CN116555132A - Modified corynebacterium microorganism and application and construction method for producing threonine thereof - Google Patents

Modified corynebacterium microorganism and application and construction method for producing threonine thereof Download PDF

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CN116555132A
CN116555132A CN202210108388.1A CN202210108388A CN116555132A CN 116555132 A CN116555132 A CN 116555132A CN 202210108388 A CN202210108388 A CN 202210108388A CN 116555132 A CN116555132 A CN 116555132A
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enzyme
microorganism
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康培
刘涛
宫卫波
何君
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention relates to the technical field of microbial engineering, and in particular discloses a modified corynebacterium microorganism and an application and construction method for producing threonine. The modified coryneform microorganism of the present invention, which assumes reduced or no expression of membrane proteins as compared to an unmodified microorganism, has enhanced threonine productivity as compared to an unmodified microorganism. The present invention improves threonine production yield of the strain by inactivating the putative membrane protein. Provides a new idea for threonine production.

Description

Modified corynebacterium microorganism and application and construction method for producing threonine thereof
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a modified corynebacterium microorganism and application and construction method for producing threonine.
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, aspartokinase (lysC-encoded), aspartate semialdehyde dehydrogenase (asd-encoded), homoserine dehydrogenase (hom-encoded), homoserine kinase (thrB-encoded) and threonine synthase (thrC-encoded), are required for threonine production from oxaloacetic acid. 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.), the 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 samm, lothar Eggling has further explored in the field to attenuate the coding gene glyA in the threonine-utilizing pathway, while overexpressing threonine-carrying protein ThrE, so that threonine production increases 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.).
However, the current report of threonine production by Corynebacterium glutamicum is mainly focused on threonine synthesis pathways, and few reports on nonessential gene knockout and the like are made. Research on genome minimization has focused mainly on whether genome simplification would result in slow strain growth, auxotrophy, and whether the simplification would be advantageous for genetic engineering. There is no report on the genomic simplification related to threonine production. Therefore, further studies on threonine production by Corynebacterium glutamicum are necessary.
Disclosure of Invention
The invention aims to improve the threonine producing capacity of a strain by reducing the expression of a postulated membrane protein, thereby providing a threonine (L-threonine) producing strain, a construction method and application thereof.
The technical scheme of the invention is as follows:
to achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which assumes reduced or no expression of a membrane protein as compared to an unmodified microorganism and which has enhanced threonine productivity as compared to an unmodified microorganism. Preferably, the membrane protein is assumed to have a reference sequence number CAF21557 at NCBI, or an amino acid sequence with 90% similarity thereto.
Further, the reduction or non-expression of the putative membrane protein in the microorganism is achieved by reducing the expression of the gene encoding the putative membrane protein or knocking out the endogenous gene encoding the putative membrane protein.
Mutagenesis, site-directed mutagenesis, or homologous recombination methods may be employed to reduce expression of the gene encoding the putative membrane protein or to knock-out the endogenous gene encoding the putative membrane protein.
Further, the microorganism has an increased and/or decreased activity of an enzyme associated with threonine synthesis in vivo as compared to the unmodified microorganism; wherein the enzyme associated with threonine synthesis with enhanced activity is selected from at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase, a mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and homoserine dehydrogenase; the enzyme associated with threonine synthesis with reduced activity is selected from at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citric acid synthase; preferably, their reference sequence numbers on NCBI are wp_011013497.1, wp_011014465.1, fob93_04945, wp_003854900.1, wp_011014792.1, wp_011013914.1, 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 (6):
(1) a microorganism which has reduced or no expression of a membrane protein and has enhanced homoserine dehydrogenase activity;
(2) a microorganism which is presumed to have reduced or no expression of the membrane protein and has enhanced homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase activity;
(3) a microorganism which assumes reduced or no expression of the membrane protein and enhanced homoserine dehydrogenase, phosphoenolpyruvate carboxylase and/or aspartate aminotransferase activity;
(4) a microorganism which assumes reduced or no expression of membrane proteins and enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase;
(5) a microorganism having enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and reduced activity of 4-hydroxytetrahydropyridine dicarboxylic acid synthase, assuming reduced or no expression of the membrane protein;
(6) a microorganism having enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and reduced activity of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and/or citrate synthase, assuming reduced or no expression of the membrane protein.
