CN117417871A - Recombinant corynebacterium crenatum and method for producing L-arginine - Google Patents
Recombinant corynebacterium crenatum and method for producing L-arginine Download PDFInfo
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- 241000424760 Corynebacterium crenatum Species 0.000 title claims abstract description 37
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 title claims abstract description 23
- 229930064664 L-arginine Natural products 0.000 title claims abstract description 23
- 235000014852 L-arginine Nutrition 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 23
- 239000008103 glucose Substances 0.000 claims abstract description 23
- 102000030595 Glucokinase Human genes 0.000 claims abstract description 16
- 108010021582 Glucokinase Proteins 0.000 claims abstract description 16
- 230000002018 overexpression Effects 0.000 claims abstract description 14
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960000367 inositol Drugs 0.000 claims abstract description 13
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 13
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 13
- 102000034356 gene-regulatory proteins Human genes 0.000 claims abstract description 12
- 108091006104 gene-regulatory proteins Proteins 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 238000005728 strengthening Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims description 16
- 238000003780 insertion Methods 0.000 claims description 14
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 5
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 3
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 235000010344 sodium nitrate Nutrition 0.000 claims description 3
- 239000004317 sodium nitrate Substances 0.000 claims description 3
- 235000019157 thiamine Nutrition 0.000 claims description 3
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 3
- 229960003495 thiamine Drugs 0.000 claims description 3
- 239000011721 thiamine Substances 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000013028 medium composition Substances 0.000 claims 1
- 230000006377 glucose transport Effects 0.000 abstract description 6
- 241000186216 Corynebacterium Species 0.000 abstract description 2
- 230000003014 reinforcing effect Effects 0.000 abstract 1
- 108010078791 Carrier Proteins Proteins 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000012795 verification Methods 0.000 description 8
- 238000012216 screening Methods 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 4
- 101100391174 Dictyostelium discoideum forC gene Proteins 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 108010011619 6-Phytase Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 229940085127 phytase Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- NDVRKEKNSBMTAX-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphoric acid Chemical compound OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O NDVRKEKNSBMTAX-BTVCFUMJSA-N 0.000 description 1
- 101100508893 Bacillus subtilis (strain 168) iolR gene Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 108010071189 phosphoenolpyruvate-glucose phosphotransferase Proteins 0.000 description 1
- 101150086745 pre gene Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
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Abstract
The invention relates to a recombinant corynebacterium crenatumCorynebacterium crenatumThe recombinant corynebacterium crenatum is prepared by MT-M4ΔTSCPΔptaΔproBΔcgl2310Δncgl1221Coding gene of glucose specific transporter through strengthening PTS system for starting strainptsGCoding base for overexpression of IPGS system key enzyme inositol permeaseBecause ofiolT1Coding gene of overexpression glucokinase PpgKcg2091And knock out coding gene of negative regulatory protein IolRiolRThe obtained product. According to the invention, by reinforcing the PTS system and the IPGS system of two glucose transport paths simultaneously, the utilization rate and the utilization efficiency of the corynebacterium crenatum on glucose are greatly improved, and the efficiency of producing L-arginine by fermentation is further improved.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a recombinant corynebacterium crenatum and a method for producing L-arginine.
Background
The total fermentation cost of the microbial fermentation method has higher proportion of raw material cost, and the carbon source is the weight of the fermentation raw material. In addition, the efficiency of carbon source utilization affects the yield of the final desired product. Corynebacterium crenatum (corynebacterium crenatum) is a spore-free, blunt-tooth gram-positive bacterium isolated from soil, and its mutant strain is widely used in various amino acid production in China, such as L-arginine, L-ornithine, L-glutamic acid, L-citrulline, etc., but has been studied relatively little in genetic background. Generally, L-arginine is produced by microbial fermentation using glucose as a carbon source. Therefore, if the sugar metabolism pathway of corynebacterium crenatum can be improved, the efficient utilization of the carbon source is necessarily advantageous for improving the production efficiency of L-arginine.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a recombinant corynebacterium crenatum and a method for producing L-arginine, and the present invention starts from enhancing the utilization efficiency of carbon sources, and by simultaneously enhancing two glucose transport paths PTS system and IPGS system, the utilization rate and efficiency of corynebacterium crenatum on glucose are greatly improved, and further the efficiency of producing L-arginine by fermentation is improved.
