CN117417954A - Recombinant microorganism and construction method and application thereof - Google Patents

Recombinant microorganism and construction method and application thereof Download PDF

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CN117417954A
CN117417954A CN202210851613.0A CN202210851613A CN117417954A CN 117417954 A CN117417954 A CN 117417954A CN 202210851613 A CN202210851613 A CN 202210851613A CN 117417954 A CN117417954 A CN 117417954A
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gene
promoter
expression
inactivating
gapn
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王亚迪
胡丹
冯帆
赵津津
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention relates to the technical field of genetic engineering, and particularly discloses a recombinant microorganism and a construction method and application thereof. The construction method of the recombinant microorganism optimizes the expression of specific genes related to lysine synthesis through metabolic engineering means, and comprises the steps of enabling an original strain to strengthen expression genes lysC, asd, argS-lysA and ddh and inactivating or weakening expression genes hom and poxB. Further comprising the step of allowing the starting strain to express genes ppc, aspB, acs, gapN, ppnk and ptsG in an enriched manner, and inactivating or attenuating the expressed genes sucC, aceE, ack, cgl, 2680, dld and NCgl 2493. The finally obtained genetically engineered strain can be used for producing lysine by efficient fermentation.

Description

Recombinant microorganism and construction method and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant microorganism and a construction method and application thereof.
Background
Corynebacterium glutamicum (Corynebacterium glutamicum) is a gram-positive microorganism with the characteristics of fast growth rate, non-pathogenic, and weak ability to degrade self-metabolites. As a conventional industrial microorganism, corynebacterium glutamicum is widely used for the production of various amino acids, nucleotides and other organic acids.
The history of the use of corynebacterium glutamicum for producing amino acids can be traced to the 60 th century, corynebacterium glutamicum can produce glutamic acid in natural environment, and the mutagenized corynebacterium glutamicum can also produce various amino acids such as lysine and valine. Along with the rapid development of metabolic engineering technology and genome sequencing technology, the metabolic pathway of corynebacterium glutamicum is studied more and more clearly, researchers successively identify a plurality of genetically high-yield mechanisms, and high-efficiency production of various metabolites is realized.
Aspartic acid and amino acids of the aspartic acid family have wide application in the fields of feed, food, medicine, chemical industry and the like. Wherein L-lysine is a basic essential amino acid with the molecular formula of C 6 H 14 N 2 O 2 The appearance is white or nearly white crystalline powder. Darkening at 210 ℃, decomposing at 224.5 ℃, being readily soluble in water, slightly soluble in alcohol, insoluble in ether. L-lysine is widely used in animal feed, medicine and food industry, of which about 90% is used in feed industry and 10% is used in food and medicine industry. The L-lysine can promote the animal body to absorb other amino acids, so as to improve the quality of the feed.
At present, the main production method of L-lysine is a microbial fermentation method, and the microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like. The industrial production strains of lysine comprise escherichia coli and corynebacterium glutamicum, and the corynebacterium glutamicum has better market competitive advantage than the escherichia coli because of the biological safety of the corynebacterium glutamicum and no need of considering endotoxin residue problem when preparing low-content feed products. Corynebacterium glutamicum (Corynebacterium glutamicum) is a gram-positive bacterium with the characteristics of fast growth rate, non-pathogenic, and weak ability to degrade self-metabolites. As a conventional industrial microorganism, corynebacterium glutamicum is widely used for the production of various amino acids, nucleotides and other organic acids.
Natural corynebacterium glutamicum is useful for the production of amino acids such as glutamate and lysine, and high yielding strains are typically obtained by random mutagenesis with UV light or chemical mutagens and subsequent strain selection. However, the potential unknown random mutation in the strain has certain side effects on the growth of the strain and the synthesis of products. Further metabolic operations and process control of the strain cannot be optimized to the most perfect state, and the strain cannot always reach the highest conversion rate.
Therefore, further studies on the fermentative production of amino acids by Corynebacterium glutamicum are necessary.
Disclosure of Invention
The invention aims to provide a recombinant microorganism which has stable performance and can efficiently produce lysine.
The technical scheme of the invention is as follows:
a method of constructing a recombinant microorganism comprising the steps of allowing an initial strain to express genes lysC, asd, argS-lysA and ddh in an enhanced manner, and inactivating or attenuating the expressed genes hom and poxB.
The construction method of the invention also comprises the steps of enabling the original strain to strengthen the expression genes ppc and aspB, introducing acs genes exogenously, and inactivating or weakening the expression genes sucC, aceE and ack.
The construction method of the invention further comprises the steps of enabling the original strain to carry out intensified expression on genes gapN and ppnk and inactivating or weakening the expressed genes Cgl2680 and dld.
The construction method of the invention also comprises the steps of enabling the original strain to express the gene ptsG in a strengthening mode and inactivating or weakening the expressed gene NCgl 2493.