The enhancement of the activity of an enzyme involved in threonine synthesis 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) Enhancement by modification of the amino acid sequence of the enzyme;
the reduction of the activity of an enzyme involved in threonine synthesis in the microorganism is achieved by a member selected from the group consisting of the following 6) to 10), or an optional combination:
6) Reduced by introducing a plasmid having a mutant encoding gene for the enzyme;
7) By reducing the copy number of the gene encoding the enzyme on the chromosome;
8) Reduced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
9) Reduced by operably linking a weak promoter to a gene encoding the enzyme;
10 Reduced by changing the amino acid sequence of the 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 threonine-producing strain, the method comprising:
A. weakening a gene encoding a putative membrane protein in a coryneform bacterium having an amino acid-producing ability to obtain a gene-weakened strain; the attenuation includes knocking out or reducing expression of a putative membrane protein encoding gene; and/or
B. Enhancing and/or reducing the activity of an enzyme associated with threonine synthesis in the attenuated strain of the gene of step A, to obtain a strain having enhanced and/or reduced enzyme activity;
the enhanced pathway is selected from the following 1) to 5), 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;
the reduced pathway is selected from the following 6) to 10), or an optional combination:
6) Reduced by introducing a plasmid having a mutant encoding gene for the enzyme;
7) By reducing the copy number of the gene encoding the enzyme on the chromosome;
8) Reduced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
9) Reduced by operably linking a weak promoter to a gene encoding the enzyme;
10 Reduced by altering the amino acid sequence of the enzyme;
wherein the enzyme associated with threonine synthesis with enhanced activity is selected from at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase, a mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and homoserine dehydrogenase; the enzyme associated with threonine synthesis having reduced activity is selected from at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase.
In a third aspect, the present invention provides a method for producing threonine, the method comprising the steps of:
a) Culturing the 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 invention provides the use of a knockout or reduced expression of a gene encoding a putative membrane protein in threonine fermentation production or in improving threonine fermentation production.
Further, the fermentation yield of threonine is improved by inactivating the putative membrane protein in coryneform bacteria (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 coryneform microorganism or the threonine-producing strain constructed according to the above-mentioned method 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 metabolic engineering of 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.
Preferably, in the modified coryneform microorganism of the present invention, the expression and/or the increase of the enzymatic activity of at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase and homoserine dehydrogenase is achieved by causing transcription of a gene encoding at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase and homoserine dehydrogenase by a strong promoter having stronger activity than its natural promoter. The increase in the expression and/or enzymatic activity of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans is achieved by inserting the gapN gene transcribed by Ptuf initiation at the Cgl1705 position of the chromosome of the starting strain.
Furthermore, the expression and/or the enzymatic activity of homoserine dehydrogenase are improved by mutating the gene hom encoding homoserine dehydrogenase, so that the encoded protein carries the G378E mutation. The expression and/or the increase in the enzymatic activity of the phosphoenolpyruvate carboxylase is also achieved by mutating the gene ppc encoding the phosphoenolpyruvate carboxylase, so that the encoded protein carries a D299N mutation.
Preferably, the coding gene of homoserine dehydrogenase is transcribed from PcspB, the nucleotide sequence of which is shown in SEQ ID NO. 43.
The coding gene of aspartate aminotransferase is transcribed from Psod whose nucleotide sequence is shown in SEQ ID NO. 44.
The coding gene of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans is transcribed by Ptuf, and the coding gene of phosphoenolpyruvate carboxylase is transcribed by Ptuf. The nucleotide sequence of Ptuf is shown as SEQ ID NO. 45.
Preferably, in the recombinant microorganism of the present invention, the expression and/or the enzymatic activity of at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase is reduced by mutating a gene encoding at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase.
More preferably, the gene dapA encoding 4-hydroxytetrahydropyridine dicarboxylic acid synthase is mutated such that its initiation codon is mutated from ATG to GTG, and the gene gltA encoding citrate synthase is mutated such that its initiation codon is mutated from ATG to GTG.
