CN108277189A - A kind of engineered strain producing riboflavin and its application - Google Patents
A kind of engineered strain producing riboflavin and its application Download PDFInfo
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- CN108277189A CN108277189A CN201710008300.8A CN201710008300A CN108277189A CN 108277189 A CN108277189 A CN 108277189A CN 201710008300 A CN201710008300 A CN 201710008300A CN 108277189 A CN108277189 A CN 108277189A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
Abstract
The present invention provides a kind of engineered strain producing riboflavin and its applications.Specifically, the present invention provides the engineered bacterials that one kind is obtained by genetic modification (gene knockout or gene integration), include the transformation to riboflavin synthetic gene and its promoter, which goes out stable riboflavin production capacity, can be used for the production of riboflavin.
Description
Technical field
The present invention relates to biotechnology, relate more specifically to a kind of engineered strain producing riboflavin and its application.
Background technology
Riboflavin (Riboflavin), i.e. vitamin B2 (VB2) are a kind of water-soluble B family vitamins, are flavine enzyme
Coenzyme component part, breathe and biological oxidation in play an important role, be a kind of vitamin necessary to human and animal,
Medicine, food and feed industry are mainly used in as food, feed addictive and edible natural pigment etc..
Before nineteen seventies, Production by Microorganism Fermentation riboflavin is mainly with cotton capsule Ah's Shu Shi yeast (Ashbya
Gossypii), proteolysis Candida (Candida famata), Ah's Shu Shi vacation capsules yeast (Eremothecium ashbyii) etc.
Based on fungi.Build the riboflavin-produced engineering bacterias such as successful Bacillus subtilis in succession, because it is short with fermentation period,
The advantages that raw material is cheap and easy to get, prokaryotic cell technique for gene engineering is ripe, becomes primary study object.In addition, in bacillus
In category, many bacterial strains including Bacillus subtilis have reliable safety, their tunning is in food
With existing long-term application in feed industry.
Riboflavin production bacterium remodeling method traditional at present is to carry out induction mutation of bacterium, such as physical mutagenesis, mutagenesis etc..
Physical mutagenesis, mutagenesis method carry out strain transformation, randomness is bigger, causes screening operation amount huge, and time-consuming, sees
Effect is slow.And the genetic stability of strain gene is poor, and gene may restore to be mutated in succeeding generations, and strain riboflavin is caused to synthesize
Ability declines.Therefore, there is an urgent need in the art to build the riboflavin of stable, high yield to produce bacterium, to carry out riboflavin production.
Invention content
The purpose of the present invention is to provide a kind of riboflavin of stabilization, high yield production bacterium and its applications.
In the first aspect of the present invention, a kind of engineered strain producing riboflavin is provided, the bacterial strain is bacillus
(Bacillus), it and in the genome of the bacterial strain is integrated with exogenous gene expression box, the exogenous gene expression box contains
There is following elements：P15 promoters and riboflavin operon gene.
In another preferred example, the exogenous gene expression box includes the first exogenous gene expression box and the second external source base
Because of expression cassette.
In another preferred example, the first exogenous gene expression box also contains AmyE Genetic elements.
In another preferred example, the AmyE genes include AmyE genetic fragments.
In another preferred example, the AmyE genetic fragments are the nucleotide that the length at AmyE gene 5 's end is 600bp
Sequence.
In another preferred example, the first exogenous gene expression box is integrated in the AmyE of the engineered strain genome
Downstream of gene.
In another preferred example, the first exogenous gene expression box and the second exogenous gene expression box also contain resistant
Genetic elements.
In another preferred example, the resistance gene element and the second external source base that the first exogenous gene expression box contains
Because the resistance gene element that expression cassette contains is different.
In another preferred example, the resistant gene is selected from the group：Tetracycline resistance gene, kalamycin resistance gene,
Ammonia benzyl mycin resistant gene, or combinations thereof.
