CN111549045B - Vitamin K improvement by utilizing recombinant bacillus natto 2 Method for producing yield - Google Patents
Vitamin K improvement by utilizing recombinant bacillus natto 2 Method for producing yield Download PDFInfo
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- CN111549045B CN111549045B CN202010051111.0A CN202010051111A CN111549045B CN 111549045 B CN111549045 B CN 111549045B CN 202010051111 A CN202010051111 A CN 202010051111A CN 111549045 B CN111549045 B CN 111549045B
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 83
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 83
- 229930003448 Vitamin K Natural products 0.000 title claims abstract description 16
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 235000019168 vitamin K Nutrition 0.000 title claims abstract description 16
- 239000011712 vitamin K Substances 0.000 title claims abstract description 16
- 150000003721 vitamin K derivatives Chemical class 0.000 title claims abstract description 16
- 229940046010 vitamin k Drugs 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 230000006872 improvement Effects 0.000 title claims description 4
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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
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- 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
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- C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
- C12Y602/01—Acid-Thiol Ligases (6.2.1)
- C12Y602/01026—O-Succinylbenzoate-CoA ligase (6.2.1.26)
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Abstract
The invention discloses a method for improving vitamin K by utilizing recombinant bacillus natto 2 Method for production by constitutive promotersP 43 Over-expressing the flutolosine synthase gene mqnA on the chromosome of bacillus natto, and based on the obtained strain NA1, utilizing P 43 The promoter over-expresses the gene of deoxyxanthine flutazocine synthetase on bacillus natto chromosome, and based on the obtained strain NA2, the gene is utilizedP hbs The promoter replaces the natural promoter of O-succinylbenzoic acid-CoA ligase gene in bacillus natto, and based on the obtained strain NA3, the promoter containsP hbs The 1, 4-dihydroxyl-6-naphthoic acid synthase gene of the promoter from the streptomyces coelicolor replaces an isochorismate gene on a bacillus natto chromosome, and seed solution of the recombinant bacteria is inoculated into a fermentation culture medium according to an inoculation amount of 3% -6%. The invention constructs 4 recombinant strains NA 1-NA 4, wherein NA 3-NA 4 enables vitamin K to be obtained 2 The yield of (2) is significantly improved.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for improving vitamin K by utilizing recombinant bacillus natto 2 A method of production.
Background
Vitamin K 2 Is one of active substances of vitamin K, is an important fat-soluble vitamin formed by connecting a menaquinone parent ring and n isoprene side chains, is used as a cofactor of gamma-glutamate carboxylase, and has the characteristics of long half-life and good bioavailability. Has effects in resisting arterial calcification and osteoarthritis, resisting tumor, resisting oxidation, delaying aging, caring skin, treating hepatitis, regulating digestive tract, relieving smooth muscle spasm, treating and preventing osteoporosis, preventing liver cirrhosis from progressing to liver cancer, and treating vitamin K 2 The Chinese medicinal composition has important effects in treating deficiency hemorrhage, promoting urination, strengthening liver, removing toxic substances, reducing blood pressure, and the like. More attention is paid to menatetrenone because of its better affinity for the human body and longer half-life.
Vitamin K is a generic term for a series of compounds whose core structure is a 2-methyl-1, 4-menaquinone ring, but whose side chain structure varies in length and saturation. In bacillus natto, chorismate can form a skeleton structure menaquinone under the action of mqn gene cluster, and mqn gene cluster mainly comprises mqnF, mqnD, ytxM, mqnC, mqnE, mqnB, yuxO 7 enzymes. Then under the action of 1, 4-dihydroxy-2-naphthoic acid heptapentenyl transferase (mqnA) to generate the normenaquinone-7, and finally under the action of menaquinone methyltransferase (mqnH) to generate the heptamenaquinone. The synthesis of the core structure in nature involves mainly two ways, one being the mqn gene cluster and the other being the mqn gene cluster found in small numbers of bacteria such as Streptomyces coelicolor (Streptomyces coelicolor). Among the Mqn gene clusters, four genes, mqna, mqnB, mqnC, mqnD, were mainly included. Precursor chorismate can synthesize precursor 1, 4-dimethyl-naphthoic acid under the action of Mqn gene cluster, and has the advantages of small catalysis step, reduced consumption of intermediate metabolites and the like compared with mqn gene cluster.
Bacillus natto (Bacillus subtilis) is commonly used in vitamin K 2 Is produced with a product that is FDA certified as "generally regarded as safe" (GRAS) security level. Therefore, the construction of recombinant bacillus natto by using metabolic engineering means is efficient in synthesizing vitamin K 2 Is an effective way of (a) is provided. However, vitamin K 2 The synthesis route of the bacillus natto is very complex, and vitamin K is synthesized by bacillus natto 2 Is insufficient in metabolic flux and will seriously affect vitamin K 2 Is a synthesis of (a). How to adjust the metabolic flux supply of bacillus natto and increase vitamin K 2 Is a problem worth going deep into question.
Disclosure of Invention
The invention aims to provide a method for improving vitamin K by utilizing recombinant bacillus natto 2 The method for producing the soybean sprout bacillus comprises the steps of taking bacillus natto (Bacillus subtilis natto) as an initial strain, and utilizing a P43 promoter to heterologously express a fluorine tamsulosin synthase gene mqnA and a deoxidized xanthine fluorine tamsulosin synthase gene mqnB on a genome of the bacillus natto; expressing O-succinylbenzoic acid-CoA ligase gene mqnC and 1, 4-dihydroxy-6-naphthoic acid synthase mqnD on chromosome by utilizing Phbs promoter, and constructing 4 recombinant strains NA 1-NA 4, wherein NA 3-NA 4 enables vitamin K 2 The yield of (c) is significantly improved to solve the problems set forth in the background art described above.