(II) technical scheme
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a recombinant corynebacterium crenatum as MT-M4ΔTSCPΔptaΔproBΔcgl2310Δncgl1221Coding gene of glucose specific transporter through strengthening PTS system for starting strainptsGCoding gene for overexpression of IPGS system key enzyme inositol permeaseiolT1Coding gene of overexpression glucokinase PpgKcg2091And knock out coding gene of negative regulatory protein IolRiolRThe obtained product.
According to a preferred embodiment of the invention, the grape of the PTS systemCoding gene of sugar specific transport proteinptsGThe nucleotide sequence of (2) is shown as SEQ ID NO. 1.
According to a preferred embodiment of the present invention, the genes encoding glucose-specific transporters of the enhanced (overexpressed) PTS systemptsGThe method of (1) is as follows: in the genome of the starting strain, genes encoding glucose-specific transporters in the PTS systemptsGThe upstream region of (sequence shown as SEQ ID NO. 1) is inserted with a strong promoter Psod, the nucleotide sequence of which is shown as SEQ ID NO. 5.
According to a preferred embodiment of the invention, the gene encoding the key enzyme, the phytase of the IPGS systemiolT1The nucleotide sequence of (2) is shown as SEQ ID NO. 2.
According to a preferred embodiment of the invention, the gene encoding the key enzyme, the inositol permease, of the IPGS system is overexpressediolT1The method of (1) is as follows: in the genome of the original strain, the gene encoding the key enzyme, the inositol permease, in the IPGS systemiolT1Insertion of a strong promoter P into the upstream region (sequence shown in SEQ ID No. 2) NCgl0824 Strong promoter P NCgl0824 The nucleotide sequence of (2) is shown as SEQ ID NO. 6.
According to a preferred embodiment of the present invention, the glucokinase PpgK encoding genecg2091The nucleotide sequence of (2) is shown as SEQ ID NO. 3.
According to a preferred embodiment of the present invention, the gene encoding glucokinase PpgK is overexpressedcg2091The method of (1) is as follows: in the genome of the starting strain, the gene encoding the glucokinase PpgKcg2091A strong promoter Psod is inserted into the upstream region (the sequence shown in SEQ ID NO. 3), and the nucleotide sequence of the strong promoter Psod is shown in SEQ ID NO. 5.
According to a preferred embodiment of the present invention, the gene encoding the negative regulatory protein IolRiolRThe nucleotide sequence of (2) is shown as SEQ ID NO. 4.
According to a preferred embodiment of the present invention, the recombinant corynebacterium crenatum is prepared as follows:
(1) Glucose-specific transporter encoding genes through PTS system in CCM01 genomeptsGInsertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM02 strain;
(2) Inositol permease encoding gene in CCM02 genomeiolT1Insertion of a strong promoter P in the upstream region of (E) NCgl0824 ，Successfully constructing CCM03 strain;
(3) Glucokinase PpgK coding gene in CCM03 genomecg2091Insertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM04 strain;
(4) Coding gene of negative regulatory protein IolR of CCM04 genome knocked outiolR，Successfully constructing CCM05 strain; recombinant CCM05 is the target recombinant corynebacterium crenatum.
In a second aspect, the present invention provides a method for producing L-arginine, comprising:
inoculating the recombinant corynebacterium crenatum into a fermentation culture medium for fermentation culture, and harvesting L-arginine in a culture solution.
According to the preferred embodiment of the invention, the culture temperature is 28-31 ℃ and the rotating speed is 100-180r/min; the initial inoculation concentration is that the seed solution is inoculated in the fermentation culture medium according to 5-15%.
Preferably, the seed solution is prepared by picking single colony from an activated solid culture medium, inoculating the single colony into an LBG culture medium, carrying out shaking culture at 28-31 ℃ for 22-26 hours, then transferring the single colony into a seed culture medium, and carrying out culture at 28-31 ℃ for 22-26 hours, wherein the seed culture medium comprises the following components: glucose 100-150g/L, corn steep liquor 20-30g/L, ammonium sulfate 30-50g/L, potassium chloride 0.5-1.5g/L, potassium dihydrogen phosphate 1-1.5g/L, magnesium sulfate heptahydrate 0.3-0.6g/L, and deionized water to prepare pH7.0-7.2.
