cggcattcaa 540
tccgcccaaa ttaaggcgat tgaaaacgac caccactggc acgaaggcaa ctactacgaa 600
tccggctgca acccagccac cggactcggc gccgcccgac gcatcgccca cctcacctac 660
cgtggcgaac tagaaatcga cgaacgcttc ggcaccaaag cccaaaagaa cgaaaaccca 720
ctcggtccct accgcaagcc cgaccagcgc ttcgccgtgg aatcctactt ggactaccaa 780
gcagacaagc tagtacagcg tttcgacgcc ggctcctacg tcttgctcac cgacgccctc 840
aaccgccacg acattggtcg cgaccgcgga ggcctcaaca aggcactcga atccatcaaa 900
gttccagtcc ttgtcgcagg cgtagatacc gatattttgt acccctacca ccagcaagaa 960
cacctctcca gaaacctggg aaatctactg gcaatggcaa aaatcgtatc ccctgtcggc 1020
cacgatgctt tcctcaccga aagccgccaa atggatcgca tcgtgaggaa cttcttcagc 1080
ctcatctccc cagacgaaga caacccttcg acctacatcg agttctacat ctaa 1134

Claims (9)

1. A modified corynebacterium microorganism, characterized in that the activity of GntR family regulatory factor iolR is reduced or lost as compared to an unmodified microorganism, and the microorganism has enhanced threonine productivity as compared to an unmodified microorganism.
2. The microorganism according to claim 1, wherein the decrease or loss of activity of GntR family regulator iolR in the microorganism is achieved by decreasing expression of a gene encoding GntR family regulator iolR or knocking out an endogenous gene encoding GntR family regulator iolR.
3. The microorganism according to claim 2, wherein the expression of the gene encoding GntR family regulatory factor iolR is reduced or the endogenous gene encoding GntR family regulatory factor iolR is knocked out by means of mutagenesis, site-directed mutagenesis or homologous recombination.
4. The microorganism of claim 1, wherein the microorganism has an increased activity of an enzyme associated with the threonine synthesis pathway in vivo as compared to an unmodified microorganism; or alternatively, the first and second heat exchangers may be,
the microorganism has reduced or lost enzymatic activity of competing pathways associated with threonine synthesis in vivo, as compared to the unmodified microorganism; or alternatively, the first and second heat exchangers may be,
the microorganism has enhanced activity of an enzyme associated with threonine synthesis pathway in vivo, while having reduced or lost activity of an enzyme associated with competing and degrading pathways associated with threonine synthesis in vivo, as compared to an unmodified microorganism;
wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, threonine synthase, homoserine kinase;
The enzyme of the competing pathway related to threonine synthesis is at least one selected from diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, homoserine acetyltransferase, threonine dehydratase;
the enzymes associated with threonine degradation pathways include threonine dehydratases.
5. The microorganism according to claim 4, wherein the microorganism is any one of the following (1) to (7):
(1) a microorganism having reduced or lost activity of GntR family regulatory factor iolR and enhanced activity of aspartate aminotransferase and aspartate semialdehyde dehydrogenase;
(2) microorganisms with reduced or lost GntR family regulator iolR activity and enhanced activity of aspartate aminotransferases, aspartate semialdehyde dehydrogenases, homoserine kinases;
(3) microorganisms with reduced or lost activity of GntR family regulatory factor iolR and enhanced activity of aspartate aminotransferase, aspartate semialdehyde dehydrogenase, homoserine kinase, threonine synthase;
(4) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase activities are enhanced, while the diaminopimelate dehydrogenase activity is reduced or lost;
(5) A microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase enzyme activities are enhanced, while the threonine dehydratase activity is reduced or lost;
(6) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase, and threonine synthase enzyme activities are enhanced, while the 4-hydroxy-tetrahydrodipicolinate synthase activity is reduced or lost;
preferably, a decrease in 4-hydroxy-tetrahydrodipicolinate synthase activity refers to a mutation of a in the nucleotide sequence initiation codon ATG encoding 4-hydroxy-tetrahydrodipicolinate synthase to G;
(7) a microorganism in which the GntR family regulatory factor iolR activity is reduced or lost and the aspartokinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase enzyme activities are enhanced, while the homoserine acetyltransferase activity is reduced or lost;
preferably, reduced homoserine acetyltransferase activity refers to a mutation of a in the initiation codon ATG of the nucleotide sequence encoding homoserine acetyltransferase to G.
6. The microorganism according to claim 4, wherein the enhancement of the activity of an enzyme involved in the threonine synthesis pathway in the microorganism is achieved by a compound 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) Enhancement by modification of the nucleotide sequence encoding the enzyme;
such attenuation includes knocking out or reducing transcription or expression of the gene or altering the amino acid, nucleotide sequence encoding the corresponding enzyme.
7. A microorganism according to any of claims 1 to 5, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
8. The construction method of the high-yield threonine genetic engineering bacteria is characterized in that the method is selected from any one of schemes i to iv:
scheme i: a strain obtained by reducing or losing the activity of the GntR family regulatory factor iolR encoding in coryneform bacteria having an amino acid-producing ability;
Scheme ii:
A. reducing or losing activity of a GntR family regulatory factor iolR encoding in corynebacteria having amino acid production ability to obtain a corresponding strain; and
B. enhancing the enzyme related to threonine synthesis pathway in the strain in the step A to obtain a strain with enhanced enzyme activity;
scheme iii:
a. reducing or losing activity of a GntR family regulatory factor iolR encoding in corynebacteria having amino acid production ability to obtain a corresponding strain; and
b. further weakening the competing pathway and degradation pathway protein activities associated with threonine synthesis in the strain of step a;
the attenuation includes knocking out or reducing gene transcription or expression or altering the amino acid and nucleotide sequences encoding the corresponding enzymes;
scheme iv:
1) Weakening a gene encoding GntR family regulatory factor iolR in coryneform bacteria having amino acid production ability to obtain a gene-weakened strain;
2) Enhancing the enzyme related to threonine synthesis pathway in the gene-attenuated strain of step 1) to obtain an enzyme activity-enhanced strain; and
3) Further attenuating the competing pathway genes associated with threonine synthesis in the enhanced strains of step 2);
the attenuation includes knocking out or reducing transcription or expression of the gene or changing the amino acid, nucleotide sequence encoding the corresponding enzyme;
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) Enhancement by modification of the nucleotide sequence encoding the enzyme;
wherein the enzyme related to threonine synthesis pathway is selected from at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, threonine synthase, homoserine kinase;
the competitive pathway protein related to threonine synthesis is at least one selected from diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, homoserine acetyltransferase, threonine dehydratase.
9. A method for producing threonine, characterized in that the method comprises the steps of:
a) Culturing the microorganism of any one of claims 1-7 to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
CN202210112898.6A 2022-01-29 2022-01-29 Construction method of high-yield threonine genetic engineering bacteria Pending CN116555135A (en)

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