CN106399218A - Bacillus subtilis engineering bacteria and application thereof - Google Patents
Bacillus subtilis engineering bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus subtilis engineering bacteria and application thereof. Whole genome sequencing comparative analysis shows that SFA-H43 and a D-ribose high-producing strain SFR-4 are higher in homology, and the nucleotide sequences of transketolase gene sequences except mutation sites are totally the same. According to the bacillus subtilis engineering bacteria disclosed by the invention, by adopting the way of homologous recombination, a transketolase gene mutation sequence of an SFR-4 strain is shifted into an SFA-43 strain so as to replace a normal sequence of transketolase genes of the SFA-43 strain, so that a transketolase mutant engineering strain SFR-43T is obtained; the transketolase mutant engineering strain SFR-43T not only maintains good fermentation properties (good stress resistance, fast glucose consumption speed, vigorous growth and difficulty in bacterial infection) of the SFA-43 strain, but also is capable of accumulating D-ribose, and the fermentation stress resistance is obviously increased.
Description
Technical field
The invention belongs to technical field of bioengineering, it is related to the breeding of industrial microorganism and the fermentable system of industrial goods
Standby, more particularly, to one bacillus subtilis engineering bacteria and its application.
Background technology
D-ribose is a kind of pentose, be life genetic materials ribonucleic acid and physiological activator ATP, NADH,
The important component part of NADPH, FADH, is the Primary Actor of biological energy i (in vivo) metabolism, has important physiological function.
D-ribose is mainly used in riboflavin Cobastab in the industry2And the production of food flavor agent, in recent years, more
Research can be as medicine intermediate, for antiviral, antineoplastic synthesis, in addition, D-ribose also may be used using D-ribose
To be used for treating myocardial ischaemia, the illnesss such as the muscle rigidity causing, DOMS of excessively straining as ancillary drug.
The production method of D-ribose include from yeast nucleic acid extract, chemical synthesis and using microbial strains fermentation
Conversion.Extraction method and chemical synthesis produce D-ribose because many reasons such as low production efficiency, complex process are not formed all the time
Large-scale production.Production by Microorganism Fermentation D-ribose production efficiency height, simple process, low production cost, make D-ribose price
Significantly decline, be truly realized D-ribose large-scale production.
D-ribose produces mostly bacterial strain is to screen transketolase deficient type mutant strain by classic mutagenesis.Sasajima in 1970 etc.
People finds a bacillus subtilis transketolase-deficient mutant accumulation D-ribose, and yield reaches 35g/L and it was found that one plant turns ketone
Enzyme lacks back mutation strain and no longer accumulates D-ribose it was demonstrated that D-ribose accumulation is the (Sasajima causing due to transketolase disappearance
K et al.Agric Biol Chem,35(4),509-517,1971).
3-Hydroxybutanone is a kind of wide variety of flavorant, additionally, 3-Hydroxybutanone is as a kind of intermediate compound
Thing, is widely used in the fields such as pharmacy, chemical industry and daily use chemicals food.The mode of production of 3-Hydroxybutanone and D-ribose includes chemistry conjunction
Become, prepared by enzymatic clarification and microbe industrial fermentation, the microbe fermentation method preparation wherein with glycogen as raw material has good
Prospects for commercial application and receive extensive concern.3-Hydroxybutanone and D-ribose coproduction microbe fermentation method is realized relatively in prior art
Few, wherein CN10197444A discloses the bacillus subtilis of a kind of high yield D-ribose and coproduction 3-Hydroxybutanone, and this bacterial strain is real
On the basis of transketolase deficient bacterial strain, secondary mutagenesis obtains, and the yield of its 3-Hydroxybutanone and D-ribose is all higher, but lures
The bacterial strain that the mode becoming obtains same performance is relatively difficult to achieve, how to realize the efficient coproduction of 3-Hydroxybutanone and D-ribose, is still
The subject matter that those skilled in the art face.
Content of the invention
Inventor's early stage obtains one plant of transketolase-deficient mutant SFR-4 by the means seed selection of classic mutagenesis, through condition
After optimization this bacterial strain D-ribose yield can reach 62.3g/L (Zhao Xiangying etc., food and medicine, 7 (3):23-26,2005), but
Inventor further study show that the negative mutation accumulation producing due to random mutagenesis, and bacterial strain SFR-4 environmental resistance is poor, in work
There is larger microbiological contamination risk, this becomes and limits the lifting of this bacterial strain D-ribose fermentation efficiency, reduces cost in industry production process
One major reason.
The problems referred to above existing for prior art, poor, raw in particular for the bacterial strain SFR-4 environmental resistance finding
There is larger microbiological contamination risk, the present invention provides one plant of strong stress resistance, is difficult the D-ribose fermentation of microbiological contamination during product
Bacillus subtilis engineering bacteria, the present invention is based on existing classic mutagenesis seed selection and obtains transketolase deficient type D-ribose high yield withered grass
The problem of bacillus resistance difference, obtains a plant height by molecular biology method and produces D-ribose bacterial strain, have degeneration-resistant simultaneously
Property good, consume sugared fireballing feature.Additionally, the present invention also aims to providing this bacillus subtilis engineering bacterium fermentation to produce
The application of D-ribose and/3-Hydroxybutanone and fermentation process.
Bacterial strain SFR-4 has been carried out genome sequencing by inventor, and for sequencing result, inventor chances on bacterial strain
SFR-4 and the accumulation 3-Hydroxybutanone of another plant of inventor screening bacillus subtilis strain SFA-H43 (Zhao Xiangying etc., one
Bacillus and its application of 3-Hydroxybutanone, Chinese patent CN 103333842A, preserving number CCTCC No are produced in strain:
M2013181) genome functions genomic constitution is highly similar, and relatively large with the conventional 168 Strain comparison differences of bacillus subtilis
(see accompanying drawing 1).
Further, by the tkt gene comparison to three bacterial strains, find the rear 50bp of SFR-4 bacterial strain tkt gene
Gene order there occurs obvious variation (see accompanying drawing 2) compared with SFA-H43 bacterial strain.Inventor passes through to two plants of bacterium (SFR-4, SFA-
H43) it is compared research, find that bacterial strain SFA-H43 is stronger than bacterial strain SFR-4 to the tolerance of environment, and grow vigorous, consumption sugar speed
Degree is fast, and sweat is difficult microbiological contamination.Based on above-mentioned analysis, inventor courageously expects, if bacterial strain SFA-H43 is normally turned ketone
Enzyme gene is substituted for the tkt gene that bacterial strain SFR-4 there occurs variation, you can obtain the SFA-H43 work that transketolase morphs
Journey bacterial strain, it is contemplated that this engineered strain can accumulate D-ribose, remains in that other good characteristics of bacterial strain SFA-H43 simultaneously, improves
The resistance of D-ribose fermentation, minimizing fermentation microbiological contamination risk.
Based on above-mentioned analysis and test, specifically, the present invention relates to technical scheme below：
First, the invention provides a kind of bacillus subtilis engineering bacteria for D-ribose production, described engineering bacteria is
The engineering that in bacillus subtilis SFA-H43 bacterial strain, tkt gene obtains after replacing with gene order shown in SEQ ID NO.7
Bacterium.
Preferably, gene order shown in SEQ ID NO.7 is derived from the tkt gene of bacillus subtilis SFR-4 bacterial strain.
Described bacillus subtilis SFA-H43 bacterial strain, preserving number is CCTCC No:M2013181.
Described bacillus subtilis SFR-4 bacterial strain, is the transketolase-deficient mutant that inventor screens previously, referring to Zhao
Auspicious grain husk etc., food and medicine, 7 (3):23-26,2005；The public can be obtained with reasonable approach.
Specifically, the bacillus subtilis engineering bacteria for D-ribose production of the present invention, is bacillus subtilis
500bp sequence before transketolase gene end codon in SFA-H43 bacterial strain, replaces with turning of bacillus subtilis SFR-4 bacterial strain
The engineering bacteria that before ketolase gene end codon, 500bp sequence (comprising mutational site) obtains.
