CN110951662B - Coryneform bacterium for high lysine production and construction method and application thereof - Google Patents

Coryneform bacterium for high lysine production and construction method and application thereof Download PDF

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CN110951662B
CN110951662B CN201911368724.0A CN201911368724A CN110951662B CN 110951662 B CN110951662 B CN 110951662B CN 201911368724 A CN201911368724 A CN 201911368724A CN 110951662 B CN110951662 B CN 110951662B
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胡丹
王成
薛婷莉
李岩
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Xinjiang Meihua Amino Acid Co ltd
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Abstract

The invention belongs to the technical field of bioengineering, and discloses coryneform bacteria for high-yield lysine as well as a construction method and application thereof. The coryneform bacterium having high lysine productivity according to the present invention has L-lysine productivity and has enhanced activity of at least one of promoters of genes encoding key enzymes of an intracellular L-lysine synthesis pathway and/or a TCA cycle make-up pathway. The coryneform bacterium for high lysine production has higher activity than an endogenous promoter by modifying the sequence of the promoter of the coding gene of key enzymes of a lysine biosynthesis pathway or a TCA (ternary polymerization reaction) circulatory compensation pathway, so that the transcription initiation efficiency is enhanced, the lysine production level is further improved, and the production capacity of the L-lysine strain is improved. Experiments show that the coryneform bacteria provided by the invention remarkably improve the yield and conversion rate of the L-lysine, lay a foundation for industrial production of the L-lysine, and have wide industrial application prospects.

Description

Coryneform bacterium for high lysine production and construction method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a coryneform bacterium for high-yield lysine as well as a construction method and application thereof.
Background
Lysine (Lysine) is known by the chemical name 2, 6-diaminohexanoic acid. Lysine is a basic essential amino acid. The first limiting amino acid is referred to as being very low in lysine content in cereal foods and is easily destroyed and absent during processing. The term lysine is generally used to refer to the L-form. L-lysine is in the form of needle crystals, darkens at 210 ℃, breaks down at 224.5 ℃, is readily soluble in water, slightly soluble in alcohol, and insoluble in ether.
Lysine is one of the essential amino acids of humans and mammals, and the body cannot synthesize itself and must be supplemented from food. Lysine is mainly present in animal foods and beans, and the content of lysine in cereal foods is very low. Lysine has positive nutritional significance in the aspects of promoting the growth and development of human bodies, enhancing the immunity of organisms, resisting viruses, promoting fat oxidation, relieving anxiety and emotion and the like, can promote the absorption of certain nutrients, can cooperate with certain nutrients, and can better exert the physiological functions of various nutrients. Lysine can regulate the metabolic balance of the human body, lysine provides a structural component for the synthesis of carnitine, which promotes the synthesis of fatty acids in cells. The food is added with a small amount of lysine, can stimulate the secretion of pepsin and gastric acid, improves the gastric secretion efficacy, and plays roles of stimulating appetite and promoting the growth and development of infants. Lysine also increases calcium absorption and accumulation in the body, and accelerates bone growth. If lysine is lacking, anorexia and nutritional anemia can occur due to insufficient gastric secretion, which results in central nerve obstruction and dysplasia. Lysine can be used as an auxiliary drug of diuretics in medicine to treat lead poisoning caused by chloride reduction in blood, and can also be used for generating salt with acidic drugs (such as salicylic acid and the like) to relieve adverse reactions, and can be used for inhibiting severe hypertension when being used with methionine.
L-lysine is the second largest amino acid production variety worldwide, and is widely used in animal feed, medicine and food industries. About 90% of them are used in the feed industry and 10% are used in the food and pharmaceutical industry. The L-lysine can help the organism absorb other amino acids when being used as an animal feed additive, thereby improving the quality of the feed.
The microbial fermentation method for producing L-lysine has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like, and is the most main method for producing L-lysine at present. However, the fermentation performance of the existing L-lysine strain is still poor, the conversion rate of the L-lysine is still low, and the requirement of large-scale industrial production cannot be met.
