CN117887649A - Leucine production strain, construction method and application thereof - Google Patents
Leucine production strain, construction method and application thereof Download PDFInfo
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- CN117887649A CN117887649A CN202410109218.4A CN202410109218A CN117887649A CN 117887649 A CN117887649 A CN 117887649A CN 202410109218 A CN202410109218 A CN 202410109218A CN 117887649 A CN117887649 A CN 117887649A
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- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 45
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Abstract
A leucine production strain is Corynebacterium glutamicum (Corynebacterium glutamicum) Leu07, which is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 12 months and 11 days, and the strain preservation number is CGMCC No.29285. The leucine production strain is obtained by adopting a directional transformation method of de novo synthesis through a leucine metabolic synthesis way, glucose is used as a carbon source, an alpha-ketoisohexide substrate is not required to be added, the production cost is low, the production rate is high, the fermentation period is short, the stability of the strain is high, the economic benefit is very high, and a foundation is laid for realizing the large-scale production of leucine.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a leucine production strain, a construction method and application thereof.
Background
L-leucine (L-Leucine) belongs to one of eight amino acids essential to human body, and is commonly called as three-branched-chain amino acid with L-isoleucine and L-valine. Leucine has a chemical formula of C 6H13NO2, is white glossy hexahedral crystal or white crystalline powder at room temperature, and has a solubility of 22.4g/L at 20 . Leucine can supply energy to organism, regulate protein metabolism, repair muscle, promote healing of skin, wound and bone, etc.
At present, the leucine synthesis method mainly comprises a chemical synthesis method, an enzyme catalysis method and a microbial fermentation method. The chemical synthesis method has the problems of larger environmental pollution and lower conversion rate; the enzyme catalysis method is to catalyze alpha-ketoisohexide substrate to produce leucine by taking leucine transaminase as key enzyme, the method has little pollution to the environment and mild reaction condition, but the alpha-ketoisohexide substrate needs to be added in the catalysis process, thus increasing the production cost; the microbial fermentation method takes glucose as an energy source for microbial growth, and the microorganism synthesizes leucine from the head without adding a substrate, so that the production cost is reduced, the fermentation process condition is mild, the method has potential for industrial production, but the strain for producing leucine by utilizing corynebacterium glutamicum is generally obtained by a mutagenesis method at present, and the method has the characteristics of auxotrophy and slow growth.
Disclosure of Invention
The invention aims to provide a leucine production strain, and a construction method and application thereof.
To achieve the above and other related objects, the present invention provides the following technical solutions: a leucine producing strain is corynebacterium glutamicum (Corynebacterium glutamicum) Leu07, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 12 months and 11 days, and the strain preservation number is CGMCC No.29285.
The preferable technical scheme is as follows: the strain Corynebacterium glutamicum ATCC 13032 is obtained by modifying an original strain Corynebacterium glutamicum ATCC 13032 by a metabolic engineering modification method, and specifically, the expression intensities of acetohydroxyacid synthase gene ilvBN fbr, isopropyl malate synthase gene leuA fbr, alpha-isopropyl malate isomerase leuD, alpha-isopropyl malate isomerase leuDleuC, beta-isopropyl malate dehydrogenase leuB, leucine transaminase gene Ncgl2204, pyruvate dehydrogenase gene Ncgl2610 and lactate dehydrogenase gene Ncgl0901 are regulated.
The preferable technical scheme is as follows: the acetohydroxyacid synthase gene ilvBN fbr is derived from corynebacterium glutamicum; the isopropyl malate synthase gene leuA fbr is derived from corynebacterium glutamicum; the alpha-isopropyl malate isomerase leuD is derived from bacillus subtilis; the beta-isopropyl malate dehydrogenase leuB and the alpha-isopropyl malate isomerase leuC are derived from bacillus subtilis; the leucine aminotransferase gene Ncgl2204 is derived from corynebacterium glutamicum; the pyruvate dehydrogenase gene Ncgl2610 is derived from corynebacterium glutamicum; the lactate dehydrogenase gene Ncgl0901 is derived from Corynebacterium glutamicum.
The preferable technical scheme is as follows: the corynebacterium glutamicum ATCC 13032 is taken as an original strain, and the control of the overexpression of the acetohydroxyacid synthase gene ilvBN fbr is carried out at a pseudogene locus Ncgl1995 by using a Ptuf promoter to obtain a strain Leu01; taking the strain Leu01 as an original strain, and controlling the overexpression of isopropyl malate synthase gene leuA fbr by using Ptuf promoter at pseudogene locus Ncgl1960 to obtain Leu02; taking the bacterial strain Leu02 as an original bacterial strain, and controlling the overexpression of alpha-isopropyl malate isomerase leuD by using Ptuf promoter at a pseudo-gene locus Ncgl2001 to obtain Leu03; taking the strain Leu03 as an original strain, and controlling the overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC by using Ptuf promoter at a pseudo-gene locus Ncgl2000 to obtain Leu04; taking the strain Leu04 as an original strain, and controlling the overexpression of a leucine aminotransferase gene Ncgl2204 at a pseudo-gene locus Ncgl2037 by using a Ptuf promoter to obtain Leu05; taking a strain Leu05 as an original strain, knocking out a pyruvate dehydrogenase gene Ncgl2610 on a genome to ensure that the gene is not expressed, and controlling double copies of an acetohydroxyacid synthase gene ilvBN fbr by using a Ptuf promoter at a site of the gene to obtain Leu06; taking the strain Leu06 as an original strain, knocking out the lactate dehydrogenase gene Ncgl0901 on a genome to ensure that the lactate dehydrogenase gene Ncgl0901 is not expressed, and controlling double copies of the leucine aminotransferase gene Ncgl2204 by using a Ptuf promoter at a site of the lactate dehydrogenase gene Ncgl0901 to obtain a target strain.