In order to achieve the above purpose, the present invention provides the following technical solutions:
vitamin K improvement by utilizing recombinant bacillus natto 2 A method of yield comprising the steps of:
step 1: construction of recombinant NA1
By constitutive promotersP 43 Over-expressing a flutoloxin synthetase gene mqnA on a bacillus natto chromosome;
step 2: construction of recombinant NA2
Based on the strain NA1 obtained in step 1, P was used in a similar manner to step 1 43 Overexpression of deoxyxanthine on chromosome of bacillus natto by promoterFluostatin synthetasemqnBGenebank ID:1099767 A) a gene;
step 3: construction of recombinant NA3
Based on the strain NA2 obtained in the step 2, a method similar to the step 1 is adopted, and the method is utilizedP hbs The promoter replaces O-succinylbenzoic acid-CoA ligase in bacillus nattomqnCGenebank ID:1099990 A natural promoter of a gene;
step 4: construction of recombinant bacterium BS4
Based on the strain NA3 obtained in step 3, the method similar to step 1 was used with a strain containingP hbs Substitution of the promoter 1, 4-dihydroxy-6-naphthoic acid synthase (mqnD, genebank ID: 1099766) gene derived from Streptomyces coelicolor for isochoric acid on the chromosome of Bacillus nattodhbBGenebank ID:936582 A) a gene;
step 5: production of vitamin K by bacterial strain fermentation 2
Inoculating the seed solution of the recombinant bacteria into a fermentation medium according to the inoculation amount of 3% -6%.
The sequence of the P43 promoter is shown in SEQ ID NO. 1; the sequence of the Phbs promoter is shown as SEQ ID NO. 2. The saidmqnAThe sequence of the gene is as genebank ID: 1099946; the saidmqnBThe sequence of the gene is as genebank ID: 1099767; the saidmqnCThe sequence of the gene is as genebank ID: 1099990; the saidmqnDThe sequence of the gene is as genebank ID: 1099766.
Further, the recombinant bacterium isBacillus subtilis natto, P 43 -mqnAIt is a sequence inserted into the amyE locus of chromosome, and the sequence includes promoterP 43 Fusion of mqnAGene to enhance the Flutatrioxin synthetasemqnAGenebank ID:1099946 Gene expression, and designated NA1.
Furthermore, the recombinant bacteria are modified based on NA1 as follows: by P 43 Over-expression of deoxyxanthine flutazocine synthetase on bacillus natto chromosome by promoter（mqnBGenebank ID:1099767 A) gene to obtain a strainBacillus subtilis natto, P 43 -mqnA P 43 -mqnBThis was designated NA2.
Furthermore, the recombinant bacteria are modified based on NA2 as follows: by means ofP hbs The promoter (the sequence is shown as SEQ ID NO. 2) over-expresses O-succinylbenzoic acid-CoA ligase on the bacillus natto genomemqnCGenebank ID:1099990 A) gene, to obtainBacillus subtilis natto,P 43 -mqnA P 43 -mqnB P hbs - mqnCThis was designated NA3.
Furthermore, the recombinant bacteria are modified based on NA4 as follows: by using a composition containingP hbs Substitution of the isochorismate on the chromosome of Bacillus natto with the promoter overexpressing the 1, 4-dihydroxy-6-naphthoate synthase (mqnD, genebank ID: 1099766) genedhbBGenebank ID:936582 Gene to obtain strainBacillus subtilis natto,P 43 - mqnA P 43 -mqnB P hbs -mqnC P hbs -mqnD ΔdhbBThis was designated NA4.
Further, the seed culture medium formula of the strain comprises the following raw materials in percentage by mass: 1% of tryptone, 0.5% of yeast extract and 1% of sodium chloride;
the fermentation medium formula comprises the following raw materials in percentage by mass: 3% of yeast extract, 4% of soybean peptone, 3% of glucose and 2% of sucrose.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention constructs 4 recombinant strains NA 1-NA 4 through modifying related genes of vitamin K2 biosynthesis pathway, wherein NA 3-NA 4 obviously improves the yield of vitamin K2, and respectively reaches 160 mg/L and 220mg/L, which are wild bacillus natto of original strainsBacillus subtilis natto) The yields were 3.2 and 4.4 times.
(2) The invention relates to a method for constructing vitamin K 2 Provides a theoretical basis for the high-yield strain of the strain.
Drawings
FIG. 1 is a gel diagram of the genes mqnA, mqnB, mqnC, mqnD of the present invention;
FIG. 2 shows the results of PCR validation of NA1 colonies of the invention; m: a marker;1: colony PCR results; 2: colony PCR results;
FIG. 3 is a graph showing the effect of the enhanced mqn gene cluster of the invention on yield;
FIG. 4 is a graph of transcript levels of the present invention over-expressing mqn gene clusters, mqnA, mqnB, mqnC, mqnD.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The bacillus natto of the inventionBacillus subtilis natto) Purchased from China center for type culture Collection of microorganisms, and the preservation number is CICC No.10260.