In another preferred example, the external source riboflavin operon gene is selected from the group：RibB genes, ribG genes,
RibA genes, ribH genes, ribTD genes, or combinations thereof.
In another preferred example, the engineered strain is the recon obtained through protoplast fusion.
In another preferred example, the copy number of foreign gene (riboflavin operon gene) is in the genome of the bacterial strain
10-50, preferably 20-40.
In another preferred example, 10-50 are integrated in the genome of the bacterial strain, it is preferably outer described in 20-40
Source expression casette.
In another preferred example, be integrated in the genome of the bacterial strain 30-50 copyribGGene.
In another preferred example, the ribA genes of 20-25 copy are integrated in the genome of the bacterial strain.
In another preferred example, the bacterial strain is selected from the group：It is Bacillus subtillis Bacillus subtilis, waxy
Bacillus cereus, bacillus amyloliquefaciens Bacillus amyloliquefaciens, stearothermophilus brood cell
Bacillus Bacillus stearothermophilus, Alkaliphilic bacillus belong to Bacillus halodurans and lichenoid form gemma
Bacillus Bacillus licheniformis.
In another preferred example, the bacterial strain is Bacillus subtillis Bacillus subtilis, preferably
Bacillus subtilis SMY.The Bacillus subtilis VKPM B2116 used in Examples below belong to
Bacillus subtilis SMY Pseudomonas, is preferred.
In the second aspect of the present invention, a kind of method producing riboflavin, including step are provided：
(i) engineered strain described in first aspect present invention is cultivated, to obtain the tunning containing riboflavin；With
(ii) riboflavin is isolated from the tunning.
In the third aspect of the present invention, a kind of method building engineered strain described in first aspect present invention, packet are provided
Include step：
(a) carrier containing the first exogenous gene expression box is built, the first exogenous gene expression box has following member
Part：AmyE genes, P15 promoters and riboflavin operon gene；
(b) carrier containing the second exogenous gene expression box is built, the second exogenous gene expression box has following member
Part：P15 promoters and riboflavin operon gene；
(c) carrier that step (a) and step (b) obtain is transferred to F-strain respectively, obtaining genome conformity has first
The first recombinant bacterial strain and genome conformity of exogenous gene expression box have the second recombinant bacterial strain of the second exogenous gene expression box；
(d) protoplast fusion is carried out to the first recombinant bacterial strain and the second recombinant bacterial strain, obtains and is integrated with the in genome
The recon of one exogenous gene expression box and the second exogenous gene expression box, the recon are first aspect present invention institute
The engineered strain stated.
In another preferred example, the method further includes step (e)：The gene for the recon that PCR verification steps (d) obtain
Type；And/or
Step (f)：The riboflavin production for the recon that fermentation detecting step (d) obtains.
In another preferred example, the carrier is plasmid and/or nucleic acid fragment.
In another preferred example, the also resistant Genetic elements of the carrier.
In another preferred example, the carrier, which also has, terminates subcomponent.
In another preferred example, the F-strain is Bacillus subtillis Bacillus subtilis, preferably
Bacillus subtilis SMY。
In another preferred example, the engineered strain is with method selected from the group below by the vector integration to recipient bacterium
In the genome of strain：Conversion, homologous recombination.
In the fourth aspect of the present invention, a kind of purposes of engineered strain described in first aspect present invention, the bacterium are provided
Strain is used as the engineering bacteria of fermenting and producing riboflavin.
In the fifth aspect of the present invention, a kind of method preparing food compositions, including step are provided：
Riboflavin prepared by second aspect of the present invention the method is added in food compositions, contains core to obtain
The food compositions of flavine.
In another preferred example, the food compositions include fodder compound.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows the structure collection of illustrative plates of pBR25 plasmids.
Fig. 2 shows the structure collection of illustrative plates of pBR31 plasmids.
Fig. 3 shows that VBB8662 and VBB8978 carries out protoplast fusion, and the fusant bacterial strain of screening carries out shake flask fermentation
Experiment, the riboflavin production of each fusant bacterial strain.