The preferable technical scheme is as follows: the Ptuf promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 1; the ilvBN fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 2; the leuA fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3; the leuD gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 4; the leuBC gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5; the Ncgl2204 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 6; the Ncgl2610 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 7; the Ncgl0901 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 8.
To achieve the above and other related objects, the present invention provides the following technical solutions: a method for constructing a leucine producing strain, comprising the steps of:
Step 1: the corynebacterium glutamicum ATCC 13032 is taken as an original strain, and a Ptuf promoter is used for controlling the overexpression of acetohydroxyacid synthase ilvBN fbr gene at a pseudogene locus Ncgl1995 to obtain a strain Leu01;
Step 2: taking the strain Leu01 as an original strain, and controlling the overexpression of isopropyl malate synthase leuA fbr genes at a pseudogene locus Ncgl1960 by using a Ptuf promoter to obtain the strain Leu02;
step 3: taking the strain Leu02 as an original strain, and controlling the overexpression of alpha-isopropyl malate isomerase leuD by using Ptuf promoter at a pseudo-gene locus Ncgl2001 to obtain the strain Leu03;
Step 4: taking the strain Leu03 as an original strain, and controlling the overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC by using Ptuf promoter at a pseudo-gene locus Ncgl2000 to obtain the strain Leu04;
Step 5: taking the strain Leu04 as an original strain, and controlling the overexpression of a leucine aminotransferase Ncgl2204 gene by using a Ptuf promoter at a pseudo-gene locus Ncgl2037 to obtain a strain Leu05;
step 6: taking the strain Leu05 as an original strain, knocking out a pyruvic acid dehydrogenase Ncgl2610 gene on a genome, and controlling acetohydroxy acid synthase ilvBN fbr gene to be overexpressed at a site of the gene by using a Ptuf promoter to obtain a strain Leu06;
Step 7: taking the strain Leu06 as an original strain, knocking out the lactate dehydrogenase Ncgl0901 gene on a genome, and controlling the overexpression of the leucine aminotransferase Ncgl2204 gene by using Ptuf promoter at the locus to obtain the strain Leu07, wherein the strain Leu07 is the target strain after the transformation is successful.
To achieve the above and other related objects, the present invention provides the following technical solutions: the leucine production strain is applied to the preparation of L-leucine by fermentation.
The preferable technical scheme is as follows: the method comprises the following specific steps:
(1) Seed culture: at the culture temperature of 35-39 , the pH value of the culture is maintained at 6.7+/-0.2 by automatically feeding ammonia water solution with mass fraction of 20-28%, the dissolved oxygen value of the culture is maintained at 25-35% by adjusting the stirring speed or ventilation, and the inoculation requirement is met when the OD 600nm is 20;
(2) Fermentation culture: according to the inoculation amount of 10-25%, the culture temperature is 35-39 , the pH value of the culture is maintained at 6.7+/-0.2 by automatically feeding 20-28% ammonia water solution, the dissolved oxygen value of the culture is maintained at 25-35% by adjusting the stirring speed or ventilation, the glucose concentration in the tank is controlled to be less than or equal to 1.5g/L by feeding 60-85% glucose solution, and the fermentation period is less than or equal to 45h.
The preferable technical scheme is as follows: seed culture medium used in seed culture: 20g/L of glucose, 3g/L of yeast, 1g/L of peptone, 15g/L of corn steep liquor dry powder, 1.5g/L of bean concentrate 15ml/L,(NH4)2SO4 1g/L,K2HPO43H2O2g/L,MgSO47H2O 2g/L, citric acid and 10mg/L of MnSO 4H2O 10mg/L,FeSO47H2 O; fermentation medium used in fermentation culture: glucose 20g/L, yeast powder 3.5g/L, peptone 1g/L, corn steep liquor dry powder 10g/L, bean concentrate 5ml/L,(NH4)2SO42g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, glutamic acid 2g/L, methionine 0.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O30 mg/L.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the leucine production strain is obtained by adopting a directional transformation method of de novo synthesis through a leucine metabolic synthesis way, glucose is used as a carbon source, an alpha-ketoisohexide substrate is not required to be added, the production cost is low, the production rate is high, the fermentation period is short, the stability of the strain is high, the economic benefit is very high, and a foundation is laid for realizing the large-scale production of leucine.
Drawings
FIG. 1 is a diagram of the process of genetic engineering of leucine producing strains from the head synthesis pathway.
FIG. 2 shows a map of pK18mobsacB-Ncgl1995: ptufilvBN fbr.
FIG. 3 is a map of pK18mobsacB-Ncgl1960: ptufleuA fbr.
FIG. 4 shows a map of pK18mobsacB-Ncgl2001: ptufleuD.
FIG. 5 is a map of pK18mobsacB-Ncgl2000:: ptufleuBC.
FIG. 6 is a map of pK18mobsacB-Ncgl2037: ptufNcgl 2204.
FIG. 7 is a map of pK18 mobsacB-. DELTA.Ncgl 2610:: ptufilvBN fbr.
FIG. 8 is a pK18mobsacB-. DELTA.Ncgl 0901: ptufNcgl2204 map.
FIG. 9 is a pXT01 plasmid map.
Detailed Description
Further advantages and effects of the present invention will be readily apparent to those skilled in the art from the following disclosure of the present invention by reference to the specific embodiments.
Please refer to fig. 1-9. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are shown only in the drawings and should not be taken as limiting the invention to those having ordinary skill in the art, since modifications, changes in proportions, or adjustments of sizes, etc. could be made without departing from the spirit or essential characteristics of the invention. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
Preservation of biological material:
Corynebacterium glutamicum (Corynebacterium glutamicum) Leu07 (Corynebacterium glutamicum Leu 07) is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 12 and 11 days, and the strain preservation number is CGMCC No.29285. China general microbiological culture Collection center address: the korean district , beijing city, part No. 1, no. 3.