In the preferred embodiment of the invention, disclose one plant of bacillus subtilis for D-ribose production
(Bacillus subtilis) engineering bacteria SFR-43T, this bacterial strain was preserved in Chinese Typical Representative culture and protects on November 22nd, 2016
Tibetan center, preservation address is：Luojiashan, Wuchang, Wuhan City, Hubei Province, preserving number is：CCTCCM 2016666.
The described bacillus subtilis engineered strain SFR-43T for D-ribose production, 37 DEG C on LB culture medium flat plate
Culture culture 12h starts bacterium colony, 24 hours colony diameter about 1.0-1.5mm, and bacterium colony is rounded, and edge is irregular, milky white
Color, flat, smooth surface, opaque, it is clearly distinguished from bacterial strain SFA-H43 bacterium colony；In LB fluid nutrient medium, 37 DEG C of shaking tables
Culture 12-20 hour, cell is in direct rod shape, exists in single or short chain, Gram-positive, sporiparous ability reduces.
Described bacterial strain SFR-43T, is transketolase mutation engineered strain, (cultivation temperature, pH, molten in suitable condition of culture
Solution oxygen etc.) under, provide appropriate nutrient media components (carbon source, nitrogen source, inorganic salts etc.) for it, this bacterial strain can amass in nutrient solution
Tired D-ribose.
Described bacterial strain SFR-43T is to be obtained by the method for molecular biology, and its parental plant is SFA-H43 bacterial strain, occurs
The tkt gene of mutation is derived from D-ribose and produces bacterial strain SFR-4.
Secondly, the present invention provides a kind of construction method of the bacillus subtilis engineering bacteria for D-ribose production, and it leads to
The method crossing homologous recombination, tkt gene in bacillus subtilis SFA-H43 bacterial strain is replaced with base shown in SEQ ID NO.7
Because of sequence.
Preferably, gene order shown in SEQ ID NO.7 is derived from the tkt gene of bacillus subtilis SFR-4 bacterial strain.
Specifically, in the construction method of the bacillus subtilis engineering bacteria SFR-43T producing for D-ribose, homologous recombination
The genetic fragment replaced is 500bp sequence replacement before transketolase gene end codon in bacillus subtilis SFA-H43 bacterial strain
For 500bp sequence before the tkt gene terminator codon of bacillus subtilis SFR-4 bacterial strain.
In the preferred embodiment of the invention, described bacterial strain SFR-43T is (to build stream by following step is built-up
Journey is referring to accompanying drawing 3)：
(1) with bacterial strain SFR-4 genome as template, expand 500bp fragment before this bacterial strain tkt gene terminator codon
(comprising mutational site), referred to as L-tkt (its sequence is as shown in SEQ ID NO.10), wherein downstream primer contains plasmid p7Z6's
Lox71-zeo-lox66 fragment front end 25bp complementary series；Preferably, the primer sequence is SEQ ID NO.1 and SEQ ID
Shown in NO.2；
(2) with bacterial strain SFA-H43 genome as template, expand its tkt gene downstream 500bp fragment, be R-tkt (its
Sequence is as shown in SEQ ID NO.11), wherein upstream primer contains the lox71-zeo-lox66 end 25bp complementation of plasmid p7Z6
Sequence；Preferably, the primer sequence is shown in SEQ ID NO.3 and SEQ ID NO.4；
(3) with plasmid p7Z6 as template, amplification carries blasticidin resistance gene fragment lox71-zeo- in lox site
lox66；Preferably, the primer sequence is shown in SEQ ID NO.5 and SEQ ID NO.6；
(4) adopt fusion DNA vaccine method, L-tkt, lox71-zeo-lox66, R-tkt are merged；Preferably, fusion DNA vaccine institute
Be SEQ ID NO.1 with primer sequence and SEQ ID NO.4 shown in；
(5) fragment conversion bacterial strain SFA-H43 will be merged, by blasticidin resistance plate screening integrated recombination mutation bacterium
Strain, obtains the engineered strain of the bacterial strain SFA-H43 containing bacterial strain SFR-4 transketolase mutational site, and adopts Cre/lox system
Method knocks out resistant gene zeo (the Xin Yan et al.Appled and Environmental in engineered strain
Microbiology,2008,74(17):5556-5562.), obtain the safe transketolase mutation work without antibiotics resistance gene
Journey bacterial strain, is named as SFR-43T.
Additionally, the invention also discloses above-mentioned bacillus subtilis engineering bacteria prepares the application in D-ribose in fermentation.
Preferably, above-mentioned application goes back fermenting and producing 3-Hydroxybutanone while being additionally included in production D-ribose.
Another object of the present invention is to providing a kind of bacillus subtilis engineering bacterium fermentation to produce D-ribose and/3- hydroxyl
The method of butanone.
For achieving the above object, specifically, the present invention relates to technical scheme below：
A kind of bacillus subtilis engineered strain fermenting and producing D-ribose and the method for/3-Hydroxybutanone, it adopts as follows
Shake flask fermentation and/ferment tank mode：
(1) Spawn incubation：It is inoculated into seed culture medium culture after engineered strain is activated, obtain first order seed and/bis- grades
Seed；
(2) shake flask fermentation：Cultured for step (1) first order seed is inoculated into equipped with the shaking flask of fermentation medium, shakes
Bottle is cultivated and is not further added by D-ribose content in zymotic fluid, terminates fermentation；
(3) ferment tank：Cultured for step (1) one-level or secondary seed are inoculated in fermentation tank, are aerated
Stir culture, in incubation, controls pH, by adjusting speed of agitator and ventilating ratio, controls dissolved oxygen to be fermented.
In preferred embodiment, in step (1), Spawn incubation is：Take the fresh of 2-3 days engineered strains of 35-40 DEG C of culture
Inclined-plane, takes 1-2 ring slant strains with oese, is inoculated in 50ml liquid seed culture medium, 35-40 DEG C, 150-200rpm, shakes
Bottle culture 10-16 hour, is for first order seed.First order seed is inoculated seed culture medium with (percentage by volume) of 1-10%,
35-40 DEG C of culture 6-12 hour, is for secondary seed；
In preferred embodiment, (2) shake flask fermentation：By cultured for step (1) first order seed with 1-10% (volume hundred
Fraction) inoculum concentration be inoculated into equipped with the shaking flask of fermentation medium, 35-40 DEG C, 180-220rpm, Shaking culture is to zymotic fluid
Middle D-ribose content is not further added by, and terminates fermentation；
In preferred embodiment, (3) ferment tank：By cultured for step (1) one-level or secondary seed with 1-
The inoculum concentration of 10% (percentage by volume) is inoculated in fermentation tank (liquid amount of 65-75%), and 35-40 DEG C is aerated stirring training
Support, in incubation, control pH in 5-7, by adjusting speed of agitator and ventilating ratio, keep Comparative dissolution oxygen concentration in zymotic fluid
5-30%.
Further, the consisting of of the slant strains culture medium used by above-mentioned inclined-plane culture：Yeast extract 5g/L, albumen
Peptone 10g/L, NaCl 3g/L, agar 20g/L, remaining is water, pH7.0-7.2.
Further, the consisting of of aforesaid liquid primary-seed medium：Glucose 20-40g/L, yeast extract 2-
10g/L, corn steep liquor 8-12g/L, pH 7.0-7.2.
Further, the consisting of of above-mentioned secondary seed medium：Glucose 30-60g/L, yeast extract 2-10g/L,
Corn steep liquor 8-12g/L, (NH4)2SO42-3g/L, MgSO42g/L, pH 7.0-7.2.
Further, above-mentioned Medium of shaking flask fermentation consists of：Glucose 120-150g/L, yeast extract 2-10g/L,
Corn steep liquor 5-15g/L, (NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH7.0-7.2.
Further, above-mentioned ferment tank initial medium consists of：Glucose 120-150g/L, yeast extract 3-
6g/L, corn steep liquor 5-10g/L, (NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH 6.0-7.2.