TABLE 1 genotype of strains
| Strain | Features (e.g. a character) |
| bacillus subtilis natto | Wild type |
| NA1 | Bacillus subtilis natto, P 43 -mqnA |
| NA2 | Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB |
| NA3 | Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB P hbs -mqnC |
| NA4 | Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB P hbs -mqnC P hbs -mqnD ΔdhbB |
Table 2 sequence listing
| Sequence(s) | Numbering device |
| P 43 Promoter sequence | SEQ ID NO.1 |
| P hbs Promoters | SEQ ID NO.2 |
| lox71-zeo-lox66 box | SEQ ID NO.3 |
| MqnA gene fragment | SEQ ID NO.4 |
| mqnB gene fragment | SEQ ID NO.5 |
| mqnCGene fragment | SEQ ID NO.6 |
| mqnDGene fragment | SEQ ID NO.7 |
Example 1: construction of recombinant NA1
The flutalocine synthase gene mqnA (mqnA, genebank ID: 1099946) was overexpressed on the chromosome of Bacillus natto by means of a constitutive promoter P43. Using a marker-free genetic modification strategy, see articles (Yan, x., yu, h. -j., hong, q., li, s.p., 2008. Cre/lox system and PCR-based genome engineering in Bacillus peptides, appl Environ microb, 74, 5556-5562), the specific construction procedure is as follows:
(1) Cloning of genes
(1) The bacillus natto genome is used as a template, and the primers mqnA-up. For and mqnA-up. REV are adopted to amplify to obtain an upstream homologous arm sequence mqnA-up of the mqnA gene.
(2) The lox71-zeo-lox66 cassette containing bleomycin gene is synthesized artificially (the sequence is shown as SEQ ID NO. 3).
(3) The bacillus natto genome is used as a template, and primers P43.for and P43.Rev are adopted for amplification to obtain a P43 promoter sequence (the sequence is shown as SEQ ID NO. 1).
(4) The genome of Streptomyces coelicolor is used as a template, and the primers mqnA.FOR and mqnA.REV are adopted to amplify to obtain the mqnA gene fragment (the sequence is shown as SEQ ID NO. 4).
And (3) taking the bacillus natto genome as a template, and adopting primers mqnA-down. For and mqnA-down. REV to amplify to obtain a downstream homologous arm sequence mqnA-down of the mqnA gene.
(2) Fusion fragment acquisition
Overlapping extension PCR is carried out on five fragments mqnA-up, lox71-zeo-lox66 box, P43 promoter sequence and mqnA-down gene fragment obtained in the step (1), and PCR conditions are as follows: pre-denaturing at 98 ℃ for 5 min, then performing denaturation at 98 ℃, 10s,55 ℃, annealing at 5s,72 ℃ and extending for 2min for 30 cycles in total, and cutting gel to recover fragments with correct sizes to obtain fusion gene fragments mqnAbp-lox 71-zeo-lox 66-P43-mqnA-mqnAldown.
(3) Homologous recombination
Transforming the fusion fragment obtained in the step (2) into competent cells of wild-type strain Bacillus subtilis natto. Since the upstream sequence of mqnA in the fusion fragment is homologous to the upstream sequence gene of amyE on the chromosome of bacillus natto, the downstream sequence gene of mqnA in the fusion fragment is homologous to the downstream sequence gene of amyE on the chromosome of bacillus natto, and the amyE locus on the chromosome of bacillus natto is replaced by the bleomycin resistance gene zeo and the P43 promoter in the fusion fragment through homologous recombination, and the primer sequences are shown in Table 3. The method comprises the following specific steps:
(1) electrically transforming competent cells of bacillus natto by the fusion fragments constructed in the step (2), wherein the addition amount of the fusion fragments is 100-300ng, and the electric transformation conditions are as follows: the voltage is 2.5kV, the electric shock time is 5ms, the electric shock is carried out at 37 ℃ for 5h, the electric shock is applied to a bleomycin resistant LB plate with the final concentration of 10 mug/mL, and the electric shock is cultured for 48h at 37 ℃ in an anaerobic mode. The positive bleomycin resistance is the successfully transformed bacillus natto.
(2) Single colonies grown on the plates were picked and subjected to colony PCR verification using primers NA1 YZ.FOR and NA1 YZ.REV, and the fragment length amplified after substitution was 1000bp (see FIG. 1). And sequencing, wherein the correct sequencing is that lox71-zeo-lox66-P43 fusion gene is successfully substituted for amyE locus on bacillus natto chromosome. Finally, the bleomycin resistance gene zeo is knocked out by a Cre/lox recombination system, and a strain Bacillus subtilis natto, P43-mqnA, which is named NA1, is finally obtained.
TABLE 3 primer sequence listing
| Primer(s) | Sequence (5 '-3') | Numbering device |
| mqnA-up.for | GGATGGCACGAAGCATTTCCGTTAT | SEQ ID NO.8 |
| mqnA-up.rev | TCTTGACACTCCTTATTTGATTTTTTGAAGAC | SEQ ID NO.9 |
| mqnA-down,for | ATTACTGAAACCGTAAGTTCCAATACGGAGAAATCCTGCAGGATA | SEQ ID NO.10 |
| mqnA-down.rev | CCCGCTCCGATTAAAGCTACTTTAT | SEQ ID NO.11 |
| mqnA-YZ.for | CAATTGCGGTCGCCTTTGC | SEQ ID NO.12 |
| mqnA-YZ.rev | TCATATTGGCGCGAAGGGCAG | SEQ ID NO.13 |
| mqnA.for | TAGGTAAGAGAGGAATGTACACGTGGATAATTCTAGAACACGCCCGAGA | SEQ ID NO.14 |
| mqnA.rev | TTTCTCCGTATTGGAACTTACGGTTTCAGTAATTCAACTTTGACATC | SEQ ID NO.15 |
Example 2: construction of recombinant NA2
Based on the strain NA1 obtained in example 1, P was used in a similar manner to example 1 43 The promoter over-expresses deoxyxanthine flutazocine synthetase on bacillus natto chromosomemqnBGenebank ID:1099767 The specific construction process of the gene is as follows:
(1) Fusion fragment acquisition
Respectively amplifying the bacillus natto genome serving as a template to obtainmqnBUpstream homology arm sequence of genemqnB- up、mqnBGene fragment and P 43 The promoter sequence is synthesized into lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2), and then four pieces are addedSegment mqnB-up, lox71-zeo-lox66 box, P 43 A promoter sequence,mqnBCarrying out overlap extension PCR on the gene fragments to obtain fusion gene fragmentsmqnB up -lox71-zeo-lox66-P 43 -mqnB-mqnB down 。
(2) Homologous recombination
The fusion fragment obtained in step (1) was transformed into BS1 competent cells and plated on bleomycin-resistant LB plates, single colonies grown on the plates were picked, colony PCR verified and sequenced as shown in fig. 2. Finally, the bleomycin resistance gene in the strain is knocked out through a Cre/lox recombination systemzeoFinally obtaining the strainBacillus subtilis natto, P 43 -mqnA P 43 -mqnBThis was designated NA2.