The reagents and materials described in the examples below are commercially available unless otherwise indicated.
Example 1:
This example is intended to illustrate the construction of the plasmid vector pK18mobsacB-Ncgl1995: ptufilvBN fbr and the genomic integration of ilvBN fbr gene procedure, which is described in detail as follows:
1. construction of plasmid vector of pK18mobsacB-Ncgl1995: ptufilvBN fbr
The upstream homology arm, the downstream homology arm and the target gene were obtained by PCR amplification using the HS enzyme using the Corynebacterium glutamicum ATCC 13032 genome as a template and the Ncgl1995-U-S, ncgl1995-U-A, ncgl1995-D-S, ncgl1995-D-A and ilvBN-S, ilvBN-A as primers, respectively, the PCR reaction system is shown in Table 3 and the PCR reaction procedure is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufilvBN-S, ptufilvBN-A as a primer. PtufilvBN fbr overlapping fragments were obtained and recovered by HS enzyme overlap PCR using the fragments as templates, the gene integration fragments consisted of the Ncgl1995 upstream homology arm, ptuf promoter, ilvBN fbr objective gene and the Ncgl1995 downstream homology arm, the overlap PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl1995: ptufilvBN fbr overlapping fragment obtained in step was transferred to DH 5. Alpha. Transduction competent cells by carrying it with pK18mobsacB line, plated on a kanamycin resistance plate at a concentration of 0.05mg/mL, screened to obtain positive transformants, and plasmid pK18mobsacB-Ncgl1995: ptufilvBN fbr was extracted.
2. Corynebacterium glutamicum genome integration ilvBN fbr Gene manipulation
(1) PtufilvBN fb electric shock transformation of the constructed plasmid pK18mobsacB-Ncgl 1995:3835 into ATCC 13032 competent cells, plating on kanamycin resistance plates at a concentration of 0.01mg/mL, and culturing at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu01 with double exchange and successful integration.
Example 2
This example is intended to illustrate the plasmid vector construction of pK18mobsacB-Ncgl 1960:. PtufleuA fbr and the genomic integration leuA fbr gene procedure, which is specifically as follows:
1. Construction of plasmid vector pK18mobsacB-Ncgl1960: ptufleuA fbr
The upstream homology arm, the downstream homology arm and the target gene are obtained by PCR amplification using HS enzyme by using the Corynebacterium glutamicum ATCC 13032 genome as a template and Ncgl1960-U-S, ncgl1960-U-A, ncgl1960-D-S, ncgl1960-D-A and leuA-S, leuA-a as primers, respectively, wherein the PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufleuA-S, ptufleuA-A as a primer. PtufleuA fbr overlapping fragments were obtained and recovered by HS enzyme overlap PCR using the fragments as templates, the gene integration fragments consisted of the upstream homology arm of Ncgl1960, ptuf promoter, leuA fbr objective gene and the downstream homology arm of Ncgl1960, the overlap PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl1960: ptufleuA fbr overlapping fragment obtained in the step was line-loaded with pK18mobsacB to DH 5. Alpha. Transformed competent cells, plated on a kanamycin resistance plate at a concentration of 0.05mg/mL, and positive transformants were obtained by selection, and plasmid pK18mobsacB-Ncgl1960: ptufleuA fbr was extracted.
2. Corynebacterium glutamicum genome integration leuA fbr Gene manipulation
(1) The constructed plasmid pK18mobsacB-Ncgl1960:: ptufleuA fbr was transformed into Leu01 competent cells by electric shock, plated on kanamycin resistance plates at a concentration of 0.01mg/mL, and incubated at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu02 with double exchange and successful integration.
Example 3
This example is intended to illustrate the construction of plasmid vector and genomic integration leuD gene procedure for pK18mobsacB-Ncgl2001: ptufleuD, as follows:
1. construction of plasmid vector pK18mobsacB-Ncgl2001: ptufleuD
The upstream homology arm, the downstream homology arm and the target gene are obtained by PCR amplification using HS enzyme by using the Corynebacterium glutamicum ATCC 13032 genome as a template and Ncgl2001-U-S, ncgl2001-U-A, ncgl2001-D-S, ncgl2001-D-A and leuD-S, leuD-A as primers, respectively, wherein the PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufleuD-S, ptufleuD-A as a primer. PtufleuD overlapping fragments are obtained and recovered by HS enzyme overlapping PCR with fragments as templates, wherein the gene integration fragments consist of an upstream homology arm of the Ncgl2001, a Ptuf promoter, a leuD target gene and a downstream homology arm of the Ncgl2001, the overlapping PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl2001:: ptufleuD overlapping fragment obtained in step was transferred to DH 5. Alpha. Transduction competent cells by carrying out transformation with pK18mobsacB line, plated on a kanamycin resistance plate at a concentration of 0.05mg/mL, and positive transformants were obtained by selection, and plasmid pK18mobsacB-Ncgl 2001::: ptufleuD was extracted.
2. Corynebacterium glutamicum genome integration leuD Gene manipulation
(1) PtufleuD electric shock transformation of the constructed plasmid pK18mobsacB-Ncgl2001 into Leu02 competent cells, plating on kanamycin resistance plates at a concentration of 0.01mg/mL, and culturing at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu03 with double exchange and successful integration.