Preferably, the time of described primary-seed medium culture is 12-14 hour.
Preferably, the time of described secondary seed medium culture is 6-8 hour.
Preferably, the cultivation temperature of described shake flask fermentation or ferment tank is 36-38 DEG C.
Preferably, the pH of described ferment tank controls in 5.5-6.5.
Preferably, the dissolved oxygen concentration of described ferment tank is maintained at 20-30%.
Preferably, glucose 130-150g/L in described fermentation medium, yeast extract 3-6g/L, corn steep liquor 6-10g/
L, (NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH 7.0-7.2.
Preferably, fermentation tank is improved fermentation efficiency by way of feed supplement and increases fermentation production rate.
Preferably, fed-batch fermentation, initial glucose concentration is 120-150g/L.
Preferably, feed supplement adopts a feed profile.
Preferably, the feed supplement time is that in zymotic fluid, concentration of glucose is down to 20-40g/L.
Preferably, feed supplement amount is 40-60g/L glucose.
Preferably, feed supplement concentration of glucose is 600-800g/L, yeast extract 0.5g/L.
The present invention achieves advantages below and good effect：
(1) present invention based on the bacterial strain SFR-4 discovery highly similar to bacterial strain SFA-H43 genome functions genomic constitution with
And SFR-4 bacterial strain tkt gene 50bp gene order there occurs the feature of obvious variation compared with SFA-H43 bacterial strain, by SFR-4 bacterium
Strain is combined with the advantage of SFA-H43 bacterial strain；The conversion parental plant SFA-H43 that the present invention selects is the accumulation 3- of Natural Selection
The bacterial strain of hydroxy butanone, transketolase base higher with D-ribose superior strain SFR-4 homology through genome sequencing comparative analysis
Because of sequence, in addition to mutational site, nucleotide sequence is identical, and the present invention adopts the method for homologous recombination by bacterial strain SFR-4 bacterial strain
Tkt gene mutant nucleotide sequence proceeds to bacterial strain SFA-43, replaces the normal sequence of this bacterial strain tkt gene, obtains one plant and turns ketone
The engineered strain SFR-43T that enzyme morphs, fermented verification experimental verification, engineered strain SFR-43T had both maintained bacterial strain SFA-43
Other premium properties (good stress resistance, the sugared speed of consumption is fast, growth is vigorous, be difficult microbiological contamination), the D- of high concentration can be accumulated simultaneously
Ribose, fermentation resistance significantly improves.
(2) SFA-H43 bacterial strain be inventor's early stage from white wine Daqu brick separation screening to Physiology and biochemistry property be suitable to
Metabolism produces the bacterial strain of 3-Hydroxybutanone, highly similar to bacterial strain SFA-H43 genome functions genomic constitution based on bacterial strain SFR-4
Feature, tkt gene in SFA-H43 bacterial strain is replaced with after the tkt gene of SFR-4 bacterial strain, engineering of the present invention
Bacterium can improve, using high concentration glucose come high yield D-ribose co-production 3-Hydroxybutanone, the valency that engineering bacteria produces D-ribose
Value.
Brief description：
Fig. 1 bacterial strain SFR-4, bacterial strain SFA-H43, bacterial strain 168 genome functions gene similarity analysis
Fig. 2 bacterial strain SFR-4, bacterial strain SFA-H43, the comparison in bacterial strain 168 tkt gene sequence difference site
The structure route map of Fig. 3 engineering bacteria SFR-43T
The electrophoretogram of L-tkt, R-tkt, lox71-zeo-lox66 fragment that Fig. 4 PCR obtains, 1.DL5000Marker；
2.L-tkt genetic fragment；3.R-tkt genetic fragment；4.zeo genetic fragment
L-tkt, lox71-zeo-lox66, R-tkt recombination fragment electrophoretic figure that Fig. 5 fusion DNA vaccine obtains,
1.DL5000Marker；2. recombination fragment
Fig. 6 engineering bacteria SFR-43T course of fermentation
Fig. 7 bacterial strain SFR-4 course of fermentation
Fig. 8 engineering bacteria SFR-43T fermentation tank fed-batch fermentation process
Specific embodiment：
Method in following embodiments, if no special instructions, is conventional method, the primer synthesis is purchased from Hua Da base
Cause, plasmid p7Z6 used and plasmid pDGC is gifted (Xin Yan et al.Appled and by Agricultural University Of Nanjing
Environmental Microbiology,74(17):5556-5562,2008.).
Embodiment 1 bacterial strain SFR-43T builds
(1) with bacterial strain SFR-4 genome as template, expand 500bp fragment before this bacterial strain tkt gene terminator codon
(comprising mutational site), referred to as L-tkt (its sequence is as shown in SEQ ID NO.10).Primer contains lox71-zeo- downstream
Lox66 front end 25bp complementary series.
Template：Bacterial strain SFR-4 genome
Upstream primer：GCAGCATGGAAGCTTGCAG(SEQ ID NO.1)
Downstream primer：TATAATGTATGCTATACGAACGGTATTATCTTGATACTATATAGAAACATCTCAAGG
(SEQ ID NO.2)
PCR reaction system (50uL)：Bacterial strain SFR-4 genomic DNA template 1uL, upstream primer 1uL, downstream primer 1uL,
2*Buffer 25uL, dNTP 4uL, High fidelity PCR enzyme PrimeSTAR 0.5uL, sterilized water 17.5uL.
PCR reaction condition：95 DEG C of denaturation 5min, 98 DEG C of 10s, 60.3 DEG C of 30s, 72 DEG C of 30s, 33 circulations.
Amplified production is detected by 1% agarose gel electrophoresis and collects.
(2) with bacterial strain SFA-H43 genome as template, expand its tkt gene downstream 500bp fragment, be R-tkt (its
Sequence is as shown in SEQ ID NO.11).Its upstream primer contains lox71-zeo-lox66 end 25bp complementary series.
Template：Bacterial strain SFA-H43 genome
Upstream primer：
ATAACTTCGTATAGCATACATTATAGCTTTTGAAAGAGGATGAGTCAAAT(SEQ ID NO.3)
Downstream primer：AAAGAATATCGTTTCTGCCTCGTAA(SEQ ID NO.4)
PCR reaction system (50uL)：Bacterial strain SFA-H43 genomic DNA template 1uL, upstream primer 1uL, downstream primer
1uL, 2*Buffer 25uL, dNTP 4uL, High fidelity PCR enzyme PrimeSTAR 0.5uL, sterilized water 17.5uL.
PCR reaction condition：95 DEG C of denaturation 5min, 98 DEG C of 10s, 60.8 DEG C of 30s, 72 DEG C of 30s, 33 circulations.
Amplified production is detected by 1% agarose gel electrophoresis and collects.
(3) with plasmid p7Z6 as template, amplification carries blasticidin resistance gene (zeo) fragment in lox site
lox71-zeo-lox66.
Template：Plasmid p7Z6
Upstream primer:TACCGTTCGTATAGCATACATTATACG(SEQ ID NO.5)
Downstream primer:TATAATGTATGCTATACGAAGTTATTCAGTC(SEQ ID NO.6)
PCR reaction system (50uL)：Bacterial strain SFA-H43 genomic DNA template 1uL, upstream primer 1uL, downstream primer
1uL, 2*Buffer 25uL, dNTP 4uL, High fidelity PCR enzyme PrimeSTAR 0.5uL, sterilized water 8.5uL.
PCR reaction condition：95 DEG C of denaturation 5min, 98 DEG C of 10s, 59.2 DEG C of 30s, 72 DEG C of 30s, 33 circulations.
Amplified production is detected by 1% agarose gel electrophoresis and collects.
L-tkt, R-tkt, lox71-zeo-lox66zeo genetic fragment electrophoretogram that PCR obtains is shown in accompanying drawing 4.
(4) adopt fusion DNA vaccine method, preparation L-tkt, lox71-zeo-lox66, R-tkt merge fragment.