Fig. 4 shows in fermentation shake flask experiment that fusant bacterial strain of the riboflavin production more than 5g/L carries out monoclonal screening, obtains
The monoclonal colonies obtained carry out shake flask fermentation experiment, the riboflavin production of each fusant bacterial strain.
Fig. 5 shows the fermentation tank experimental conditions of high-yield monoclonal VBB11863.
Fig. 6 shows the fermentation tank experimental conditions of high-yield monoclonal VBB11866.
Specific implementation mode
The present inventor is surprised to find that a kind of exogenous gene expression of special construction for the first time by depth studying extensively
Box, the exogenous gene expression box include P15 promoters and riboflavin operon gene.Utilize the exogenous gene expression box
Bacillus subtillis is converted, recycles Protoplast Fusion Technique to carry out the fusion of protoplast, riboflavin production can be obtained
The engineered strain significantly improved.What the foreign gene of the engineered strain can be stablized carries out heredity, expression in genome, and again
Renaturation is good, it can be achieved that scale industrial production.
Specifically, the present invention provides a kind of gene order of recombinant bacteria, the gene order is preferred：Including with one
The external source riboflavin operon gene order and tetracycline resistance gene sequence of a P15 promoters, the riboflavin operon base
Because sequence includes：RibB, ribG, ribA, ribH and ribTD；Including there are one the external source riboflavin of P15 promoters for another band
Operon gene sequence and kalamycin resistance gene sequence, the riboflavin operon gene order include：ribB、ribG、
RibA, ribH and ribTD；Said gene sequence is introduced into host cell chromosome by modes such as conversion, homologous recombinations；
RibA, ribG gene exist in host cell chromosome in the form of multicopy, the copy number of gene >=10, the external source core
Flavine operon gene sequence is synthetic gene sequence.
Microorganism of the present invention suitable for riboflavin biosynthesis includes following microbial cells, and the microorganism is thin
Born of the same parents can produce riboflavin (such as given carbon source is converted to riboflavin), and carry unmodified rib operons, packet
Unmodified leader or its equivalent or homologue are included (then in a manner of causing riboflavin production to improve as described herein
It is mutated), or the rib operons/rib leaders or its equivalent through modification version are introduced thereto.With it is such not
The suitable microorganism of modified rib operons/rib leaders or its equivalent can be selected from bacterium, such as Gram-negative
And gram-positive bacterium, it is wild-type strain, by the mutant strain of classical mutagenesis and selection method acquisition, or recombination
Bacterial strain.Preferably, microorganism or host cell are selected from the group being made up of：Bacillus subtillis Bacillus subtilis,
Bacillus cereus Bacillus cereus, bacillus amyloliquefaciens Bacillus amyloliquefaciens, stearothermophilus
Bacillus Bacillus stearothermophilus, Alkaliphilic bacillus belong to Bacillus halodurans, lichenoid form
Bacillus licheniformis.More preferably Bacillus subtilis, especially Bacillus
subtilis SMY。
The strain remodeling method of the present invention is by the gene integration of transformation to strain genome one or more site, gene
What can be stablized carries out heredity, expression in genome, and improved strain has stable riboflavin ability to express.
As used herein, " engineered strain ", " riboflavin production strains ", " recombinant bacteria " are used interchangeably,
Refer to the engineered strain for producing riboflavin of the present invention, i.e. bacterial strain described in first aspect present invention.
As used herein, " AmyE genes " is bacillus subtilis amylase gene, and the gene is for instructing the application's
Exogenous gene expression box is in bacillus gene group site-directed integration.
Exogenous gene expression box
As used herein, term " exogenous gene expression box " refers to the expression alien gene (riboflavin with P15 promoters
Operon gene) expression cassette.The exogenous gene expression box includes the first exogenous gene expression box and the second exogenous gene expression
Box.Wherein, the first exogenous gene expression box also contains AmyE genes in addition to containing P15 promoters and riboflavin operon gene
Segment, for instructing the first exogenous gene expression box site-directed integration in the downstream of bacillus gene group AmyE genes.Outside second
Source expression casette random integration is in bacillus gene group.