Example 4
This example is intended to illustrate the construction of plasmid vector and genomic integration leuBC gene procedure for pK18mobsacB-Ncgl2000: ptufleuBC, as follows:
1. Construction of plasmid vector pK18mobsacB-Ncgl2000: ptufleuBC
The upstream homology arm, the downstream homology arm and the target gene are obtained by PCR amplification using the HS enzyme by using the Corynebacterium glutamicum ATCC 13032 genome as a template and using Ncgl2000-U-S, ncgl2000-U-A, ncgl2000-D-S, ncgl2000-D-A and leuBC-S, leuBC-A as primers, respectively, wherein the PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufleuBC-S, ptufleuBC-A as a primer. PtufleuBC overlapping fragments are obtained by HS enzyme overlapping PCR by taking fragments as templates, and the gene integration fragments are recovered, wherein the gene integration fragments consist of an upstream homology arm of the Ncgl2000, a Ptuf promoter, a leuBC target gene and a downstream homology arm of the Ncgl2000, the overlapping PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl2000:: ptufleuBC overlapping fragment obtained in step was transferred to DH 5. Alpha. Transduction competent cells by carrying out transformation with pK18mobsacB line, plated on a kanamycin resistance plate at a concentration of 0.05mg/mL, and positive transformants were obtained by selection, and plasmid pK18mobsacB-Ncgl2000:: ptufleuBC was extracted.
2. Genomic integration leuBC Gene manipulation of Corynebacterium glutamicum
(1) The constructed plasmid pK18mobsacB-Ncgl2000: ptufleuBC was transformed into Leu03 competent cells by electric shock, plated on kanamycin resistance plates at a concentration of 0.01mg/mL and incubated at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu04 with double exchange and successful integration.
Example 5
This example is intended to illustrate the construction of plasmid vector PtufNcgl2204 and the procedure for genomic integration of Ncgl2204 gene by pK18mobsacB-Ncgl2037, the specific steps are as follows:
1. Construction of plasmid vector pK18mobsacB-Ncgl2037: ptufNcgl2204
The genome of Corynebacterium glutamicum ATCC 13032 is used as a template, ncgl2037-U-S, ncgl2037-U-A, ncgl2037-D-S, ncgl2037-D-A and Ncgl2204-S, ncgl2204-A are respectively used as primers, an HS enzyme is used for PCR amplification to obtain an upstream homology arm, a downstream homology arm and a target gene, the PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufNcgl2204-S, ptufNcgl2204-A as a primer. PtufNcgl2204 overlapped fragments are obtained by HS enzyme overlapped PCR by taking fragments as templates, and the gene integrated fragments consist of an upstream homology arm of the Ncgl2037, a Ptuf promoter, a Ncgl2204 target gene and a downstream homology arm of the Ncgl2037, wherein the overlapped PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl2037: ptufNcgl2204 overlapping fragment obtained in the step is carried by a pK18mobsacB line and transferred into DH5 alphation transformation competent cells, the cells are coated on a kanamycin resistance plate with the concentration of 0.05mg/mL, positive transformants are obtained by screening, and a plasmid pK18mobsacB-Ncgl2037: ptufNcgl2204 is extracted.
2. Corynebacterium glutamicum genome integration Ncgl2204 Gene manipulation
(1) The constructed plasmid pK18mobsacB-Ncgl2037: ptufNcgl2204 was transformed into Leu04 competent cells by electric shock, plated on kanamycin resistance plates at a concentration of 0.01mg/mL and incubated at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu 05 with double exchange and successful integration.
Example 6
This example is intended to illustrate the procedures for construction of plasmid vector PtufilvBN fbr and the genomic knockout of the Ncgl2610 gene and integration of ilvBN fbr gene at its site as follows:
1. construction of plasmid vector pK18 mobsacB-. DELTA.Ncgl2610: ptufilvBN fbr
The upstream homology arm, the downstream homology arm and the target gene are obtained by PCR amplification with HS enzyme by using the Corynebacterium glutamicum ATCC 13032 genome as A template and using Ncgl2610-U-S, ncgl2610-U-A, ncgl2610-D-S, ncgl2610-D-A as A primer and ilvBN-S-2 as A primer respectively, wherein the PCR reaction system is shown in Table 3 and the PCR reaction program is shown in Table 4. The Ptuf promoter fragment was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufilvBN-S-2, ptufilvBN-A-2 as primers. PtufilvBN fbr overlapping fragments are obtained by HS enzyme overlapping PCR by taking fragments as templates, and the gene integration fragments are recovered, wherein the gene integration fragments consist of an upstream homology arm of the Ncgl2610, a Ptuf promoter, an ilvBN fbr target gene and a downstream homology arm of the Ncgl2610, the overlapping PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl2610: ptufilvBN fbr overlapping fragment obtained in the step was line-loaded with pK18mobsacB to DH 5. Alpha. Transformed competent cells, which were plated on kanamycin-resistant plates at a concentration of 0.05mg/mL, and positive transformants were obtained by selection, and plasmid pK18mobsacB- Ncgl2610: ptufilvBN fbr was extracted.
2. Operation of knocking out Ncgl2610 Gene from Corynebacterium glutamicum genome and integrating ilvBN fbr Gene
(1) PtufilvBN fbr electric shock transformation of the constructed plasmid pK18 mobsacB-. DELTA.Ncgl 2610 into Leu05 competent cells, plating on kanamycin resistance plates at a concentration of 0.01mg/mL, and incubation at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. And (3) inoculating the single colony to a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, picking a single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL, performing colony PCR, and detecting that the PCR fragment is correct by agarose gel electrophoresis, thus obtaining the single colony Leu06 with double exchange and successful integration.