1. fusion reaction
Fusion DNA vaccine reaction system：L-tkt genetic fragment 3uL, R-tkt genetic fragment 3uL, lox71-zeo-lox66zeo
Genetic fragment 3uL, 2*Buffer 25uL, dNTP 4uL, High fidelity PCR enzyme PrimeSTAR 0.5uL, sterilized water 11.5uL.
Fusion DNA vaccine reaction condition：95 DEG C of denaturation 5min, 98 DEG C of 10s, 59.2 DEG C of 30s, 72 DEG C of 90s, 10 circulations.
2. amplified reaction
Template：Merge fragment
Upstream primer：GCAGCATGGAAGCTTGCAG(SEQ ID NO.1)
Downstream primer：AAAGAATATCGTTTCTGCCTCGTAA(SEQ ID NO.4)
Reaction system：Fusion fragment 10uL, upstream primer 1uL, downstream primer 1uL, 2*Buffer 25uL, dNTP 4uL,
High fidelity PCR enzyme PrimeSTAR 0.5uL, sterilized water 8.5uL.
Reaction condition：95 DEG C of denaturation 5min, 98 DEG C of 10s, 59.2 DEG C of 30s, 72 DEG C of 90s, 33 circulations.
Amplified production is detected by 1% agarose gel electrophoresis and collects.Accompanying drawing 5 obtains recombination piece for fusion DNA vaccine
Section electrophoretogram.
(5) this fusion fragment is converted by SFA-H43 bacterial strain using electric robin, by blasticidin resistance flat board (25ug/
ML) screen 8 plants of resistant strains, obtain 4 plants of inheritance stability bacterial strains through repeatedly passing on, find there are 3 plants of bacterium accumulation through shake flask fermentation
D-ribose, selects wherein one plant and carries out next step resistant gene zeo knockout.
(6) in the recombinant strain being obtained plasmid pDGC importing (5) using electric robin, using in IPTG inducing plasmid
The expression of cre gene, knocks out the zeo gene in recombinant using Cre/lox systems approach, obtains one through screening and fermentation test
Strain carries bacterial strain SFR-4 tkt gene mutant nucleotide sequence and the transketolase without resistant gene is mutated engineered strain, is named as
SFR-43T.
This bacterial strain is preserved in China typical culture collection center on November 22nd, 2016, and preservation address is：Hubei Province
Wuchang, wuhan Luo Jia Shan, preserving number is：CCTCC M 2016666.
Embodiment 2 bacterial strain SFR-43T product analysis
Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, take 1-2 ring slant strains with oese, be inoculated into liquid
In seed culture medium, 37 DEG C, 180rpm shaking table culture 14 hours, 5% inoculum concentration inoculation fermentation culture medium, 37 DEG C, 180rpm shakes
Bottle culture 72 hours.Zymotic fluid is centrifuged 10min through 4500rpm, takes supernatant, carries out product analysis, and result shows bacterial strain SFR-43T
Detect 3-Hydroxybutanone and D-ribose component in zymotic fluid simultaneously.
Fermentation medium (g/L)：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 10g/L, (NH4)2SO4
2.5g/L, MgSO42g/L, MnSO40.2g/L, pH6.5.
Embodiment 3 fermentation medium optimization
Culture based on bacterial strain SFR-4D- ribose fermentation medium, to bacterial strain SFR-43T fermentation D-ribose for the present invention
The components such as base nitrogen source are optimized further.
Nitrogen source is preferred：Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, with oese picking 1-2 ring, be inoculated into
In liquid seed culture medium, 37 DEG C, 180rpm shaking table culture 14 hours, inoculum concentration 5%, it is inoculated into the shaking flask containing different nitrogen sources and send out
(nitrogen source is respectively peptone, yeast extract, Dried Corn Steep Liquor Powder, ammonium nitrate, diammonium hydrogen phosphate, ammonium sulfate, concentration to ferment culture medium
It is 10g/L, carbon source is glucose, other components are identical), 37 DEG C, 180rpm Shaking culture 72 hours.Zymotic fluid goes through centrifugation
Thalline, supernatant is used for D-ribose assay.Result display bacterial strain SFR-43T is individually with peptone, yeast extract, jade
Rice ＆ peanut milk dry powder, ammonium nitrate (NH4NO3), diammonium hydrogen phosphate ((NH4)2HPO4), ammonium sulfate ((NH4)2SO4) all can produce D- for nitrogen source
Ribose, is nitrogen source wherein individually with yeast extract, D-ribose yield highest.Higher in view of yeast extract price, this
Invention is combined preferably to each composition of multiple compound nitrogen sources, and result works as yeast extract 3-6g/L, corn steep liquor 6-10g/L,
(NH4)2SO4During 2-3g/L, bacterial strain SFR-43T fermentation D-ribose yield highest.
Concentration of glucose is preferred：Take the fresh inclined-planes of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, with oese picking 1-2 ring,
It is inoculated in liquid seed culture medium, 37 DEG C, 180rpm shaking table culture 14 hours, inoculum concentration 5%, be inoculated into Portugal containing variable concentrations
The Medium of shaking flask fermentation of grape sugar, 37 DEG C, 180rpm Shaking culture 72 hours.Zymotic fluid removes thalline through centrifugation, and supernatant is used for
D-ribose assay.Result shows, concentration of glucose in the range of 120-150g/L, bacterial strain D-ribose yield and conversion ratio
Higher.
Inorganic ions is preferred：Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, with oese picking 1-2 ring, connect
Plant in liquid seed culture medium, 37 DEG C, 180rpm shaking table culture 14 hours, inoculum concentration 5%, be inoculated into containing different inorganic ions
Medium of shaking flask fermentation (inorganic ions is respectively magnesium sulfate, ferric sulfate, manganese sulfate, sodium sulphate, zinc sulfate, and concentration is 1g/
L, carbon source is glucose, and nitrogen source is yeast extract 2-10g/L, corn steep liquor 5-15g/L, (NH4)2SO42-3g/L, other components
Identical), 37 DEG C, 180rpm Shaking culture 72 hours.Zymotic fluid removes thalline through centrifugation, and supernatant is used for D-ribose assay.
Zymotic fluid is centrifuged 15min through 4500rpm, and supernatant exists through liquid chromatogram measuring D-ribose content, result display bacterial strain SFR-43T
It is added with magnesium sulfate and the culture medium of manganese sulfate, D-ribose content is higher, through this inorganic ions of composite optimization, result shows,
Work as MgSO42g/L, MnSO4During 0.2g/L, bacterial strain SFR-43T fermentation D-ribose yield is higher.
Embodiment 4 bacterial strain SFR-43T and bacterial strain SFR-4 contrast fermentation
The present embodiment is used for the D-ribose fermentation of contrast provided genetic comparison engineering bacteria SFR-43T and bacterial strain SFR-4
Feature.
Engineering bacteria SFR-43T and bacterial strain SFR-4 access shake-flask seed culture medium, after be respectively connected to culture in 5L fermentation tank,
8h samples once, and the pH of ferment tank controls 6.5, and ventilation is 2L/min, and by adjusting rotating speed, dissolved oxygen concentration keeps
In 20-30%.Relatively biomass, remaining sugar concentration, D-ribose yield, and draw course of fermentation curve, as shown in accompanying drawing 6, Fig. 7,
Compared to natural mutagenic and breeding bacterial strain SFR-4, although engineering bacteria SFR-43T D-ribose yield slightly reduces, thalli growth is relatively
Hurry up, fermentation period is shorter.
Inclined-plane culture based formulas：Yeast extract 5g/L, peptone 10g/L, NaCl 3g/L, agar 20g/L, pH7.0.
Liquid seed culture medium fills a prescription (g/L)：Glucose 20g/L, yeast extract 5g/L, corn steep liquor 10g/L, pH7.0.
Bacterial strain SFR-43T fermentation medium (g/L)：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 10g/L,
(NH4)2SO42.0g/L, MgSO42g/L, MnSO40.2g/L, pH6.5.
Bacterial strain SFR-4 fermentation medium (g/L)：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 3g/L, (NH4)2SO43g/L, MgSO42g/L, CaCO35g/L, pH6.5.