Disclosure of Invention
In view of the above, the present invention aims to provide a coryneform bacterium capable of producing L-lysine with high yield, and a construction method and application thereof, aiming at the defects existing in the prior art.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a coryneform bacterium which has a high lysine-producing ability and has an enhanced activity of at least one of promoters of genes encoding key enzymes of an intracellular L-lysine synthesis pathway and/or a TCA cycle make-up pathway.
The invention modifies the sequence of the promoter of the coding gene of the key enzyme of the lysine biosynthesis pathway or the TCA circulation compensation pathway to ensure that the promoter has higher activity than an endogenous promoter and provides a reliable initial binding site for RNA polymerase, thereby enhancing the initial transcription efficiency and further improving the lysine production level of the RNA polymerase, and further obtaining the genetic engineering bacteria with high yield of L-lysine.
In the present invention, the key enzyme is aspartokinase (lysC) or pyruvate carboxylase (pyc).
In the present invention, the activity enhancement is to enhance the promoter activity by altering the promoter-10 region and its extension sequence.
In an embodiment of the present invention, the sequence comprising the aspartokinase activity-enhancing promoter is shown as SEQ ID No.17 or SEQ ID No. 18; the sequence of the promoter containing the pyruvate carboxylase activity enhancement is shown as SEQ ID No.19 or SEQ ID No. 20.
The nucleic acid molecules according to the invention having promoter activity can be used as promoters for the gene expression of prokaryotes, in particular of E.coli or coryneform bacteria. Wherein the coryneform bacterium refers to a microorganism belonging to the genus Corynebacterium or Brevibacterium. Examples of coryneform bacteria useful in the present invention include, but are not limited to: corynebacterium glutamicum, brevibacterium flavum, brevibacterium lactofermentum and Corynebacterium ammoniagenes.
In the present invention, the coryneform bacterium is Corynebacterium glutamicum, brevibacterium flavum, brevibacterium lactofermentum or Corynebacterium ammoniagenes.
The invention also provides a construction method of the coryneform bacterium with high lysine yield, which comprises the steps of preparing a gene fragment containing an activity enhanced promoter and a coding gene of a key enzyme, connecting the gene fragment with a vector to obtain a recombinant vector, and transforming the coryneform bacterium to obtain the coryneform bacterium with high lysine yield.
In some embodimentsIn the scheme, the method for preparing the encoding gene containing the activity enhanced promoter and the key enzyme in the construction method of the coryneform bacteria with high lysine yield comprises the following steps: ATCC13032 genome was used as a template, plysC was used mut1 -1/PlysC mut1 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut1 -up; ATCC13032 genome was used as a template, plysC was used mut1 -3/PlysC mut1 PCR amplification of the-4 primer pair to obtain fragment PlysC mut1 -dn; the mixture of the two fragments is used as a template, plysC is used mut1 -1/PlysC mut1 PCR amplification of the primer set-4 to give a mutated PlysC comprising an activity-enhancing promoter and a gene encoding aspartokinase mut1 Fragments.
In some embodiments, the method for preparing the encoding gene comprising the activity-enhancing promoter and the key enzyme in the construction method of the coryneform bacterium having high lysine productivity is specifically: ATCC13032 genome was used as a template, plysC was used mut2 -1/PlysC mut2 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut2 -up; ATCC13032 genome was used as a template, plysC was used mut2 -3/PlysC mut2 PCR amplification of the-4 primer pair to obtain fragment PlysC mut2 -dn; the mixture of the two fragments is used as a template, plysC is used mut2 -1/PlysC mut2 PCR amplification of the primer set-4 to give a mutated PlysC comprising an activity-enhancing promoter and a gene encoding aspartokinase mut2 Fragments.
