CN108456652A - A kind of Sphingol single-cell genetic engineering bacterium and its construction method and application - Google Patents
A kind of Sphingol single-cell genetic engineering bacterium and its construction method and application Download PDFInfo
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- CN108456652A CN108456652A CN201810207379.1A CN201810207379A CN108456652A CN 108456652 A CN108456652 A CN 108456652A CN 201810207379 A CN201810207379 A CN 201810207379A CN 108456652 A CN108456652 A CN 108456652A
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- 230000004151 fermentation Effects 0.000 claims abstract description 38
- 238000000855 fermentation Methods 0.000 claims abstract description 37
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention discloses a kind of Sphingol single-cell genetic engineering bacteriums, and to have imported the Sphingol single-cell of adversity gene, the adversity gene comes from extreme microorganism.The invention also discloses the methods that the method combined by three parents builds Sphingol single-cell genetic engineering bacterium.The invention also discloses application of the Sphingol single-cell genetic engineering bacterium in fermentation prepares Weilan gum or gellan gum.The Sphingol single-cell genetic engineering bacterium of the present invention significantly improves the temperature tolerance and degrees of Sphingol single-cell, ranging from 30~42 DEG C of tolerable temperature, and tolerable pH range is 4.0~10.0；Weilan gum is prepared for fermentation, not under conditions of external source regulation and control pH, microbial polysaccharide yield reaches 20~30g/L；When preparing gellan gum for fermentation, not under conditions of external source regulation and control pH, microbial polysaccharide yield reaches 15~22g/L；Enhance the production efficiency of the microbial polysaccharides such as gellan gum, Weilan gum.
Description
Technical field
The present invention relates to genetic engineering bacterium and its construction method and applications, and in particular to a kind of Sphingol single-cell gene work
Journey bacterium and its construction method and application.
Background technology
Microbial polysaccharide is a kind of typical microbial fermentation product, most important to human lives and industrial production.It is early
Ratified as important food additives by FDA in xanthans in 1969；Gellan gum in 1998 is reported as treatment eye illness most
Primary drug；Weilan gum in 1992 is reported as deep-sea and recovers the oil irreplaceable carminative.Kelco companies of the U.S. are that the whole world is maximum
Microbial polysaccharide production supplier, develop xanthans and novel microbial polysaccharide i.e. sphingan (such as gellan gum, prestige
Blue glue, sandlwood carbohydrate gum, S88, S7) etc. multiple-microorganisms polysaccharide.Research finds that these polysaccharide have structure diversity, unique rheology
Learn, physics characteristic and it is safe and non-toxic the features such as, be widely used in food, medicine, agricultural, cosmetics, building and petroleum industry
Etc. multiple fields.Therefore domestic and international researcher has been devoted to the PRODUCTION TRAITS of microbial polysaccharide, at present whole world microbial polysaccharide
Annual output is about 300,000 tons, and for annual value of production up to nearly 20,000,000,000 dollars, application prospect is very wide.
So far, in addition to xanthans production scale is larger in microbial polysaccharide, gellan gum, Weilan gum, the life of sandlwood carbohydrate gum
Production scale is little, and the country just started Weilan gum production until nearly 2 years, and poor efficiency and high production cost are to limit it greatly
The major obstacle of scale industrialization.Main cause is that microbial polysaccharide produces Sphingol single-cell to temperature extreme sensitivity, and sends out
Mechanical agitation heat and metabolic heat are generated during ferment, need a large amount of cooling waters to control temperature, process energy consumption is big, of high cost；
The fermentation process middle and later periods, with a large amount of accumulation of Weilan gum, weakly acidic feature is presented in zymotic fluid, and pH value grows as microorganism
And the important limiting factor of secondary metabolite synthesis.Exploitation high resistance to cold and diseases production Sphingol single-cell solves the above problems
Key, but the research of this aspect at present is in space state.
Invention content
Goal of the invention：In order to solve existing production microbial polysaccharide Sphingol single-cell to temperature and pH poor resistances
Problem, the present invention provides a kind of Sphingol single-cell genetic engineering bacteriums, and it is a further object of the present invention to provide sphingol lists
The construction method of born of the same parents' bacterium genetic engineering bacterium, it is still further an object of the present invention to provide Sphingol single-cell genetic engineering bacteriums to send out
Ferment produces the application in polysaccharide.
Gene transcription level adjusting is that microorganism is essential in order to maintain normal cell activity and response environment to change
Regulatory mechanism.Transcription regulatory factor (transcriptional regulators) by adjust gene expression, DNA plerosis or
Protein macromolecule, promotion form a series of biological approaches of the approach such as biomembrane influence, are played in microorganism growth metabolism
Vital effect.Such transcription regulatory factor is operated, it is made to play regulating and controlling effect in microorganism, it is high by complexity
The transcription regulatory network of effect, microorganism can make ambient pressure environment (such as pH, temperature, nutriment, osmotic pressure) in time
Response, and optimize metabolism degree to adapt to new environment.In addition, temperature substantially change or has heavy metal, toxic chemical, oxidation
Under the unfavorable conditions such as agent presence and pathogenic microorganism infection, the adaptive change on a kind of shared molecular level of biology is base
Because of substantially changeing for express spectra, wherein important one kind is referred to as heat shock protein (Heat shock proteins, Hsps)
Protein families synthesize conspicuousness increase, as a kind of molecular chaperones, Hsps participate in albumen it is correct folding, polymerization, transport and
The important physiology courses such as signal transmission.Their a large amount of synthesis can guarantee the survival of the cell under stressed condition, otherwise will cause
The irreversible damage of cell simultaneously leads to death.Since long adaptation in extreme circumstances is evolved, extreme microorganism has bright
The aobvious anti-adversity better than other Sphingol single-cells；It will be from the heat shock of extreme microorganism using molecular biology method
Albumen, which is applied to industrial microorganism tolerance performance transformation, has feasibility in theory.