The engineered strain of the application is by by " recombinant bacterium for integrating the first exogenous gene expression box " and " outside integration second
The recombinant bacterium of source expression casette " carries out what protoplast fusion obtained.The engineered strain is integrated with the first foreign gene simultaneously
Expression cassette and the second exogenous gene expression box.Wherein, in the downstream of AmyE genes the first exogenous gene expression box of integration and arbitrarily
Integrate the second exogenous gene expression box in position.Foreign gene (riboflavin operon gene) copies in the genome of the engineered strain
Shellfish number is 10-50, and preferably 20-40, the copy number of ribA, ribG gene is >=10.
In the preferred embodiment of the application, VKPM B2116 strains are transferred to pBR25 plasmids, which contains band P15 and open
The riboflavin synthetic gene and tetracycline resistance gene of mover, bacterial strain after conversion carry out monoclonal screening, high-yield monoclonal into
Row EMS mutagenesis filters out riboflavin superior strain VBB8662 using the tetracycline of various concentration.VKPM B2116 species strains
Riboflavin synthetic gene knock out after, pBR31 plasmids are transformed into the bacterial strain, the plasmid contain AmyE 5 ' hold 600bp genes
Segment, riboflavin synthetic gene and kalamycin resistance gene with P15 promoters, the bacterial strain of conversion carry out monoclonal screening and
Kanamycins concentration screening filters out superior strain VBB8978.VBB8662 and VBB8978 is subjected to protoplast fusion, is melted
Bacterial strain after conjunction carries out monoclonal screening, filters out superior strain VBB11863, VBB11866.
P15 promoters
Promoter is a component part of gene (gene), initial time and the expression of control gene expression (transcription)
Degree can activate RNA polymerase, be allowed to that the specificity of transcription initiation is accurately combined and had with template DNA.P15 promoters
It is a kind of strong promoter, has very high-affinity to RNA polymerase, it can instructs to synthesize a large amount of mRNA, promote core yellow plain gene
Expression, improve the riboflavin generative capacity of thalline.
Main advantages of the present invention include：
(a) exogenous origin gene integrator for including in engineered strain of the present invention is in genome, and genetic stability is high, which can
The production of stable progress riboflavin；
(b) its riboflavin production of the riboflavin engineered strain of process transformation is significantly improved；
(c) engineered strain of the present invention use P15 promoters, the promoter be strong promoter, can efficiently promotor gene turn
Record, improves the expression efficiency of gene.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Embodiment 1
The structure of riboflavin recombinant bacterium
Riboflavin synthetic gene (ribB, ribG, ribA, ribH and ribTD) of the synthesis with P15 promoters, utilizes enzyme
The gene is introduced into pUC19 plasmids by enzyme site, and carries tetracycline resistance gene, which is named as pBR25 plasmids (see figure
1).PBR25 plasmids are transformed into host cell (commercially available Bacillus subtilis VKPM B2116 strains, Bacillus
Subtilis VKPM B2116) in, gene is introduced into host cell gene group using the method for homologous recombination, utilizes difference
The tetracycline of concentration is screened, and antibiotic concentration ranging from 10-100mg/L obtains the bacterial strain of high copy number genes
VBB8662。
Riboflavin synthetic gene (ribB, ribG, ribA, ribH and ribTD) of the synthesis with P15 promoters, utilizes enzyme
The gene is introduced into pUC19 plasmids by enzyme site, and carries kalamycin resistance gene and part AmyE genes, plasmid name
For pBR31 plasmids (see Fig. 2).PBR31 plasmids are transformed into the host cell (B2116 strains) for knocking out riboflavin synthetic gene
In, using homologous recombination method by gene orientation be introduced into host cell gene group, using various concentration kanamycins into
Row screening, antibiotic concentration ranging from 10-100mg/L obtain the bacterial strain VBB8978 of high copy number genes.