Example 3: construction of recombinant NA3
Based on the strain NA2 obtained in example 2, a method similar to that of example 1 was employedP hbs The promoter (the sequence is shown as SEQ ID NO. 5) replaces O-succinylbenzoic acid-CoA ligase in bacillus nattomqnCGenebank ID:1099990 A natural promoter of a gene, and the specific construction process is as follows:
(1) Fusion fragment acquisition
Respectively amplifying the bacillus natto genome serving as a template to obtainmqnCUpstream homology arm sequence of genemqnC- up、mqnCGene fragmentP hbs The promoter sequence is artificially synthesized into a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2). Then four fragments, mqnC-up, lox71-zeo-lox66 cassette,P hbs A promoter sequence,mqnCCarrying out overlap extension PCR on the gene fragments to obtain fusion gene fragmentsmqnC up -lox71-zeo-lox66-P hbs -mqnC。
(2) Homologous recombination
Transforming the fusion fragment obtained in the step (1) into NA2 competent cells, and knocking out bleomycin resistance genes in the strain through a Cre/lox recombination systemzeoObtaining the strainBacillus subtilis,P 43 -mqnA P 43 -mqnB P hbs -mqnCThis was designated BS3.
Example 4: construction of recombinant bacterium BS4
Based on the strain NA3 obtained in example 3, the method similar to example 1 was used with a strain containingP hbs Substitution of the promoter 1, 4-dihydroxy-6-naphthoic acid synthase (mqnD, genebank ID: 1099766) gene derived from Streptomyces coelicolor for isochoric acid on the chromosome of Bacillus nattodhbBGenebank ID:936582 A) gene. The specific construction process is as follows:
(1) Fusion fragment acquisition
Respectively amplifying the bacillus natto genome serving as a template to obtaindhbBUpstream homology arm sequence of genedhbB- up、dhbBDownstream homology arm sequencesdhbB-down、P 43 A promoter sequence; artificially synthesizing a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2); amplification is carried out by taking Streptomyces coelicolor genome as templatemqnDGene sequence, five fragments were then useddhbB-upA lox71-zeo-lox66 cassette,P 43 A promoter sequence,mqnDA gene sequence,dhbB-downCarrying out overlap extension PCR on the gene fragments to obtain fusion gene fragmentsdhbB up -lox71-zeo-lox66-P 43 -entC-dhbB down 。
(2) Homologous recombination
The fusion fragment obtained in the step (1) is transformed into NA3 competent cells, and then the bleomycin resistance gene zeo in the strain is knocked out through a Cre/lox recombination system to obtain a strain Bacillus subtilis, P43-mqnF P43-mqnB Phbs-mqnC Phbs-mqnD delta dhbB, which is named as NA4.
Example 5: production of vitamin K by bacterial strain fermentation 2
Seed culture medium formula (mass percent): tryptone 1%, yeast extract 0.5%, sodium chloride 1%.
Fermentation medium formula (mass percent): 3% of yeast extract, 4% of soybean peptone, 3% of glucose and 2% of sucrose.
(1) Seed liquid preparation
The wild strain bacillus natto and the recombinant bacteria NA 1-4 constructed in the examples 1-4 are respectively inoculated into a seed culture medium, and are cultured at 37 ℃ and 220 rpm for 12 h to obtain bacillus natto seed liquid.
(2) Fermentation culture
Transferring the seed solution obtained in the step (1) into a fermentation medium at an inoculum size of 5%, culturing at 41 deg.C and 220 rpm for 4 days, and collecting the fermentation solution to measure vitamin K 2 Is contained (see table 4).
Table 4 fermentation production of vitamin K by bacterial strains 2
| Strain | VK2 yield (mg/L) | Relative MK-7 content |
| BS natto (original Strain) | 50 | 1 |
| NA1 | 60 | 1.2 |
| NA2 | 80 | 1.6 |
| NA3 | 160 | 3.2 |
| NA4 | 220 | 4.4 |
Vitamin K 2 The detection method comprises the following steps: adding 4 times volume of isopropyl alcohol and n-hexane mixture (1:2V/V) into the fermentation broth, extracting for 30 min by vortex oscillation, filtering out the extract, centrifuging for 15 min at 8000 r/min. Collecting supernatant, dissolving MK-7 in the phase, freezing at-80deg.C, crystallizing lipid substances, collecting filtrate, and detecting MK-7 content by HPLC.
