Recombinant Bacteria for Producing L-Lysine, Method for Constructing the Same, and Method for Producing L-Lysine

Abstract

The present invention relates to a recombinant bacterium for producing L-lysine, a method for constructing the same, and a method for producing L-lysine. Compared with the starting bacterium, which is a strain capable of accumulating lysine, the recombinant bacterium has increased expression and / or activity of asparaginase. The recombinant bacterium has been observed to have an effect of superposing and increasing the yield during fermentation culture, and significantly increases the yield of L-lysine.

Description

Technical Field

[0001] The present invention generally relates to the field of microbial fermentation, and particularly relates to a recombinant bacterium for producing L-lysine, a method for constructing the same, and a method for producing L-lysine. Background Art

[0002] L-lysine is one of the nine essential amino acids in the human body and has various physiological functions such as regulating the body's metabolic balance and promoting growth and development. It is widely used in the fields of food, feed, and medicine. In the feed industry, lysine is the first limiting amino acid for the growth of pigs and poultry. Adding L-lysine to feed can improve the utilization rate of amino acids and proteins in the feed, improve the nutritional value of the feed, and promote the growth of livestock and poultry. In the food industry, L-lysine is mainly used as a nutritional fortifier and deodorant. In the medical field, L-lysine is one of the main components of compound amino acid preparations. Currently, the lysine industry is the second largest amino acid industry after glutamic acid. Therefore, the industrial production research of L-lysine is of great significance.

[0003] Currently, L-lysine is mainly produced by direct microbial fermentation. The fermentation production performance of lysine-producing bacteria is the key factor affecting the production cost of the fermentation method.

[0004] The breeding methods of high-lysine-producing strains mainly include traditional mutagenesis and metabolic engineering modification.

[0005] The strains obtained by mutagenesis screening will accumulate a large number of negative-effect mutations, resulting in problems such as slow strain growth, reduced environmental tolerance, and increased nutrient requirements. These defects limit the industrial application of the strains.

[0006] As Figure 1 shown, in the lysine synthesis metabolic pathway of Corynebacterium glutamicum, the precursor of lysine synthesis is oxaloacetate in the tricarboxylic acid cycle (TCA cycle). Oxaloacetate is transaminated to aspartic acid and enters the lysine synthesis pathway. Therefore, the existing technologies for metabolic engineering modification of lysine-producing strains mainly focus on the modification of key genes in the lysine terminal synthesis pathway, the glycolysis pathway for providing synthesis precursors, the TCA cycle, and the pentose phosphate pathway for providing cofactor NADPH. Specifically, it mainly improves the synthesis of oxaloacetate and thus the accumulation of lysine by enhancing the expression of pyruvate carboxylase gene (pyc gene) and weakening the expression of phosphoenolpyruvate carboxykinase gene (pck gene). However, so far, no existing technology has carried out metabolic engineering modification of lysine-producing strains from the perspective of affecting the supply of aspartic acid. Summary of the Invention

[0007] In previous studies, the present inventors found that the supply of aspartic acid is also a key factor affecting lysine synthesis. Improving the synthesis of aspartic acid can ensure the supply of precursor substances for the large-scale synthesis of lysine and improve the lysine synthesis efficiency of the strain. During the metabolism of aspartic acid, aspartic acid is catalyzed by asparagine synthetase to generate asparagine, and asparagine is then catalyzed by asparaginase to hydrolyze into aspartic acid and ammonia.

[0008] The object of the present invention is to provide a recombinant bacterium for producing L-lysine by metabolic engineering transformation of lysine-producing bacteria.

[0009] The present invention provides a recombinant bacterium for producing L-lysine, wherein the recombinant bacterium has increased expression and / or activity of asparaginase (EC 3.5.1.1 asparaginase) compared with the parental bacterium, and the parental bacterium is a strain capable of accumulating lysine.

[0010] Preferably, according to the aforementioned recombinant bacterium, wherein the recombinant bacterium has at least two copies of the asparaginase-encoding gene, and / or the expression of the asparaginase-encoding gene of the recombinant bacterium is mediated by a regulatory element with high transcriptional or high expression activity. Preferably, the regulatory element is a strong promoter. More preferably, the strong promoter is the P tuf promoter of the parental bacterium.

[0011] Preferably, according to the aforementioned recombinant bacterium, wherein the recombinant bacterium has decreased expression and / or activity of homoserine dehydrogenase (Hom) compared with the parental bacterium. The decrease in homoserine dehydrogenase expression is achieved by at least one of the following methods: (A) inactivation of the homoserine dehydrogenase-encoding gene of the recombinant bacterium, (B) the expression of the homoserine dehydrogenase-encoding gene of the recombinant bacterium is mediated by a regulatory element with low transcriptional or low expression activity. The decrease in homoserine dehydrogenase activity is achieved by the following method: valine at position 59 of the homoserine dehydrogenase of the recombinant bacterium is mutated to alanine, and the homoserine dehydrogenase-encoding gene of the recombinant bacterium is preferably as shown in SEQ ID NO.1.

[0012] Preferably, according to the aforementioned recombinant bacterium, wherein the recombinant bacterium has increased expression and / or activity of pyruvate carboxylase (pyc) compared with the parental bacterium. Preferably, the increase in pyruvate carboxylase expression is achieved by at least one of the following methods: (C) the recombinant bacterium has at least two copies of the pyruvate carboxylase-encoding gene, (D) the expression of the pyruvate carboxylase-encoding gene of the recombinant bacterium is mediated by a regulatory element with high transcriptional or high expression activity. The increase in pyruvate carboxylase activity is achieved by the following method: proline at position 458 of the pyruvate carboxylase of the recombinant bacterium is mutated to serine, and the pyruvate carboxylase-encoding gene of the recombinant bacterium is preferably as shown in SEQ ID NO.8.

[0013] Preferably, according to the recombinant bacterium described above, wherein the recombinant bacterium has reduced expression and / or activity of phosphoenolpyruvate carboxykinase (pck) compared with the parental bacterium. Preferably, the phosphoenolpyruvate carboxykinase-encoding gene of the recombinant bacterium is inactivated, and / or the expression of the phosphoenolpyruvate carboxykinase-encoding gene is mediated by a regulatory element with low transcriptional or low expression activity. More preferably, the inactivation is knocking out the phosphoenolpyruvate carboxykinase-encoding gene of the recombinant bacterium.

[0014] Preferably, according to the recombinant bacterium described above, wherein the recombinant bacterium has increased expression and / or activity of dihydropicolinate reductase (dapB) compared with the parental bacterium. Preferably, the recombinant bacterium has at least two copies of the dihydropicolinate reductase-encoding gene, and / or the expression of the dihydropicolinate reductase-encoding gene is mediated by a regulatory element with high transcriptional or high expression activity. More preferably, the regulatory element is a strong promoter. Most preferably, the strong promoter is the P tuf promoter.

[0015] Preferably, according to the recombinant bacterium described above, wherein the recombinant bacterium has increased expression and / or activity of aspartokinase (lysC), diaminopimelate dehydrogenase (ddh) and / or diaminopimelate decarboxylase (lysA) compared with the parental bacterium. Preferably, the recombinant bacterium has at least two copies of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene, and / or the expression of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene is mediated by a regulatory element with high transcriptional or high expression activity. More preferably, the regulatory element is a strong promoter. Most preferably, the strong promoter is the P tuf promoter.

[0016] Alternatively preferably, according to the recombinant bacterium described above, wherein the parental bacterium is a bacterium selected from the group consisting of Corynebacterium, Brevibacterium, Bacillus, Bifidobacterium and Lactobacillus or a fungus selected from yeast.

[0017] The bacterium of the genus Corynebacterium is selected from one bacterium of Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium aminogenes, Corynebacterium lilium, Corynebacterium callunae, and Corynebacterium herculis.

[0018] The bacterium of the genus Brevibacterium is selected from one bacterium of Brevibacteriaceae flvum, Brevibacteriaceae lactofermentum, and Brevibacteriaceae ammoniagenes.

[0019] The bacterium of the genus Bacillus is selected from one bacterium of Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus.

[0020] The bacterium of the genus Bifidobacterium is selected from one bacterium of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium adolescentis.

[0021] The bacterium of the genus Lactobacillus is selected from one bacterium of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp, and Lactobacillus fermentum.

[0022] The yeast fungus is a strain of fungus selected from Candida utilis, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0023] The present invention also provides a method for constructing the above-mentioned recombinant bacterium, which includes the following steps: increasing the expression and / or activity of asparaginase in the starting bacterium. Specifically, increasing the expression and / or activity of asparaginase in the starting bacterium is achieved by at least one of the following methods: (E) increasing the copy number of the asparaginase-encoding gene in the starting bacterium, (F) replacing the regulatory element of the asparaginase-encoding gene in the starting bacterium with a regulatory element having high transcriptional or high expression activity.

[0024] Preferably, the construction method further includes the following step: reducing the expression and / or activity of homoserine dehydrogenase in the starting bacterium.

[0025] Preferably, the construction method further includes the following step: increasing the expression and / or activity of pyruvate carboxylase in the starting bacterium.

[0026] Preferably, the construction method further includes the following step: reducing the expression and / or activity of phosphoenolpyruvate carboxykinase in the starting bacterium. Specifically, reducing the expression and / or activity of phosphoenolpyruvate carboxykinase in the starting bacterium is achieved by at least one of the following methods: (G) inactivating the phosphoenolpyruvate carboxykinase-encoding gene on the chromosome of the starting bacterium, and the inactivation is preferably knockout, (H) replacing the regulatory element of the phosphoenolpyruvate carboxykinase-encoding gene in the starting bacterium with a regulatory element having low transcriptional or low expression activity.