According to the invention, through strain research and development and production analysis for many years, the metabolic engineering means, preferably ten steps of genetic operation, are utilized to realize specific optimization of expression of nineteen key genes related to lysine synthesis in the original strain, and finally the obtained genetic engineering strain can be used for efficient fermentation production of lysine.
First, the present invention preferentially enhances the terminal synthesis pathway, including expression enhancement of the following genes lysC, asd, argS-lysA, ddh, and inactivation modification of hom and poxB genes.
Secondly, the central metabolism is optimized, and the purpose is to pull metabolic flux balance to aspartic acid nodes, so as to strengthen the synthesis supply of lysine. The related genes comprise glycolysis and citric acid circulation, preferably the expression enhancement of the following genes ppc and aspB is carried out, and the external introduction of acs genes is carried out, and the attenuation or inactivation modification of the following genes sucC, aceE and ack is carried out.
Again, the supply of reducing cofactors was enhanced, including exogenously introduced codon optimized gapN genes, and the following genes gapN, ppnk were expression enhanced. The following genes Cgl2680 and dld were attenuated or inactivated.
Finally, the glucose phosphotransferase activity is enhanced, including enhancement of the expressed gene ptsG and inactivation or attenuation of the expressed gene NCgl2493.
In the construction method of the present invention, the means for enhancing expression of the gene may be selected from one or more of the following (1) to (3):
(1) Mutating or replacing the endogenous promoter of the gene with a stronger promoter;
(2) Increasing the copy number of the gene;
(3) Mutating the coding region of the gene (making it change at the amino acid level);
and/or the manner of inactivating or attenuating the expressed gene may be selected from one or more of the following (1) - (3):
(1) Mutating or replacing the endogenous promoter of the gene with a weaker promoter;
(2) Reducing the copy number of the gene;
(3) The coding region of the gene is mutated (changed at the amino acid level).
The inactivation modification of the gene is preferably to carry out deletion mutation on a coding region of the gene or simultaneously carry out insertion modification of other genes, so that the inactivation modification of the target deletion gene and the ectopic insertion modification of the target over-expressed gene are realized in one step.
Preferably, in the construction method of the present invention, the hom gene is inactivated and expression of the lysC-asd gene is enhanced simultaneously by deleting the hom gene and adding a copy of the lysC-asd gene at the site where the hom gene is deleted;
and/or, enhancing expression of the ddh gene by introducing an artificial strong promoter before the initiation codon of the ddh gene;
and/or, inactivating the poxB gene and enhancing the expression of the argS-lysA gene simultaneously by deleting the poxB gene and adding one copy of the argS-lysA gene at the poxB gene deletion;
and/or inactivating the sucC gene and enhancing expression of the ppc gene simultaneously by deleting the sucC gene and adding a mutated copy of the ppc gene at the point of deletion of the sucC gene;
And/or, enhancing expression of the aspB gene by introducing a strong promoter before the start codon of the aspB gene;
and/or inactivating or attenuating expression of the aceE gene by introducing a weak promoter before the initiation codon of the aceE gene;
and/or inactivating the ack gene and enhancing expression of the acs gene simultaneously by deleting the ack gene and adding one copy of the acs gene at the ack gene deletion;
and/or inactivating the Cgl2680 gene and enhancing the expression of the gapN gene simultaneously by deleting the Cgl2680 gene and adding a copy of the gapN gene at the position where the Cgl2680 gene is deleted, and changing the start codon of the gapN gene and introducing an artificial strong promoter before the start codon of the gapN gene;
and/or inactivating the dld gene and enhancing expression of the ppnk gene simultaneously by deleting the dld gene and adding a ppnk gene copy at the site of the dld gene deletion;
and/or, inactivating the NCgl2493 gene and enhancing expression of the ptsG gene simultaneously by deleting the NCgl2493 gene and adding a copy of the ptsG gene at the point where the NCgl2493 gene is deleted, and introducing a strong promoter before the start codon of the ptsG gene.
In the construction method of the invention, when the expression of ddh gene is enhanced, a promoter of pyc gene containing point mutation is introduced before the start codon of ddh gene, wherein the promoter of pyc gene containing point mutation takes the promoter of wild pyc gene as a reference sequence, and the 209 th to 221 th sites are mutated into TGTGGTATAATGG (SEQ ID No. 71);
When the expression of the ppc gene is enhanced, the mutated ppc gene takes the wild-type ppc gene as a reference sequence, and the 299 th position is mutated into N;
when the expression of aspB gene is enhanced, the introduced strong promoter is PcspB;
the exogenously introduced acs gene is derived from E.coli;
when the expression of aceE gene is inactivated or weakened, the weak promoter is PdapB;
when the expression of gapN gene is enhanced, the increased gapN gene is from streptococcus mutans, the starting codon of the gapN gene is replaced by ATG, and a promoter of pyc gene containing point mutation is introduced before the starting codon of the gapN gene, wherein the promoter of pyc gene containing point mutation takes the promoter of wild pyc gene as a reference sequence, and the 209 th to 221 th are mutated into TGTGGTATAATGG (SEQ ID No. 71);
when the expression of the ptsG gene is enhanced, the introduced strong promoter is Pgap;
and/or, the starting strain is corynebacterium glutamicum.