Example 5 engineering bacteria SFR-43T shake flask fermentation
Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, oese takes 1 ring slant strains, is inoculated into 50ml/
In 250mL liquid seed culture medium, cultivate 14 hours for 36 DEG C, 50ml/250mL fermentation medium is inoculated into 5% inoculum concentration
In, 37 DEG C, 180rpm shake flask fermentation, every 12h sampling, to 72 hours, glucose exhausted cultivation cycle, terminated fermentation, D- core
Candy output reaches 39.35g/L, 3-Hydroxybutanone yield 35.7g/L.
Inclined-plane culture based formulas：Yeast extract 5g/L, peptone 10g/L, NaCl 3g/L, agar 20g/L, pH7.0.
Liquid seed culture medium is filled a prescription：Glucose 20g/L, yeast extract 5g/L, corn steep liquor 10g/L, pH7.0.
Medium of shaking flask fermentation is filled a prescription：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 10g/L, (NH4)2SO4
2.0g/L, MgSO42g/L, MnSO40.2g/L, pH7.0.
Example 6 engineering bacteria SFR-43T shake flask fermentation
Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, oese takes 1 ring slant strains, is inoculated into 50ml/
In 250mL liquid seed culture medium, cultivate 12 hours for 37 DEG C, 50ml/250mL fermentation medium is inoculated into 10% inoculum concentration
In, 37 DEG C, 200rpm shake flask fermentation, every 12h sampling, to 72 hours, glucose exhausted cultivation cycle, terminated fermentation, D- core
Candy output reaches 36.28g/L, 3-Hydroxybutanone yield 33.65g/L.
Inclined-plane culture based formulas：Yeast extract 5g/L, peptone 10g/L, NaCl 3g/L, agar 20g/L, pH7.0.
Liquid seed culture medium is filled a prescription：Glucose 20g/L, yeast extract 5g/L, corn steep liquor 8g/L, pH7.0.
Medium of shaking flask fermentation is filled a prescription：Glucose 140g/L, yeast extract 6g/L, corn steep liquor 8g/L, (NH4)2SO4
3.0g/L, MgSO42g/L, MnSO40.2g/L, pH7.0.
Example 7 bacterial strain SFR-43T ferment tank
Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, oese takes 1 ring slant strains, is inoculated into 50ml/
In 250mL liquid seed culture medium, cultivate 14 hours for 36 DEG C, 5L fermentation tank is inoculated into the inoculum concentration of 5% (percentage by volume)
In (liquid amount be 3L), 37 DEG C of ventilation stir culture, ventilation is 2L/min, controls pH5.5-6.0.Turned by adjusting stirring
Speed, keeps Comparative dissolution oxygen concentration in zymotic fluid to be 20% about, 68h fermentation ends, D-ribose yield reaches 40.5g/L, 3- hydroxyl
Base butanone yield is 35.6g/L.
Liquid seed culture medium is filled a prescription：Glucose 50g/L, yeast extract 6g/L, corn steep liquor 10g/L, (NH4)2SO4
2.0g/L, MgSO42g/L, pH7.0.
Ferment tank culture medium prescription：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 10g/L, (NH4)2SO4
2.0g/L, MgSO42g/L, MnSO40.2g/L, pH6.5.
Example 8 bacterial strain SFR-43T ferment tank
Take the fresh inclined-plane of 2 days bacterial strain SFR-43T of 37 DEG C of cultures, oese takes 1 ring slant strains, is inoculated into 50ml/
In 250mL liquid seed culture medium, cultivate 12 hours for 37 DEG C, 800ml/5L is inoculated into the inoculum concentration of 5% (percentage by volume)
In triangular flask, 37 DEG C of shaken cultivation 8 hours, 50L fermentation tank is inoculated with 10% inoculum concentration, stir culture of divulging information, ventilating ratio
0.5-1.0, controls pH6.0 ± 0.2.By adjusting speed of agitator, Comparative dissolution oxygen concentration in zymotic fluid is kept to be 20-40%,
64h fermentation ends, D-ribose yield reaches 42.5g/L, and 3-Hydroxybutanone yield is 33.6g/L.
Level liquid seed culture based formulas：Glucose 20g/L, yeast extract 8g/L, corn steep liquor 8g/L, pH7.0.
Secondary liquid seed culture based formulas：Glucose 60g/L, yeast extract 6g/L, corn steep liquor 10g/L, (NH4)2SO42.0g/L, MgSO42g/L, pH7.0.
Ferment tank culture medium prescription：Glucose 150g/L, yeast extract 5g/L, corn steep liquor 10g/L, (NH4)2SO4
2.5g/L, MgSO42g/L, MnSO40.2g/L, pH6.5.
Example 9 bacterial strain SFR-43T fermentation tank fed-batch fermentation
37 DEG C of first order seed is cultivated 14 hours, is inoculated into secondary seed medium with the inoculum concentration of 5% (percentage by volume)
In, cultivate 8 hours for 37 DEG C, 10% inoculum concentration inoculation 50L fermentation tank (liquid amount 30L), stir culture of divulging information.Control fermentation temperature
Degree 36-37 DEG C, ventilating ratio 0.5-0.8, control pH6.0 ± 0.2, by adjusting speed of agitator, keep Comparative dissolution in zymotic fluid
Oxygen concentration is 20-40%, 44h feed supplement 1 time, feed supplement 40g/L, 80 hours fermentation ends, and D-ribose yield reaches 51.5g/L, 3- hydroxyl
Base butanone yield 35.1g/L, fermentation results are as shown in Figure 8.
Ferment tank initial incubation based formulas：Glucose 120g/L, yeast extract 5g/L, corn steep liquor 10g/L,
(NH4)2SO42.5g/L, MgSO42g/L, MnSO40.2g/L, pH6.5.Ferment tank process streams add material composition：Grape
Sugared 800g/L, yeast extract 0.5g/L.
Embodiment described above is only that the preferred embodiment of the present invention is described, the not model to the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, the technical side to the present invention for the those of ordinary skill in the art
Various modifications and improvement that case is made, all should fall in the protection domain of claims of the present invention determination.
SEQUENCE LISTING
<110>Shandong Food Fermentative Industry Research and Design Inst.