In some embodiments, the method for preparing the encoding gene comprising the activity-enhancing promoter and the key enzyme in the construction method of the coryneform bacterium having high lysine productivity is specifically: using ATCC13032 genome as a template and Ppyc mut1 -1/PlysC mut1 PCR amplification of the-2 primer pair to give the upstream fragment Ppyc mut1 -up; using ATCC13032 genome as a template and Ppyc mut1 -3/Ppyc mut1 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut1 -dn; the mixture of the two fragments is used as a template, ppyc is used as a template mut1 -1/Ppyc mut1 PCR amplification of the primer set-4 to yield a primer set comprising an activity-enhancing promoterAnd mutant Ppyc of the gene encoding pyruvate carboxylase mut1 Fragments.
In some embodiments, the method for preparing the encoding gene comprising the activity-enhancing promoter and the key enzyme in the construction method of the coryneform bacterium having high lysine productivity is specifically: using ATCC13032 genome as a template and Ppyc mut2 -1/PlysC mut2 PCR amplification of the-2 primer pair to give the upstream fragment Ppyc mut2 -up; using ATCC13032 genome as a template and Ppyc mut2 -3/Ppyc mut2 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut2 -dn; the mixture of the two fragments is used as a template, ppyc is used as a template mut2 -1/Ppyc mut2 PCR amplification of the primer pair to obtain a mutated Ppyc comprising the activity-enhancing promoter and the gene encoding the pyruvate carboxylase mut2 Fragments.
Wherein the PlysC mut1 The sequence of the-1 is shown as SEQ ID No. 1;
the PlysC mut1 The sequence of-2 is shown as SEQ ID No. 2;
the PlysC mut1 The sequence of the-3 is shown as SEQ ID No. 3;
the PlysC mut1 -4 has the sequence shown in SEQ ID No. 4;
the PlysC mut2 The sequence of the-1 is shown as SEQ ID No. 5;
the PlysC mut2 The sequence of the-2 is shown as SEQ ID No. 6;
the PlysC mut2 The sequence of the-3 is shown as SEQ ID No. 7;
the PlysC mut2 The sequence of-4 is shown as SEQ ID No. 8;
the Ppyc mut1 The sequence of the-1 is shown as SEQ ID No. 9;
the Ppyc mut1 The sequence of-2 is shown as SEQ ID No. 10;
the Ppyc mut1 The sequence of-3 is shown as SEQ ID No. 11;
the Ppyc mut1 The sequence of-4 is shown as SEQ ID No. 12;
the Ppyc mut2 The sequence of-1 is shown as SEQ ID No.13；
The Ppyc mut2 The sequence of-2 is shown as SEQ ID No. 14;
the Ppyc mut2 The sequence of-3 is shown as SEQ ID No. 15;
the Ppyc mut2 The sequence of-4 is shown as SEQ ID No. 16.
In some embodiments, the coryneform bacterium of the method of constructing a lysine-producing coryneform bacterium is Corynebacterium glutamicum MHZ-0912-1 strain. The strain is obtained by taking ATCC13032 as a starting strain and introducing a point mutation T311I into lysC to release feedback inhibition of aspartokinase (see Chinese patent application No. 201610119394.1). The person skilled in the art can prepare MHZ-0912-1 competent cells according to the classical method of cereal bars (C.glutamicumhandbook, charpter 23).
In some embodiments, the vector in the method of constructing a coryneform bacterium that produces lysine is pK18mobsacB.
In some embodiments, recombinant vectors comprising an activity-enhancing promoter and a gene encoding a key enzyme are electroporated to transform MHZ-0912-1 competent cells, and transformants are selected on selection medium containing 15mg/L kanamycin, wherein the gene of interest is inserted into the chromosome due to homology.
Further, in some embodiments, the screened transformants are grown overnight in normal liquid brain heart infusion medium at a temperature of 33℃and shaking culture at 220rpm on a rotary shaker. 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. The strain grown on 10% sucrose brain heart infusion medium did not carry the inserted vector sequence in its genome. The target sequence is amplified by PCR, and the target mutant strains are named MHZ-0914-1, MHZ-0914-3 and MHZ-0914-4 respectively through nucleotide sequencing analysis.