[0027] Preferably, the construction method further includes the following step: increasing the expression and / or activity of dihydropicolinate reductase in the starting bacterium. Specifically, increasing the expression and / or activity of dihydropicolinate reductase in the starting bacterium is achieved by at least one of the following methods: (I) increasing the copy number of the dihydropicolinate reductase-encoding gene in the starting bacterium, (J) replacing the regulatory element of the dihydropicolinate reductase in the starting bacterium with a regulatory element having high transcriptional or high expression activity.

[0028] Alternatively, or preferably, the construction method further comprises the following steps: enhancing the expression and / or activity of aspartokinase, diaminopimelate dehydrogenase and / or diaminopimelate decarboxylase in the starting bacterium. Specifically, the enhancement of the expression and / or activity of aspartokinase, diaminopimelate dehydrogenase and / or diaminopimelate decarboxylase in the starting bacterium is achieved by at least one of the following means: (L) increasing the copy number of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene in the starting bacterium, (M) replacing the regulatory elements of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene in the starting bacterium with regulatory elements having high transcriptional or high expression activity.

[0029] The present invention also provides a method for producing L-lysine, which comprises the following step: fermentatively culturing the above-mentioned recombinant bacterium.

[0030] The recombinant bacterium for producing L-lysine provided by the present invention has been observed to have an effect of superposing and increasing the yield through fermentative culture, and significantly increases the yield of L-lysine. The lysine production intensity at 48 hours of fermentation is 0.05 - 5 g / L / h, and the lysine yield at the end of fermentation is 1 - 300 g / L.

[0031] The present invention first proposes a metabolic engineering modification strategy for enhancing the supply of aspartic acid, a precursor for lysine synthesis, by enhancing the expression of asparaginase, significantly increasing the yield of lysine, and thus can be practically used for the bacterial fermentation production of lysine. It has opened up and practically proved a new method for increasing the fermentation yield of lysine, and an effect of superposing and increasing the yield has been observed, so that it can be practically used for the bacterial fermentation production of lysine and is convenient for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the lysine synthesis metabolic pathway of Corynebacterium glutamicum;

[0033] Figure 2 It is a schematic diagram of the recombinant plasmid YZ022;

[0034] Figure 3 It is a schematic diagram of the recombinant plasmid YZ023;

[0035] Figure 4 It is a schematic diagram of the recombinant plasmid YZ025;

[0036] Figure 5 It is a schematic diagram of the recombinant plasmid YE019;

[0037] Figure 6 It is a schematic diagram of the recombinant plasmid YZ037;

[0038] Figure 7Schematic diagram of recombinant plasmid YZ039; and

[0039] Figure 8 Schematic diagram of recombinant plasmid YZ035. Detailed implementation manners

[0040] The following further elaborates on the detailed implementation manners of the present invention in conjunction with the accompanying drawings and examples, so as to better understand the solution of the present invention and the advantages of its various aspects. However, the following described detailed implementation manners and examples are for illustrative purposes only and are not limitations on the present invention.

[0041] The present invention relates to a recombinant bacterium for producing L-lysine, wherein the recombinant bacterium has increased expression and / or activity of asparaginase compared with the starting bacterium, and the starting bacterium is a strain capable of accumulating lysine.

[0042] The increased expression and / or activity of asparaginase can be based on various factors, including increased copy number of the encoding gene, replacement of the native promoter with a more effective strong promoter, and artificial mutations aimed at increasing the activity. Specifically, the gene copy number can be increased by the introduction and / or amplification of endogenous and / or exogenous alleles. Regarding the replacement of the gene promoter, examples thereof include the introduction of endogenous and / or exogenous promoters, and the used promoter has effective activity to effectively enhance the expression of the downstream structural gene.

[0043] In one embodiment, the recombinant bacterium has at least two copies of the asparaginase-encoding gene. Specifically, the recombinant bacterium has one or more copies of the asparaginase-encoding gene from endogenous and / or exogenous sources in addition to one copy of the endogenous asparaginase-encoding gene in its nuclear DNA. More specifically, the nucleotide sequence of the asparaginase-encoding gene can be as shown in SEQ ID NO.39.

[0044] In one embodiment, the expression of the asparaginase-encoding gene in the recombinant bacterium is mediated by a regulatory element with high transcriptional or high expression activity. Preferably, the regulatory element is a strong promoter. More preferably, the strong promoter is the P tuf promoter of the starting bacterium. Specifically, an effective endogenous and / or exogenous strong promoter is present upstream of the asparaginase-encoding gene in the nuclear DNA of the recombinant bacterium, thereby effectively increasing the expression of the asparaginase-encoding gene.

[0045] The "starting bacterium" (or also referred to as "starting strain") described in the present invention refers to the initial strain used in the gene modification strategy of the present invention. This strain can be a naturally occurring strain or a strain selected by means such as mutagenesis or genetic engineering modification.

[0046] "Inactivation" as described in the present invention refers to the change of the corresponding modified object to achieve certain effects, including but not limited to, site-directed mutagenesis, insertional inactivation, and / or knockout.

[0047] The methods of gene knockout, gene insertion, promoter replacement, and site-directed mutagenesis described in the present invention can be achieved by homologous recombination of the homologous arms carrying the modified target gene through a vector.

[0048] The introduction of a certain gene or the increase of the copy number of a certain gene in the present invention can be achieved by constructing a recombinant plasmid containing the gene and then introducing the recombinant plasmid into the starting bacterium, or by directly inserting a certain gene into a suitable site on the chromosome of the starting bacterium.

[0049] Although examples of regulatory elements with high transcriptional or high expression activity are given in the present invention, there are no particular limitations on the regulatory elements with high transcriptional or high expression activity in the present invention, as long as they can enhance the expression of the initiated gene. Regulatory elements that can be listed for use in the present invention include P of the starting bacterium 45 、P eftu 、P sod 、P glyA 、P pck 、P pgk promoters, etc., but not limited to these. For regulatory elements with low transcriptional or low expression activity, there are also no particular limitations in the present invention, as long as they can reduce the expression of the initiated gene.

[0050] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0051] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. Reference can be made to "Molecular Cloning: A Laboratory Manual (3rd Edition)" (Science Press), "Microbiology Experiments (4th Edition)" (Higher Education Press), and the manufacturer's instructions of the corresponding instruments and reagents, etc. The instruments, equipment, and reagents used in the examples are commercially available common instruments and reagents. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0052] Example 1 Construction of Lysine Chassis Engineering Bacteria

[0053] In this example, the wild-type Corynebacterium glutamicum ATCC13032 was used as the starting strain. By site-directed mutagenesis of the hom (homoserine dehydrogenase, GenBank: CAF19887.1) gene, the metabolic flux of the threonine synthesis pathway in the branched pathway was weakened; by site-directed mutagenesis of the pyc (pyruvate carboxylase, GenBank: CAF19394.1) gene, the supply of oxaloacetate, a precursor for lysine synthesis, was increased; the pck (phosphoenolpyruvate carboxykinase, GenBank: CAF20888.1) gene was knocked out and the copy numbers of the pyc* and dapB (dihydrodipicolinate reductase, GenBank: CAF20314.1) genes were increased simultaneously; the lysC (aspartokinase, GenBank: CAF18822.1), ddh (diaminopimelate dehydrogenase, GenBank: CAF21279.1), and lysA (diaminopimelate decarboxylase, GenBank: CAF19884.1) genes were overexpressed on a plasmid to further enhance the lysine synthesis pathway, and a chassis engineering bacterium for lysine production was constructed.

[0054] (1) Site-directed mutagenesis of the chromosomal hom gene

[0055] Primers were designed based on the hom gene of Corynebacterium glutamicum ATCC13032 and its upstream and downstream sequences in Genbank.

[0056] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers were designed at the mutation site to amplify two parts of the hom gene upstream and downstream of the mutation site. The upper half of the hom gene was amplified with P1 and P2 as primers, and the lower half of the hom gene was amplified with P3 and P4 as primers. Then, using the purified above-mentioned PCR products as templates and P1 and P4 as primers, overlap extension PCR technology (SOE) was used for amplification to obtain a 1638 bp PCR product, which was the hom gene (SEQ ID NO.1) containing the mutation of valine at position 59 to alanine (V59A mutation).

[0057] The above-mentioned 1638 bp PCR product was double-digested with Xba I and EcoR I and then ligated to the homologous recombination vector pK18mobsacB (purchased from the American Type Culture Collection ATCC, catalog number 87097) that had been treated with the same double digestion. The ligation product was transformed into Escherichia coli DH5α by chemical transformation method. Transformants were screened on an LB plate containing kanamycin (50 μg / mL). After the transformants were subcultured for three generations, colony PCR was used to identify the transformants with P5 and P6 as primers. For the correctly identified transformants, plasmids were extracted and double-digested with Xba I and EcoR I for identification. A 1638 bp product was positive.

[0058] The positive plasmid was sent for sequencing. The result showed that the plasmid was a recombinant plasmid obtained by inserting the nucleotide shown in SEQ ID NO.1 in the sequence listing into the vector pK18mobsacB, named YE019, and its structure was as Figure 5 shown.

[0059] Table 1

[0060]

[0061]

[0062] The correctly sequenced homologous recombinant plasmid YE019 was electrotransformed into the wild-type Corynebacterium glutamicum ATCC13032. Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Through sucrose counter-selection, positive colonies that had undergone two homologous recombinations were obtained. The positive colonies were identified by PCR amplification using P5 and P6 as primers, and the correctly identified recombinant bacteria were named Corynebacterium glutamicum EPCG1000.

[0063] The genomic DNA of this recombinant bacterium was extracted for sequencing. The result confirmed that the hom gene in the wild-type Corynebacterium glutamicum ATCC13032 had been successfully replaced with the hom gene of V59A, and Corynebacterium glutamicum EPCG1000 was successfully constructed.