Preferably, the fermentation medium consists of: 100-150g/L of glucose, 20-30g/L of corn steep liquor, 30-50g/L of ammonium sulfate, 0.5-1.5g/L of potassium chloride, 1-1.5g/L of monopotassium phosphate, 20-40g/L of calcium carbonate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 5-8g/L of sodium nitrate, 300-800 mu g/L of thiamine, 50-200 mu g/L of biotin, and the preparation of the corn steep liquor with deionized water and the pH of 7.0-7.2.
The invention also relates to application of the recombinant corynebacterium crenatum in fermentation production of L-arginine.
In this application: the starting strain is MT-M4ΔTSCPΔAptaΔproBΔcgl2310Δncgl1221It has been reported in the literature as TSCP of Corynebacterium crenatum MT-M4,pta、proB、cgl2310、ncgl1221Engineering bacteria with gene deletion. The pre-gene "delta" indicates that the gene is deleted.
(III) beneficial effects
The recombinant corynebacterium crenatum with the PTS system and the IPGS system for strengthening two glucose transport paths simultaneously is successfully constructed, the glucose transport paths are greatly optimized, the recombinant corynebacterium crenatum CCM05 is fermented to 120h finally, the yield of L-arginine reaches 12.98g/L, and compared with the original strain CCM01, the yield of L-arginine is improved by 23.35%, and the yield and the production speed of L-arginine are improved. Meanwhile, the recombinant corynebacterium crenatum CCM05 also increases the glucose consumption, which shows that the PTS system and the IPGS system for enhancing glucose transport have obvious effects on improving the yield and the production speed of L-arginine.
Detailed Description
The invention is described in detail below in connection with specific embodiments for better understanding of the invention.
By studying the sugar metabolism pathway of Corynebacterium crenatum, it was found that there are two glucose transport pathways of Corynebacterium crenatum, the first being the PTS system, in whichptsGEncoded specific transporter EII glu Glucose is taken up. The second is the IPGS system, where glucose enters the cell under the action of IolT and glucose phosphate is produced under the action of glucokinase Glk or PpgK, but the negative regulatory protein IolR normally represses the pathway.
In order to improve the sugar metabolism path of corynebacterium crenatum, improve the utilization efficiency of carbon sources in the fermentation culture process and further improve the yield of L-arginine, the invention strengthens the coding gene of the glucose specific transport protein of the PTS system of the original strainptsGCoding gene for overexpression of IPGS system key enzyme inositol permeaseiolT1Coding gene of overexpression glucokinase PpgKcg2091Coding gene for simultaneously knocking out negative regulatory protein IolRiolRObtaining a recombinant corynebacterium crenatum, in particular, the recombinant corynebacterium crenatumThe preparation method of the corynebacterium crenatum comprises the following steps:
(1) Glucose-specific transporter encoding genes through PTS system in CCM01 genomeptsGInsertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM02 strain;
(2) Inositol permease encoding gene in CCM02 genomeiolT1Insertion of a strong promoter P in the upstream region of (E) NCgl0824 ，Successfully constructing CCM03 strain;
(3) Glucokinase PpgK coding gene in CCM03 genomecg2091Insertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM04 strain;
(4) Coding gene of negative regulatory protein IolR of CCM04 genome knocked outiolR，Successfully constructing CCM05 strain; recombinant CCM05 is the target recombinant corynebacterium crenatum.
The invention is further illustrated by the following examples.
Example 1
The present example corresponds to step (1) of the above preparation method, in which the PTS system glucose-specific transporter encoding gene of CCM01 genomeptsGInsertion of a strong promoter P into the upstream region of (E)sod，To construct CCM02 strain. The method comprises the following specific steps:
(1) Preparing a BHI culture medium: peptone 10g/L, yeast extract 5g, sodium chloride 10g, bovine brain heart extract 25g/L.