The invention also provides coryneform bacteria obtained by the construction method.
The coryneform bacteria obtained by the invention can be used for fermentation production, so that effective accumulation of L-lysine can be obtained, and a foundation is laid for industrial production of L-lysine. The invention therefore also provides the use of the coryneform bacteria in the fermentative preparation of L-lysine.
In some embodiments, the coryneform bacterium is Corynebacterium glutamicum MHZ-1012-3.
Furthermore, the invention also provides a production method of the L-lysine, and the coryneform bacteria with high lysine yield are inoculated into a fermentation medium for fermentation culture.
In the present invention, the fermentation medium comprises the following components: glucose 60g/L, (NH) 4 ) 2 SO 4 25g/L，KH 2 PO 4 2.0g/L，MgSO 4 ·7H 2 O1.0 g/L, 10g/L of soybean meal hydrolysate and CaCO 3 30g/L，pH7.0。
Further, in the present invention, the fermentation culture is performed at 33℃for 14 to 15 hours.
According to the technical scheme, the invention provides a coryneform bacterium for high lysine production, and a construction method and application thereof. The coryneform bacterium having high lysine productivity according to the present invention has L-lysine productivity and has enhanced activity of at least one of promoters of genes encoding key enzymes of an intracellular L-lysine synthesis pathway and/or a TCA cycle make-up pathway. The coryneform bacterium for high lysine production has higher activity than an endogenous promoter by modifying the sequence of the promoter of the coding gene of key enzymes of a lysine biosynthesis pathway or a TCA (ternary polymerization reaction) circulatory compensation pathway, and provides a reliable initial binding site for RNA (ribonucleic acid) polymerase, so that the initial transcription efficiency is enhanced, the lysine production level is further improved, and the production capacity of an L-lysine strain is improved. Experiments show that the coryneform bacteria provided by the invention remarkably improve the yield and conversion rate of the L-lysine, lay a foundation for industrial production of the L-lysine, and have wide industrial application prospects.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a recombinant plasmid pK18mobsacB-PlysC mut1 Schematic of (2);
FIG. 2 is a recombinant plasmid pK18mobsacB-PlysC mut2 Schematic of (2);
FIG. 3 is a recombinant plasmid pK18mobsacB-Ppyc mut1 Schematic of (2);
FIG. 4 is a recombinant plasmid pK18mobsacB-Ppyc mut2 Is a schematic diagram of (a).
Detailed Description
The invention discloses a coryneform bacterium for high lysine production, a construction method and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the invention has been described with reference to preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods described herein, and the application of the techniques of the invention without departing from the spirit and scope of the invention.
For a further understanding of the present invention, the present invention will be described in detail with reference to the following examples, which are commercially available and commercially available, unless otherwise specified. The ATCC13032 strain is a corynebacterium glutamicum standard strain, and can be obtained through commercial purchase. The formula of the common liquid brain heart infusion culture medium is 3.7% brain heart infusion powder solution, and the formula of the common solid brain heart infusion culture medium is 3.7% brain heart infusion powder solution and 1.8% agar powder.