In the present invention, each wild-type gene is a gene known in the art, and as described in NCBI, the wild-type hom gene is NCgl1136, the wild-type lysC gene is NCgl0247, the wild-type asd gene is NCgl0248, the wild-type ddh gene is NCgl2528, the wild-type poxB gene is NCgl2521, the wild-type argS gene is NCgl1131, the wild-type lysA gene is NCgl1132, the wild-type sucC gene is NCgl2476, the wild-type ppc gene is NCgl1523, the wild-type aspB gene is NCgl0237, the wild-type aceE gene is NCgl2167, the wild-type ack gene is NCgl2656, the wild-type acs gene is 948572, the encoded protein ID of the wild-type gapN gene is WP 002262986.1, the wild-type dld gene is NCgl0865, the wild-type ppnk gene is NCgl1358, and the wild-type ptsG gene is NCgl.
The promoter sequence of the wild-type pyc gene is shown in SEQ ID No. 67; the sequence of the promoter PcspB is shown as SEQ ID No.68, the sequence of the promoter PdapB is shown as SEQ ID No.69, and the sequence of the promoter Pgap is shown as SEQ ID No. 70.
According to the invention, the Corynebacterium glutamicum model strain ATCC 13032 is taken as an initial strain, 19 genes related to lysine synthesis are screened out through analysis of metabolic pathways, and specific expression optimization is carried out on the genes, preferably, the effect of rapid construction is achieved. Finally, through 10 rounds of genetic operation, the specific combination optimization of 19 genes can be realized, and finally the obtained genetically engineered bacteria have the effect of high lysine yield through 5L tank test.
The invention also provides a recombinant microorganism which is constructed by the construction method.
The invention also provides an application of any one of the recombinant microorganisms as follows:
(1) The application in producing lysine by fermentation;
(2) Use in genetic breeding of microorganisms for producing lysine;
(3) The application of the method in improving the performance of synthesizing lysine by a biological method.
The present invention also provides a method for producing lysine by fermentation, which comprises the step of culturing the recombinant microorganism described above.
The invention has the advantages that:
the invention provides a new construction method of high-performance lysine production strains, which has the characteristics of repeatability, fewer construction steps and short period, and the obtained genetic engineering strains have excellent production performance.
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. In particular, the examples are not to be construed as specific techniques or conditions, as described in the literature in this field, or as product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The primer sequence information used in the examples of the present invention is shown in Table 1. The target points of the genetically modified genes according to the embodiment of the invention are shown in table 2.
TABLE 1 primer sequence information (SEQ ID No. 3-66)
TABLE 2 examples and genetic engineering Gene target information
EXAMPLE 1 deletion of hom Gene and addition of a copy of lysC-asd at the position of the hom Gene deletion
1.1 construction of engineering plasmid pK18-hom:: lysC-asd
The genomic sequence of ATCC 13032 was published and queried from NCBI website, and PCR amplification was performed using hom-1f/hom-1r primer set to obtain the upstream homology arm fragment hom-up of gene recombination, using ATCC 13032 genome as a template (Corynebacterium glutamicum (Corynebacterium glutamicum) model strain ATCC 13032 is available from public sources). PCR amplification was performed using ATCC 13032 genome as a template and a hom-2f/hom-2r primer pair to obtain a downstream homology arm fragment hom-dn of the gene recombination. The ATCC 13032 genome is used as a template, and a lysC-asd-f/lysC-asd-r primer pair is used for PCR amplification to obtain a target inserted gene fragment lysC-asd, wherein the fragment comprises a coding region of the lysC-asd and a natural promoter region of the coding region. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment after enzyme digestion and gel recovery and the vector are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and is named pK18-hom:: lysC-asd.
1.2 construction of engineering Strain 13032, hom:: lysC-asd
Competent cells of Corynebacterium glutamicum ATCC13032 were prepared and subjected to shock transformation and screening of recombinant strains according to the method described in Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23).
The recombinant plasmid pK18-hom obtained above was transformed into ATCC13032 competent cells by electric shock, and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) Thin, thinThe release solution was spread on a normal BHI solid medium containing 10% sucrose, and was allowed to stand at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair hom-1f/hom-2 r) and nucleotide sequencing analysis. The genotype of the finally obtained target modified strain is 13032, hom:: lysC-asd, and the strain is named SCK001.
EXAMPLE 2 introduction of an Artificial strong promoter before the initiation codon of the ddh Gene
2.1 construction of the engineering plasmid pK 18-ddhPyc
The sequence of the artificial strong promoter is shown in SEQ ID No.1, which is a promoter containing the point mutated pyc gene. According to the invention, through promoter activity measurement, the mutant promoter is found to have stronger expression activity than other known strong promoters. The sequence SEQ ID No.1 can be synthesized in full sequence by a third party gene synthesis company, and can also be introduced with the target mutation by a specific primer PCR amplification and fragment fusion mode. In this example, the full-sequence synthesis was performed by a third party gene synthesis company.