<120>One bacillus subtilis engineering bacteria and its application
<130> 11
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial primer sequence
<400> 1
gcagcatgga agcttgcag 19
<210> 2
<211> 57
<212> DNA
<213>Artificial primer sequence
<400> 2
tataatgtat gctatacgaa cggtattatc ttgatactat atagaaacat ctcaagg 57
<210> 3
<211> 50
<212> DNA
<213>Artificial primer sequence
<400> 3
ataacttcgt atagcataca ttatagcttt tgaaagagga tgagtcaaat 50
<210> 4
<211> 25
<212> DNA
<213>Artificial primer sequence
<400> 4
aaagaatatc gtttctgcct cgtaa 25
<210> 5
<211> 27
<212> DNA
<213>Artificial primer sequence
<400> 5
taccgttcgt atagcataca ttatacg 27
<210> 6
<211> 31
<212> DNA
<213>Artificial primer sequence
<400> 6
tataatgtat gctatacgaa gttattcagt c 31
<210> 7
<211> 2004
<212> DNA
<213> SFR-4 tkt
<400> 7
atggatacaa ttgaaaagaa atcagttgct accattcgca cactgtcaat agacgctatt 60
gaaaaagcaa attctggtca cccagggatg ccgatgggag ccgctccaat ggcatacacg 120
ctgtggacaa aatttatgaa cgtaagtccg gcaaaccctg gctggtttaa ccgtgaccgt 180
tttgttttat ctgctggaca cgggtcagca ctattataca gcatgcttca tttaagcggg 240
tttgatctta gtattgaaga tcttaaggga ttccgccagt ggggcagcaa aacaccagga 300
catccggaat tcggacatac tgccggtgtt gatgctacaa caggtccgct tggccaagga 360
attgccatgg cagtcggtat ggcaattgct gaacgccatt tagcggaaac atacaaccgc 420
gattcattta acgtagtcga tcattataca tacagtattt gcggtgatgg tgatttaatg 480
gaaggtattt cttctgaagc cgcttcactc gcaggccatc ttcagcttgg ccgtctgatc 540
gtactatacg attctaatga catctctctt gatggagacc tcgaccgttc attctctgaa 600
aacgtgaaac agcgttttga agcaatgaat tgggaagttc tttatgttga ggatggaaac 660
aatattgaag aattaacagc ggctatcgaa aaagcacgcc aaaatgaaaa gaaacctaca 720
ttaattgaag tgaaaacgac aatcggattc ggttcaccta accgtgccgg tacatccggt 780
gttcacggtg cgccgcttgg taaagaggaa agcaaattaa caaaagaagc ttacgcgtgg 840
acatatgaag aagacttcta cgttccgtca gaagtttatg agcatttcgc tgcagctgtt 900
aaagaatcag gtgagaaaaa agaacaagaa tggaatgctc aattcgctaa atataaagaa 960
gtttatcctg aacttgctga acagcttgaa ctggcaatca aaggagagct tccgaaggac 1020
tgggatcaag aggttcctgt gtatgaaaaa ggaagcagcc tggcatcccg tgcatcttcc 1080
ggtgaagttc tcaacggact tgcgaaaaaa attcctttct ttgtcggagg ttctgctgac 1140
ctagcgggat cgaacaaaac gactattaaa aatgccggtg attttacagc ggttgattac 1200
tcaggcaaaa acttctggtt tggtgtacgt gaatttgcga tgggtgcagc cttaaacggt 1260
atggcgcttc atggcggtct tcgtgtattc ggcggaactt tctttgtctt ctctgattac 1320
ctgcgtcctg cgattcgcct tgcagcgtta atgggccttc ctgtgacata tgtcttcaca 1380
catgacagta ttgcggttgg tgaagacggt ccgacgcacg agcctgttga acagcttgct 1440
tcactccgtg cgatgcctaa cctttctttg atccgtccag cagacggcaa tgagacagca 1500
gcagcatgga agcttgcagt gcaaagcact gaccacccaa cagcgctagt gcttacacgt 1560
caaaaccttc ctaccatcga tcaaacagct gaagaagcat tggcaggagt agaaaaaggt 1620
gcatatgtcg tttctaaatc taaaaacgaa acacctgacg ctcttctcat cgcttccgga 1680
tcagaggtag gtcttgcaat tgaagcgcag gctgaattgg caaaagaaaa tatcgatgtt 1740
tctgttgtca gcatgccttc aatggaccgt tttgagaaac aatctgatga atacaaaaac 1800
gaagtccttc ctgcagatgt gaaaaaacgt cttgcaattg aaatgggctc atcatttgga 1860
tggggcaaat acacggggct tgaaggtgac gttctcggca tagaccgatt cggtgcatct 1920
gctccaggtg aaaccatcat taacgaatac gggatggggg gctatttttt cgccttgaga 1980
tgtttctata tagtatcaag ataa 2004
<210> 8
<211> 2004
<212> DNA
<213>SFA-H43 tkt gene
<400> 8
atggatacaa ttgaaaagaa atcagttgct accattcgca cactgtcaat agacgctatt 60
gaaaaagcaa attctggtca cccagggatg ccgatgggag ccgctccaat ggcatacacg 120
ctgtggacaa aatttatgaa cgtaagtccg gcaaaccctg gctggtttaa ccgtgaccgt 180
tttgttttat ctgctggaca cgggtcagca ctattataca gcatgcttca tttaagcggg 240
tttgatctta gtattgaaga tcttaaggga ttccgccagt ggggcagcaa aacaccagga 300
catccggaat tcggacatac tgccggtgtt gatgctacaa caggtccgct tggccaagga 360
attgccatgg cagtcggtat ggcaattgct gaacgccatt tagcggaaac atacaaccgc 420
gattcattta acgtagtcga tcattataca tacagtattt gcggtgatgg tgatttaatg 480
gaaggtattt cttctgaagc cgcttcactc gcaggccatc ttcagcttgg ccgtctgatc 540
gtactatacg attctaatga catctctctt gatggagacc tcgaccgttc attctctgaa 600
aacgtgaaac agcgttttga agcaatgaat tgggaagttc tttatgttga ggatggaaac 660
aatattgaag aattaacagc ggctatcgaa aaagcacgcc aaaatgaaaa gaaacctaca 720
ttaattgaag tgaaaacgac aatcggattc ggttcaccta accgtgccgg tacatccggt 780
gttcacggtg cgccgcttgg taaagaggaa agcaaattaa caaaagaagc ttacgcgtgg 840
acatatgaag aagacttcta cgttccgtca gaagtttatg agcatttcgc tgcagctgtt 900
aaagaatcag gtgagaaaaa agaacaagaa tggaatgctc aattcgctaa atataaagaa 960
gtttatcctg aacttgctga acagcttgaa ctggcaatca aaggagagct tccgaaggac 1020
tgggatcaag aggttcctgt gtatgaaaaa ggaagcagcc tggcatcccg tgcatcttcc 1080
ggtgaagttc tcaacggact tgcgaaaaaa attcctttct ttgtcggagg ttctgctgac 1140
ctagcgggat cgaacaaaac gactattaaa aatgccggtg attttacagc ggttgattac 1200
tcaggcaaaa acttctggtt tggtgtacgt gaatttgcga tgggtgcagc cttaaacggt 1260
atggcgcttc atggcggtct tcgtgtattc ggcggaactt tctttgtctt ctctgattac 1320
ctgcgtcctg cgattcgcct tgcagcgtta atgggccttc ctgtgacata tgtcttcaca 1380
catgacagta ttgcggttgg tgaagacggt ccgacgcacg agcctgttga acagcttgct 1440
tcactccgtg cgatgcctaa cctttctttg atccgtccag cagacggcaa tgagacagca 1500
gcagcatgga agcttgcagt gcaaagcact gaccacccaa cagcgctagt gcttacacgt 1560
caaaaccttc ctaccatcga tcaaacagct gaagaagcat tggcaggagt agaaaaaggt 1620
gcatatgtcg tttctaaatc taaaaacgaa acacctgacg ctcttctcat cgcttccgga 1680
tcagaggtag gtcttgcaat tgaagcgcag gctgaattgg caaaagaaaa tatcgatgtt 1740
tctgttgtca gcatgccttc aatggaccgt tttgagaaac aatctgatga atacaaaaac 1800
gaagtccttc ctgcagatgt gaaaaaacgt cttgcaattg aaatgggctc atcatttgga 1860
tggggcaaat acacggggct tgaaggtgac gttctcggca tagaccgatt cggtgcatct 1920
gctccaggtg aaaccatcat taacgaatac ggcttctcag ttccgaacgt agtgaatcga 1980
gttaaggcat taatcaataa gtaa 2004
<210> 9
<211> 2004
<212> DNA
<213>168 bacterial strain tkt gene orders
<400> 9
atggatacaa ttgaaaagaa atcagttgct accattcgca cactgtcaat agacgctatt 60
gaaaaagcaa attctggtca cccagggatg ccgatgggag ccgctccaat ggcatacacg 120
ctgtggacaa aatttatgaa cgtaagtccg gcaaaccctg gctggtttaa ccgtgaccgt 180
tttgttttat ctgctggaca cgggtcagca ctattataca gcatgcttca tttaagcggg 240
tttgatctta gtattgaaga tcttaaggga ttccgccagt ggggcagcaa aacaccagga 300
catccggaat tcggacatac tgccggtgtt gatgctacaa caggtccgct tggccaagga 360
attgccatgg cagtcggtat ggcaattgct gaacgccatt tagcggaaac atacaaccgc 420
gattcattta acgtagtcga tcattataca tacagtattt gcggtgatgg tgatttaatg 480
gaaggtattt cttctgaagc cgcttcactc gcaggccatc ttcagcttgg ccgtctgatc 540
gtactatacg attctaatga catctctctt gatggagacc tcgaccgttc attctctgaa 600
aacgtgaaac agcgttttga agcaatgaat tgggaagttc tttatgttga ggatggaaac 660