The primer sequence information used in the examples is shown in Table 1
TABLE 1 primer sequence information
EXAMPLE 1 construction of recombinant vector comprising lysC promoter with type 1 mutation and introduction into Corynebacterium glutamicum
ATCC13032 genome was used as a template, plysC was used mut1 -1/PlysC mut1 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut1 -up; ATCC13032 genome was used as a template, plysC was used mut1 -3/PlysC mut1 PCR amplification of the-4 primer pair to obtain fragment PlysC mut1 Dn. The mixture of the two fragments is used as a template, plysC is used mut1 -1/PlysC mut1 PCR amplification of the primer pair-4 to obtain PlysC containing the desired mutation mut1 Fragments. The fragment was digested with BamHI and PstI, and vector pK18mobsacB was digested with the same enzymes. The two enzyme digestion products are connected by T4 DNA Ligase, and Trans1T1 competent cells are transformed to obtain recombinant plasmid pK18mobsacB-lysC mut1 。
MHZ-0912-1 competent cells were prepared according to the classical method of cereal bars (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-lysC mut1 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 33℃and shaking culture at 220rpm with a rotary 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 desired mutant strain was obtained by PCR amplification of the desired sequence and nucleotide sequencing analysis, and was designated MHZ-0914-1.
EXAMPLE 2 construction of recombinant vector comprising lysC promoter with type 2 mutation and introduction into Corynebacterium glutamicum
ATCC13032 genome was used as a template, plysC was used mut2 -1/PlysC mut2 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut2 -up; ATCC13032 genome was used as a template, plysC was used mut2 -3/PlysC mut2 PCR amplification of the-4 primer pair to obtain fragment PlysC mut2 Dn. The mixture of the two fragments is used as a template, plysC is used mut2 -1/PlysC mut2 PCR amplification of the primer pair-4 to obtain PlysC containing the desired mutation mut2 Fragments. The fragment was digested with BamHI and PstI, and vector pK18mobsacB was digested with the same enzymes. The two enzyme digestion products are connected by T4 DNA Ligase, and Trans1T1 competent cells are transformed to obtain recombinant plasmid pK18mobsacB-lysC mut2 。
MHZ-0912-1 competent cells were prepared according to the classical method of cereal bars (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-lysC mut2 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 33℃and shaking culture at 220rpm with a rotary 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 desired mutant strain was obtained by PCR amplification of the desired sequence and nucleotide sequencing analysis, and was designated MHZ-0914-2.
EXAMPLE 3 construction of recombinant vector containing type 1 mutated pyc promoter and introduction in Corynebacterium glutamicum
Using ATCC13032 genome as a template and Ppyc mut1 -1/PlysC mut1 -2 primer pair for PCR amplificationObtaining the upstream fragment Ppyc mut1 -up; using ATCC13032 genome as a template and Ppyc mut1 -3/Ppyc mut1 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut1 Dn. The mixture of the two fragments is used as a template, ppyc is used as a template mut1 -1/Ppyc mut1 PCR amplification of the primer pair-4 to obtain Ppyc containing the desired mutation mut1 Fragments. The fragment was digested with BamHI and PstI, and vector pK18mobsacB was digested with the same enzymes. The two enzyme cutting products are connected by T4 DNA Ligase to transform Trans1T1 competent cells to obtain recombinant plasmid pK18mobsacB-pyc mut1 。
MHZ-0912-1 competent cells were prepared according to the classical method of cereal bars (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-pyc mut1 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 33℃and shaking culture at 220rpm with a rotary 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 desired mutant strain was obtained by PCR amplification of the desired sequence and nucleotide sequencing analysis, and was designated MHZ-0914-3.
EXAMPLE 4 construction of recombinant vector containing type 2 mutated pyc promoter and introduction in Corynebacterium glutamicum
Using ATCC13032 genome as a template and Ppyc mut2 -1/PlysC mut2 PCR amplification of the-2 primer pair to give the upstream fragment Ppyc mut2 -up; using ATCC13032 genome as a template and Ppyc mut2 -3/Ppyc mut2 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut2 Dn. The mixture of the two fragments is used as a template, and Pp is used as a templateyc mut2 -1/Ppyc mut2 PCR amplification of the primer pair-4 to obtain Ppyc containing the desired mutation mut2 Fragments. The fragment was digested with BamHI and PstI, and vector pK18mobsacB was digested with the same enzymes. The two enzyme cutting products are connected by T4 DNA Ligase to transform Trans1T1 competent cells to obtain recombinant plasmid pK18mobsacB-pyc mut2 。
MHZ-0912-1 competent cells were prepared according to the classical method of cereal bars (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-pyc mut2 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 33℃and shaking culture at 220rpm with a rotary 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 desired mutant strain was obtained by amplifying the desired sequence by PCR and analyzing by nucleotide sequencing and was designated MHZ-0914-4.