PCR amplification was performed using ATCC 13032 genome as a template and ddh-1f/ddh-1r primer pairs to obtain a gene recombinant upstream homology arm fragment ddh-up. PCR amplification was performed using ATCC 13032 genome as a template and ddh-2f/ddh-2r primer pairs to obtain a downstream homology arm fragment ddh-dn of the gene recombination. PCR amplification was performed using the sequence SEQ ID No.1 as a template and a Ppyc-f/Ppyc-r primer pair to obtain the target artificial promoter fragment Ppyc carrying the fusion region. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and vector after cleavage and gel recovery were ligated with T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid was sequenced and confirmed and designated pK 18-ddhPyc.
2.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc
Competent cells of the strain SCK001 obtained in example 1 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of model bacteria 13032 described in example 1.
The recombinant plasmid pK18-ddhPpyc obtained above was transformed into SCK001 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair ddh-1f/ddh-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc, strain designated SCK002.
EXAMPLE 3 deletion of poxB Gene and addition of an argS-lysA copy at the poxB Gene deletion
3.1 construction of the engineering plasmid pK18-poxB: argS-lysA
PCR amplification was performed using ATCC 13032 genome as a template and a poxB-1f/poxB-1r primer pair to obtain a recombinant upstream homology arm fragment poxB-up. PCR amplification was performed using ATCC 13032 genome as a template and a poxB-2f/poxB-2r primer pair to obtain a downstream homology arm fragment poxB-dn of gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and the argS-lysA-f/argS-lysA-r primer pair to obtain an insert gene fragment of interest argS-lysA, which comprises the coding region of argS-lysA and its natural promoter region. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment after digestion and gel recovery and the vector are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and named pK18-poxB:: argS-lysA.
3.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc:, poxB::: argS-lysA
Competent cells of the strain SCK002 obtained in example 2 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of model bacteria 13032 described in example 1.
The recombinant plasmid pK18-poxB obtained above was transformed into SCK002 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair poxB-1f/poxB-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB::: argS-lysA, strain designated SCK003.
The above examples are examples of enhancement of the terminal synthesis pathway, including enhancement of expression of the following genes lysC, asd, argS-lysA, ddh, and inactivation modification of hom and poxB genes. The enhancement of the gene may be by mutation of the endogenous promoter of the gene, or by substitution with another strong promoter, or by increasing the copy number of the gene, or by a combination of the above. The inactivation modification of the gene is preferably to carry out deletion mutation on a coding region of the gene or simultaneously carry out insertion modification of other genes, so that the inactivation modification of the target deletion gene and the ectopic insertion modification of the target over-expressed gene are realized in one step. The next step is to optimize the central metabolism.
EXAMPLE 4 deletion of the sucC Gene and addition of a ppc copy at the position of the sucC Gene deletion, and introduction of the coding region of the ppc Gene into the mutant D299N
4.1 construction of the engineering plasmid pK18-sucC:: ppcD299N
PCR amplification was performed using ATCC 13032 genome as a template and a sucC-1f/sucC-1r primer pair to obtain a gene recombinant upstream homology arm fragment sucC-up. PCR amplification was performed using ATCC 13032 genome as a template and a sucC-2f/sucC-2r primer pair to obtain a downstream homology arm fragment sucC-dn of the gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and ppc-1f/ppc-1r primer pairs to obtain ppcD299N-up upstream of the target substitution fragment. PCR amplification was performed using ATCC 13032 genome as a template and ppc-2f/ppc-2r primer pairs to obtain ppcD299N-dn downstream of the target substitution fragment. And carrying out fusion PCR on the four fragments to obtain the full-length fragment fused with the 4 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and the vector after enzyme digestion and gel recovery are connected by T4 DNA Ligase and transformed into Trans1T1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and is named pK18-sucC:: ppcD299N.
4.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc: poxB:: argS-lysA, sucC::: ppcD299N
Competent cells of the strain SCK003 obtained in example 3 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model bacteria 13032 described in example 1.
The recombinant plasmid pK18-sucC obtained above was transformed into SCK003 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, transformants developAnd a second recombination step, wherein the vector sequence is removed from the genome by gene exchange and the target mutation is introduced. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. Further confirmation of successful recombination was achieved by PCR amplification (using primer pair sucC-1f/sucC-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC::: ppcD299N, strain was designated SCK004.
EXAMPLE 5 introduction of a strong promoter before the initiation codon of aspB Gene
5.1 construction of the engineering plasmid pK 18-asppBPcspB
PCR amplification was performed using ATCC 13032 genome as a template and aspB-1f/aspB-1r primer pair to obtain an upstream homology arm fragment aspB-up of gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and aspB-2f/aspB-2r primer pairs to obtain a downstream homology arm fragment aspB-dn of gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and PcspB-f/PcspB-r primer pairs to obtain a promoter fragment PcspB. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment obtained after cleavage and gel recovery was ligated with the vector using T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid was confirmed by sequencing and designated pK 18-asppBPcspB.