The invention has the advantages that:
the invention enhances the growth speed, the adaptability and the protein expression capacity of the strain by inactivating the postulated membrane protein (knocking out the non-essential gene cg 1746), and improves the threonine production yield of the strain compared with the strain before modification. The recombinant microorganism can be applied to threonine production, so that better production efficiency can be obtained, and a new idea is provided for threonine production.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The protein and the coding gene thereof related to the invention are as follows:
assuming membrane proteins, NCBI accession numbers: cg1746, cgl1548, NCgl1490;
aspartate aminotransferase, coding gene name aspB, NCBI accession number: cg0294, cgl0240, NCgl0237;
homoserine dehydrogenase, coding gene name hom, NCBI accession number: cg1337, cgl1183, NCgl1136;
phosphoenolpyruvate carboxylase, coding gene name ppc, NCBI accession number: cg1787, cgl1588, NCgl1523;
NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from streptococcus mutans, encoding the gene name gapN, NCBI accession No.: FOB93_04945;
4-Hydroxytetrahydropyridine dicarboxylic acid synthase, coding gene name dapA, NCBI accession number: cg2161, cgl1971, NCgl1895;
citrate synthase, encoding the gene name gltA, NCBI accession No.: cg0949, cgl0829, NCgl0795.
The invention constructs a series of threonine producing bacteria SMCT211, SMCT212, SMCT213, SMCT214, SMCT215 and SMCT216 by taking a model strain ATCC13032 as an initial strain, and constructs corresponding cg1746 inactivated strains SMCT218, SMCT219, SMCT220, SMCT221, SMCT222 and SMCT223, wherein the threonine yield is respectively improved by 20%, 26.7%, 36%, 38.9%, 50% and 56.6%, and the threonine yield is further improved when the inactivation of cg1746 is combined with protein modification related to a threonine synthesis path.
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; expression attenuation in the modification process comprises means such as replacement of a promoter, initial codon change, ribosome binding site change, gene knockout, gene mutation and the like. All of the above means are well known to those skilled in the art. The above means are not exhaustive, and therefore, the examples of the present invention will be described with reference to expression enhancement by using promoter substitution as a representative, and expression attenuation by using gene knockout and gene mutation as a representative.
EXAMPLE 1 construction of plasmid for genome engineering of Strain
1) cg1746 knockout plasmid pK18mobsacB- Δcg1746
PCR amplification was performed using ATCC13032 genome as a template, using PCT84/PCT85 primer pair to obtain upstream homology arm up, and using PCT86/PCT87 primer pair to obtain downstream homology arm dn. Fusion PCR was performed using the up/dn fragment as template with PCT84/PCT87 primer pair to obtain 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 cg1746.
2) Homoserine dehydrogenase expression enhancing plasmid pK18mobsacB-PcspB-hom G378E
Performing PCR amplification with ATCC13032 genome as template, P29/P30 primer pair to obtain upstream homology arm up, P35/P36 primer pair to obtain downstream homology arm dn, and P33/P34 primer pair to obtain hom G378E Fragments. The PcspB fragment was amplified using the ATCC14067 genome as a template and a P31/P32 primer pair. Up, dn, pcspB fragment, hom using P29/P36 as primer G378E Fusion PCR is carried out by taking the fragment as a template to obtain a full-length fragment up-PcspB-hom G378E 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-PcspB-hom G378E 。
3) Asparagus aminotransferase expression enhancing plasmid pK18mobsacB-Psod-aspB
The ATCC13032 genome is used as a template, the P103/P104 primer pair is used for PCR amplification to obtain an upstream homology arm up, the P107/P108 primer pair is used for PCR amplification to obtain a downstream homology arm dn, and the P105/P106 primer pair is used for amplification to obtain a Psod fragment. Fusion PCR was performed using the up, dn, psod fragment as a template and P103/P108 to obtain the full-length fragment 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-aspB.
4) NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression enhancing plasmid pK18mobsacB-Ptuf-gapN derived from Streptococcus mutans
PCR amplification was performed using ATCC13032 genome as a template, a P137/P138 primer pair to obtain an upstream homology arm up, a P143/P144 primer pair to obtain a downstream homology arm dn, a P139/P140 primer pair to obtain a Ptuf fragment, and a P141/P142 primer pair to obtain a gapN fragment. Fusion PCR is carried out by using up and Ptuf as templates and P137/P140 to obtain up-Ptuf, and fusion PCR is carried out by using up-Ptuf, gapN, dn segment as template and P137/P144 primer to obtain full-length segment up-Ptuf-gpaN-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-gapN.