EXAMPLE 5L-lysine Gene engineering bacterium fermentation production of L-lysine
The genetically engineered strains constructed in examples 1 to 4 were fermented in 500ml triangular flasks at a temperature of 33℃for 14 to 15 hours, three replicates were set per set of experiments. The components of the fermentation medium are as follows: glucose 60g/L, (NH) 4 ) 2 SO 4 25g/L，KH 2 PO 4 2.0g/L，MgSO 4 ·7H 2 O1.0 g/L, 10g/L of soybean meal hydrolysate and CaCO 3 30g/L, naOH adjusts pH=7.0.
The results of L-lysine production by fermentation of each genetically engineered strain are shown in Table 2.
TABLE 2 results of L-lysine production by fermentation of genetically engineered strains
As is clear from Table 2, the lysine productivity of the strain can be significantly improved by changing the promoter-10 region and the-10 region extension sequences. Compared with the original strain, the-10 region of the lysC promoter and the extension sequence thereof are mutated, and the obtained mutant promoter has higher activity, so that the expression of the lysC gene can be enhanced. The obtained mutant strain is subjected to shake flask fermentation test, and compared with the original strain, the lysine yield (acid production and conversion rate) of the mutant strain is remarkably improved.
Compared with the original strain, the-10 region of the pyc promoter and the extension sequence thereof are mutated, and the obtained mutant promoter has higher activity, so that the expression of the pyc gene can be enhanced. The pyruvic carboxylase encoded by the pyc gene catalyzes the conversion of pyruvic acid to oxaloacetic acid, an important precursor for lysine biosynthesis. The obtained mutant strain is subjected to shake flask fermentation test, and compared with the original strain, the lysine yield (acid production and conversion rate) of the mutant strain is remarkably improved.
In summary, by modifying the promoters of lysC and pyc genes, which are lysine synthesis-related genes, the expression activities of the corresponding genes can be greatly improved, thereby obtaining a lysine-producing strain with a high conversion rate.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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<213> Artificial sequence (Artificial sequence)
<400> 2
agttacccgc tcaattatac ctttataaac tgtgtctacc 40
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ggtagacaca gtttataaag gtataattga gcgggtaact 40
<210> 4
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
aactgcagca gctgccaacg caactgca 28
<210> 5
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
gaggatcccc cagaagattt cagttcgg 28
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
agttacccgc tcaattatac cacaataaac tgtgtctacc 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ggtagacaca gtttattgtg gtataattga gcgggtaact 40
<210> 8
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
aactgcagca gctgccaacg caactgca 28
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
gaggatcctt ctgccgccgt aactctgg 28
<210> 10
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
ctgcgtccta gtattatacc acacgattcc cccaatcaaa 40
<210> 11
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
tttgattggg ggaatcgtgt ggtataatac taggacgcag 40
<210> 12
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
cccaagcttc ttttaacgat ctcatcgat 29