[0064] (2) Site-directed mutagenesis of the chromosomal pyc gene

[0065] Primers were designed according to the pyc gene of Corynebacterium glutamicum ATCC13032 and its upstream and downstream sequences in Genbank respectively.

[0066] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers were designed at the mutation site to amplify two parts of the pyc gene upstream and downstream of the mutation site respectively. The upper half of the pyc gene was amplified using P7 and P8 as primers, and the lower half of the pyc gene was amplified using P9 and P10 as primers. Then, using the purified above-mentioned PCR products as templates and P7 and P10 as primers, amplification was carried out by the overlap extension PCR technique (SOE) to obtain a 3423 bp PCR product, which was the pyc gene (SEQ ID NO.8) containing the proline at position 458 mutated to serine (P458S mutation), that is, the pyc* gene.

[0067] The above-mentioned 3423 bp PCR product was digested with Xba I and Hind III, and then ligated to the homologous recombination vector pK18mobsacB (purchased from the American Type Culture Collection ATCC, catalog number 87097) that had been digested with the same two enzymes. The ligation product was transformed into Escherichia coli DH5α by the chemical transformation method. Transformants were screened on an LB plate containing kanamycin (50 μg / mL). After the transformants were subcultured for three generations, P11 and P12 were used as primers for colony PCR to identify the transformants. For the correctly identified transformants, plasmids were extracted and digested with Xba I and Hind III for identification. A plasmid with a 3423 bp band was considered positive.

[0068] The positive plasmid was sent for sequencing. The result showed that this plasmid was a recombinant plasmid obtained by inserting the nucleotide shown in SEQ ID NO.8 in the sequence listing into the vector pK18mobsacB, named YZ037, and its structure was as Figure 6 shown.

[0069] Table 2

[0070]

[0071] The homologous recombination plasmid YZ037 with correct sequence determination was electrotransformed into Corynebacterium glutamicum EPCG1000. Colonies with the recombinant plasmid integrated into the chromosome were obtained through positive screening with kanamycin resistance. Through sucrose counterselection, positive colonies that had undergone two homologous recombinations were obtained. The positive colonies were identified by PCR amplification using P11 and P12 as primers, and the correctly identified recombinant bacterium was named Corynebacterium glutamicum EPCG1007.

[0072] The genomic DNA of this recombinant bacterium was extracted for sequencing. The result confirmed that the pyc gene in Corynebacterium glutamicum EPCG1000 had been successfully replaced with the pyc* gene with a P458S mutation, and Corynebacterium glutamicum EPCG1007 was successfully constructed.

[0073] (3) Knockout of the pck gene and increase in the copy number of pyc*-dapB

[0074] Primers were designed according to the pck gene of Corynebacterium glutamicum ATCC13032 in Genbank and its upstream and downstream sequences respectively.

[0075] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the upstream sequence of the pck gene (SEQ ID NO.15) was amplified with P13 and P14 as primers, the promoter of the pyc gene was amplified with P15 and P16 as primers, and the downstream sequence of the pck gene (SEQ ID NO.16) was amplified with P21 and P22 as primers. Using the purified above-mentioned PCR products as the upstream and downstream homologous arms of the pyc*-dapB operon respectively, when integrating into the genome of Corynebacterium glutamicum ATCC13032, the purpose of knocking out pck can be achieved.

[0076] Using the constructed pyc* gene with point mutations and the genomic DNA of Corynebacterium glutamicum ATCC13032 in (2) as templates respectively, primers were designed to amplify pyc* and dapB (SEQ ID NO.17). The base information of the relevant gene sequences was obtained from the NCBI database, and a total of six pairs of primers were designed to construct the pyc*-dapB gene fragment (Table 3).

[0077] Table 3

[0078]

[0079] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, genes with lengths of 750bp, 244bp, 3423bp, 896bp and 767bp were amplified with P13 and P14, P15 and P16, P17 and P18, P19 and P20, P21 and P22 respectively, which were the upstream sequence of the pck gene, the promoter sequence of the pyc gene (SEQ ID NO.57), the pyc* gene sequence, the dapB gene sequence and the downstream sequence of the pck gene respectively.

[0080] The purified above-mentioned PCR products were mixed with the Escherichia coli cloning vector pK18mobsacB, and were ligated and assembled using NEbuilder (NEBuilder HiFi DNA Assembly Cloning Kit), and then transformed into Escherichia coli DH5α. Transformants were screened on LB plates containing kanamycin (50μg / mL). After the transformants were subcultured for three generations, colony PCR was performed with P23 and P'24 as primers to identify the transformants, and plasmids were extracted from the correctly identified transformants.

[0081] The positive plasmid was sent for sequencing, and the result showed that this plasmid was a recombinant plasmid obtained by inserting pyc*-dapB into the vector pK18mobsacB, named YZ039, and its structure was as Figure 7 shown.

[0082] The correctly sequenced homologous recombination plasmid YZ039 was electrotransformed into Corynebacterium glutamicum EPCG1007. Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies that had undergone two homologous recombinations were obtained by negative screening with sucrose. The positive colonies were identified by PCR amplification using P23 and P24 as primers, and the correctly identified recombinant strain was named Corynebacterium glutamicum EPCG1009.

[0083] The genomic DNA of this recombinant strain was extracted for sequencing. The results confirmed that the pck gene in Corynebacterium glutamicum EPCG1007 had been successfully knocked out, and at the same time, the pyc*-dapB gene fragment had been inserted, and Corynebacterium glutamicum EPCG1009 was successfully constructed.

[0084] (4) Increasing the copy numbers of lysC, ddh, and lysA genes

[0085] Primers were designed according to the lysC (SEQ ID NO.30), ddh (SEQ ID NO.31), lysA (SEQ ID NO.32) genes and their upstream and downstream sequences of Corynebacterium glutamicum ATCC13032 in Genbank.

[0086] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, primers were designed to amplify the lysC, ddh, and lysA genes respectively.

[0087] Table 4

[0088]

[0089] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, genes with lengths of 1266bp, 963bp, and 1338bp were amplified using P25 and P26, P27 and P28, P29 and P30 respectively.

[0090] The purified above-mentioned PCR products were mixed with the Escherichia coli cloning vector pXMJ19, and were ligated and assembled using NEbuilder (NEBuilder HiFi DNA Assembly Cloning Kit), and then transformed into Escherichia coli DH5α. Transformants were screened on LB plates containing chloramphenicol (20 μg / mL). After the transformants were subcultured for three generations, colony PCR was used with P25 and P30 as primers to identify the transformants, and plasmids were extracted from the correctly identified transformants.

[0091] The positive plasmid was sent for sequencing. The result showed that this plasmid was a recombinant plasmid with lysC, ddh, and lysA inserted into the vector pXMJ19, named YZ035, and its structure was as Figure 8 shown.

[0092] The homologous recombination plasmid YZ035 with correct sequence determination was electrotransformed into Corynebacterium glutamicum EPCG1009. The positive colonies that could grow on the resistance plate were identified by PCR amplification using P25 and P30 as primers, and the correctly identified recombinant bacteria were obtained and named Corynebacterium glutamicum EPCG1010.

[0093] The genomic DNA of this recombinant bacterium was extracted for sequencing, and the results confirmed that the free plasmid YZ035 had been successfully introduced into Corynebacterium glutamicum EPCG1009, and Corynebacterium glutamicum EPCG1010 was successfully constructed.

[0094] Example 2 Replacement of the promoter of the asparaginase-encoding gene NCgl2062 in the lysine chassis engineering bacterium

[0095] According to the upstream and downstream sequences of the NCgl2062 gene promoter of Corynebacterium glutamicum ATCC13032 in Genbank and the P tuf promoter sequence (SEQ ID NO.40), primers were designed respectively.

[0096] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P31 and P32 as primers, the upstream homologous arm of the NCgl2062 gene promoter was amplified by PCR; using P33 and P34 as primers to amplify the promoter P tuf ; using P35 and P36 as primers to amplify the downstream homologous arm of the NCgl2062 gene promoter. Using the purified above-mentioned PCR products as templates and P31 and P36 as primers, overlapping extension PCR technology (SOE) was used for amplification to obtain a 1800bp PCR product, which was a fragment containing the replaced promoter P tuf and the upstream and downstream homologous arms of the promoter to be replaced.

[0097] The above 1800bp PCR product was double-digested with Xba I and EcoR I, and then ligated with the homologous recombination vector pK18mobsacB (purchased from the American Type Culture Collection ATCC, catalog number 87097) that had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli DH5α by chemical transformation method, and the transformants were screened on an LB plate containing kanamycin (50 μg / mL). After the transformants were subcultured for three generations, the transformants were identified by colony PCR using P31 and P36 as primers. The positive transformants were those with a 1800bp band. The plasmids of the correctly identified transformants were extracted and double-digested with Xba I and EcoR I for identification. Those with a 1800bp band were positive.

[0098] The positive plasmid was sent for sequencing, and the result showed that the plasmid contained the strong promoter P with upstream and downstream homologous arms tufThe recombinant plasmid obtained by inserting into the vector pK18mobsacB was named YZ022, and its structure is as Figure 2 shown.

[0099] Table 6

[0100]

[0101] The correctly sequenced homologous recombination plasmid YZ022 was electrotransformed into Corynebacterium glutamicum EPCG1010. Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies with two homologous recombinations were obtained by sucrose counter-selection. The positive colonies were identified by PCR amplification using P31 and P36 as primers, and those with a 1800 bp band were recombinant bacteria, named Corynebacterium glutamicum EPCG1036.

[0102] The genomic DNA was extracted from this recombinant bacterium for sequencing, and the result confirmed that the promoter of NCgl2062 in Corynebacterium glutamicum EPCG1010 had been successfully replaced with the endogenous strong promoter P tuf of Corynebacterium glutamicum, and Corynebacterium glutamicum EPCG1036 was successfully constructed.