5) Phosphoenolpyruvate carboxylase expression enhancing plasmid pK18mobsacB-Ptuf-ppc D299N
Using ATCC13032 genome as a template, performing PCR amplification by using a P53/P54 primer pair to obtain an upstream homology arm up, performing PCR amplification by using a P59/P60 primer pair to obtain a downstream homology arm dn, performing PCR amplification by using a P55/P56 primer pair to obtain a Ptuf fragment, and performing PCR amplification by using a P57/P58 primer pair to obtain ppc D299N Fragments. Fusion PCR was performed with P53/P56 using up and Ptuf as templates to obtain up-Ptuf, and up-Ptuf, ppc D299N Fusion PCR was performed using the dn fragment as a template and the P53/P60 primer to obtain the full-length fragment up-Ptuf-ppc D299N Dn. pK18mobsacB was digested with BamHI/HindIII. Assembling the two with a seamless cloning kit, and transforming a Trans 1T 1 competent cell to obtain a recombinant plasmid pK18mobsacB-Ptuf-ppc D299N 。
6) 4-hydroxy tetrahydropyridine dicarboxylic acid synthase expression attenuation plasmid pK18mobsacB-dapA a1g
The ATCC13032 genome is used as a template, the P75/P76 primer pair is used for PCR amplification to obtain an upstream homology arm up, and the P77/P78 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR was performed using the up/dn fragment as template with the P75/P78 primer pair to obtain the full-length fragment 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 。
7) Citrate synthase expression attenuation plasmid pK18mobsacB-gltA a1g
The ATCC13032 genome is used as a template, the P153/P154 primer pair is used for PCR amplification to obtain an upstream homology arm up, and the P155/P156 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR was performed using the up/dn fragment as template with the P153/P156 primer pair to obtain the full-length fragment 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-gltA a1g 。
The primers used in the plasmid construction procedure are shown in Table 1 below:
TABLE 1
Note that: the corresponding bases of the primers in Table 1 into which the point mutations were introduced were bold underlined bases.
EXAMPLE 2 construction of wild-type Strain genome engineering Strain
1) Construction of homoserine dehydrogenase-enhanced expression Strain
According to the classical method of cereal bars (C.glutamicum Handbook, charpter 23) to prepare ATCC13032 competent cells. Recombinant plasmid pK18mobsacB-PcspB-hom G378E 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 SMCT211.
2) Construction of phosphoenolpyruvate carboxylase-enhanced expression Strain
Strain construction method referring to 1) above, modification of phosphoenolpyruvate carboxylase expression enhancement was performed using SMCT211 as a starting strain (pK 18mobsacB-Ptuf-ppc was used) D299N Introduction of SMCT 211), the engineered strain obtained was named SMCT212.
3) Construction of aspartate aminotransferase-enhanced expression Strain
Method for constructing Strain referring to 1) above, modification of enhanced expression of aspartate aminotransferase (pK 18mobsacB-Psod-aspB was introduced into SMCT 212) was performed using SMCT212 as a starting strain, and the obtained modified strain was designated as SMCT213.
4) Construction of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase-enriched expression Strain derived from Streptococcus mutans
Method for constructing Strain referring to 1) above, the modified strain obtained by performing modification of the enhanced expression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans (introduction of pK18mobsacB-Ptuf-gapN into SMCT 213) using SMCT213 as a starting strain was designated as SMCT214.
5) Construction of 4-hydroxytetrahydropyridine dicarboxylic acid synthase attenuated expression strains
Strain construction method referring to 1) above, modification of 4-hydroxytetrahydropyridine dicarboxylic acid synthase expression attenuation was performed using SMCT214 as starting strain (pK 18mobsacB-dapA was modified a1g Introducing SMCT 214), the resulting engineered strain was designated SMCT215.
6) Construction of citrate synthase attenuated expression strains
Strain construction method referring to 1) above, modification of expression attenuation of citrate synthase was performed using SMCT215 as starting strain (pK 18mobsacB-gltA was modified a1g Introducing SMCT 215), the resulting engineered strain was designated SMCT216.
The obtained strains are listed in table 2 below.
TABLE 2
Strain name Genotype of the type
SMCT211 ATCC13032，PcspB-hom G378E
SMCT212 ATCC13032，PcspB-hom G378E ，Ptuf-ppc D299N
SMCT213 ATCC13032，PcspB-hom G378E ，Ptuf-ppc D299N ，Psod-aspB
SMCT214 ATCC13032，PcspB-hom G378E ，Ptuf-ppc D299N ，Psod-aspB，Ptuf-gapN
SMCT215 ATCC13032，PcspB-hom G378E ，Ptuf-ppc D299N ，Psod-aspB，Ptuf-gapN，dapA a1g
SMCT216 ATCC13032，PcspB-hom G378E ，Ptuf-ppc D299N ，Psod-aspB，Ptuf-gapN，dapA a1g ，gltA a1g
EXAMPLE 3 construction of a genomic engineered Strain of the cg1746 knockout Strain
1) construction of cg1746 knockout Strain
ATCC13032 competent cells were prepared according to the classical method of cereal bars (c.glutamicum Handbook, charter 23). The recombinant plasmid pK18 mobsacB-. DELTA.cg 1746 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 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 SMCT217.