Example 7
This example is intended to illustrate the procedures for construction of plasmid vector pK18 mobsacB-. DELTA.Ncgl 0901: ptufNcgl2204 and for genomic knockout of the Ncgl0901 gene and integration of the Ncgl2204 gene at its site as follows:
1. construction of plasmid vector pK18 mobsacB-. DELTA.Ncgl0901: ptufNcgl2204
The genome of Corynebacterium glutamicum ATCC 13032 is used as a template, ncgl0901-U-S, ncgl0901-U-A, ncgl0901-D-S, ncgl0901-D-A and Ncgl2204-S-2 and Ncgl2204-A-2 are respectively used as primers, an HS enzyme is used for PCR amplification to obtain an upstream homology arm, a downstream homology arm and a target gene, the PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. The promoter fragment Ptuf was obtained by HS enzyme PCR amplification using the pXT01 plasmid (plasmid map see FIG. 9) as a template and PtufNcgl2204-S-2, ptufNcgl2204-A-2 as primers. PtufNcgl2204 overlapping fragments are obtained by HS enzyme overlapping PCR by taking fragments as templates, and the gene integration fragments are recovered, wherein the gene integration fragments consist of an upstream homology arm of the Ncgl0901, a Ptuf promoter, a Ncgl2204 target gene and a downstream homology arm of the Ncgl0901, the overlapping PCR reaction system is shown in table 5, and the PCR reaction program is shown in table 4.
The pK18mobsacB plasmid was extracted, and the plasmid was digested with XbaI and EcoRI to form a linearized vector and recovered.
The Ncgl0901: ptufNcgl2204 overlapping fragment obtained in step was line-loaded with pK18mobsacB into DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, screened for positive transformants, and plasmid pK18mobsacB- Ncgl0901: ptufNcgl2204 was extracted.
2. Manipulation of knockout of the Ncgl0901 Gene from Corynebacterium glutamicum genome and integration of the Ncgl2204 Gene
(1) PtufNcgl2204 of the constructed plasmid pK18 mobsacB-. DELTA.Ncgl 0901 was transformed into Leu06 competent cells by electric shock, plated on kanamycin resistance plates at a concentration of 0.01mg/mL, and cultured at 32for 24 hours. Single colonies were selected for PCR, and the PCR fragments were checked by agarose gel electrophoresis, and colonies grown on kanamycin-resistant plates were single colonies in which single exchange occurred.
(2) The single colony with single exchange is inoculated into BHI shake tube and cultured at 32 . 50. Mu.L of fermentation broth was applied to BHI plates containing 20% sucrose at 2h, 4h and 6h, respectively, and incubated at 32for 24h. The single colony is inoculated on a BHI plate containing 20% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, the single colony which grows on the BHI plate containing 20% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL is selected, colony PCR is carried out, and the correct PCR fragment is detected by agarose gel electrophoresis, so that the single colony Leu07 which is subjected to double exchange and successfully integrated is obtained.
Example 8
Leu07 was used as a leucine production strain, and this example is intended to illustrate a method for producing leucine using the production strain, and the specific culturing method is as follows:
Seed culture: using a 5L mechanical stirring type fermentation tank, wherein the culture temperature is 37 , the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 30% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD 600nm is 20; the seed culture medium adopted is as follows: glucose 20g/L, yeast 3g/L, peptone 1g/L, corn steep liquor dry powder 15g/L, bean concentrate 15ml/L,(NH4)2SO4 1g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, citric acid 1.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O10 mg/L.
Fermentation culture: using a 5L mechanical stirring type fermentation tank, wherein the fermentation inoculation amount is 20%, the culture temperature is 37 , the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 30% by adjusting the stirring rotation speed or ventilation, the glucose concentration in the tank is controlled to be less than or equal to 1g/L by feeding 80% glucose solution, and the fermentation period is 35 hours; the fermentation medium adopted is: 20g/L glucose, 3.5g/L yeast powder, 1g/L peptone, 10g/L corn steep liquor dry powder, 5ml/L bean concentrate, (NH 4) 2SO4 g/L,
K 2HPO43H2O 2g/L,MgSO47H2 O2 g/L, glutamic acid 2g/L, methionine 0.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O30 mg/L.
The genotype of the leucine bacterium corynebacterium glutamicum constructed by the invention is ATCC 13032
Ncgl1995::PtufilvBNfbr Ncgl1960::PtufleuAfbr Ncgl2001::PtufleuD
Ncgl2000:: ptufleuBC Ncgl2037:: ptufNcgl2204Ncgl2610:: ptufilvBN fbr Ncgl0901:: ptufNcgl2204, designated Leu07 (corresponding to example 7).
The fermentation experiment of the 5L fermentation tank proves that the leucine yield can reach 50.2g/L, the sugar acid conversion rate is 25.1%, and the fermentation period is less than or equal to 35h. The strain has good production capacity and higher conversion rate, and lays a foundation for the industrialized production of leucine.
The primer sequences used for constructing the strains in the above examples are shown in Table 1.
The plasmids described in the above examples are shown in Table 2.
The HS enzyme PCR system described in the above examples is shown in Table 3.
The PCR reaction procedure described in the above examples is shown in Table 4.
The HS enzyme overlap PCR system described in the above examples is shown in Table 5.
The results of the fermentation production of leucine by each of the above strains in the examples are shown in Table 6.