The structure of table 1.pBR25 plasmids and pBR31 plasmids
Embodiment 2
Protoplast fusion
1) preparation of culture medium：
Regeneration culture medium：Sodium succinate 81g, network Argine Monohydrochloride 5g, yeast powder 10g, K2HPO4 4.6g are weighed,
KH2PO4 1.5g, L-Trp 0.1g, Mgc l2.6H2O 4.06g, BSA 0.1g, glucose 10g are added to the water, after dissolving
Water is added to be dissolved to 1L (every liter of solid medium plus 15~16g agar powders), 121 DEG C of autoclave sterilization 20min.
SMM solution：Sorbierite 0.75M, pH6.5 maleic acid solution 0.02M, Mgc l2.6H2O 4.06g/L.
Lysozyme：It is configured to 20mg/mL, filtration sterilization, -20 DEG C of preservations after packing with aseptic double-distilled water.
2) two plants of bacterial strains VBB8662 and VBB8978 for obtaining embodiment 1, are inoculated in 4ml GM fluid nutrient mediums respectively
In, 37 DEG C of shaken cultivations are stayed overnight.
3) 4ml cultures bacterium solution is taken to be transferred in 50ml GM fluid nutrient mediums, 37 DEG C of shaken cultivation 3-4h to OD600 are
0.6-0.8。
4) 5000rpm centrifuges 10min, and thalline is suspended in 4-5ml SMM solution after abandoning supernatant, is added final concentration of
The lysozyme of 0.25mg/mL keeps the temperature 20-30min in 37 DEG C of water-baths (10min or so observations are primary).Immediately with 4000rpm from
Heart 10min abandons supernatant to remove lysozyme, then with 3-5ml SMM washing thallines.
5) 4000rpm centrifuges 10min, abandons supernatant, protoplast is resuspended in 1-2ml SMM, and VBB8662 is made
With the hypertonic suspension of protoplast of VBB8978.
6) each 1ml of both the above protoplast profundity suspension is taken to mix static 5-10min, 4000rpm centrifuges 10min,
Supernatant is abandoned, 10%PEG solution, 36 DEG C of water-baths is added, rush melts 10min, and 4000rpm centrifuges 10min, abandons supernatant, and 2ml regeneration is added
Culture medium is coated on the solid regenerated culture medium containing tetracycline and kanamycins, 36 DEG C, is inverted culture 3-4 days.
7) monoclonal colonies of cultured on solid medium are stored in 10% glycerine, -80 DEG C of preservations respectively.
The present embodiment increases the riboflavin gene copy number of thalline using Protoplast Fusion Technique, improves the core yellow of thalline
The riboflavin expression quantity of plain generative capacity, thalline is significantly improved.
Embodiment 3
The screening of Riboflavinoverproducstrains recombinant bacteria
1) preparation of culture medium：
1L solid mediums：Tryptone：5-10g；Yeast Extract：1-5g；NaCl：1-5g；Maltose：10-
20g；Agar：20g；Tetracycline：10-100mg；Kanamycins：10-100mg/L.