<210> 13
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
gaggatcctt ctgccgccgt aactctgg 28
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
ctgcgtccta gtatcatacc acacgattcc cccaatcaaa 40
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
tttgattggg ggaatcgtgt ggtatgatac taggacgcag 40
<210> 16
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
cccaagcttc ttttaacgat ctcatcgat 29
<210> 17
<211> 379
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
tgagtaaagg tgagctcctt agggagccat cttttggggt gcggagcgcg atccggtgtc 60
tgaccacggt gccccatgcg attgttaatg ccgatgctag ggcgaaaagc acggcgagca 120
gattgctttg cacttgattc agggtagttg actaaagagt tgctcgcgaa gtagcacctg 180
tcacttttgt ctcaaatatt aaatcgaata tcaatatatg gtctgtttat tggaacgcgt 240
cccagtggct gagacgcatc cgctaaagcc ccaggaaccc tgtgcagaaa gaaaacactc 300
ctctggctag gtagacacag tttataaagg tataattgag cgggtaactg tcagcacgta 360
gatcgaaagg tgcacaaag 379
<210> 18
<211> 379
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tgagtaaagg tgagctcctt agggagccat cttttggggt gcggagcgcg atccggtgtc 60
tgaccacggt gccccatgcg attgttaatg ccgatgctag ggcgaaaagc acggcgagca 120
gattgctttg cacttgattc agggtagttg actaaagagt tgctcgcgaa gtagcacctg 180
tcacttttgt ctcaaatatt aaatcgaata tcaatatatg gtctgtttat tggaacgcgt 240
cccagtggct gagacgcatc cgctaaagcc ccaggaaccc tgtgcagaaa gaaaacactc 300
ctctggctag gtagacacag tttattgtgg tataattgag cgggtaactg tcagcacgta 360
gatcgaaagg tgcacaaag 379
<210> 19
<211> 281
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
ttaagaggta agatgtctgc aggtggaagc gtttaaatgc gttaaacttg gccaaatgtg 60
gcaacctttg caaggtgaaa aactggggcg gggttagatc ctggggggtt tatttcattc 120
actttggctt gaagtcgtgc aggtcagggg agtgttgccc gaaaacattg agaggaaaac 180
aaaaaccgat gtttgattgg gggaatcgtg tggtataata ctaggacgca gtgactgcta 240
tcacccttgg cggtctcttg ttgaaaggaa taattactct a 281
<210> 20
<211> 281
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
ttaagaggta agatgtctgc aggtggaagc gtttaaatgc gttaaacttg gccaaatgtg 60
gcaacctttg caaggtgaaa aactggggcg gggttagatc ctggggggtt tatttcattc 120
actttggctt gaagtcgtgc aggtcagggg agtgttgccc gaaaacattg agaggaaaac 180
aaaaaccgat gtttgattgg gggaatcgtg tggtatgata ctaggacgca gtgactgcta 240
tcacccttgg cggtctcttg ttgaaaggaa taattactct a 281

Claims (6)

1. A coryneform bacterium having a high lysine-producing ability, characterized in that an activity of at least one of promoters of genes encoding key enzymes of an intracellular L-lysine synthesis pathway and/or TCA cycle anaplerotic pathway is enhanced;
the key enzyme is aspartokinase or pyruvate carboxylase; the activity enhancement is to enhance the promoter activity by changing the promoter-10 region and the extension sequence thereof;
the sequence of the aspartokinase activity enhanced promoter is shown as SEQ ID No.17 or SEQ ID No. 18; the sequence of the pyruvate carboxylase activity enhancing promoter is shown as SEQ ID No.19 or SEQ ID No. 20;
the coryneform bacterium is Corynebacterium glutamicum.
2. The method for constructing coryneform bacteria according to claim 1, wherein a gene fragment comprising a gene encoding an activity-enhancing promoter and a key enzyme is prepared, and the gene fragment is ligated with a vector to obtain a recombinant vector, and the coryneform bacteria are transformed to obtain coryneform lysine-producing coryneform bacteria.