TABLE 1 primers involved in the construction of strains
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TABLE 2 plasmids involved in the construction of strains
TABLE 3HS enzyme PCR amplification system
TABLE 4PCR amplification procedure
TABLE 5 overlap PCR amplification System
TABLE 6 fermentation results of Leu production by Strain
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Example 9: leucine production strain, construction method and application thereof
A leucine producing strain is obtained by further modifying an original strain C.glutamicum by a metabolic engineering modification method, in particular to the regulation of the expression intensity of acetohydroxy acid synthase genes ilvBN fbr, isopropyl malate synthase genes leuA fbr, alpha-isopropyl malate isomerase leuD, beta-isopropyl malate dehydrogenase leuB, alpha-isopropyl malate isomerase leuC, leucine transaminase genes Ncgl2204, pyruvate dehydrogenase genes Ncgl2610 and lactic acid dehydrogenase genes Ncgl0901, wherein:
The acetohydroxyacid synthase gene ilvBN fbr is overexpressed at pseudolocus Ncgl1995 using Ptuf promoter;
The isopropyl malate synthase gene leuA fbr is controlled to be overexpressed at a pseudogene locus Ncgl1960 by using a Ptuf promoter;
The -isopropylmalate isomerase leuD overexpression was controlled at pseudolocus Ncgl2001 using Ptuf promoter;
the -isopropyl malate dehydrogenase leuB and -isopropyl malate isomerase leuC overexpression was controlled at pseudolocus Ncgl2000 using Ptuf promoter;
The leucine dehydrogenase gene Ncgl2204 is controlled to be overexpressed at the pseudogene locus Ncgl2037 by using Ptuf promoter;
Knocking out the pyruvate dehydrogenase gene Ncgl2610 on the genome so as to ensure that the pyruvate dehydrogenase gene Ncgl2610 is not expressed, and controlling the overexpression of the acetohydroxyacid synthase gene ilvBN fbr by using a Ptuf promoter at the locus of the pyruvate dehydrogenase gene Ncgl 2610;
the lactate dehydrogenase gene Ncgl0901 was knocked out of the genome so that it was not expressed and the isopropyl malate synthase gene leuA fbr was overexpressed at its locus using the Ptuf promoter.
Preferably, the above leucine producing strain, the metabolic engineering method is a homologous recombination double-crossover gene editing technology.
The starting strain was C.glutamicum, accession number ATCC 13032.
Preferably, the acetohydroxyacid synthase gene ilvBN fbr is derived from Corynebacterium glutamicum, is a gene after gene mutation (China patent: CN 201611248621.7), and is a key enzyme gene of a leucine biosynthesis pathway.
Preferably, the leucine production strain is characterized in that the isopropyl malate synthase gene leuA fbr is derived from Corynebacterium glutamicum and is a gene after gene mutation (Chinese patent: ZL 201910825591. X).
Preferably, the above leucine producing strain, the alpha-isopropyl malate isomerase leuD and leuC, are derived from Bacillus subtilis, are heterologous expressed genes.
Preferably, the leucine production strain, the beta-isopropyl malate dehydrogenase leuB, is derived from bacillus subtilis and is a heterologous expression gene.
Preferably, the leucine production strain, wherein the leucine transaminase gene Ncgl2204 is derived from Corynebacterium glutamicum and is a key enzyme gene of a leucine biosynthesis pathway.
Preferably, in the leucine production strain, the pyruvate dehydrogenase gene Ncgl2610 is derived from Corynebacterium glutamicum, and the deletion of the gene enhances the accumulation of leucine precursor pyruvic acid.
Preferably, in the leucine production strain, the lactate dehydrogenase gene Ncgl0901 is derived from Corynebacterium glutamicum, and the leucine precursor pyruvate accumulation is enhanced by knocking out the gene.
The construction method of the leucine production strain is further subjected to directional transformation on the basis of an original strain C.glutamicum, and comprises the following specific steps:
(1) Taking C.glutamicum as an original strain, and controlling the overexpression of acetohydroxyacid synthase ilvBN fbr gene at a pseudogene locus Ncgl1995 by using Ptuf promoter to obtain a strain Leu01;
(2) Taking the strain Leu01 as an original strain, and controlling the overexpression of isopropyl malate synthase leuA fbr genes at a pseudogene locus Ncgl1960 by using a Ptuf promoter to obtain the strain Leu02;
(3) Taking the strain Leu02 as an original strain, and controlling the overexpression of alpha-isopropyl malate isomerase leuD by using Ptuf promoter at a pseudo-gene locus Ncgl2001 to obtain the strain Leu03;
(4) Taking the strain Leu03 as an original strain, and controlling the overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC by using Ptuf promoter at a pseudo-gene locus Ncgl2000 to obtain the strain Leu04;
(5) Taking the strain Leu04 as an original strain, and controlling the overexpression of a leucine aminotransferase Ncgl2204 gene by using a Ptuf promoter at a pseudo-gene locus Ncgl2037 to obtain a strain Leu05;
(6) Taking the strain Leu05 as an original strain, knocking out the pyruvic dehydrogenase Ncgl2610 gene on a genome, and controlling the overexpression of the isopropyl malate synthase LeuA fbr gene by using a Ptuf promoter at the locus to obtain the strain Leu06.
(7) Taking the strain Leu06 as an original strain, knocking out the lactate dehydrogenase Ncgl0901 gene on a genome, and controlling the overexpression of the leucine aminotransferase Ncgl2204 gene by using Ptuf promoter at the locus to obtain the strain Leu07, wherein the strain Leu07 is the target strain after the transformation is successful.
Preferably, the above construction method of the leucine-producing Corynebacterium glutamicum strain is carried out by constructing pK18mobsacB-Ncgl1995: ptufilvBN fbr, electrotransforming the constructed plasmid into a competent cell of the strain C.glutamicum, performing two-step homologous recombination, single-crossover and double-crossover (see example 1 for detailed procedures), and controlling acetohydroxyacid synthase ilvBN fbr gene overexpression at the pseudolocus Ncgl1995 using Ptuf promoter to construct the strain Leu01.
Preferably, the construction method of the leucine-producing Corynebacterium glutamicum strain is carried out by constructing pK18mobsacB-Ncgl1960: ptufleuA fbr, electrotransforming the constructed plasmid into competent cells of strain Leu01, performing two-step homologous recombination, single-crossover and double-crossover (see example 2 for detailed steps), and using Ptuf promoter to control overexpression of isopropyl malate synthase LeuA fbr gene at pseudogene locus Ncgl1960 to construct strain Leu02.