1L shake-flask seed culture mediums：Glucose：5-10g；Corn steep liquor：10-20g；Blackstrap：10-20g；Yeast extract：5-
10g；MgSO4：1-5g；(NH4)2SO4:5-10g；
1L Medium of shaking flask fermentation：Glucose：50-100g；Corn steep liquor：1-10g；Bean cake powder：10-50g；
MgSO4.7H2O：1-5g；Urea:5-10g；
2) the monoclonal colonies glycerine bacterium solution spread plate culture medium for the recombinant bacteria that embodiment 2 obtains is cultivated,
Condition of culture：37 DEG C, 14-16hr；
3) 5ml sterile waters is utilized to collect the thalline on tablet, it is spare after mixing；
4) it is inoculated into seed flask and is cultivated with 1% inoculum concentration, condition of culture：37 DEG C, 220rpm, 14-16hr；
5) bacterium solution of overnight incubation is inoculated into 10% inoculum concentration in fermentation medium, is cultivated, condition of culture：37
DEG C, 220rpm, 48hr；
6) riboflavin content in HPLC detection shaking flasks is utilized；
7) multiple riboflavin high yield fusant bacterial strains are obtained after the method for utilizing embodiment 2, VBB8662 and VBB8978 fusions,
Bacterial strain after fusion carries out shake flask fermentation experiment, and experimental result is shown in Fig. 3, riboflavin production reaches as high as 6g/L
8) bacterial strain by the core yellow cellulose content 7) obtained higher than 5g/L is coated on solid medium, carries out monoclonal sieve
Choosing repeats step 2) -6)；
9) riboflavin content in HPLC detection shaking flasks is utilized；
10) pass through monoclonal to screen, obtain multiple riboflavin high-yield monoclonal bacterial strains, monoclonal colonies carry out shake flask fermentation
Experiment, experimental result is shown in Fig. 4, riboflavin production reaches as high as 8-9g/L.
Embodiment 4
The fermentation tank of riboflavin recombinant bacteria is tested
1) preparation of culture medium：
1L solid mediums：Tryptone：5-10g；Yeast Extract：1-5g；NaCl：1-5g；Maltose：10-
20g；Agar：20g；Tetracycline：10-100mg；Kanamycins：10-100mg/L.
1L shake-flask seed culture mediums：Glucose：5-10g；Corn steep liquor：10-20g；Blackstrap：10-20g；Yeast extract：5-
10g；MgSO4：1-5g；(NH4)2SO4:5-10g；
1L Medium of shaking flask fermentation：Glucose：50-100g；Corn steep liquor：1-10g；Bean cake powder：10-50g；
MgSO4.7H2O：1-5g；Urea:5-10g；
2) the glycerine bacterium solution spread plate culture medium for recombinant bacteria VBB11863, the VBB11866 for obtaining embodiment 2 into
Row culture, condition of culture：37 DEG C, 14-16hr；
3) 20% glycerine of 5ml is utilized to collect the thalline on tablet, it is spare after mixing；
4) it is inoculated into seed flask and is cultivated with 1% inoculum concentration, condition of culture：37 DEG C, 220rpm, 14-16hr；
5) bacterium solution of overnight incubation is inoculated into 10% inoculum concentration in 5L fermentation tanks and is cultivated, condition of culture：37 DEG C,
Dissolved oxygen controls 15-20%, fermentation time 48hr；
6) riboflavin content in HPLC detection shaking flasks is utilized.
As a result as seen in figs. 5-6, through aforementioned riboflavin bacterial strain be transformed, in 5L fermentation tanks, ferment 70 hours, core yellow
Plain yield can reach 20g/L or more, and wherein recombinant bacterial strain VBB11863 is under the action of P15 promoters, when 70hr, riboflavin production
Amount is up to 22.5g/L (see Fig. 5), and recombinant bacterial strain VBB11866 is under the action of P15 promoters, and when 50hr, riboflavin production can
Up to 20.8g/L (see Fig. 6), the riboflavin generation of thalline is significantly improved.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of engineered strain producing riboflavin, which is characterized in that the bacterial strain is bacillus (Bacillus), and institute
It states and is integrated with exogenous gene expression box in the genome of bacterial strain, the exogenous gene expression box contains following elements：P15 starts
Son and riboflavin operon gene.
2. engineered strain as described in claim 1, which is characterized in that the exogenous gene expression box includes the first external source base
Because of expression cassette and the second exogenous gene expression box.
3. engineered strain as described in claim 1, which is characterized in that the first exogenous gene expression box also contains AmyE
Genetic elements.
4. engineered strain as described in claim 1, which is characterized in that the engineered strain is obtained through protoplast fusion
Recon.