(2) Constructing a recombinant plasmid carrying a strong promoter: to be used forC. crenatumGenome is used as a template, and is utilizedptsG-up-F (sequence shown as SEQ ID NO. 7),ptsG-up-R (sequence shown as SEQ ID NO. 8)ptsG-Down-F (sequence shown as SEQ ID NO. 9),ptsGAmplification of Down-R (sequence shown in SEQ ID NO. 10)ptsG sod Upper and lower homologous arms of (C)ptsG-sodF (sequence shown as SEQ ID NO. 11),ptsG-sodR (sequence shown as SEQ ID NO. 12) to obtain a strong promoterP sod The fragment was then subjected to overlap PCR to obtain a fragment length of 1239 bpptsG sod Fusion arm, connected to pK18mobsacBVector to obtain pK18mobsacB-ptsG sod pK18mobsacB-ptsG sod Sequencing, and comparing the sequences, wherein the result shows that the sequences are correct.
(3) Electrotransformation yields CCM02: pK18mobsacB-ptsG sod The recombinant plasmid is electrically transferred into an original strain CCM01, resuscitated in a BHI culture medium for 3 hours, then the recombinant plasmid is coated on a kanamycin resistance plate, the single colony is amplified and cultured after verification, the single colony is transferred onto a plate containing 10% of sucrose for screening, colony PCR is subjected to double-exchange verification, and the glucose specific transporter coding gene of the overexpression PTS system is obtained after screeningptsGIs designated CCM02.
Example 2
This example corresponds to the step (2) of the above preparation method, and the gene encoding the phytase in CCM02 genomeiolT1Insertion of a strong promoter P in the upstream region of (E) NCgl0824 ，The CCM03 strain was successfully constructed. The method comprises the following specific steps:
(1) Preparing a BHI culture medium: peptone 10g/L, yeast extract 5g, sodium chloride 10g, bovine brain heart extract 25g/L.
(2) Constructing a recombinant plasmid carrying a strong promoter: to be used forC. crenatumGenome is used as a template, and is obtained by amplificationiolT1 NCgl0824 Upper and lower homology arms of (a) and strong promotersP NCgl0824 Fragments, obtained by overlapping PCRiolT1 NCgl0824 Fusion arm, connected to pK18mobsacBVector to obtain pK18mobsacB- iolT1 NCgl0824 pK18mobsacB- iolT1 NCgl0824 Sequencing, and comparing the sequences, wherein the result shows that the sequences are correct.
(3) Electrotransformation yields CCM03: pK18mobsacB- iolT1 NCgl0824 The recombinant plasmid is electrically transferred into CCM02, resuscitated in BHI culture medium for 3 hr, then spread on kanamycin resistance plate, and the single colony is amplified after verification, transferred onto 10% sucrose-containing plate for screening, colony PCR is double-exchanged for verification, screeningObtaining the overexpression inositol permease coding geneiolT1Is designated CCM03.
Example 3
This example corresponds to step (3) of the above preparation method, in which a strong promoter P is inserted into the CCM03 genome sod Overexpression of glucokinase PpgK coding genecg2091The CCM04 strain was successfully constructed. The specific method comprises the following steps:
(1) Preparing a BHI culture medium: 10g/L peptone, 5g yeast extract, 10g sodium chloride and 25g/L bovine brain heart extract.
(2) Constructing a recombinant plasmid carrying a strong promoter: to be used forC. crenatumGenome is used as a template, and is utilizedcg2091-up-F (sequence shown as SEQ ID NO. 13),cg2091-up-R (sequence shown as SEQ ID NO. 14)cg2091-Down-F (sequence shown as SEQ ID NO. 15),cg2091Amplification of Down-R (SEQ ID NO. 16)cg2091 sod Upper and lower homologous arms of (C)cg2091-sodF (sequence shown as SEQ ID NO. 17),cg2091-sodR (sequence shown as SEQ ID NO. 18) to obtain a strong promoterP sod Fragments, and obtaining 1812bp long by overlapping PCRcg2091 sod Fusion arm, connected to pK18mobsacBVector to obtain pK18mobsacB-cg2091 sod pK18mobsacB- cg2091 sod Sequencing, and comparing the sequences, wherein the result shows that the sequences are correct.