[0103] Example 3 Increasing the copy number of the asparaginase-encoding gene NCgl2062 in the lysine chassis engineering bacterium

[0104] Primers were designed according to the NCgl2062 gene and its upstream and downstream sequences of Corynebacterium glutamicum ATCC13032 in Genbank.

[0105] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and P37 and P38 as primers, the upstream sequence of the target insertion site was amplified by PCR as the upstream homologous arm for increasing the copy number of the NCgl2062 gene; the NCgl2062 gene was amplified using P39 and P40 as primers; the downstream sequence of the target insertion site was amplified using P41 and P42 as primers as the downstream homologous arm for increasing the copy number of the NCgl2062 gene. Using the purified above-mentioned PCR products as a template and P37 and P42 as primers, amplification was carried out by the overlap extension PCR technique (SOE) to obtain a 2778 bp PCR product, which was a fragment containing the upstream and downstream homologous arms of the target insertion site and the NCgl2062 gene.

[0106] The above 2778-bp PCR product was digested with Xba I and Nhe I, and then ligated to the homologous recombination vector pK18mobsacB (purchased from American Type Culture Collection, ATCC, catalog number 87097) that had been digested with the same two enzymes. The ligation product was transformed into Escherichia coli DH5α by chemical transformation method. Transformants were screened on LB plates containing kanamycin (50 μg / mL). After the transformants were subcultured for three generations, P37 and P42 were used as primers for colony PCR to identify the transformants. Those with a 2778-bp band were positive transformants. Plasmids were extracted from the correctly identified transformants and digested with Xba I and Nhe I for identification. Those with a 2778-bp band were positive.

[0107] The positive plasmid was sent for sequencing. The result showed that this plasmid was a recombinant plasmid obtained by inserting the homologous arms upstream and downstream of the target insertion site and the NCgl2062 gene into the vector pK18mobsacB, named YZ023, and its structure is as Figure 3 shown.

[0108] Table 7

[0109]

[0110] The correctly sequenced homologous recombination plasmid YZ023 was electrotransformed into Corynebacterium glutamicum EPCG1010. Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies that had undergone two homologous recombinations were obtained by sucrose counterselection. The positive colonies were identified by PCR amplification using P37 and P42 as primers. Those with a 2778-bp band were recombinant bacteria, named Corynebacterium glutamicum EPCG1039.

[0111] Genomic DNA was extracted from this recombinant bacterium for sequencing. The result confirmed that a copy of the NCgl2062 gene had been successfully inserted into the target site in Corynebacterium glutamicum EPCG1010, and Corynebacterium glutamicum EPCG1039 was successfully constructed.

[0112] Example 4. Knockout of the asparaginase-encoding gene NCgl2062 in the lysine chassis engineering bacterium

[0113] Primers were designed according to the NCgl2062 gene and its upstream and downstream sequences of Corynebacterium glutamicum ATCC13032 in Genbank.

[0114] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P43 and P44 as primers, the upstream homologous arm of the NCgl2062 gene was amplified by PCR; the downstream homologous arm of the NCgl2062 gene was amplified using P45 and P46 as primers. Using the purified above-mentioned PCR products as templates and P43 and P46 as primers, amplification was carried out by the overlap extension PCR technique (SOE) to obtain a 1600bp PCR product, which is a fragment containing the upstream and downstream homologous arms of the NCgl2062 gene.

[0115] The above-mentioned 1600bp PCR product was double-digested with EcoR I and Nhe I and then ligated to the homologous recombination vector pK18mobsacB (purchased from the American Type Culture Collection ATCC, catalog number 87097) that had been treated with the same double digestion. The ligation product was transformed into Escherichia coli DH5α by the chemical transformation method, and transformants were screened on an LB plate containing kanamycin (50 μg / mL). After the transformants were subcultured for three generations, colony PCR was performed using P43 and P46 as primers to identify the transformants. Those with a 1600bp product were positive transformants. The plasmids were extracted from the correctly identified transformants, and the plasmids were double-digested with EcoR I and Nhe I for identification. Those with a 1600bp product were positive.

[0116] The positive plasmid was sent for sequencing. The result showed that this plasmid is a recombinant plasmid obtained by inserting the fragment containing the upstream and downstream homologous arms of the NCgl2062 gene into the vector pK18mobsacB, named YZ025, and its structure is as Figure 4 shown.

[0117] Table 8

[0118]

[0119] The correctly sequenced homologous recombination plasmid YZ025 was electrotransformed into Corynebacterium glutamicum EPCG1010. Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies that had undergone two homologous recombinations were obtained by sucrose counter-selection. The positive colonies were identified by PCR amplification using P43 and P46 as primers. Those with a 1600bp product were recombinant bacteria, named Corynebacterium glutamicum EPCG1038.

[0120] The genomic DNA was extracted from this recombinant bacterium for sequencing. The result confirmed that the NCgl2062 gene had been successfully knocked out in Corynebacterium glutamicum EPCG1010, and Corynebacterium glutamicum EPCG1038 was successfully constructed.

[0121] Example 5 Application of Corynebacterium glutamicum lysine engineering bacteria in lysine fermentation production

[0122] The Corynebacterium glutamicum EPCG1036, EPCG1038, EPCG1039 constructed in Examples 2 to 4 and the starting strain EPCG1010 were cultured at the shake flask level and the 3 L fermenter level respectively to produce L-lysine, and the method is as described below.

[0123] (1) Shake flask fermentation:

[0124] Corynebacterium glutamicum EPCG1036, EPCG1038, EPCG1039 and EPCG1010 were inoculated into 500 ml Erlenmeyer flasks containing 50 ml of the following seed medium and cultured with shaking at 220 rpm at 30 °C for 8 - 9 h. Then, 5 ml of each seed culture was inoculated into 500 ml baffled flasks containing 50 ml of the following fermentation medium and cultured with shaking at 220 rpm at 37 °C for 42 - 46 h. And isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mmol / L was added at 6 h of fermentation culture for the induced expression of the target gene. Concentrated ammonia water was intermittently added to control the pH of the fermentation broth between 7.0 - 7.2. According to the residual sugar situation, a glucose mother liquor with a concentration of 400 g / L was added to control the residual sugar in the fermentation broth at 5 - 10 g / L.

[0125] (2) 3 L fermenter fermentation:

[0126] Corynebacterium glutamicum EPCG1036, EPCG1038, EPCG1039 and EPCG1010 were inoculated into 1000 ml Erlenmeyer flasks containing 100 ml of the following seed medium and cultured with shaking at 220 rpm at 30 °C for 8 - 9 h. Then, each seed culture was inoculated into a 3 L fermenter containing 900 ml of the following fermentation medium and cultured at 37 °C and a tank pressure of 0.01 MPa for 42 - 46 h. The seed liquid was inoculated into the fermentation medium containing chloramphenicol with a final concentration of 10 μg / ml at a volume percentage of 10%. The fermenter used was a 3 L fermenter: equipped with a constant-speed programmable pump inside, which can achieve constant-speed feeding. During the fermentation process, 600 g / L glucose was supplemented by a peristaltic pump to control the glucose concentration in the fermentation system at 5 - 10 g / L, and the fermentation temperature was maintained at 30 °C by a heating jacket and cooling water; air was introduced to provide dissolved oxygen, and the dissolved oxygen was maintained at 30% by cascade control of the rotation speed and the dissolved oxygen signal; concentrated ammonia water was added to regulate the pH, maintaining it at about 6.9. The fermentation was carried out continuously for 52 h. When OD 600 = 4 - 5, IPTG (isopropylthiogalactoside, final concentration 0.1 mmol / L) was added to induce the expression of the gene carried by the recombinant plasmid.

[0127] The seed medium and the fermentation medium are as described below:

[0128] Seed medium (pH 7.0)

[0129] 20 g of sucrose, 10 g of peptone, 5 g of yeast extract, 3.5 g of urea, 4 g of potassium dihydrogen phosphate, 10 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.2 mg of biotin, 1.5 mg of vitamin B1, 2 mg of calcium D-pantothenate, 3 mg of nicotinamide (dissolved in 1 liter of distilled water).

[0130] Fermentation medium (pH 7.0)

[0131] 40 g of glucose, 20 g of molasses, 0.4 g of phosphoric acid, 15 g of ammonium sulfate, 0.87 g of magnesium sulfate heptahydrate, 0.88 mg of biotin, 6.3 mg of vitamin B1, 6.3 mg of calcium D-pantothenate, 42 mg of nicotinamide (dissolved in 1 liter of distilled water).

[0132] (3) Detection of lysine production

[0133] HPLC method:

[0134] 1. Mobile phase:

[0135] Organic phase: methanol: acetonitrile: water = 45:45:10 (V / V);

[0136] Aqueous phase: 12.436 g of NaH2PO4·2H2O is dissolved in 2 L of ultrapure water, and the pH is adjusted to 7.8 with NaOH.

[0137] 2. Elution program:

[0138]

[0139]

[0140] Prepare standard concentration solutions with lysine standard, with concentrations of 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1.6 g / L respectively. Draw a standard curve based on the peak area, and calculate the lysine concentration in the fermentation broth as follows:

[0141] Table 9

[0142]

[0143] The results of the shake flask fermentation experiment showed that enhancing the expression of the asparaginase gene significantly increased the lysine production; after knocking out this gene, the lysine production decreased significantly.

[0144] Corresponding to the results of shake flask fermentation, at the 3L fermenter level, increasing one copy of the asparaginase-encoding gene increased lysine production by 59.48%; replacing the promoter of the asparaginase-encoding gene with a strong promoter increased lysine production by 14.83%; while knocking out the asparaginase-encoding gene decreased lysine production by 27.39%.