5. engineered strain as described in claim 1, which is characterized in that foreign gene (riboflavin in the genome of the bacterial strain
Operon gene) copy number be 10-50, preferably 20-40.
6. a kind of method producing riboflavin, which is characterized in that including step：
(i) engineered strain described in claim 1 is cultivated, to obtain the tunning containing riboflavin；With
(ii) riboflavin is isolated from the tunning.
7. a kind of method of engineered strain described in structure claim 1, which is characterized in that including step：
(a) carrier containing the first exogenous gene expression box is built, the first exogenous gene expression box has following elements：
AmyE genes, P15 promoters and riboflavin operon gene；
(b) carrier containing the second exogenous gene expression box is built, the second exogenous gene expression box has following elements：
P15 promoters and riboflavin operon gene；
(c) carrier that step (a) and step (b) obtain is transferred to F-strain respectively, obtaining genome conformity has the first external source
The first recombinant bacterial strain and genome conformity of expression casette have the second recombinant bacterial strain of the second exogenous gene expression box；
(d) protoplast fusion is carried out to the first recombinant bacterial strain and the second recombinant bacterial strain, obtains and is integrated with outside first in genome
The recon of source expression casette and the second exogenous gene expression box, the recon are engineering described in claim 1
Bacterial strain.
8. the method for claim 7, which is characterized in that the F-strain is Bacillus subtillis Bacillus
Subtilis, preferably Bacillus subtilis SMY.
9. the purposes of engineered strain described in a kind of claim 1, which is characterized in that the bacterial strain is used as fermenting and producing riboflavin
Engineering bacteria.
10. a kind of method preparing food compositions, which is characterized in that including step：
Riboflavin prepared by claim 2 the method is added in food compositions, to obtain the food containing riboflavin
Product composition.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710008300.8A CN108277189A (en) | 2017-01-05 | 2017-01-05 | A kind of engineered strain producing riboflavin and its application |
| PCT/CN2017/075585 WO2018126527A1 (en) | 2017-01-05 | 2017-03-03 | Engineering strain for producing riboflavin and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710008300.8A CN108277189A (en) | 2017-01-05 | 2017-01-05 | A kind of engineered strain producing riboflavin and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182438A (en) * | 2018-10-15 | 2019-01-11 | 宁夏启元药业有限公司 | Vitamin B is produced using fermentation of bacillus2Culture medium and cultural method |
| CN110591990A (en) * | 2019-07-05 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | High-riboflavin-yield engineering strain and application thereof |
| CN113249261A (en) * | 2021-05-26 | 2021-08-13 | 浙江新和成股份有限公司 | Bacillus subtilis and application thereof in production of riboflavin |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109868253B (en) * | 2019-01-11 | 2020-03-13 | 齐鲁工业大学 | Bacillus licheniformis engineering bacteria for inhibiting bacterial autolysis and construction method and application thereof |
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- 2017-01-05 CN CN201710008300.8A patent/CN108277189A/en not_active Withdrawn
- 2017-03-03 WO PCT/CN2017/075585 patent/WO2018126527A1/en active Application Filing
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182438A (en) * | 2018-10-15 | 2019-01-11 | 宁夏启元药业有限公司 | Vitamin B is produced using fermentation of bacillus2Culture medium and cultural method |
| CN109182438B (en) * | 2018-10-15 | 2021-12-07 | 宁夏启元药业有限公司 | Production of vitamin B by fermentation of bacillus2Culture medium and culture method |
| CN110591990A (en) * | 2019-07-05 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | High-riboflavin-yield engineering strain and application thereof |
| CN110591990B (en) * | 2019-07-05 | 2021-09-07 | 中国科学院天津工业生物技术研究所 | High-riboflavin-yield engineering strain and application thereof |
| CN113249261A (en) * | 2021-05-26 | 2021-08-13 | 浙江新和成股份有限公司 | Bacillus subtilis and application thereof in production of riboflavin |
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| WO2018126527A1 (en) | 2018-07-12 |
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