(3) Electrotransformation gives CCM04: pK18mobsacB-cg2091 sod The recombinant plasmid is electrically transferred into CCM03, resuscitated in BHI culture medium for 3h, then coated on kanamycin resistance plate, the single colony is amplified and cultured after verification, transferred onto a plate containing 10% sucrose for screening, colony PCR is subjected to double-exchange verification, and the overexpression glucokinase PpgK coding gene is obtained after screeningcg2091Is designated CCM04.
Example 4
The present example corresponds to step (4) of the above preparation method, in which the coding gene of the negative regulatory protein IolR of CCM04 genome is knocked outiolR，Successful construction of CCM05 bacteriumA strain. The specific method comprises the following steps:
(1) Preparing a BHI culture medium: 10g/L peptone, 5g yeast extract, 10g sodium chloride and 25g/L bovine brain heart extract.
(2) Constructing a recombinant plasmid carrying a strong promoter: to be used forC. crenatumGenome is used as a template, and is obtained by amplificationiolRUpper and lower homologous arms of (2), and obtaining father through overlap PCRiolRFusion arm, connected to pK18mobsacBVector to obtain pK18mobsacB-∆iolR，pK18mobsacB- ∆iolRSequencing, and comparing the sequences, wherein the result shows that the sequences are correct.
(3) Electric conversion: pK18mobsacB-∆iolRThe recombinant plasmid is electrically transferred into CCM04, resuscitated in BHI culture medium for 3h, then coated on kanamycin resistance plate, the single colony is amplified and cultured after verification, transferred onto a plate containing 10% sucrose for screening, colony PCR is subjected to double-exchange verification, and the coding gene of knocked-out negative regulatory protein IolR is obtained after screeningiolRIs designated CCM05.
Example 5
This example produces L-arginine by culturing recombinant Corynebacterium crenatum CCM05 in fermentation medium as follows:
(1) Seed culture
Single colonies of recombinant corynebacterium crenatum CCM05 and a starting strain CCM01 are picked from an activation plate, inoculated into 5-10mL of LBG culture medium in parallel, cultured for 24 hours at 30 ℃ in a shaking mode, and then all inoculated into 50mL of seed culture medium and cultured for 24 hours at 30 ℃.
(2) Fermentation culture
The fermentation culture volume is 25mL, and the adopted fermentation culture medium comprises the following components:
fermentation medium components: 100-150g/L of glucose, 20-30g/L of corn steep liquor, 30-50g/L of ammonium sulfate, 0.5-1.5g/L of potassium chloride, 1-1.5g/L of monopotassium phosphate, 20-40g/L of calcium carbonate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 5-8g/L of sodium nitrate, 300-800 mu g/L of thiamine, 50-200 mu g/L of biotin and pH of 7.0-7.2.
Fermentation conditions: inoculating the cultured seed liquid into a fermentation culture medium according to 10% of inoculum size respectively for fermentation culture, and controlling fermentation conditions in parallel: the fermentation temperature is 30 ℃, the pH is 7.0, the rotating speed is 200r/min, and the fermentation time is 120h.
During fermentation, samples were taken every 12 hours, glucose content was measured by a biosensing analyzer (academy of sciences of Shandong), cell OD was measured at A562 by an enzyme-labeled instrument, and L-arginine was measured by HPLC (Agilent 1100, USA). Compared with the original strain CCM01, the method has the advantages that the growth rate and sugar consumption of the recombinant strain are both accelerated, the fermentation is carried out for 120 hours, the L-arginine yield of the recombinant strain CCM05 reaches 12.98+/-0.375 g/L, the yield is improved by 23.35% compared with the original strain, and the L-arginine yield is improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A recombinant corynebacterium crenatum is characterized in that the recombinant corynebacterium crenatum is prepared by MT-M4ΔTSCPΔptaΔproBΔcgl2310Δncgl1221Coding gene of glucose specific transporter through strengthening PTS system for starting strainptsGCoding gene for overexpression of IPGS system key enzyme inositol permeaseiolT1Coding gene of overexpression glucokinase PpgKcg2091And knock out coding gene of negative regulatory protein IolRiolRThe obtained product.