aatattgaag aattaacagc ggctatcgaa aaagcacgcc aaaatgaaaa gaaacctaca 720
ttaattgaag tgaaaacgac aatcggattc ggttcaccta accgtgccgg tacatccggt 780
gttcacggtg cgccgcttgg taaagaagaa agcaaattaa caaaagaagc ttacgcgtgg 840
acatatgaag aagacttcta cgttccgtca gaagtttatg agcatttcgc tgtagctgtt 900
aaagaatcag gtgagaaaaa agaacaagaa tggaatgctc aattcgctaa atataaagaa 960
gtttatcctg aacttgctga acagcttgaa ctggcaatca aaggagagct tccgaaggac 1020
tgggatcaag aggttcctgt gtatgaaaaa ggaagcagtt tggcatcccg tgcatcttcc 1080
ggtgaagttc tcaacggact tgcgaaaaaa attcctttct ttgtcggagg ttctgctgac 1140
ctagcgggat cgaacaaaac gactattaaa aatgccggtg attttacagc ggttgattac 1200
tcaggcaaaa acttctggtt tggtgtacgt gaatttgcga tgggtgcggc cttaaacggt 1260
atggcgcttc atggcggtct tcgtgtattc ggcggaactt tctttgtctt ctctgattac 1320
ctgcgtcctg cgattcgcct tgcagcgtta atgggccttc ctgtgacata tgtcttcaca 1380
catgacagta ttgcggttgg tgaagacggt ccgacgcacg agcctgttga acagcttgct 1440
tcactccgtg cgatgcctaa cctttctttg atccgtccag cagacggcaa tgagacagca 1500
gcagcatgga agcttgcagt gcaaagcact gaccacccaa cagcgctagt gcttacacgt 1560
caaaaccttc ctaccatcga tcaaacatct gaagaagcat tggcaggagt agaaaaaggt 1620
gcatatgtcg tttctaaatc taaaaacgaa acacctgacg ctcttctcat cgcttccgga 1680
tcagaggtag gtcttgcaat tgaagcgcag gctgaattgg caaaagaaaa tatcgatgtt 1740
tctgttgtca gcatgccttc aatggaccgt tttgagaaac aatctgatga atacaaaaac 1800
gaagtccttc ctgcagatgt gaaaaaacgt cttgcaattg aaatgggctc atcatttgga 1860
tggggcaaat acacggggct tgaaggtgac gttctcggca tagaccgatt cggtgcatct 1920
gctcctggtg aaaccatcat taacgaatac ggcttctcag ttccgaacgt agtgaatcga 1980
gttaaggcat taatcaataa gtaa 2004
<210> 10
<211> 500
<212> DNA
<213>500bp sequence before bacterial strain SFR-4 tkt gene terminator
<400> 10
catggaagct tgcagtgcaa agcactgacc acccaacagc gctagtgctt acacgtcaaa 60
accttcctac catcgatcaa acagctgaag aagcattggc aggagtagaa aaaggtgcat 120
atgtcgtttc taaatctaaa aacgaaacac ctgacgctct tctcatcgct tccggatcag 180
aggtaggtct tgcaattgaa gcgcaggctg aattggcaaa agaaaatatc gatgtttctg 240
ttgtcagcat gccttcaatg gaccgttttg agaaacaatc tgatgaatac aaaaacgaag 300
tccttcctgc agatgtgaaa aaacgtcttg caattgaaat gggctcatca tttggatggg 360
gcaaatacac ggggcttgaa ggtgacgttc tcggcataga ccgattcggt gcatctgctc 420
caggtgaaac catcattaac gaatacggga tggggggcta ttttttcgcc ttgagatgtt 480
tctatatagt atcaagataa 500
<210> 11
<211> 500
<212> DNA
<213>500bp sequence after bacterial strain SFA-H43 tkt gene terminator
<400> 11
gcttttgaaa gaggatgagt caaatcatcc tctttttctt gtttatccga caaaaatagt 60
attggctgtt taacagtaat agacaattct ttcttttctt cttttgtata atagagagca 120
aatgaaatgc tgctggaaaa ggggatgaaa agatggaacg tcactactat acgtacctga 180
tcaaagagga atttgccaat cactatttcg gccgggaatc ggttatgttt gagctgtttc 240
aagactatca ttggacaagc cttgaaaagc agcagtatga aatgacagag aaacagattc 300
aatatattac acaaccaatc ccgattttac atatgcatca gcggttaaaa atgaatttaa 360
acaagacgga ttacaggcag ctggattata tttatagaat agctttgccg aaagcaaaag 420
gccacgcgac gtttatgatg aaggagcaca tgatagaaat tgtggcttcg ggagattacg 480
aggcagaaac gatattcttt 500
Claims (10)
1. a kind of bacillus subtilis engineering bacteria for D-ribose production is it is characterised in that described engineering bacteria is withered grass gemma
The engineering bacteria that in bacillus SFA-H43 bacterial strain, tkt gene obtains after replacing with gene order shown in SEQ ID NO.7.
2. a kind of bacillus subtilis engineering bacteria for D-ribose production is it is characterised in that bacillus subtilis SFA-H43 bacterium
500bp sequence before transketolase gene end codon in strain, replaces with the tkt gene of bacillus subtilis SFR-4 bacterial strain eventually
The engineering bacteria that only before codon, 500bp sequence obtains.
3. one plant is used for bacillus subtilis (Bacillus subtilis) the engineering bacteria SFR-43T that D-ribose produces, this bacterial strain
It is preserved in China typical culture collection center on November 22nd, 2016, preservation address is：Wuhan City, Hubei Province Wuchang Luo Ka
Mountain, preserving number is：CCTCC M 2016666.
4. a kind of construction method of the bacillus subtilis engineering bacteria for D-ribose production is it is characterised in that pass through homology weight
The method of group, tkt gene in bacillus subtilis SFA-H43 bacterial strain is replaced with gene shown in SEQ ID NO.7.
5. method according to claim 4 is it is characterised in that gene order shown in SEQ ID NO.7 is derived from withered grass gemma
Bacillus SFR-4 bacterial strain.
6. method according to claim 5 is it is characterised in that in the method for homologous recombination, bacillus subtilis SFA-H43
Before transketolase gene end codon in bacterial strain, 500bp sequence replaces with the tkt gene of bacillus subtilis SFR-4 bacterial strain
500bp sequence before terminator codon.
7. method according to claim 5 is it is characterised in that the construction method of described engineering bacteria is：
(1) with bacterial strain SFR-4 genome as template, expand 500bp fragment before this bacterial strain tkt gene terminator codon, referred to as
L-tkt, wherein downstream primer contain the lox71-zeo-lox66 fragment front end 25bp complementary series of plasmid p7Z6；Preferably, institute
Be SEQ ID NO.1 with primer sequence and SEQ ID NO.2 shown in；
(2) with bacterial strain SFA-H43 genome as template, expand its tkt gene downstream 500bp fragment, be R-tkt, wherein go up
Trip primer contains the lox71-zeo-lox66 end 25bp complementary series of plasmid p7Z6；Preferably, the primer sequence is SEQ
Shown in ID NO.3 and SEQ ID NO.4；
(3) with plasmid p7Z6 as template, amplification carries blasticidin resistance gene fragment lox71-zeo- in lox site
lox66；Preferably, the primer sequence is shown in SEQ ID NO.5 and SEQ ID NO.6；
(4) adopt fusion DNA vaccine method, L-tkt, lox71-zeo-lox66, R-tkt are merged；Preferably, draw used by fusion DNA vaccine
Thing sequence is shown in SEQ ID NO.1 and SEQ ID NO.4；
(5) fragment conversion bacterial strain SFA-H43 will be merged, by blasticidin resistance plate screening integrated recombination mutation bacterial strain,
Obtain the engineered strain of the bacterial strain SFA-H43 containing bacterial strain SFR-4 transketolase mutational site, and adopt Cre/lox systems approach
Knock out the resistant gene zeo in engineered strain, obtain the safe transketolase mutation engineered strain without antibiotics resistance gene, life
Entitled SFR-43T.