[0145] Example 6 Enhanced expression of the asparaginase-encoding gene NCgl2062 in Corynebacterium pekinense 1.563

[0146] Using Corynebacterium pekinense AS1.563, which can accumulate lysine, as the starting strain, the effect of the expression of the asparaginase-encoding gene on lysine accumulation was analyzed.

[0147] (1) Replacement of the strong promoter of the NCgl2062 gene

[0148] The recombinant vector YZ022 constructed in Example 2 was transformed into Corynebacterium pekinense AS1.563 (China Center for Industrial Culture Collection of Microorganisms, CICC10178) to replace the promoter of the NCgl2062 gene with the strong promoter Ptuf.

[0149] Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies with two homologous recombinations were obtained by sucrose counter-selection. The positive colonies were identified by PCR amplification using P31 and P36 as primers. The recombinant bacteria with a 1800bp band were named Corynebacterium glutamicum CP1008.

[0150] Genomic DNA was extracted from the recombinant bacteria and sequenced. The results confirmed that the promoter of NCgl2062 in Corynebacterium pekinense AS1.563 had been successfully replaced with the endogenous strong promoter Ptuf of Corynebacterium glutamicum, and Corynebacterium glutamicum CP1008 was successfully constructed.

[0151] (2) Increasing the copy number of the NCgl2062 gene

[0152] The recombinant vector YZ023 constructed in Example 3 was transformed into Corynebacterium pekinense AS1.563 to increase the copy number of the NCgl2062 gene.

[0153] Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies with two homologous recombinations were obtained by sucrose counter-selection. The positive colonies were identified by PCR amplification using P37 and P42 as primers. The recombinant bacteria with a 2778bp band were named Corynebacterium glutamicum CP1009.

[0154] The genomic DNA was extracted from the recombinant bacterium for sequencing, and the results confirmed that a copy of the NCgl2062 gene had been successfully inserted at the target site in Corynebacterium pekinense AS1.563, and Corynebacterium pekinense CP1009 was successfully constructed.

[0155] (3) Knockout of the asparaginase-encoding gene NCgl2062 in Corynebacterium pekinense AS1.563

[0156] The recombinant vector YZ025 constructed in Example 4 was transformed into Corynebacterium pekinense AS1.563 to achieve the knockout of the NCgl2062 gene.

[0157] Colonies with the recombinant plasmid integrated into the chromosome were obtained by positive screening with kanamycin resistance. Positive colonies that had undergone two homologous recombinations were obtained by sucrose counter-selection. The positive colonies were identified by PCR amplification using P43 and P46 as primers. The recombinant bacterium with a 1600bp band was named Corynebacterium glutamicum CP1010.

[0158] The genomic DNA was extracted from the recombinant bacterium for sequencing, and the results confirmed that the NCgl2062 gene had been successfully knocked out in Corynebacterium pekinense AS1.563, and Corynebacterium pekinense CP1010 was successfully constructed.

[0159] Example 7 Application of Corynebacterium pekinense lysine engineering bacteria in lysine fermentation production

[0160] The L-lysine-producing strains Corynebacterium pekinense CP1008, CP1009, CP1010 constructed in Example 6 and the starting strain AS1.563 were cultured at the shake flask level and 3L fermentor level respectively to produce L-lysine, and the method is described as follows.

[0161] (1) Shake flask fermentation:

[0162] Corynebacterium pekinense CP1008, CP1009, CP1010 and AS1.563 were inoculated into 500 ml Erlenmeyer flasks containing 50 ml of the following seed medium and cultured at 30 °C with shaking at 220 rpm for 8 - 9 hours. Then, 5 ml of each seed culture was inoculated into 500 ml baffled flasks containing 50 ml of the following fermentation medium and cultured at 37 °C with shaking at 220 rpm for 42 - 46 hours. Concentrated ammonia water was intermittently added to control the pH of the fermentation broth between 7.0 - 7.2. According to the residual sugar situation, a glucose mother liquor with a concentration of 400 g / L was added to control the residual sugar in the fermentation broth at 5 - 10 g / L.

[0163] (2) 3L fermentor fermentation:

[0164] Corynebacterium pekinense CP1008, CP1009, CP1010 and AS1.563 were inoculated into 1000 ml Erlenmeyer flasks containing 100 ml of the following seed medium and cultured with shaking at 220 rpm at 30 °C for 8 - 9 hours. Then, each seed culture was inoculated into a 3 L fermenter containing 900 ml of the following fermentation medium and cultured at 37 °C and a tank pressure of 0.01 MPa for 42 - 46 hours. The fermenter used was a 3 L fermenter: equipped with a constant-speed programmable pump, enabling constant-rate feeding. During the fermentation process, 600 g / L glucose was added via a peristaltic pump to control the glucose concentration in the fermentation system at 5 - 10 g / L, the fermentation temperature was maintained at 30 °C by a heating jacket and cooling water; air was introduced to provide dissolved oxygen, and the dissolved oxygen was controlled at 30% by cascading the rotation speed and the dissolved oxygen signal; concentrated ammonia water was added to regulate the pH, maintaining it at around 6.9. The fermentation was carried out continuously for 52 h.

[0165] The seed medium and fermentation medium are as follows:

[0166] Seed medium (pH 7.0)

[0167] Sucrose 20 g, peptone 10 g, yeast extract 5 g, urea 3.5 g, potassium dihydrogen phosphate 4 g, dipotassium hydrogen phosphate 10 g, magnesium sulfate heptahydrate 0.5 g, biotin 0.2 mg, vitamin B1 1.5 mg, calcium D-pantothenate 2 mg, nicotinamide 3 mg (dissolved in 1 liter of distilled water).

[0168] Production medium (pH 7.0)

[0169] Glucose 40 g, molasses 20 g, phosphoric acid 0.4 g, ammonium sulfate 15 g, magnesium sulfate heptahydrate 0.87 g, biotin 0.88 mg, vitamin B1 6.3 mg, calcium D-pantothenate 6.3 mg, nicotinamide 42 mg (dissolved in 1 liter of distilled water).

[0170] After the cultivation was completed, HPLC analysis was carried out to determine the content of L-lysine produced by the strains. The L-lysine concentrations in the cultures of Corynebacterium pekinense CP1008, CP1009, CP1010 and AS1.563 are shown in Table 10.

[0171] Table 10

[0172]

[0173] As can be seen from the above table, significant differences were shown among the modified strains both at the shake flask level and at the 3 L fermenter level.

[0174] The differences between flask fermentation and fermenter fermentation show a consistent trend. That is, after enhancing the expression of the asparaginase gene, the lysine yield increased. Correspondingly, after knocking out this gene, the lysine yield decreased significantly.

[0175] Regarding the acid production data of fermenter fermentation, compared with AS1.563, the lysine yield of CP1008 increased by 86.6%, and compared with AS1.563, the lysine yield of CP1009 increased by 36.3%. While compared with AS1.563, the lysine yield of CP1010 decreased by 29.5%.