2) Construction of homoserine dehydrogenase-enhanced expression Strain
Strain construction method referring to 1) above, modification of homoserine dehydrogenase expression enhancement was performed using SMCT217 as a starting strain (pK 18mobsacB-PcspB-hom was used G378E Introduction of SMCT 217), the engineered strain obtained was designated SMCT218.
3) Construction of phosphoenolpyruvate carboxylase-enhanced expression Strain
Strain construction method referring to 1) above, modification of phosphoenolpyruvate carboxylase expression enhancement was performed using SMCT218 as a starting strain (pK 18mobsacB-Ptuf-ppc was modified) D299N Introducing SMCT 218), the engineered strain obtained was named SMCT219.
4) Construction of aspartate aminotransferase-enhanced expression Strain
Method for constructing Strain referring to 1) above, modification of enhanced expression of aspartate aminotransferase was performed using SMCT219 as a starting strain (pK 18mobsacB-Psod-aspB was introduced into SMCT 219), and the obtained modified strain was designated as SMCT220.
5) Construction of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase-enriched expression Strain derived from Streptococcus mutans
Method for constructing Strain referring to 1) above, the modified strain obtained by performing modification of enhanced expression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Streptococcus mutans (introduction of pK18mobsacB-Ptuf-gapN into SMCT 220) using SMCT220 as starting strain was designated as SMCT221.
6) Construction of 4-hydroxytetrahydropyridine dicarboxylic acid synthase attenuated expression strains
Strain construction method referring to 1) above, modification of 4-hydroxytetrahydropyridine dicarboxylic acid synthase expression attenuation was performed using SMCT221 as starting strain (pK 18mobsacB-dapA was modified a1g The resulting engineered strain was designated SMCT222.
7) Construction of citrate synthase attenuated expression strains
Strain construction method referring to 1) above, modification of expression attenuation of citrate synthase was performed using SMCT222 as starting strain (pK 18mobsacB-gltA was modified a1g Introduction of SMCT 222), the engineered strain obtained was named SMCT223.
The obtained strain list is shown in table 3 below.
TABLE 3 Table 3
EXAMPLE 4 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 & HCl40mg/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: ATCC13032, smtt 211, smtt 212, smtt 213, smtt 214, smtt 215, smtt 216, smtt 217, smtt 218, smtt 219, smtt 220, smtt 221, smtt 222, smtt 223, slant seed 1 was looped into a 500mL flask containing 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, and the concentrations thereof were shown in Table 4 below.
TABLE 4 comparison of threonine-producing Capacity of Corynebacterium glutamicum
As can be seen from the above table, the modified strain inactivated cg1746 has a different improvement in threonine yield compared with the non-inactivated strain, and the threonine yield is improved by 20% -56.6%. In addition, when cg1746 was combined with at least one site of enhanced expression, 4-hydroxytetrahydropyridine dicarboxylic acid synthase, and citrate synthase, which are expressed in a weakened manner, of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, and mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, it was found that cg1746 was also advantageous for threonine production in combination with the above sites.
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> a modified microorganism of Corynebacterium genus, use and construction method for threonine production thereof
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cattcgcagg gtaacggcca agggtgttgg cgtgcatgag 40
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<212> DNA
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<212> DNA
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<212> DNA
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tagctgccaa ttattccggg cttgt 25
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<210> 32
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<212> DNA
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
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<212> DNA
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gattttttac cc 192
<210> 44
<211> 260
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<400> 44
acctgcgttt ataaagaaat gtaaacgtga tcggatcgat ataaaagaaa cagtttgtac 60
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tttctcaacg acaggctgct aagctgctag ttcggtggcc tagtgagtgg cgtttacttg 180
gataaaagta atcccatgtc gtgatcagcc attttgggtt gtttccatag catccaaagg 240
tttcgtcttt cgatacctat 260
<210> 45
<211> 200
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
tggccgttac cctgcgaatg tccacagggt agctggtagt ttgaaaatca acgccgttgc 60
ccttaggatt cagtaactgg cacattttgt aatgcgctag atctgtgtgc tcagtcttcc 120
aggctgctta tcacagtgaa agcaaaacca attcgtggct gcgaaagtcg tagccaccac 180
gaagtccagg aggacataca 200

Claims (9)

1. A modified coryneform microorganism, characterized in that the microorganism has reduced or no expression of a membrane protein as compared to an unmodified microorganism and the microorganism has enhanced threonine-producing ability as compared to an unmodified microorganism.