8. application in fermenting and producing D-ribose and/3-Hydroxybutanone for the engineering bacteria described in any one of claim 1-3.
9. the method that engineering bacterium fermentation described in a kind of utilization any one of claim 1-3 produces D-ribose and/3-Hydroxybutanone, its
Using following shake flask fermentation and/ferment tank mode：
(1) Spawn incubation：It is inoculated into seed culture medium culture after engineered strain is activated, obtain first order seed and/secondary seed；
(2) shake flask fermentation：Cultured for step (1) first order seed is inoculated into equipped with the shaking flask of fermentation medium, shaking flask is trained
Support and be not further added by D-ribose content in zymotic fluid, terminate fermentation；
(3) ferment tank：Cultured for step (1) one-level or secondary seed are inoculated in fermentation tank, are aerated stirring
Culture, in incubation, controls pH, by adjusting speed of agitator and ventilating ratio, controls dissolved oxygen to be fermented.
10. method according to claim 9 it is characterised in that
In step (1), Spawn incubation is：Take the fresh inclined-plane of 2-3 days engineered strains of 35-40 DEG C of culture, take 1-2 ring with oese
Slant strains, are inoculated in 50ml liquid seed culture medium, 35-40 DEG C, 150-200rpm, Shaking culture 10-16 hour, be for
First order seed；First order seed is inoculated seed culture medium with (percentage by volume) of 1-10%, cultivates 6-12 hour for 35-40 DEG C,
It is for secondary seed；
Or, step (2) shake flask fermentation：By cultured for step (1) first order seed with the inoculation of 1-10% (percentage by volume)
Amount is inoculated into equipped with the shaking flask of fermentation medium, 35-40 DEG C, 180-220rpm, and in Shaking culture to zymotic fluid, D-ribose contains
Amount is not further added by, and terminates fermentation；
Or, step (3) ferment tank：By cultured for step (1) one-level or secondary seed with 1-10% (volume basis
Number) inoculum concentration be inoculated in fermentation tank (liquid amount of 65-75%), 35-40 DEG C is aerated stir culture, incubation
In, control pH in 5-7, by adjusting speed of agitator and ventilating ratio, keep Comparative dissolution oxygen concentration 5-30% in zymotic fluid；
Preferably, the consisting of of the slant strains culture medium used by inclined-plane culture：Yeast extract 5g/L, peptone 10g/L,
NaCl 3g/L, agar 20g/L, remaining is water, pH7.0-7.2；
Preferably, the consisting of of liquid primary-seed medium：Glucose 20-40g/L, yeast extract 2-10g/L, corn steep liquor
8-12g/L, pH 7.0-7.2；
Preferably, the consisting of of secondary seed medium：Glucose 30-60g/L, yeast extract 2-10g/L, corn steep liquor 8-
12g/L, (NH4)2SO42-3g/L, MgSO42g/L, pH 7.0-7.2；
Preferably, Medium of shaking flask fermentation consists of：Glucose 120-150g/L, yeast extract 2-10g/L, corn steep liquor 5-
15g/L, (NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH 7.0-7.2；
Preferably, ferment tank initial medium consists of：Glucose 120-150g/L, yeast extract 3-6g/L, corn
Slurry 5-10g/L, (NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH 6.0-7.2；
Preferably, the time of primary-seed medium culture is 12-14 hour；
Preferably, the time of secondary seed medium culture is 6-8 hour；
Preferably, the cultivation temperature of shake flask fermentation or ferment tank is 36-38 DEG C；
Preferably, the pH of ferment tank controls in 5.5-6.5；
Preferably, the dissolved oxygen concentration of ferment tank is maintained at 20-30%；
It is furthermore preferred that glucose 130-150g/L in fermentation medium, yeast extract 3-6g/L, corn steep liquor 6-10g/L,
(NH4)2SO42-3g/L, MgSO42g/L, MnSO40.2g/L, pH 7.0-7.2；
Preferably, ferment tank is improved fermentation efficiency by way of feed supplement and increases fermentation production rate；
Preferably, fed-batch fermentation, initial glucose concentration is 120-150g/L；
Preferably, feed supplement adopts a feed profile；
Preferably, the feed supplement time is that in zymotic fluid, concentration of glucose is down to 20-40g/L；
Preferably, feed supplement amount is 40-60g/L glucose；
Preferably, feed supplement concentration of glucose is 600-800g/L, yeast extract 0.5g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828710A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of unmarked multiple-sites integration expression glutaminase bacterial strain and its construction method |
| CN108913706A (en) * | 2018-07-10 | 2018-11-30 | 郑州轻工业学院 | A kind of bacillus subtilis glycerokinase mutated gene glpK and its application |
| CN111647485A (en) * | 2020-06-16 | 2020-09-11 | 宜春市庆春硒黄酒酿造有限公司 | High-activity selenium-rich cardamine hirsute distiller's yeast and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007051552A1 (en) * | 2005-11-02 | 2007-05-10 | Dsm Ip Assets B.V. | Modified transketolase and use thereof |
| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
| CN101974444A (en) * | 2010-05-13 | 2011-02-16 | 南京工业大学 | Bacillus subtilis for highly producing D-ribose and co-producing acetoin |
| CN103146595A (en) * | 2012-12-03 | 2013-06-12 | 山东邦奥创业生物科技有限公司 | Bacillus subtilis and method for fermentation production of D- ribose |
| CN103333842A (en) * | 2013-07-10 | 2013-10-02 | 山东省食品发酵工业研究设计院 | Bacillus subtilis producing 3-hydroxybutanone and application thereof |
-
2016
- 2016-12-16 CN CN201611169915.0A patent/CN106399218A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007051552A1 (en) * | 2005-11-02 | 2007-05-10 | Dsm Ip Assets B.V. | Modified transketolase and use thereof |
| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
| CN101974444A (en) * | 2010-05-13 | 2011-02-16 | 南京工业大学 | Bacillus subtilis for highly producing D-ribose and co-producing acetoin |
| CN103146595A (en) * | 2012-12-03 | 2013-06-12 | 山东邦奥创业生物科技有限公司 | Bacillus subtilis and method for fermentation production of D- ribose |
| CN103333842A (en) * | 2013-07-10 | 2013-10-02 | 山东省食品发酵工业研究设计院 | Bacillus subtilis producing 3-hydroxybutanone and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| P. DE WULF等: "Optimization of D-ribose production with a transketolaseaffected Bacillus subtilis mutant strain in glucose and gluconic acid-based media", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| P.DE WULF等: "Production of D-ribose by fermentation", 《APPL. MICROBIOL. BIOTECHNOL.》 * |
| 赵祥颖等: "D-核糖性质、应用及其生产菌种的选育", 《食品与药品》 * |
| 赵祥颖等: "发酵法生产D-核糖技术的研究（III）——生物发酵法生产D-乳糖", 《山东食品发酵》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828710A (en) * | 2017-12-13 | 2018-03-23 | 江南大学 | A kind of unmarked multiple-sites integration expression glutaminase bacterial strain and its construction method |
| CN107828710B (en) * | 2017-12-13 | 2020-07-03 | 江南大学 | Mark-free multi-site integrated expression glutaminase strain and construction method thereof |
| CN108913706A (en) * | 2018-07-10 | 2018-11-30 | 郑州轻工业学院 | A kind of bacillus subtilis glycerokinase mutated gene glpK and its application |
| CN108913706B (en) * | 2018-07-10 | 2021-04-09 | 郑州轻工业学院 | Bacillus subtilis glycerol kinase mutant gene glpK and application thereof |
| CN111647485A (en) * | 2020-06-16 | 2020-09-11 | 宜春市庆春硒黄酒酿造有限公司 | High-activity selenium-rich cardamine hirsute distiller's yeast and preparation method thereof |
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