3. The construction method according to claim 2, wherein the coryneform bacterium is a strain of Corynebacterium glutamicum MHZ-0912-1; the vector is pK18mobsacB;
the method for preparing the coding gene containing the activity enhanced promoter and the key enzyme comprises the following steps:
（1）ATCC13032 genome as template with PlysC mut1 -1/ PlysC mut1 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut1 -up; ATCC13032 genome was used as a template, plysC was used mut1 -3/ PlysC mut1 PCR amplification of the-4 primer pair to obtain fragment PlysC mut1 -dn; the mixture of the two fragments is used as a template, plysC is used mut1 -1/ PlysC mut1 PCR amplification of the primer set-4 to give a mutated PlysC comprising an activity-enhancing promoter and a gene encoding aspartokinase mut1 Fragments; or (b)
(2) ATCC13032 genome was used as a template, plysC was used mut2 -1/ PlysC mut2 PCR amplification of the primer pair-2 to obtain the upstream fragment PlysC mut2 -up; ATCC13032 genome was used as a template, plysC was used mut2 -3/ PlysC mut2 PCR amplification of the-4 primer pair to obtain fragment PlysC mut2 -dn; the mixture of the two fragments is used as a template, plysC is used mut2 -1/ PlysC mut2 PCR amplification of the primer set-4 to give a mutated PlysC comprising an activity-enhancing promoter and a gene encoding aspartokinase mut2 Fragments; or (b)
(3) Using ATCC13032 genome as a template and Ppyc mut1 -1/ PlysC mut1 PCR amplification of the-2 primer pair to give the upstream fragment Ppyc mut1 -up; using ATCC13032 genome as a template and Ppyc mut1 -3/ Ppyc mut1 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut1 -dn; the mixture of the two fragments is used as a template, ppyc is used as a template mut1 -1/ Ppyc mut1 -4 primer pair for PCR amplification to obtain said mutated Ppyc comprising an activity-enhancing promoter and a gene encoding a pyruvate carboxylase enzyme mut1 Fragments; or (b)
(4) Using ATCC13032 genome as a template and Ppyc mut2 -1/ PlysC mut2 PCR amplification of the-2 primer pair to give the upstream fragment Ppyc mut2 -up; using ATCC13032 genome as a template and Ppyc mut2 -3/ Ppyc mut2 PCR amplification of the-4 primer pair to obtain fragment Ppyc mut2 -dn; the mixture of the two fragments is used as a template, and Pp is used as a templateyc mut2 -1/ Ppyc mut2 -4 primer pair for PCR amplification to obtain said mutated Ppyc comprising an activity-enhancing promoter and a gene encoding a pyruvate carboxylase enzyme mut2 Fragments;
the PlysC mut1 The sequence of the-1 is shown as SEQ ID No. 1;
the PlysC mut1 The sequence of-2 is shown as SEQ ID No. 2;
the PlysC mut1 The sequence of the-3 is shown as SEQ ID No. 3;
the PlysC mut1 -4 has the sequence shown in SEQ ID No. 4;
the PlysC mut2 The sequence of the-1 is shown as SEQ ID No. 5;
the PlysC mut2 The sequence of the-2 is shown as SEQ ID No. 6;
the PlysC mut2 The sequence of the-3 is shown as SEQ ID No. 7;
the PlysC mut2 The sequence of-4 is shown as SEQ ID No. 8;
the Ppyc mut1 The sequence of the-1 is shown as SEQ ID No. 9;
the Ppyc mut1 The sequence of-2 is shown as SEQ ID No. 10;
the Ppyc mut1 The sequence of-3 is shown as SEQ ID No. 11;
the Ppyc mut1 The sequence of-4 is shown as SEQ ID No. 12;
the Ppyc mut2 The sequence of the-1 is shown as SEQ ID No. 13;
the Ppyc mut2 The sequence of-2 is shown as SEQ ID No. 14;
the Ppyc mut2 The sequence of-3 is shown as SEQ ID No. 15;
the Ppyc mut2 The sequence of-4 is shown as SEQ ID No. 16.
4. A corynebacterium glutamicum obtained by the construction method of claim 2 or 3.
5. Use of the corynebacterium glutamicum according to claim 1 or 4 for the fermentative production of L-lysine.
6. A process for producing L-lysine, which comprises inoculating the corynebacterium glutamicum according to claim 1 or 4 to a fermentation medium and culturing the same in fermentation medium.
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