5.2 construction of engineering bacteria 13032, hom:: lysC-asd, ddhPpyc: poxB:: argS-lysA, sucC::: ppcD299N, asppBPcspB
Competent cells of the strain SCK004 obtained in example 4 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model bacteria 13032 described in example 1.
By electricityThe recombinant plasmid pK 18-asppBPcspB obtained above was transformed into SCK004 competent cells by the method of knocking, and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of the recombination was further confirmed by PCR amplification (using primer pair aspB-1f/aspB-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPpyc:, poxB:: argS-lysA, sucC::: ppcD299N, aspB Pcspb, strain designated SCK005.
EXAMPLE 6 introduction of a weak promoter before the initiation codon of the aceE Gene
6.1 construction of the engineering plasmid pK18-aceEPdapB
And (3) taking the ATCC 13032 genome as a template, and carrying out PCR amplification by using an aceE-1f/aceE-1r primer pair to obtain an upstream homologous arm fragment aceE-up of the gene recombination. And (3) taking the ATCC 13032 genome as a template, and carrying out PCR amplification by using aceE-2f/aceE-2r primer pairs to obtain a downstream homologous arm fragment aceE-dn of the gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and PdapB-f/PdapB-r primer pairs to obtain a promoter fragment PdapB. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and vector after cleavage and gel recovery were ligated with T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid was sequenced and confirmed and designated pK18-aceEPdapB.
6.2 construction of engineering bacteria 13032, hom:: lysC-asd, ddhPyc: poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB
Competent cells of the strain SCK005 obtained in example 5 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model strain 13032 described in example 1.
The recombinant plasmid pK18-aceEPdapB obtained above was transformed into SCK005 competent cells by electric shock, and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair aceE-1f/aceE-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC::: ppcD299N, aspBPcspB, aceEPdapB, strain designated SCK006.
EXAMPLE 7 deletion of ack Gene and addition of one acs copy at the ack Gene deletion
7.1 construction of the engineering plasmid pK 18-ack::: acs
In this example, an exogenous acs gene was inserted at the ack gene. The insertion and inactivation of the gene were achieved in one step in the same way as in the previous examples 1 and 3.
Firstly, constructing a plasmid, and carrying out PCR amplification by using ATCC 13032 genome as a template and ack-1f/ack-1r primer pairs to obtain an upstream homologous arm fragment ack-up of gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and ack-2f/ack-2r primer pair to obtain downstream homology arm fragment ack-dn of gene recombination. And (3) taking the escherichia coli MG1655 genome as a template, and carrying out PCR amplification by using an acs-f/acs-r primer pair to obtain a target inserted gene fragment acs, wherein the fragment comprises an acs coding region and a natural promoter region thereof. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and the vector after enzyme digestion and glue recovery are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and is named pK18-ack:: acs.
7.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc: poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack::: acs
Competent cells of the strain SCK006 obtained in example 6 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model bacteria 13032 described in example 1.
The recombinant plasmid pK18-ack obtained above was transformed into SCK006 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair ack-1f/ack-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, strain designated SCK007.
The above examples optimize the central metabolism with the aim of pulling the metabolic flux balance to the aspartic acid node, thus enhancing the synthetic supply of lysine. The genes involved include glycolysis and citric acid cycle, and expression enhancement is preferably performed on the following genes ppc, aspB and acs, and attenuation or inactivation modification is performed on the following genes sucC, aceE, ack. The enhancement of the gene may be by mutation of the endogenous promoter of the gene, or by substitution with another strong promoter, or by increasing the copy number of the gene, or by introducing mutations in the coding region that alter the amino acid level, or by a combination of the above. The attenuation or inactivation modification can be replaced by other weak promoters, or the coding region of the gene is subjected to deletion mutation, or insertion modification of other genes is simultaneously carried out, so that the inactivation modification of the target deletion gene and the ectopic insertion modification of the target over-expressed gene are realized in one step. The next step is to intensify the supply of the reducing power cofactor.
EXAMPLE 8 deletion of Cgl2680 Gene and addition of one copy of gapN at the deletion of Cgl2680 Gene, and modification of the initiation codon of gapN Gene and introduction of an artificial strong promoter before it
8.1 construction of the engineering plasmid pK18-Cgl2680:: gapN
The sequence of the artificial strong promoter is shown in SEQ ID No.1, which is a promoter containing the point mutated pyc gene. According to the invention, through promoter activity measurement, the mutant promoter is found to have stronger expression activity than other known strong promoters. The sequence SEQ ID No.1 can be synthesized in full sequence by a third party gene synthesis company, and can also be introduced with the target mutation by a specific primer PCR amplification and fragment fusion mode. In this example, the full-sequence synthesis was performed by a third party gene synthesis company. The gapN gene derived from streptococcus mutans and subjected to initial codon mutation is shown as a sequence SEQ ID No.2, wherein the sequence comprises a complete coding region, and the initial codon of the coding region is ATG. The sequence SEQ ID No.2 can be synthesized in full sequence by a third party gene synthesis company, and can also be introduced into target mutation by a specific primer PCR amplification and multiple fragment fusion mode. In this example, the full-sequence synthesis was performed by a third party gene synthesis company.