Preferably, the above construction method of the leucine-producing Corynebacterium glutamicum strain comprises the steps of constructing pK18mobsacB-Ncgl2001: ptufleuD, electrotransforming the constructed plasmid into competent cells of the strain Leu02, performing two-step homologous recombination, single-crossover and double-crossover (see example 3 for detailed steps), and controlling over-expression of the alpha-isopropyl malate isomerase LeuD gene at the pseudogene locus Ncgl2001 by using Ptuf promoter to construct the strain Leu03.
Preferably, the construction method of the leucine-producing Corynebacterium glutamicum strain is carried out by constructing pK18mobsacB-Ncgl2000: ptufleuBC, electrotransforming the constructed plasmid into competent cells of strain Leu03, performing two-step homologous recombination, single-crossover and double-crossover (see example 4 for detailed steps), and controlling overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC genes at pseudogene locus Ncgl2000 by using Ptuf promoter to construct strain Leu04.
Preferably, the construction method of the leucine-producing corynebacterium glutamicum strain comprises the steps of constructing a pK18mobsacB-Ncgl2037: ptufNcgl 2204.4, electrically transforming the constructed plasmid into competent cells of a strain Leu04, performing two-step homologous recombination, single exchange and double exchange (see example 5 for detailed steps), and controlling the overexpression of a leucine aminotransferase Ncgl2204 gene at a pseudogene locus Ncgl2037 by using a Ptuf promoter to construct the strain Leu05.
Preferably, the construction method of the leucine-producing Corynebacterium glutamicum strain is carried out by constructing pK18 mobsacB-DeltaNcgl 2610: ptufilvBN fbr, electrically transforming the constructed plasmid into competent cells of strain Leu05, carrying out two-step homologous recombination, single-exchange and double-exchange (see the implementation 6 for detailed steps), knocking out Ncgl2610 gene, and double-copying ilvBN fbr gene at the site thereof to construct strain Leu06.
Preferably, the construction method of the leucine-producing corynebacterium glutamicum strain comprises the steps of constructing a pK18 mobsacB-delta Ncgl0901: ptufNcgl 2204-2204, electrically transforming the constructed plasmid into competent cells of a strain Leu06, performing two-step homologous recombination, single exchange and double exchange (see example 7 for detailed steps), knocking out the Ncgl0901 gene, and double-copying the Ncgl2204 gene at the site of the gene, thereby constructing a strain Leu07, wherein the strain Leu07 is the target bacterium after successful transformation.
Preferably, in the construction method of the leucine production strain, the Ptuf promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 1; the ilvBN fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 2; the leuA fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3; the leuD gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 4; the leuBC gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5; the Ncgl2204 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 6; the Ncgl2610 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 7; the Ncgl0901 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 8.
The leucine production strain is applied to the fermentation production of leucine.
Preferably, the leucine production strain is applied by the following specific steps:
(1) Seed culture: using a 5L mechanical stirring type fermentation tank, wherein the culture temperature is 37 , the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 30% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD 600nm is 20;
(2) Fermentation culture: A5L mechanical stirring type fermentation tank is used, the inoculation amount is 20%, the culture temperature is 37 , the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 30% by adjusting the stirring speed or ventilation, the glucose concentration in the tank is controlled to be less than or equal to 1g/L by feeding 80% glucose solution, and the fermentation period is less than or equal to 35 hours.
The leucine production strain is used for efficiently and stably synthesizing leucine from the head by using glucose as a substrate, and producing leucine by fermenting for 35 hours at a speed of 50.2g/L.
Preferably, the leucine production strain is applied, and a seed culture medium is used in the seed culture: glucose 20g/L, yeast 3g/L, peptone 1g/L, corn steep liquor dry powder 15g/L, bean concentrate 15ml/L,(NH4)2SO4 1g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, citric acid 1.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O10 mg/L.
Preferably, the leucine production strain is applied, and a fermentation medium is adopted in the fermentation culture: glucose 20g/L, yeast powder 3.5g/L, peptone 1g/L, corn steep liquor dry powder 10g/L, bean concentrate 5ml/L,(NH4)2SO4 2g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, glutamic acid 2g/L, methionine 0.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O30 mg/L.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.
Claims (9)
1. A leucine producing strain, characterized in that: the leucine production strain is corynebacterium glutamicum (Corynebacterium glutamicum) Leu07, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 12 and 11 days, and the strain preservation number is CGMCC No.29285.
2. The leucine producing strain according to claim 1, wherein: the strain Corynebacterium glutamicum ATCC 13032 is obtained by modifying an original strain Corynebacterium glutamicum ATCC 13032 by a metabolic engineering modification method, and specifically, the expression intensities of acetohydroxyacid synthase gene ilvBN fbr, isopropyl malate synthase gene leuA fbr, alpha-isopropyl malate isomerase leuD, alpha-isopropyl malate isomerase leuDleuC, beta-isopropyl malate dehydrogenase leuB, leucine transaminase gene Ncgl2204, pyruvate dehydrogenase gene Ncgl2610 and lactate dehydrogenase gene Ncgl0901 are regulated.
3. The leucine producing strain according to claim 2, wherein: the acetohydroxyacid synthase gene ilvBN fbr is derived from corynebacterium glutamicum; the isopropyl malate synthase gene leuA fbr is derived from corynebacterium glutamicum; the alpha-isopropyl malate isomerase leuD is derived from bacillus subtilis; the beta-isopropyl malate dehydrogenase leuB and the alpha-isopropyl malate isomerase leuC are derived from bacillus subtilis; the leucine aminotransferase gene Ncgl2204 is derived from corynebacterium glutamicum; the pyruvate dehydrogenase gene Ncgl2610 is derived from corynebacterium glutamicum; the lactate dehydrogenase gene Ncgl0901 is derived from Corynebacterium glutamicum.