2. The microorganism of claim 1, wherein the reduced or no expression of the putative membrane protein in the microorganism is achieved by reducing expression of a gene encoding the putative membrane protein or knocking out an endogenous gene encoding the putative membrane protein.
3. The microorganism of claim 2, wherein the expression of the gene encoding the putative membrane protein is reduced or the endogenous gene encoding the putative membrane protein is knocked out by mutagenesis, site-directed mutagenesis or homologous recombination.
4. The microorganism according to claim 1, characterized in that the activity of an enzyme involved in threonine synthesis in vivo is increased and/or decreased as compared to the unmodified microorganism;
wherein the enzyme associated with threonine synthesis with enhanced activity is selected from at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase, a mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and homoserine dehydrogenase; the enzyme associated with threonine synthesis having reduced activity is selected from at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase.
5. The microorganism according to claim 4, wherein the microorganism is any one of the following (1) to (6):
(1) a microorganism which has reduced or no expression of a membrane protein and has enhanced homoserine dehydrogenase activity;
(2) a microorganism which is presumed to have reduced or no expression of the membrane protein and has enhanced homoserine dehydrogenase and/or phosphoenolpyruvate carboxylase activity;
(3) a microorganism which assumes reduced or no expression of the membrane protein and enhanced homoserine dehydrogenase, phosphoenolpyruvate carboxylase and/or aspartate aminotransferase activity;
(4) a microorganism which assumes reduced or no expression of membrane proteins and enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase;
(5) a microorganism having enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and reduced activity of 4-hydroxytetrahydropyridine dicarboxylic acid synthase, assuming reduced or no expression of the membrane protein;
(6) a microorganism having enhanced activity of homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and/or mutans streptococcus derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and reduced activity of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and/or citrate synthase, assuming reduced or no expression of the membrane protein.
6. The microorganism according to claim 4, wherein the enhancement of the activity of an enzyme involved in threonine synthesis in the microorganism is achieved by a compound selected from the group consisting of 1) to 5), 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;
the reduction of the activity of an enzyme involved in threonine synthesis in the microorganism is achieved by a member selected from the group consisting of the following 6) to 10), or an optional combination:
6) Reduced by introducing a plasmid having a mutant encoding gene for the enzyme;
7) By reducing the copy number of the gene encoding the enzyme on the chromosome;
8) Reduced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
9) Reduced by operably linking a weak promoter to a gene encoding the enzyme;
10 Reduced by changing the amino acid sequence of the enzyme.
7. A microorganism according to any of claims 1 to 5, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
8. A method for constructing a threonine-producing strain, the method comprising:
A. weakening a gene encoding a putative membrane protein in a coryneform bacterium having an amino acid-producing ability to obtain a gene-weakened strain; the attenuation includes knocking out or reducing expression of a putative membrane protein encoding gene; and/or
B. Enhancing and/or reducing the activity of an enzyme associated with threonine synthesis in the attenuated strain of the gene of step A, to obtain a strain having enhanced and/or reduced enzyme activity;
the enhanced pathway is selected from the following 1) to 5), 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;
the reduced pathway is selected from the following 6) to 10), or an optional combination:
6) Reduced by introducing a plasmid having a mutant encoding gene for the enzyme;
7) By reducing the copy number of the gene encoding the enzyme on the chromosome;
8) Reduced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
9) Reduced by operably linking a weak promoter to a gene encoding the enzyme;
10 Reduced by altering the amino acid sequence of the enzyme;
wherein the enzyme associated with threonine synthesis with enhanced activity is selected from at least one of aspartate aminotransferase, phosphoenolpyruvate carboxylase, a mutans streptococcus-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and homoserine dehydrogenase; the enzyme associated with threonine synthesis having reduced activity is selected from at least one of 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase.
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).
CN202210108388.1A 2022-01-28 2022-01-28 Modified corynebacterium microorganism and application and construction method for producing threonine thereof Pending CN116555132A (en)

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CN113322218B (en) * 2020-02-28 2022-11-22 廊坊梅花生物技术开发有限公司 Recombinant corynebacterium glutamicum and method for producing L-threonine

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