[0176] Finally, it should be noted that: Obviously, the above embodiments are only examples for clearly explaining the present invention, rather than limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention. Sequence Listing <110> Institute of Microbiology, Chinese Academy of Sciences <120> Recombinant Bacteria for Producing L-Lysine, Method for Constructing the Same, and Method for Producing L-Lysine <130> 170710CI <141> 2017-11-01 <160> 57 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1638 <212> DNA <213> Corynebacterium glutamicum <400> 1 ctgcgacagc atggaactca gtgcaatggc tgtaaggcct gcaccaacaa tgattgagcg 60 aagctccaaa atgtcctccc cgggttgata ttagatttca taaatatact aaaaatcttg 120 agagtttttc cgttgaaaac taaaaagctg ggaaggtgaa tcgaatttcg gggctttaaa 180 gcaaaaatga acagcttggt ctatagtggc taggtaccct ttttgttttg gacacatgta 240 gggtggccga aacaaagtaa taggacaaca acgctcgacc gcgattattt ttggagaatc 300 atgacctcag catctgcccc aagctttaac cccggcaagg gtcccggctc agcagtcgga 360 attgcccttt taggattcgg aacagtcggc actgaggtga tgcgtctgat gaccgagtac 420 ggtgatgaac ttgcgcaccg cattggtggc ccactggagg ttcgtggcat tgctgcttct 480 gatatctcaa agccacgtga aggcgttgca cctgagctgc tcactgagga cgcttttgca 540 ctcatcgagc gcgaggatgt tgacatcgtc gttgaggtta tcggcggcat tgagtaccca 600 cgtgaggtag ttctcgcagc tctgaaggcc ggcaagtctg ttgttaccgc caataaggct 660 cttgttgcag ctcactctgc tgagcttgct gatgcagcgg aagccgcaaa cgttgacctg 720 tacttcgagg ctgctgttgc aggcgcaatt ccagtggttg gcccactgcg tcgctccctg 780 gctggcgatc agatccagtc tgtgatgggc atcgttaacg gcaccaccaa cttcatcttg 840 gacgccatgg attccaccgg cgctgactat gcagattctt tggctgaggc aactcgtttg 900 ggttacgccg aagctgatcc aactgcagac gtcgaaggcc atgacgccgc atccaaggct 960 gcaattttgg catccatcgc tttccacacc cgtgttaccg cggatgatgt gtactgcgaa 1020 ggtatcagca acatcagcgc tgccgacatt gaggcagcac agcaggcagg ccacaccatc 1080 aagttgttgg ccatctgtga gaagttcacc aacaaggaag gaaagtcggc tatttctgct 1140 cgcgtgcacc cgactctatt acctgtgtcc cacccactgg cgtcggtaaa caagtccttt 1200 aatgcaatct ttgttgaagc agaagcagct ggtcgcctga tgttctacgg aaacggtgca 1260 ggtggcgcgc caaccgcgtc tgctgtgctt ggcgacgtcg ttggtgccgc acgaaacaag 1320 gtgcacggtg gccgtgctcc aggtgagtcc acctacgcta acctgccgat cgctgatttc 1380 ggtgagacca ccactcgtta ccacctcgac atggatgtgg aagatcgcgt gggggttttg 1440 gctgaattgg ctagcctgtt ctctgagcaa ggaatctccc tgcgtacaat ccgacaggaa 1500 gagcgcgatg atgatgcacg tctgatcgtg gtcacccact ctgcgctgga atctgatctt 1560 tcccgcaccg ttgaactgct gaaggctaag cctgttgtta aggcaatcaa cagtgtgatc 1620 cgcctcgaaa gggactaa 1638 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(26) <223> Forward primer for upstream of hom-V59A <400> 2 gctctagaag ctgtttcaca atttct 26 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(19) <223> Reverse primer for mutation site of hom-V59A <400> 3 atatcagaag cagcaatgc 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(19) <223> Forward primer for mutation site of hom-V59A <400> 4 gcattgctgc ttctgatat 19 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(27) <223> Reverse primer downstream of hom-V59A <400> 5 ccggaattcc caacaacttg atggtgt 27 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(19) <223> Forward identification primer of hom-V59A <400> 6 tctacgttgt atctcgcac 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(18) <223> Reverse identification primer of hom-V59A <400> 7 caggcgacca gctgcttc 18 <210> 8 <211> 2280 <212> DNA <213> Corynebacterium glutamicum <400> 8 aaaaccgatg tttgattggg ggaatcgggg gttacgatac taggacgcag tgactgctat 60 cacccttggc ggtctcttgt tgaaaggaat aattactcta gtgtcgactc acacatcttc 120 aacgcttcca gcattcaaaa agatcttggt agcaaaccgc ggcgaaatcg cggtccgtgc 180 tttccgtgca gcactcgaaa ccggtgcagc cacggtagct atttaccccc gtgaagatcg 240 gggatcattc caccgctctt ttgcttctga agctgtccgc attggtaccg aaggctcacc 300 agtcaaggcg tacctggaca tcgatgaaat tatcggtgca gctaaaaaag ttaaagcaga 360 tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc cagcttgccc gcgagtgtgc 420 ggaaaacggc attactttta ttggcccaac cccagaggtt cttgatctca ccggtgataa 480 gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca gttttggcgg aatccacccc 540 gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc cagacttacc ccatctttgt 600 gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt gttgcttcac ctgatgagct 660 tcgcaaatta gcaacagaag catctcgtga agctgaagcg gctttcggcg atggcgcggt 720 atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa gtgcagatcc ttggcgatca 780 cactggagaa gttgtacacc tttatgaacg tgactgctca ctgcagcgtc gtcaccaaaa 840 agttgtcgaa attgcgccag cacagcattt ggatccagaa ctgcgtgatc gcatttgtgc 900 ggatgcagta aagttctgcc gctccattgg ttaccagggc gcgggaaccg tggaattctt 960 ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac ccacgtatcc aggttgagca 1020 caccgtgact gaagaagtca ccgaggtgga cctggtgaag gcgcagatgc gcttggctgc 1080 tggtgcaacc ttgaaggaat tgggtctgac ccaagataag atcaagaccc acggtgcagc 1140 actgcagtgc cgcatcacca cggaagatcc aaacaacggc ttccgcccag ataccggaac 1200 tatcaccgcg taccgctcac caggcggagc tggcgttcgt cttgacggtg cagctcagct 1260 cggtggcgaa atcaccgcac actttgactc catgctggtg aaaatgacct gccgtggttc 1320 cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg gctgagttca ccgtgtctgg 1380 tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg gaagaggact tcacttccaa 1440 gcgcatcgcc accggattca ttgccgatca ctcgcacctc cttcaggctc cacctgctga 1500 tgatgagcag ggacgcatcc tggattactt ggcagatgtc accgtgaaca agcctcatgg 1560 tgtgcgtcca aaggatgttg cagctcctat cgataagctg cctaacatca aggatctgcc 1620 actgccacgc ggttcccgtg accgcctgaa gcagcttggc ccagccgcgt ttgctcgtga 1680 tctccgtgag caggacgcac tggcagttac tgataccacc ttccgcgatg cacaccagtc 1740 tttgcttgcg acccgagtcc gctcattcgc actgaagcct gcggcagagg ccgtcgcaaa 1800 gctgactcct gagcttttgt ccgtggaggc ctggggcggc gcgacctacg atgtggcgat 1860 gcgtttcctc tttgaggatc cgtgggacag gctcgacgag ctgcgcgagg cgatgccgaa 1920 tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg ggatacaccc cgtacccaga 1980 ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc ggcgtggaca tcttccgcat 2040 cttcgacgcg cttaacgacg tctcccagat gcgtccagca atcgacgcag tcctggagac 2100 caacaccgcg gtagccgagg tggctatggc ttattctggt gatctctctg atccaaatga 2160 aaagctctac accctggatt actacctaaa gatggcagag gagatcgtca agtctggcgc 2220 tcacatcttg gccattaagg atatggctgg tctgcttcgc ccagctgcgg taaccaagct 2280 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(28) <223> Forward primer for upstream of pyc - P458S <400> 9 cccaagcttt gactgctcac tgcagcgt 28 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(17) <223> Reverse primer for mutation site of pyc - P458S <400> 10 aggtgcgagt gatcggc 17 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(17) <223> Forward primer for mutation site of pyc - P458S <400> 11 gccgatcact cgcacct 17 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(26) <223> Reverse primer for pyc-P458S downstream <400> 12 gctctagagc gtcgattgct ggacgc 26 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(19) <223> Forward identification primer for pyc-P458S <400> 13 cgcaaattag caacagaag 19 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(18) <223> Reverse identification primer for pyc-P458S <400> 14 ccttaatggc caagatgt 18 <210> 15 <211> 750 <212> DNA <213> Corynebacterium glutamicum <400> 15 acctggccct cgatacctcg agtctggtgg cggctttgtc tgaagatatt tctggcgccg 60 gattaaatga cctgaaagtt ctcgacgtcg gcggcggacc cggatacttc gccgaagcct 120 ttgagacact gggcgccacc tacttctccg tcgaacccga cgttggcgaa atgtccgcag 180 ctggcatcga cgtccacgga tcagtccgcg gatccggcct cgacctgccg tttcttcccg 240 attcctttga cgtggtgtac tcctccaacg ttgcagaaca tgtctccgca ccgtgggaat 300 tgggagaaga aatgctccgc gtcacccgca gcggcggcct ggcaatcctg agctacacca 360 tttggttagg gcccttcggc ggccatgaaa ccggactgtg ggaacactac gttggcggag 420 aatttgcccg cgatcgctac acgaagaaac acgggcaccc gcctaagaac gttttcgggg 480 agtcactgtt taatgtgtcc tgccgggagg ggctggaatg gggagcctcc gtgggcaatg 5403] cggaattggt tgccgctttt ccccgctacc acccgtattg ggtctggtgg atggttaaag 600 tcccagtgct ccgagaattc gcggtaagta acttggtgtt ggtgtttaaa aagcactgag 660 gttttgagga attcatcgct taacgacaag aaaggctccc actttcggtg ggagcctttc 720 ttgttattta gcagttctta agcgtgaact 750 <210> 16 <211> 767 <212> DNA <213> Corynebacterium glutamicum <400> 16 tacttctcca gattttgtgt cattcgacag agttctcgcc ccctagcgta gctttcagat 60 acagaactag ttaaaacttt aggtgagaca acggacacat ttgtcattac cagtggacct 120 accccctgcc cacacgcatc tacacacttt ctttaatatg agagcacccg tttaaatagc 180 ctattttggg ggtggtttca agaattaacc tcaaccgttc tccgacagtt cattccccgt 240 ccatggccat tgggttcaga tttgggcaat tctcacacat tccaggggac aactttccca 300 gttttcccac cattaacact taacattcgg acaataggca acaaaacgcc aagaacagcg 360 gtaatggtaa tccctttccc ttaccctgcc atcacaatcc aagcactccg ctagtggccg 420 accagcacaa accggcccac tgtcagtaca caccttttta aaacaacatt tacactcaca 480 tgcatgcccg cactgtcacc acccgccctc aactaccgaa ctaaagatat gtacttgaag 540 ccaaattttt accctagatc ccccttttaa atactttgaa aattactcac acacatcccc 600 acgttacccc aaaggttata tccagttagt cgtatcaaaa agtgctctga tcttaacttt 660 gccctaccta aatacatgac cccaccacga cggccagtac taacgacaga atccactagc 720 gaacccattt attaacaaac attgcaaaca agtgttgaag tattcgc 767 <210> 17 <211> 960 <212> DNA <213> Corynebacterium glutamicum <400> 17 gctcctttta aaaaattcca cccgctgctg aaatgagcct ttacaggcct aggtaatgct 60 caagtcctac gactaggcct gggtgctgtg caatgttgcg cacacccacc aagacacctg 120 gtgcaaatga gttgcgatca taggagtcct gcttgatggt caaggtctga ccctgggtgc 180 caaagataac ttgctcgtga gcaaccatgc cggacatgcg gactgcatga accgggattc 240 catctacgct tgcgccacgg gaaccctcaa gtgcctgctc ggtcgcatct ggctgtgcgt 300 ccatgcctgc ttctttgcgt gccgcagcaa tgccctgagc agtgtggatc gcggtgcctg 360 aaggtgcatc cagcttgttg gggtggtgca gctcaataac ttcagctgat tcgaagaagc 420 gggcagcctg cttggaaaag accatggtca acaccgcaga gatagcaaag ttaggtgcga 480 tcagaacacc gacattgtct tttccttcaa gccagtcgcg aacctgctcc aaacgagcat 540 catcgaagcc cgtggttcca acaaccgcag aaatgccgtt gttgatgcag aactccaggt 600 tgcccatcac agcgttagga gtggtgaagt caacgacaac ttcagcgccg ttgtctacca 660 gaaggctcaa atcatcgtcg acgccgatct ctgcaacaag ctccagatcg tcggactcat 720 tgactgctgc cacaatagtt tgaccaacac ggcctttggc tccgagaacg ccaaccttga 780 ttcccattat gctccttcat tttcgtgggg cgaagagttt ttcaaacgcc caccagtcta 840 gacgtacccg ttcagaaggt gctgatatcc atacctaact gaccgttttg gtctgtgttg 900 ttaacgattg ttcatcagtt tgttccgttc tagggattaa tcacacaggt ggaactatgt 960 <210> 18 <211> 49 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(49) <223> Forward primer of pck upstream homologous arm <400> 18 tctagagtcg acctgcaggc atgcaagctt acctggccct cgatacctc 49 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(41) <223> Reverse primer of pck upstream homologous arm <400> 19 cctaggcctg taaagttcac gcttaagaac tgctaaataa c 41 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Forward primer of pyc promoter <400> 20 tgtgagtcga cattagagta attattcctt tcaacaagag 40 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(34) <223> Reverse primer of pyc promoter <400> 21 atctggagaa gtatgcgtta aacttggcca aatg 34 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(32) <223> Forward primer of pyc* <400> 22 tccgttctag ggattaggaa acgacgacga tc 32 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(36) <223> pyc* reverse primer <400> 23 aggaataatt actctaatgt cgactcacac atcttc 36 <210> 24 <211> 31 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(31) <223> dapB forward primer <400> 24 ttaagcgtga actttacagg cctaggtaat g 31 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(36) <223> dapB reverse primer <400> 25 tcgtcgtcgt ttcctaatcc ctagaacgga acaaac 36 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(32) <223> Forward