PCR amplification was performed using ATCC 13032 genome as a template and Cgl2680-1f/Cgl2680-1r primer set to obtain a recombinant upstream homology arm fragment Cgl2680-up. PCR amplification was performed using ATCC 13032 genome as a template and Cgl2680-2f/Cgl2680-2r primer set to obtain a downstream homology arm fragment Cgl2680-dn of gene recombination. PCR amplification is carried out by taking the sequence SEQ ID No.1 as a template and a Ppyc-f/Ppyc-r primer pair to obtain a target artificial promoter fragment Ppyc carrying a fusion region, and PCR amplification is carried out by taking the sequence SEQ ID No.2 as a template and a gapN-f/gapN-r primer pair to obtain a gapN gene. And carrying out fusion PCR on the four fragments to obtain the full-length fragment fused with the 4 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and the vector after enzyme digestion and gel recovery are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and named pK18-Cgl2680:: gapN.
8.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc: poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, cgl2680:: gapN
Competent cells of the strain SCK007 obtained in example 7 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model bacteria 13032 described in example 1.
The recombinant plasmid pK18-Cgl2680 obtained above was transformed into SCK007 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. Further amplified by PCR (using primer pair Cgl2680- 1f/Cgl2680-2 r) and nucleotide sequencing analysis confirmed successful recombination. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC::: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, cgl2680:: gapN, strain designated SCK008.
EXAMPLE 9 deletion of the dld Gene and addition of a ppnk copy at the dld Gene deletion
9.1 construction of the engineering plasmid pK18-dld:: ppnk
PCR amplification is carried out by taking ATCC 13032 genome as a template and a dld-1f/dld-1r primer pair to obtain an upstream homologous arm fragment dld-up of gene recombination. PCR amplification is carried out by taking ATCC 13032 genome as a template and a dld-2f/dld-2r primer pair to obtain a downstream homologous arm fragment dld-dn of gene recombination. PCR amplification was performed using ATCC 13032 genome as a template and ppnk-f/ppnk-r primer pairs to obtain the target insert gene fragment ppnk, which comprises the coding region of ppnk and its natural promoter region. And carrying out fusion PCR on the three fragments to obtain the full-length fragment fused with the 3 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and the vector after enzyme digestion and gel recovery are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and is named pK18-dld:: ppnk.
9.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc: poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, cgl2680:: gapN, dld::: ppnk
Competent cells of the strain SCK008 obtained in example 8 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model strain 13032 described in example 1.
The recombinant plasmid pK18-dld obtained above was transformed into SCK008 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant is cultured in a common BHI liquid culture medium overnightThe culture temperature was 33℃and the shaking culture was carried out at 220rpm on a rotary shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair dld-1f/dld-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, cgl2680:: gapN, dld::: ppnk, and the strain was designated SCK009.
The above examples are the supply of the enhanced reducing power cofactor, including the exogenous introduction of the codon-optimized gapN gene, and the expression enhancement of the following genes gapN, ppnk. The following genes Cgl2680 and dld were attenuated or inactivated. The enhancement of the gene may be by mutation of the endogenous promoter of the gene, or by substitution with another strong promoter, or by increasing the copy number of the gene, or by a combination of the above. The attenuation or inactivation modification preferably carries out deletion mutation on a coding region of the gene or simultaneously carries out insertion modification of other genes, so that the inactivation modification of the target deletion gene and the ectopic insertion modification of the target over-expressed gene are realized in one step. The next step is to enhance sugar utilization.
EXAMPLE 10 deletion of NCgl2493 Gene, addition of one ptsG copy at the deletion of NCgl2493 Gene, and introduction of a strong promoter before the initiation codon of ptsG Gene
10.1 construction of the engineering plasmid pK18-NCgl2493:: ptsG
The ATCC 13032 genome is used as a template, and the NCgl2493-1f/NCgl2493-1r primer pair is used for PCR amplification, so that the upstream homologous arm fragment NCgl2493-up of the gene recombination is obtained. The ATCC 13032 genome is used as a template, and NCgl2493-2f/NCgl2493-2r primer pair is used for PCR amplification, so that a downstream homologous arm fragment NCgl2493-dn of the gene recombination is obtained. The ATCC 13032 genome is used as a template, a ptsG-f/ptsG-r primer pair is used for PCR amplification to obtain a target inserted gene fragment ptsG, the ATCC 13032 genome is used as a template, and a Pgap-f/Pgap-r primer pair is used for PCR amplification to obtain a strong promoter fragment Pgap. And carrying out fusion PCR on the four fragments to obtain the full-length fragment fused with the 4 fragments. The full length fragment was double digested with XbaI, nheI and the product recovered using a gel recovery kit. The plasmid vector was pK18mobsacB (GenBank: FJ 1287239.1), which was digested with the same restriction enzymes and subjected to gel recovery treatment. The fragment and the vector after enzyme digestion and gel recovery are connected by T4 DNA Ligase and transformed into Trans 1T 1 competent cells, and the obtained transformant extract plasmid is subjected to sequencing confirmation and named pK18-NCgl2493:: ptsG.