HPLC detection of vitamin K 2 Yield: using a Thermo ODC-C18 separation column (5 μm, 250X 4.6 mm), the temperature was measured at 40℃and methanol was used as the mobile phase: dichloromethane (9:1, v/v), flow rate of 1 mL/min, detection wavelength 254 nm, and sample injection amount of 10. Mu.L.
Enhancement of vitamin K by mqn Gene Cluster 2 The effect of yield is shown in FIG. 3, the transcript levels of the overexpressed mqn gene clusters, mqnA, mqnB, mqnC, mqnD are shown in FIG. 4.
Taking bacillus natto (Bacillus subtilis natto) as an initial strain, and utilizing a P43 promoter to heterologously express a fluorine tamsulosin synthase gene mqnA and a deoxidized xanthine fluorine tamsulosin synthase gene mqnB on a genome of the bacillus natto; the O-succinylbenzoate-CoA ligase gene mqnC and 1, 4-dihydroxy-6-naphthoate synthase mqnD were expressed on the chromosome using the Phbs promoter.
P43 promoter sequence
TgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcGGTACCATTATAGGTAAGAGAGGAATGTACAC
Phbs promoter sequence
Ggatgatccgctctccatctggaattccgatatcttcggaaggtcgctgagctcaattgtgagagaagggattcaggcaaagctgtcattgatgcctgaaaacgcacggtataaattaaaagaaacattagaaagaatcataaacgaaggctctggcggcttaatcgccatcatcctgtaataccggtagacctctttatagaatgggaggtcttttttctttgctcttaataatggaaaaggatcaaggaataggatgaaaaaaggaaaaaaaggaatattcgttcggtaaatcaccttaaatccttgacgagcaagggattgacgctttaaaatgcttgatatggctttttatatgtgttactctacatacagaaattcttcactttgttggacaaacattcctcagagtgcagtttttcttaaaaagccgtttaattgtctttctcttacttgctctcatttttttctgagacaggtttagaatcagactgaactgtgaagaaatgataataaacgaactgaatgtatccttttgggaggaggtgaaaggc
lox71-zeo-lox66 box
gagcggataacaatttcacacaggaaacagctatgaccatgattacgaattcgagctcggtacccggggatcctctagagataccgttcgtatagcatacattatacgaagttatcttgatatggctttttatatgtgttactctacatacagaaaggaggaactaaacatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattatacgaacggtaaatcgtcgac
MqnA gene fragment
GTGGATAATTCTAGAACACGCCCGAGAGTCGGCCATATTCAATTTCTTAACTGCCTTCCGTTATATTGGGGCTTAGCGCGCACAGGAACACTGCTTGATTTTGAACTTACAAAAGATACACCGGAAAAACTGTCAGAACAGCTGGTCAGAGGCGATTTAGATATCGGACCGGTGACACTTGTCGAATTTCTGAAAAACGCAGATGATCTGGTTGCGTTTCCGGATATTGCTGTTGGCTGCGATGGACCGGTGATGAGCTGTGTTATCGTGTCTCAAGTTCCGCTTGATCGCTTAGATGGCGCAAGAGTGGCGCTGGGATCAACAAGCCGCACATCAGTCAGACTTGCTCAATTACTGCTTAGCGAACGCTTTGGCGTGCAGCCGGATTATTATACATGTCCGCCGGATCTGTCACTTATGATGCAGGAAGCTGATGCAGCGGTTCTGATTGGAGATGCTGCCCTTCGCGCCAATATGATCGATGGCCCGAGATATGGATTAGATGTGCATGATCTGGGCGCCCTTTGGAAAGAATGGACAGGACTGCCGTTTGTTTTTGCAGTGTGGGCAGCGAGACGCGATTATGCGGAACGCGAACCGGTGATTACAAGAAAAGTCCATGAAGCTTTTCTTGCCTCTCGCAACTTATCACTGGAAGAAGTCGAAAAAGTTGCAGAACAAGCTGCCAGATGGGAAGCTTTTGATGAAGATACACTGGCCAAATACTTTACAACACTGGATTTTAGATTTGGAGCCCCGCAGTTAGAAGCAGTCACAGAATTTGCGAGACGCGTTGGCCCGACAACAGGATTTCCGGCGGATGTCAAAGTTGAATTACTGAAACCGTAA
mqnB gene fragment
ATGTCCTGCTCCAGGATCTCCTTCCACAGGCTGTCTCACTGCAGGGACTTGTTGTCCGCCCGGATGTGCTCTTCCCGCTCCGCCTCCGCCGCGTCCGCCTCCCTGTCCTCTGCCGCCTTCAACAGCAGGTCCAACAACTTGATCATCTCTTCCTGTATCAGCCGCTGCTTCGCGGCCTAATCTTCCATCCGCGCCTTGATCATCTCCGACTCTATCGCCTCCGCCCACTTGAGCTCCAAGTCCGCCCACCGCTTCTTGTCTATCTCCAACTTCTGCTTCGGCTCCATCGCCAACCTGGCGTCTTCCGCGTTGATCAGCTCCGCCTCCCAGTCTTCCCGGTCCAGGTCCAGCTCCATGCTGTCCAGGGCCTCCATCTCCTGCGCCGCGTCCGGCGCCTCTCTGTTGAGCCAGAGCAACTCTAGCTCCTTGTCCTAATCTATGCAGTGCTTCGGCATCTCCTAAGCCTCCGCCGCGCACTGCATTTCTATGCTGAAATTGGGCTCTATCGCCAATGCGATCGCTTCTAGCAACCGCTCCGCCCGGTGCATCAGCTAATCTTTCGCGTCCTTGGCGGACTCCATCCGGAAGTTCGCAGCCATCTTTCAGTGTCGGAAATCTCATTAAGGCTGA
mqnC gene fragment