primer of the homologous arm downstream of pck <400> 26 caagtttaac gcatacttct ccagattttg tg 32 <210> 27 <211> 49 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(49) <223> Reverse primer of the homologous arm downstream of pck <400> 27 cgttgtaaaa cgacggccag tgccaagctt gcgaatactt caacacttg 49 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(20) <223> Forward primer for pyc*-dapB identification <400> 28 taccttgggc aggtcgtggg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(20) <223> Reverse primer for pyc*-dapB identification <400> 29 tgggagcgtt gtgcgctcga 20 <210> 30 <211> 1266 <212> DNA <213> Corynebacterium glutamicum <400> 30 atggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60 aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120 tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180 ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240 gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300 ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360 gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420 aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480 ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540 accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600 atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660 Met Leu Glu Leu Leu Leu Val Gly Pro Lys Ile Phe Gly Cys Ala Gln Val Glu Tyr Ala 660 cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720 Arg Cys Ile Gln Met Cys His Ser Arg Tyr Ala Ser Ser Tyr Val Asn Asp Pro Gly Thr Phe 720 attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780 Ile Ala Gly Leu Met Glu Glu Ile Pro Val Glu Glu Gln Ser Leu Thr Gly Val Arg Asn 780 gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840 Asp Lys Ser Glu Ala Lys Val Thr Arg Leu Gly Ile Ser Asp Lys Pro Arg Glu Ala Ala 840 aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900 Lys Val Phe Pro Cys Arg Leu Ala Asp Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val 900 tcttctgtag aagacggcac caccgacatc atcttcacct gccctcgttc cgacggccgc 960 Ser Ser Val Glu Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Leu Val Pro Asp Gly Arg 960 cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020 Arg Arg Asp Gly Asp Leu Glu Glu Ala Ser Gly Gln Trp Thr Gln Met Cys Leu Tyr 1020 gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080 Asp Asp Gln Val Gly Lys Ser Pro Ser Val Gly Ala Gly Met Ser Ser Pro Gly Val 1080 accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140 Thr Ala Glu Phe Met Glu Ala Ser Ala Asp Val Asn Val Asn Ile Glu Leu Ile Pro Thr 1140 tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200 Ser Glu Ile Arg Tyr Phe Pro Cys Asp Pro Val Glu Asp Asp Leu Asp Ala Ala His Val Gln 1200 ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Arg Val Tyr Ala Gly Thr Gly 1260 cgctaa 1266 Arg Leu 1266 <210> 31 <211> 963 <212> DNA <213> Corynebacterium glutamicum <400> 31 atgaccaaca tccgcgtagc tatcgtgggc tacggaaacc tgggacgcag cgtcgaaaag 60 cttattgcca agcagcccga catggacctt gtaggaatct tctcgcgccg ggccaccctc 120 gacacaaaga cgccagtctt tgatgtcgcc gacgtggaca agcacgccga cgacgtggac 180 gtgctgttcc tgtgcatggg ctccgccacc gacatccctg agcaggcacc aaagttcgcg 240 cagttcgcct gcaccgtaga cacctacgac aaccaccgcg acatcccacg ccaccgccag 300 gtcatgaacg aagccgccac cgcagccggc aacgttgcac tggtctctac cggctgggat 360 ccaggaatgt tctccatcaa ccgcgtctac gcagcggcag tcttagccga gcaccagcag 420 cacaccttct ggggcccagg tttgtcacag ggccactccg atgctttgcg acgcatccct 480 ggcgttcaaa aggcagtcca gtacaccctc ccatccgaag acgccctgga aaaggcccgc 540 cgcggcgaag ccggcgacct taccggaaag caaacccaca agcgccaatg cttcgtggtt 600 gccgacgcgg ccgatcacga gcgcatcgaa aacgacatcc gcaccatgcc tgattacttc 660 gttggctacg aagtcgaagt caacttcatc gacgaagcaa ccttcgactc cgagcacacc 720 ggcatgccac acggtggcca cgtgattacc accggcgaca ccggtggctt caaccacacc 780 gtggaataca tcctcaagct ggaccgaaac ccagatttca ccgcttcctc acagatcgct 840 ttcggtcgcg cagctcaccg catgaagcag cagggccaaa gcggagcttt caccgtcctc 900 gaagttgctc catacctgct ctccccagag aacttggacg atctgatcgc acgcgacgtc 960 taa 963 <210> 32 <211> 1338 <212> DNA <213> Corynebacterium glutamicum <400> 32 atggctacag ttgaaaattt caatgaactt cccgcacacg tatggccacg caatgccgtg 60 cgccaagaag acggcgttgt caccgtcgct ggtgtgcctc tgcctgacct cgctgaagaa 120 tacggaaccc cactgttcgt agtcgacgag gacgatttcc gttcccgctg tcgcgacatg 180 gctaccgcat tcggtggacc aggcaatgtg cactacgcat ctaaagcgtt cctgaccaag 240 accattgcac gttgggttga tgaagagggg ctggcactgg acattgcatc catcaacgaa 300 ctgggcattg ccctggccgc tggtttcccc gccagccgta tcaccgcgca cggcaacaac 360 aaaggcgtag agttcctgcg cgcgttggtt caaaacggtg tgggacacgt ggtgctggac 420 tccgcacagg aactagaact gttggattac gttgccgctg gtgaaggcaa gattcaggac 480 gtgttgatcc gcgtaaagcc aggcatcgaa gcacacaccc acgagttcat cgccactagc 540 cacgaagacc agaagttcgg attctccctg gcatccggtt ccgcattcga agcagcaaaa 600 gccgccaaca acgcagaaaa cctgaacctg gttggcctgc actgccacgt tggttcccag 660 gtgttcgacg ccgaaggctt caagctggca gcagaacgcg tgttgggcct gtactcacag 720 atccacagcg aactgggcgt tgcccttcct gaactggatc tcggtggcgg atacggcatt 780 gcctataccg cagctgaaga accactcaac gtcgcagaag ttgcctccga cctgctcacc 840 gcagtcggaa aaatggcagc ggaactaggc atcgacgcac caaccgtgct tgttgagccc 900 ggccgcgcta tcgcaggccc ctccaccgtg accatctacg aagtcggcac caccaaagac 960 gtccacgtag acgacgacaa aacccgccgt tacatcgccg tggacggagg catgtccgac 1020 aacatccgcc cagcactcta cggctccgaa tacgacgccc gcgtagtatc ccgcttcgcc 1080 gaaggagacc cagtaagcac ccgcatcgtg ggctcccact gcgaatccgg cgatatcctg 1140 atcaacgatg aaatctaccc atctgacatc accagcggcg acttccttgc actcgcagcc 1200 accggcgcat actgctacgc catgagctcc cgctacaacg ccttcacacg gcccgccgtc 1260 gtgtccgtcc gcgctggcag ctcccgcctc atgctgcgcc gcgaaacgct cgacgacatc 1320 ctctcactag aggcataa 1338 <210> 33 <211> 58 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(58) <223> lysC forward primer <400> 33 caggtcgact ctagaggatc cccgggaaag gaggacaacc atggccctgg tcgtacag 58 <210> 34 <211> 53 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(53) <223> lysC reverse primer <400> 34 caccgacatc atcttcacct gcgttgtcct cctttttagc gtccggtgcc tgc 53 <210> 35 <211> 43 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(43) <223> ddh forward primer <400> 35 caccggacgc taaaaaggag gacaaccatg accaacatcc gcg 43 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(32) <223> ddh reverse primer <400> 36 gttgtcctcc tttttagacg tcgcgtgcga tc 32 <210> 37 <211> 45 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(45) <223> lysA forward primer <400> 37 acgcgacgtc taaaaaggag gacaaccatg gctacagttg aaaat 45 <210> 38 <211> 49 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(49) <223> lysA reverse primer <400> 38 ctcatccgcc aaaacagcca agctgaattc ttatgcctct agtgagagg 49 <210> 39 <211> 978 <212> DNA <213> Corynebacterium glutamicum <400> 39 atgtcgaagc agcactccac accattaaac aatgatgaag aacacacttc cgctcctcaa 60 aaggttgcgg taatcaccac gggcggaacc atcgcctgta cttccgacgc aaatgggcat 120 ctgcttccca ccgtcagcgg tgcagacctg cttgcgccaa tcgcaccacg gttcaatgga 180 gcgcagatcg ctttcgaaat ccacgaaatc aaccgccttg attcctcctc catgacgttt 240 gaggatctcg attccatcat cgccacggtt cataaggtgt tggaggatcc ggatgttgtt 300 ggcgtagtag ttacccacgg caccgattcc atggaagagt ccgccatcgc cgtagacacc 360 ttccttgatg atccccgccc agtcattttc accggcgccc aaaaaccctt cgatcatccc 420 gaagccgacg gcccaaacaa ccttttcgaa gcctgcctca tcgcatccga cccctccgct 480 cgcggaattg gtgcactcat tgtcttcggt cacgccgtca tccctgctcg cggctgcgtt 540 aaatggcaca cctctgatga gctggcgttt gcaaccaacg gccctgaaga accagagcgc 600 cccgatgcgc tgcccgtagc taaattggcg gatgtctctg tcgaaatcat ccccgcatac 660 cctggtgcca ccggcgcaat ggtggaagct gccatcgctg ccggtgctca aggacttgta 720 gtggaagcaa tgggatcagg caatgttggt tcccgcatgg gtgatgccct aggtaaagca 780 cttgacgctg gaattcccgt ggtgatgagc actagggttc ctcgtggtga agtatccgga 840 gtgtatggcg gtgcaggtgg aggtgcgact ttggctgcga agggcgctgt gggatctcgc 900 tacttcagag ctggtcaggc acgtattttg ctcgcgattg ccattgcgac gggcgcacat 960 ccggtgacgc tttactaa 978 <210> 40 <211> 200 <212> DNA <213> Corynebacterium glutamicum <400> 40 tggccgttac cctgcgaatg tccacagggt agctggtagt ttgaaaatca acgccgttgc 60 ccttaggatt cagtaactgg cacattttgt aatgcgctag atctgtgtgc tcagtcttcc 120 aggctgctta tcacagtgaa agcaaaacca attcgtggct gcgaaagtcg tagccaccac 180 gaagtccagg aggacataca 200 <210> 41 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(29) <223> Insert the forward primer of the homologous arm upstream of Ptuf <400> 41 ccggaattct gctcaggagc aacagtatt 29 <210> 42 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Insert the reverse primer of the homologous arm upstream of Ptuf <400> 42 cattcgcagg gtaacggcca gcgctctagc gtatcaacta 40 <210> 43 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Forward primer of Ptuf <400> 43 tagttgatac gctagagcgc tggccgttac cctgcgaatg 40 <210> 44 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Ptuf reverse primer <400> 44 gtggagtgct gcttcgacat tgtatgtcct cctggacttc 40 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Forward primer for inserting the downstream homologous arm of Ptuf <400> 45 gaagtccagg aggacataca atgtcgaagc agcactccac 40 <210> 46 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(29) <223> Reverse primer for inserting the downstream homologous arm of Ptuf <400> 46 tgctctagac agcgatggca gcttccacc 29 <210> 47 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(29) <223> Forward primer for the upstream homologous arm of the ncgl2062 copy addition <400> 47 tgctctagaa agggcaatga gtttgtcga 29 <210> 48 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Reverse primer for the upstream homologous arm of the ncgl2062 copy addition <400> 48 gtggagtgct gcttcgacat ttagttctcc aagtagagcc 40 <210> 49 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Forward primer for ncgl2062 <400> 49 ggctctactt ggagaactaa atgtcgaagc agcactccac 40 <210> 50 <211> 39 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(39) <223> Reverse primer of ncgl2062 <400> 50 tatcagacga gatcttggat tagtaaagcg tcaccggat 39 <210> 51 <211> 39 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(39) <223> Forward primer of the downstream homologous arm for increasing the copy number of ncgl2062 <400> 51 atccggtgac gctttactaa tccaagatct cgtctgata 39 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(29) <223> Reverse primer of the downstream homologous arm for increasing the copy number of ncgl2062 <400> 52 ctagctagcg tgtggatccg agcgcgaag 29 <210> 53 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(29) <223> Forward primer of the upstream homologous arm for ncgl2062 knockout <400> 53 ccggaattct gctcaggagc aacagtatt 29 <210> 54 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Reverse primer for the upstream homologous arm of ncgl2062 knockout <400> 54 atgcaagacc aagggcgaaa gcgctctagc gtatcaacta 40 <210> 55 <211> 40 <212> DNA <21�> Artificial Sequence <220> <221> prim_bind <222> (1)..(40) <223> Forward primer for the downstream homologous arm of ncgl2062 knockout <400> 55 tagttgatac gctagagcgc tttcgccctt ggtcttgcat 40 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <2,20>` <221> prim_bind <222> (1)..(29) <223> Reverse primer for the downstream homologous arm of ncgl2062 knockout <400> 56 ctagctagct tatgaggtag gcgtgcaat 29 <210> 57 <211> 244 <212> DNA <213> Corynebacterium glutamicum <400> 57 tgcgttaaac ttggccaaat gtggcaacct ttgcaaggtg aaaaactggg gcggggttag 60 atcctggggg gtttatttca ttcactttgg cttgaagtcg tgcaggtcag gggagtgttg 120 cccgaaaaca ttgagaggaa aacaaaaacc gatgtttgat tgggggaatc gtgtggtata 180 atggtaggac gcagtgactg ctatcaccct tggcggtctc ttgttgaaag gaataattac 240 tcta 244