10.2 construction of engineering Strain 13032, hom:: lysC-asd, ddhPyc, poxB:: argS-lysA, sucC:: ppcD299N, aspBPcspB, aceEPdapB, ack::: acs, cgl2680:: gapN, dld::: ppnk, NCgl 2493::: ptsG
Competent cells of the strain SCK009 obtained in example 9 were prepared and subjected to shock transformation and screening of recombinant strains according to the method for preparing competent cells of the model bacteria 13032 described in example 1.
The recombinant plasmid pK18-NCgl2493 obtained above was transformed into SCK009 competent cells by electric shock and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, the transformant undergoes a second recombination, the vector sequence is removed from the genome by gene exchange, and the mutation of interest is introduced at the same time. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown transformants should carry the desired mutation and not carry the inserted vector sequence. The success of recombination was further confirmed by PCR amplification (using primer pair NCgl2493-1f/NCgl2493-2 r) and nucleotide sequencing analysis. The genotype of the final engineered strain of interest obtained was 13032, hom:: lysC-asd, ddhPyc:, poxB:: argS-lysA, sucC::: ppcD299N, aspBPcspB, aceEPdapB, ack:: acs, cgl 2680::: gapN, dld::: pp The NCgl 2493:ptsG, strain SCK0010.
The above examples are directed to enhancing glucose phosphotransferase activity by replacing the gene with a combination of other strong promoters and increasing the copy number of the gene.
Through 10 rounds of genetic operations, the combination optimization of 19 genes is realized, and finally the obtained genetically engineered bacteria are tested by using a 5L tank.
EXAMPLE 11 5L tank test of lysine fermentation Capacity of SCK010 Strain
The fermentation of the obtained genetically engineered strain SCK0010 was verified by a control bacterium CGMCC No.13407 (which was deposited at the China general microbiological center of the China Committee for culture Collection of microorganisms at 11 and 30 of 2016, and which was named Corynebacterium glutamicum, corynebacterium glutamicum, which was published in China patent application CN 106635944A), CGMCC No.11942 (which was deposited at the China general microbiological center of the China Committee for culture collection of microorganisms at 12 and 25 of 2015, and which was named Corynebacterium glutamicum, corynebacterium glutamicum, which was published in China patent application CN105734004B, at the national institute for microbiological center of China, which was named as national institute of sciences of China at 3 of the North West road 1 of Beijing, korea, which was named as Beijing, was published in China). And (3) using a Bailun tetrad 5L fermentation tank to synchronously verify the transformed bacteria and the control bacteria. The formulation of the fermentation medium is shown in Table 3, and the control process is shown in Table 4.
TABLE 3 fermentation Medium formulation
Composition of the components Concentration (g/L)
Glucose 30
Ammonium sulfate 10
Yeast powder 2
Peptone 4
KH 2 PO 4 1.5
MgSO 4 ·7H 2 O 2.0
FeSO 4 ·7H 2 O 0.2
MnSO 4 ·H 2 O 0.2
Nicotinamide 0.05
Calcium pantothenate 0.01
Biotin 0.001
Thiamine 0.01
Defoaming agent 0.5
Initial constant volume 2.5L
TABLE 4 Process control parameters
The results of the fermentation test are shown in Table 5. And fermenting the control bacteria and the modified bacteria in parallel respectively for three batches, taking 2mL of fermentation liquor for centrifugation (12000 rpm,2 min), collecting supernatant, detecting the lysine content in the fermentation liquor of the recombinant bacteria and the control bacteria by using HPLC, and taking the average value of the three batches as a final experimental result. 100 μl of the broth was diluted to an appropriate multiple, and the OD was measured at 562nm wavelength using a spectrophotometer, taking the average of three batches as the final experimental result.
TABLE 5L results of lysine fermentation experiments
The fermentation experimental result shows that the conversion rate of the high-performance lysine production strain SCK0010 reaches 68.3%, and the high-performance lysine production strain SCK0010 has higher sugar acid conversion rate compared with the previously reported lysine production strain. The sugar acid conversion rate of SCK0010 is respectively improved by 7.8 percent and 6.8 percent compared with that of a control group CGMCC No.13407 and CGMCC No.11942, and the growth is consistent.
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.

Claims (10)

1. A method for constructing a recombinant microorganism, comprising the steps of allowing an initial strain to express genes lysC, asd, argS-lysA and ddh in an enhanced manner, and inactivating or weakening the expressed genes hom and poxB.