GTCACAGAAAAAGCGGATCTTCAACCGATTTTAGATAGAGCAGCTGCTGGCGGAAGAATCACACCTGAAGAAGCGCTTGATTTATATAGAGATGCCCCGTTACATGCACTGGGAGCTGCAGCTGATGCTGTTAGACGCAGACGCTATGCCGGAACAGAACATATCGCAACATACATCATCGAAAGAAACATCAACTATACAAATGTTTGCGTGACAGCGTGCAAATTTTGTGCTTTTTATGCTGCCCCGAAAGATACAAAGAAAGGCTGGAGCCGCGATCTGGATGATATTCTTAGACGCTGTGCCGAAACAGTCGAATTAGGCGGAACACAAATCATGTTTCAGGGCGGACATCATCCGGATTACGGCGTTGAATACTACGAAGAACATTTTGCAGCGATTAAGAAAGAATTTCCGCAACTTGTTATCCATTCTTTAGGAGCCTCAGAAGTGGAACATATGGCAAGAATTTCAAAAGTCAGCGTTGAAGAAGCTATTAGACGCATCCATGCTGCCGGCCTTGATTCATTTGCAGGCGCGGGAGCTGAACTGCTTCCGGAAAGACCGCGCAAAGCCATCGCACCGCTTAAAGAAAGCGGAGAAAGATGGTTAGAAATTATGGAAATCGCGCATGGCCTGGGAGTTGAAAGCACATCTACAATGCTTATGGGCACAGGAGAAACAAATGCTGAACGCATTGAACATCTTAGAATGATCCGCGATGTGCAGGATAGAACAGGCGGATTTCGCGCCTTTATTCCGTACACATACCAACCGGAAAACAACCATCTGAAAGGCAGAACACAGGCAACACTGTTTGAATACCTGAGAATGATCGCGATCGCTCGCGTGTTTTTAGATAACGTCGCGCATATTCAAGGATCTTGGCTGACAACAGGCAAAGAAGTGGGACAGCTGAGCCTTCATTATGGCGCTGATGATCTGGGATCTATTATGCTTGAAGAAAATGTTGTGTCAAGCGCCGGCGCAAAACATAGATCAAACCGCCTGGAAATCATCGATCTGATCAGAAAAGCTGGAAGAGTGCCTGCTCAGAGAGCTACAACATATGAACATCTGGTCGTTCATGATGATCCGGCAAATGATCCGGTCGATGAACGCGTGGTCTCTCATATTTCTTCAACAGCGATCGAAGGCGGAACAGCTCATCCGGAACTTAAATTACTGGCACCGAACTAA
mqnD gene fragment
ATGCGCAGAAACGCCTTGTAAATCGCGAACGCCGCCTGCTCTCACAACTCCTTCATCTTCAACGCCTTCTCCGACCGCTGCATTTCTCGCGCCTCCGCCTTCCACATGGCCTTCTCTGACATCCACATCGCCCACTGCATGTCCTAAGGCAGCAAGCTGAACATGTTCAAGATGTCTGACGCCATGTTGTCCCACATCTTCGACCACCGGTCTCTGTTGTCCAGCTGCAGCTCTCTGTGCAGGAGCAGCTGCTCTCTGATGTTGTCCCGGCAGGCGAACTCTGACTTGGGCCGGAGCGCGATGTCTATGGCCAGCTAAGCGTCTGCGTCCGACCTGTTGTTCCGGCTGAGGTCTGCGCACTCCATGTCCAGCAGGATGTGCTAAATCATCATCATGGCGTTCGACAAGATCATGTCTGCCATGCGGCACCGGAAGATCAACTCTGGCCTGATGATCCACTAAGCCAGCTTCGCGAACAAGAACGACCGGTTGGACCAGTTCTCCCACATGGGCCAGCACCGGTAAAACTCCGCCCGGTTGGCGATCTCTTTCAGCGCGATCATCTCTCGGGGCTTACTGAGCTCTGCGTCTTTGTCCCGGTTCGCGCACTCTATGTGCGCCTCTATCTGCTCTTCCAGGCACAACTCTCAGTCTTCCAGGTCCCACATCATGTAAGACGCGAAGCAGATGAACTCTGCCATCTCCCACCAGAACATCCGGCTGGACATCCACAAGTTCTCCGCCGACTTCCGCCAGAACTGCGACGCGTCTATCGCTGGCCTGTTGGCCAGCGCGTCTTCCAAGTGATTCATATCTTCCTTCAGCGCGAACTCTTTGGCGTTCTCTCAG。
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Sequence listing
<110> West Bao Biotechnology (Shanghai) Co., ltd
<120> a method for improving vitamin K2 production using recombinant Bacillus natto
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>300
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>1
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300
<210>2
<211>554
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>2
ggatgatccg ctctccatct ggaattccga tatcttcgga aggtcgctga gctcaattgt 60
gagagaaggg attcaggcaa agctgtcatt gatgcctgaa aacgcacggt ataaattaaa 120
agaaacatta gaaagaatca taaacgaagg ctctggcggc ttaatcgcca tcatcctgta 180
ataccggtag acctctttat agaatgggag gtcttttttc tttgctctta ataatggaaa 240
aggatcaagg aataggatga aaaaaggaaa aaaaggaata ttcgttcggt aaatcacctt 300
aaatccttga cgagcaaggg attgacgctt taaaatgctt gatatggctt tttatatgtg 360
ttactctaca tacagaaatt cttcactttg ttggacaaac attcctcaga gtgcagtttt 420
tcttaaaaag ccgtttaatt gtctttctct tacttgctct catttttttc tgagacaggt 480
ttagaatcag actgaactgt gaagaaatga taataaacga actgaatgta tccttttggg 540
aggaggtgaa aggc 554
<210>3
<211>588
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>3
gagcggataa caatttcaca caggaaacag ctatgaccat gattacgaat tcgagctcgg 60
tacccgggga tcctctagag ataccgttcg tatagcatac attatacgaa gttatcttga 120
tatggctttt tatatgtgtt actctacata cagaaaggag gaactaaaca tggccaagtt 180
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 240
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 300
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 360
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 420
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 480
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 540
ctgaataact tcgtatagca tacattatac gaacggtaaa tcgtcgac 588
<210>4
<211>849
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>4
gtggataatt ctagaacacg cccgagagtc ggccatattc aatttcttaa ctgccttccg 60
ttatattggg gcttagcgcg cacaggaaca ctgcttgatt ttgaacttac aaaagataca 120
ccggaaaaac tgtcagaaca gctggtcaga ggcgatttag atatcggacc ggtgacactt 180
gtcgaatttc tgaaaaacgc agatgatctg gttgcgtttc cggatattgc tgttggctgc 240