Claims

1. A recombinant bacterium for producing L-lysine, characterized in that, The recombinant bacterium has increased expression and / or activity of asparaginase, decreased expression and / or activity of homoserine dehydrogenase, increased expression and / or activity of pyruvate carboxylase, decreased expression and / or activity of phosphoenolpyruvate carboxykinase, increased expression and / or activity of dihydropicolinate reductase, and increased expression and / or activity of aspartokinase, diaminopimelate dehydrogenase and / or diaminopimelate decarboxylase, compared with the parental bacterium. The parental bacterium is a strain capable of accumulating lysine. The decreased expression and / or activity of homoserine dehydrogenase is achieved by: valine at position 59 of homoserine dehydrogenase in the recombinant bacterium is mutated to alanine, and the gene sequence of the mutated homoserine dehydrogenase is as shown in SEQ ID NO.

1. The increased expression and / or activity of pyruvate carboxylase is achieved by: proline at position 458 of pyruvate carboxylase in the recombinant bacterium is mutated to serine, and the gene sequence of the mutated pyruvate carboxylase is as shown in SEQ ID NO.

8. The regulatory element for increasing gene expression is the strong promoter P tuf romoter; wherein, the parental bacterium is Corynebacterium glutamicum or Corynebacterium pekinense.

2. The recombinant bacterium according to claim 1, wherein The recombinant bacterium has at least two copies of the asparaginase-encoding gene, and / or the expression of the asparaginase-encoding gene of the recombinant bacterium is mediated by a regulatory element with high transcriptional or high expression activity.

3. The recombinant bacterium according to claim 2, wherein The regulatory element is a strong promoter.

4. The recombinant bacterium according to claim 1, wherein The phosphoenolpyruvate carboxykinase-encoding gene of the recombinant bacterium is inactivated, and / or the expression of the phosphoenolpyruvate carboxykinase-encoding gene is mediated by a regulatory element with low transcriptional or low expression activity; The inactivation is to knockout the phosphoenolpyruvate carboxykinase-encoding gene of the recombinant bacterium.

5. The recombinant bacterium according to claim 1, wherein The recombinant bacterium has at least two copies of the dihydropicolinate reductase-encoding gene, and / or the expression of the dihydropicolinate reductase-encoding gene is mediated by a regulatory element with high transcriptional or high expression activity.

6. The recombinant bacterium according to claim 5, wherein The regulatory element is a strong promoter.

7. The recombinant bacterium according to claim 6, wherein The strong promoter is the P of the starting bacterium tuf promoter.

8. The recombinant bacterium according to claim 1, wherein The recombinant bacterium has at least two copies of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene, and / or the expression of the aspartokinase-encoding gene, diaminopimelate dehydrogenase-encoding gene and / or diaminopimelate decarboxylase-encoding gene is mediated by a regulatory element with high transcriptional or high expression activity.

9. The recombinant bacterium according to claim 8, wherein, The regulatory element is a strong promoter.

10. The recombinant bacterium according to claim 9, wherein The strong promoter is the P promoter of the starting bacterium tuf promoter.

11. A method for constructing a recombinant bacterium according to any one of claims 1-10, characterized in that, Comprising the following steps: Improve the expression and / or activity of asparaginase in the starting bacterium; reduce the expression and / or activity of homoserine dehydrogenase; improve the expression and / or activity of pyruvate carboxylase; reduce the expression and / or activity of phosphoenolpyruvate carboxykinase; improve the expression and / or activity of dihydropicolinate reductase; improve the expression and / or activity of aspartokinase, diaminopimelate dehydrogenase and / or diaminopimelate decarboxylase.

12. The construction method according to claim 11, wherein, The improvement of the expression and / or activity of asparaginase in the starting bacterium is achieved by at least one of the following ways: (E) increasing the copy number of the asparaginase-encoding gene in the starting bacterium, (F) replacing the regulatory element of the asparaginase-encoding gene in the starting bacterium with a regulatory element with high transcriptional or high expression activity.

13. A method for producing L-lysine, characterized in that, Comprising the following steps: fermentatively culturing the recombinant bacterium according to any one of claims 1-10.

Images