4. The leucine producing strain according to claim 1, wherein: the corynebacterium glutamicum ATCC 13032 is taken as an original strain, and the control of the overexpression of the acetohydroxyacid synthase gene ilvBN fbr is carried out at a pseudogene locus Ncgl1995 by using a Ptuf promoter to obtain a strain Leu01; taking the strain Leu01 as an original strain, and controlling the overexpression of isopropyl malate synthase gene leuA fbr by using Ptuf promoter at pseudogene locus Ncgl1960 to obtain Leu02; taking the bacterial strain Leu02 as an original bacterial strain, and controlling the overexpression of alpha-isopropyl malate isomerase leuD by using Ptuf promoter at a pseudo-gene locus Ncgl2001 to obtain Leu03; taking the strain Leu03 as an original strain, and controlling the overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC by using Ptuf promoter at a pseudo-gene locus Ncgl2000 to obtain Leu04; taking the strain Leu04 as an original strain, and controlling the overexpression of a leucine aminotransferase gene Ncgl2204 at a pseudo-gene locus Ncgl2037 by using a Ptuf promoter to obtain Leu05; taking a strain Leu05 as an original strain, knocking out a pyruvate dehydrogenase gene Ncgl2610 on a genome to ensure that the gene is not expressed, and controlling double copies of an acetohydroxyacid synthase gene ilvBN fbr by using a Ptuf promoter at a site of the gene to obtain Leu06; taking the strain Leu06 as an original strain, knocking out the lactate dehydrogenase gene Ncgl0901 on a genome to ensure that the lactate dehydrogenase gene Ncgl0901 is not expressed, and controlling double copies of the leucine aminotransferase gene Ncgl2204 by using a Ptuf promoter at a site of the lactate dehydrogenase gene Ncgl0901 to obtain a target strain.
5. The leucine producing strain according to claim 4, wherein: the Ptuf promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 1; the ilvBN fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 2; the leuA fbr gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3; the leuD gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 4; the leuBC gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5; the Ncgl2204 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 6; the Ncgl2610 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 7; the Ncgl0901 gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 8.
6. A construction method of leucine production strains is characterized in that: comprises the following steps:
Step 1: the corynebacterium glutamicum ATCC 13032 is taken as an original strain, and a Ptuf promoter is used for controlling the overexpression of acetohydroxyacid synthase ilvBN fbr gene at a pseudogene locus Ncgl1995 to obtain a strain Leu01;
Step 2: taking the strain Leu01 as an original strain, and controlling the overexpression of isopropyl malate synthase leuA fbr genes at a pseudogene locus Ncgl1960 by using a Ptuf promoter to obtain the strain Leu02;
step 3: taking the strain Leu02 as an original strain, and controlling the overexpression of alpha-isopropyl malate isomerase leuD by using Ptuf promoter at a pseudo-gene locus Ncgl2001 to obtain the strain Leu03;
Step 4: taking the strain Leu03 as an original strain, and controlling the overexpression of beta-isopropyl malate dehydrogenase leuB and alpha-isopropyl malate isomerase leuC by using Ptuf promoter at a pseudo-gene locus Ncgl2000 to obtain the strain Leu04;
Step 5: taking the strain Leu04 as an original strain, and controlling the overexpression of a leucine aminotransferase Ncgl2204 gene by using a Ptuf promoter at a pseudo-gene locus Ncgl2037 to obtain a strain Leu05;
step 6: taking the strain Leu05 as an original strain, knocking out a pyruvic acid dehydrogenase Ncgl2610 gene on a genome, and controlling acetohydroxy acid synthase ilvBN fbr gene to be overexpressed at a site of the gene by using a Ptuf promoter to obtain a strain Leu06;
Step 7: taking the strain Leu06 as an original strain, knocking out the lactate dehydrogenase Ncgl0901 gene on a genome, and controlling the overexpression of the leucine aminotransferase Ncgl2204 gene by using Ptuf promoter at the locus to obtain the strain Leu07, wherein the strain Leu07 is the target strain after the transformation is successful.
7. Use of the leucine producing strain according to any one of claims 1-5 for the fermentative preparation of L-leucine.
8. The use of the leucine producing strain according to claim 7 for the fermentative preparation of L-leucine, wherein: the method comprises the following specific steps:
(1) Seed culture: at the culture temperature of 35-39 , the pH value of the culture is maintained at 6.7+/-0.2 by automatically feeding ammonia water solution with mass fraction of 20-28%, the dissolved oxygen value of the culture is maintained at 25-35% by adjusting the stirring speed or ventilation, and the inoculation requirement is met when the OD 600nm is 20;
(2) Fermentation culture: according to the inoculation amount of 10-25%, the culture temperature is 35-39 , the pH value of the culture is maintained at 6.7+/-0.2 by automatically feeding 20-28% ammonia water solution, the dissolved oxygen value of the culture is maintained at 25-35% by adjusting the stirring speed or ventilation, the glucose concentration in the tank is controlled to be less than or equal to 1.5g/L by feeding 60-85% glucose solution, and the fermentation period is less than or equal to 45 h.
9. The use of the leucine producing strain according to claim 8 for the fermentative preparation of L-leucine, wherein: seed culture medium used in seed culture: 20g/L of glucose, 3g/L of yeast, 1g/L of peptone, 15g/L of corn steep liquor dry powder, 1.5g/L of bean concentrate 15ml/L ,(NH4)2SO4 1g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, citric acid and 10 mg/L of MnSO 4H2O 10mg/L,FeSO47H2 O; fermentation medium used in fermentation culture: glucose 20g/L, yeast powder 3.5g/L, peptone 1g/L, corn steep liquor dry powder 10g/L, bean concentrate 5ml/L ,(NH4)2SO4 2g/L,K2HPO43H2O 2g/L,MgSO47H2O 2g/L, glutamic acid 2g/L, methionine 0.5g/L, mnSO 4H2O 10mg/L,FeSO47H2 O30 mg/L.
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