2. The construction method according to claim 1, further comprising the step of allowing the starting strain to express the genes ppc and aspB in a intensified manner, introducing the acs gene in an exogenous manner, and inactivating or weakening the expressed genes sucC, aceE and ack.
3. The method of construction according to claim 2, further comprising the step of allowing the starting strain to express the genes gapN and ppnk in an intensified manner, and inactivating or weakening the expressed genes Cgl2680 and dld.
4. A method of constructing according to claim 3, further comprising the step of allowing the starting strain to express the gene ptsG in an enhanced manner and inactivating or attenuating the expressed gene NCgl 2493.
5. The method of claim 1 to 4, wherein the means for enhancing expression of the gene is selected from one or more of the following (1) to (3):
(1) Mutating or replacing the endogenous promoter of the gene with a stronger promoter;
(2) Increasing the copy number of the gene;
(3) Mutating the coding region of the gene;
and/or the manner of inactivating or attenuating the expressed gene may be selected from one or more of the following (1) - (3):
(1) Mutating or replacing the endogenous promoter of the gene with a weaker promoter;
(2) Reducing the copy number of the gene;
(3) The coding region of the gene is mutated.
6. The construction method according to any one of claims 1 to 4, wherein the hom gene is inactivated and expression of the lysC-asd gene is enhanced simultaneously by deleting the hom gene and adding a copy of the lysC-asd gene at the site where the hom gene is deleted;
and/or, enhancing expression of the ddh gene by introducing an artificial strong promoter before the initiation codon of the ddh gene;
and/or, inactivating the poxB gene and enhancing the expression of the argS-lysA gene simultaneously by deleting the poxB gene and adding one copy of the argS-lysA gene at the poxB gene deletion;
and/or inactivating the sucC gene and enhancing expression of the ppc gene simultaneously by deleting the sucC gene and adding a mutated copy of the ppc gene at the point of deletion of the sucC gene;
And/or, enhancing expression of the aspB gene by introducing a strong promoter before the start codon of the aspB gene;
and/or inactivating or attenuating expression of the aceE gene by introducing a weak promoter before the initiation codon of the aceE gene;
and/or inactivating the ack gene and enhancing expression of the acs gene simultaneously by deleting the ack gene and adding one copy of the acs gene at the ack gene deletion;
and/or inactivating the Cgl2680 gene and enhancing the expression of the gapN gene simultaneously by deleting the Cgl2680 gene and adding a copy of the gapN gene at the position where the Cgl2680 gene is deleted, and changing the start codon of the gapN gene and introducing an artificial strong promoter before the start codon of the gapN gene;
and/or inactivating the dld gene and enhancing expression of the ppnk gene simultaneously by deleting the dld gene and adding a ppnk gene copy at the site of the dld gene deletion;
and/or, inactivating the NCgl2493 gene and enhancing expression of the ptsG gene simultaneously by deleting the NCgl2493 gene and adding a copy of the ptsG gene at the point where the NCgl2493 gene is deleted, and introducing a strong promoter before the start codon of the ptsG gene.
7. The construction method according to claim 6, wherein,
When the expression of the ddh gene is enhanced, introducing a promoter of a pyc gene containing point mutation in front of a start codon of the ddh gene, wherein the promoter of the pyc gene containing the point mutation is obtained by taking a promoter of a wild type pyc gene as a reference sequence, mutating positions 209 to 221 into TGTGGTATAATGG, and the sequence of the promoter of the wild type pyc gene is shown as SEQ ID No. 67;
when the expression of the ppc gene is enhanced, the mutated ppc gene takes the wild-type ppc gene as a reference sequence, and the 299 th position is mutated into N;
when the expression of aspB gene is enhanced, the introduced strong promoter is PcspB;
the exogenously introduced acs gene is derived from E.coli;
when the expression of aceE gene is inactivated or weakened, the weak promoter is PdapB;
when the expression of the gapN gene is enhanced, the increased gapN gene is from streptococcus mutans, the starting codon of the gapN gene is replaced by ATG, and a promoter containing a point mutation pyc gene is introduced in front of the starting codon of the gapN gene, wherein the promoter containing the point mutation is obtained by taking a promoter of a wild type pyc gene as a reference sequence, mutating positions 209 to 221 to TGTGGTATAATGG, and the sequence of the promoter of the wild type pyc gene is shown as SEQ ID No. 67;
When the expression of the ptsG gene is enhanced, the introduced strong promoter is Pgap;
and/or, the starting strain is corynebacterium glutamicum.
8. Recombinant microorganism constructed by the construction method according to any one of claims 1 to 7.
9. Use of the recombinant microorganism of claim 8 for any of the following:
(1) The application in producing lysine by fermentation;
(2) Use in genetic breeding of microorganisms for producing lysine;
(3) The application of the method in improving the performance of synthesizing lysine by a biological method.
10. A method for fermentative production of lysine comprising the step of culturing the recombinant microorganism of claim 8.
CN202210851613.0A 2022-07-19 2022-07-19 Recombinant microorganism and construction method and application thereof Pending CN117417954A (en)

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