gatggaccgg tgatgagctg tgttatcgtg tctcaagttc cgcttgatcg cttagatggc 300
gcaagagtgg cgctgggatc aacaagccgc acatcagtca gacttgctca attactgctt 360
agcgaacgct ttggcgtgca gccggattat tatacatgtc cgccggatct gtcacttatg 420
atgcaggaag ctgatgcagc ggttctgatt ggagatgctg cccttcgcgc caatatgatc 480
gatggcccga gatatggatt agatgtgcat gatctgggcg ccctttggaa agaatggaca 540
ggactgccgt ttgtttttgc agtgtgggca gcgagacgcg attatgcgga acgcgaaccg 600
gtgattacaa gaaaagtcca tgaagctttt cttgcctctc gcaacttatc actggaagaa 660
gtcgaaaaag ttgcagaaca agctgccaga tgggaagctt ttgatgaaga tacactggcc 720
aaatacttta caacactgga ttttagattt ggagccccgc agttagaagc agtcacagaa 780
tttgcgagac gcgttggccc gacaacagga tttccggcgg atgtcaaagt tgaattactg 840
aaaccgtaa 849
<210>5
<211>630
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>5
atgtcctgct ccaggatctc cttccacagg ctgtctcact gcagggactt gttgtccgcc 60
cggatgtgct cttcccgctc cgcctccgcc gcgtccgcct ccctgtcctc tgccgccttc 120
aacagcaggt ccaacaactt gatcatctct tcctgtatca gccgctgctt cgcggcctaa 180
tcttccatcc gcgccttgat catctccgac tctatcgcct ccgcccactt gagctccaag 240
tccgcccacc gcttcttgtc tatctccaac ttctgcttcg gctccatcgc caacctggcg 300
tcttccgcgt tgatcagctc cgcctcccag tcttcccggt ccaggtccag ctccatgctg 360
tccagggcct ccatctcctg cgccgcgtcc ggcgcctctc tgttgagcca gagcaactct 420
agctccttgt cctaatctat gcagtgcttc ggcatctcct aagcctccgc cgcgcactgc 480
atttctatgc tgaaattggg ctctatcgcc aatgcgatcg cttctagcaa ccgctccgcc 540
cggtgcatca gctaatcttt cgcgtccttg gcggactcca tccggaagtt cgcagccatc 600
tttcagtgtc ggaaatctca ttaaggctga 630
<210>6
<211>1200
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>6
gtcacagaaa aagcggatct tcaaccgatt ttagatagag cagctgctgg cggaagaatc 60
acacctgaag aagcgcttga tttatataga gatgccccgt tacatgcact gggagctgca 120
gctgatgctg ttagacgcag acgctatgcc ggaacagaac atatcgcaac atacatcatc 180
gaaagaaaca tcaactatac aaatgtttgc gtgacagcgt gcaaattttg tgctttttat 240
gctgccccga aagatacaaa gaaaggctgg agccgcgatc tggatgatat tcttagacgc 300
tgtgccgaaa cagtcgaatt aggcggaaca caaatcatgt ttcagggcgg acatcatccg 360
gattacggcg ttgaatacta cgaagaacat tttgcagcga ttaagaaaga atttccgcaa 420
cttgttatcc attctttagg agcctcagaa gtggaacata tggcaagaat ttcaaaagtc 480
agcgttgaag aagctattag acgcatccat gctgccggcc ttgattcatt tgcaggcgcg 540
ggagctgaac tgcttccgga aagaccgcgc aaagccatcg caccgcttaa agaaagcgga 600
gaaagatggt tagaaattat ggaaatcgcg catggcctgg gagttgaaag cacatctaca 660
atgcttatgg gcacaggaga aacaaatgct gaacgcattg aacatcttag aatgatccgc 720
gatgtgcagg atagaacagg cggatttcgc gcctttattc cgtacacata ccaaccggaa 780
aacaaccatc tgaaaggcag aacacaggca acactgtttg aatacctgag aatgatcgcg 840
atcgctcgcg tgtttttaga taacgtcgcg catattcaag gatcttggct gacaacaggc 900
aaagaagtgg gacagctgag ccttcattat ggcgctgatg atctgggatc tattatgctt 960
gaagaaaatg ttgtgtcaag cgccggcgca aaacatagat caaaccgcct ggaaatcatc 1020
gatctgatca gaaaagctgg aagagtgcct gctcagagag ctacaacata tgaacatctg 1080
gtcgttcatg atgatccggc aaatgatccg gtcgatgaac gcgtggtctc tcatatttct 1140
tcaacagcga tcgaaggcgg aacagctcat ccggaactta aattactggc accgaactaa 1200
<210>7
<211>849
<212>DNA
<213> Bacillus natto (Bacillus subtilis natto)
<400>7
atgcgcagaa acgccttgta aatcgcgaac gccgcctgct ctcacaactc cttcatcttc 60
aacgccttct ccgaccgctg catttctcgc gcctccgcct tccacatggc cttctctgac 120
atccacatcg cccactgcat gtcctaaggc agcaagctga acatgttcaa gatgtctgac 180
gccatgttgt cccacatctt cgaccaccgg tctctgttgt ccagctgcag ctctctgtgc 240
aggagcagct gctctctgat gttgtcccgg caggcgaact ctgacttggg ccggagcgcg 300
atgtctatgg ccagctaagc gtctgcgtcc gacctgttgt tccggctgag gtctgcgcac 360
tccatgtcca gcaggatgtg ctaaatcatc atcatggcgt tcgacaagat catgtctgcc 420
atgcggcacc ggaagatcaa ctctggcctg atgatccact aagccagctt cgcgaacaag 480
aacgaccggt tggaccagtt ctcccacatg ggccagcacc ggtaaaactc cgcccggttg 540
gcgatctctt tcagcgcgat catctctcgg ggcttactga gctctgcgtc tttgtcccgg 600
ttcgcgcact ctatgtgcgc ctctatctgc tcttccaggc acaactctca gtcttccagg 660