2. The recombinant corynebacterium crenatum according to claim 1, wherein a gene encoding a glucose-specific transporter of the PTS systemptsGThe nucleotide sequence of (2) is shown as SEQ ID NO. 1; coding gene of glucose specific transporter for strengthening PTS systemptsGThe method of (1) is as follows: glucose in PTS System in the genome of the starting StrainCoding gene of specific transport proteinptsGThe nucleotide sequence of the strong promoter Psod is shown in SEQ ID NO. 5.
3. The recombinant corynebacterium crenatum according to claim 1, wherein the gene encoding the key enzyme, inositol permease of the IPGS systemiolT1The nucleotide sequence of (2) is shown as SEQ ID NO. 2; coding gene for overexpression of IPGS system key enzyme inositol permeaseiolT1The method of (1) is as follows: in the genome of the original strain, the gene encoding the key enzyme, the inositol permease, in the IPGS systemiolT1Insertion of a strong promoter P into the upstream region of (E) NCgl0824 Strong promoter P NCgl0824 The nucleotide sequence of (2) is shown as SEQ ID NO. 6.
4. The recombinant corynebacterium crenatum of claim 1, wherein the glucokinase PpgK encodes a genecg2091The nucleotide sequence of (2) is shown as SEQ ID NO. 3; coding gene of overexpression glucokinase PpgKcg2091The method of (1) is as follows: in the genome of the starting strain, the gene encoding the glucokinase PpgKcg2091Insertion of a strong promoter P in the upstream region of (E)sodStrong promoter PsodThe nucleotide sequence of (2) is shown as SEQ ID NO. 5.
5. The recombinant corynebacterium crenatum according to claim 1, wherein the gene encoding the negative regulatory protein IolRiolRThe nucleotide sequence of (2) is shown as SEQ ID NO. 4.
6. The recombinant corynebacterium crenatum according to any one of claims 1 to 5, wherein the recombinant corynebacterium crenatum is prepared by:
(1) Glucose-specific transporter encoding genes through PTS system in CCM01 genomeptsGInsertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM02 strain;
(2) Inositol permease encoding gene in CCM02 genomeiolT1Insertion of a strong promoter P in the upstream region of (E) NCgl0824 ，Successfully constructing CCM03 strain;
(3) Glucokinase PpgK coding gene in CCM03 genomecg2091Insertion of a strong promoter P into the upstream region of (E)sod，Successfully constructing CCM04 strain;
(4) Coding gene of negative regulatory protein IolR of CCM04 genome knocked outiolR，Successfully constructing CCM05 strain; recombinant CCM05 is the target recombinant corynebacterium crenatum.
7. A method for producing L-arginine, comprising: inoculating the recombinant corynebacterium crenatum of any one of claims 1 to 6 into a fermentation medium for fermentation culture, and harvesting the L-arginine in the culture solution.
8. The method according to claim 7, wherein the cultivation temperature is 28-31 ℃ and the rotation speed is 100-180r/min; the initial inoculation concentration is that the seed solution is inoculated in the fermentation culture medium according to 5-15%.
9. The method of claim 7, wherein the seed solution is prepared by picking single colonies from an activated solid medium, inoculating the single colonies to an LBG medium, shaking culturing the single colonies at 28-31 ℃ for 22-26 hours, transferring the single colonies to a seed medium, and culturing the single colonies at 28-31 ℃ for 22-26 hours, wherein the seed medium comprises the following components: glucose 100-150g/L, corn steep liquor 20-30g/L, ammonium sulfate 30-50g/L, potassium chloride 0.5-1.5g/L, potassium dihydrogen phosphate 1-1.5g/L, magnesium sulfate heptahydrate 0.3-0.6g/L, and deionized water to prepare pH7.0-7.2.
10. The method of claim 7, wherein the fermentation medium composition is: 100-150g/L of glucose, 20-30g/L of corn steep liquor, 30-50g/L of ammonium sulfate, 0.5-1.5g/L of potassium chloride, 1-1.5g/L of monopotassium phosphate, 20-40g/L of calcium carbonate, 0.3-0.6g/L of magnesium sulfate heptahydrate, 5-8g/L of sodium nitrate, 300-800 mu g/L of thiamine, 50-200 mu g/L of biotin, and the preparation of the corn steep liquor with deionized water and the pH of 7.0-7.2.
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