tcccacatca tgtaagacgc gaagcagatg aactctgcca tctcccacca gaacatccgg 720
ctggacatcc acaagttctc cgccgacttc cgccagaact gcgacgcgtc tatcgctggc 780
ctgttggcca gcgcgtcttc caagtgattc atatcttcct tcagcgcgaa ctctttggcg 840
ttctctcag 849
Claims (2)
1. Vitamin K improvement by utilizing recombinant bacillus natto 2 A method of producing yield comprising the steps of:
step 1: construction of recombinant NA1
By constitutive promoter P 43 Over-expressing a flutolosine synthase gene mqnA on a bacillus natto chromosome, wherein the bacillus natto is a wild type;
step 2: construction of recombinant NA2
Based on the strain NA1 obtained in the step 1, P is utilized 43 The promoter over-expresses the gene of deoxyxanthine flutazocine synthetase mqnB on bacillus natto chromosome;
step 3: construction of recombinant NA3
Based on the strain NA2 obtained in the step 2, P is utilized hbs The promoter replaces the natural promoter of the O-succinylbenzoic acid-CoA ligase mqnC gene in bacillus natto;
step 4: construction of recombinant NA4
Based on the strain NA3 obtained in step 3, a strain containing P was used hbs The promoter 1, 4-dihydroxy-6-naphthoic acid synthase mqnD gene from Streptomyces coelicolor replaces isochorismate dhbB gene on bacillus natto chromosome;
step 5: production of vitamin K by bacterial strain fermentation 2
Inoculating the seed solution of the recombinant NA4 into a fermentation medium according to the inoculum size of 3% -6%;
wherein the recombinant NA1 in step 1 is Bacillus subtilis natto, P 43 -mqnA, which is a sequence inserted at chromosomal amyE site, the sequence comprising promoter P 43 Fusion of the mqnA gene to enhance expression of the flutoloxin synthase mqnA gene, and designated NA1;
wherein, the recombinant bacteria NA2 in the step 2 is modified based on NA1 as follows: by P 43 The promoter over-expresses the gene of deoxyxanthine flutazocine synthetase mqnB on bacillus natto chromosome to obtain strain Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB, named NA2;
wherein, the recombinant bacteria NA3 in the step 3 is modified based on NA2 as follows: by P hbs The promoter over-expresses the gene of O-succinylbenzoic acid-CoA ligase mqnC on the genome of bacillus natto to obtain Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB P hbs -mqnC, designated NA3;
wherein, the recombinant bacteria NA4 in the step 4 is modified based on NA3 as follows: by a composition containing P hbs The promoter overexpresses 1, 4-dihydroxyl-6-naphthoic acid synthase mqnD gene to replace isochorismate dhbB gene on bacillus natto chromosome to obtain strain Bacillus subtilis natto, P 43 -mqnA P 43 -mqnB P hbs -mqnC P hbs -mqnD Δdhbb, designated NA4;
the P is 43 The sequence of the promoter is shown as SEQ ID NO. 1; the P is hbs The sequence of the promoter is shown as SEQ ID NO. 2; the sequence of the flutarone synthase gene mqnA is shown in SEQ ID NO. 4; the sequence of the deoxyxanthine flutazocine synthetase mqnB is shown in SEQ ID NO. 5; the sequence of the O-succinylbenzoic acid-CoA ligase mqnC is shown in SEQ ID NO. 6; the gene sequence of the 1, 4-dihydroxy-6-naphthoic acid synthase mqnD is shown in SEQ ID NO. 7; the sequence of the isochoric acid dhbB is as genebank ID: 936582.
2. The method for increasing vitamin K using recombinant Bacillus natto of claim 1 2 The fermentation medium formula comprises the following raw materials in percentage by mass: 3% of yeast extract, 4% of soybean peptone, 3% of glucose and 2% of sucrose.
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| JP2000287676A (en) * | 1999-04-07 | 2000-10-17 | Mitsukan Group Honsha:Kk | Bacillus natto capable of highly producing vitamin k2, its breeding and fermented soybean |
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