Technical solution：A kind of Sphingol single-cell genetic engineering bacterium of the present invention, to have imported the sheath ammonia of adversity gene
Alcohol monad, the adversity gene come from extreme microorganism；Wherein, the adversity gene is the gene of encoding heat shock proteins
Or the gene of encoding global transcription regulatory factor.
Ranging from 30~42 DEG C of the Sphingol single-cell genetic engineering bacterium tolerable temperature of structure, tolerable pH range be 4.0~
10.0。
The extreme microorganism be Tengchong heat mutually bacteria (Thermoanaerobacter tengcongensis), thermophilic dwell
Hot bacterium (Thermus thermophilus), sulfolobus solfataricus (Sulfolobus solfataricus), heat color bacillus
(Flammeovirga sp.) or the unusual coccus of radiation hardness (Deinococcus radiodurans), purchased from the common micro- life of China
Object culture presevation administrative center (CGMCC).
Preferably, the adversity gene is specially any gene in following (1) to (5)：
(1) the GroES genes of encoding heat shock proteins GroES；
(2) the GroEL genes of encoding heat shock proteins GroEL；
(3) the DnaK genes of encoding heat shock proteins DnaK；
(4) the DnaJ genes of encoding heat shock proteins DnaJ；
(5) the IrrE genes of encoding global transcription regulatory factor IrrE.
The nucleotide sequence of the GroES genes such as SEQ ID NO：Shown in 1, the nucleotide sequence of the GroEL genes
Such as SEQ ID NO：Shown in 2, the nucleotide sequence such as SEQ ID NO of the DnaK genes：Shown in 3, the core of the DnaJ genes
Nucleotide sequence such as SEQ ID NO：Shown in 4, the nucleotide sequence such as SEQ ID NO of the IrrE genes：Shown in 5.
The present invention further provides the construction method of the Sphingol single-cell genetic engineering bacterium, includes the following steps：
First from genes such as the heat shock protein in ncbi database screening extreme microorganism source, global transcription regulatory factors
Sequence；
(1) it is the plasmid that sets out with pET28a, PCR amplification adversity gene builds the recombination pET28a plasmids containing adversity gene；
(2) as template, PCR amplification obtains carrying the degeneration-resistant of T7 promoters the recombination pET28a plasmids obtained using step (1)
Genetic fragment；
(3) be to set out plasmid with pBBR1MCS-5, the adversity gene segment for the carrying T7 promoters that step (2) is obtained and
PBBR1MCS-5 plasmids connect, structure recombination pBBR1MCS-5 plasmids；
(4) recombination pBBR1MCS-5 plasmids are imported in E.coli DH5 α competent cells, obtains recombination E.coliDH5
α；
(5) using Sphingol single-cell as recipient bacterium, the recombination E.coli DH5 α that step (4) obtains are donor bacterium, E.coli
HB101/pRK2013 is to assist bacterium, and the method combined by three parents, structure obtains Sphingol single-cell genetic engineering bacterium.
Sphingol single-cell described in step (5) is Alcaligenes sp.CGMCC2428 or Sphingomonas
sp.CGMCC31461；Sphingomonas is the new category that nineteen ninety Japanese scholars Yabuuchi etc. is put forward for the first time, and early stage is studied
Think that Weilan gum production bacterial strain belongs to alcaligenes (Alcaligenes sp.), rear Authenticate afresh is attributed to sphingol list
Born of the same parents Pseudomonas (Sphingomonas sp.), therefore Alcaligenes sp.CGMCC2428 are Sphingomonas in present patent application
Bacterium.
The present invention further provides application of the Sphingol single-cell genetic engineering bacterium in fermentation prepares polysaccharide.
When recipient bacterium is Alcaligenes sp.CGMCC2428, Weilan gum is prepared for fermentation；The fermentation stage
Fermentation temperature is 30~42 DEG C, and fermentation pH is 4.0~10.0, and fermentation process does not add allogene regulation and control pH.Fermented and cultured basigamy
Side is as follows：Carbon source is any one or a few in glucose, sucrose, fructose, soluble starch, molasses and starch hydrolyzate
Mixture, a concentration of 20~70g/L；The nitrogen source be peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed meal, urea,
(NH4)2SO4And NH4Any one or a few mixture in Cl, a concentration of 1~10g/L；The inorganic salts are sylvite, magnesium
Any one or a few mixture in salt, phosphate and sulfate, a concentration of 0.1~10g/L, solvent are water.
When recipient bacterium is Sphingomonas sp.CGMCC31461, gellan gum is prepared for fermentation；The fermentation rank
Section fermentation temperature is 30~42 DEG C, and fermentation pH is 4.0~10.0, and fermentation process does not add allogene regulation and control pH.Fermentation medium
Formula is as follows：Carbon source is any one or a few in glucose, sucrose, fructose, soluble starch, molasses and starch hydrolyzate
Mixture, a concentration of 20~70g/L；The nitrogen source be peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed meal, urea,
(NH4)2SO4And NH4Any one or a few mixture in Cl, a concentration of 1~10g/L；The inorganic salts are sylvite, magnesium
Any one or a few mixture in salt, phosphate and sulfate, a concentration of 0.1~10g/L, solvent are water.
Advantageous effect：The Sphingol single-cell genetic engineering bacterium of the present invention can be applied in the production of sphingan,
The temperature tolerance and degrees of fermenting Sphingomonas are significantly improved, tolerable temperature range expands from 30~36 DEG C
To 30~42 DEG C, tolerable pH range is expanded to 4.0~10.0 by 7.0 or so；Weilan gum is prepared for fermentation, external source does not regulate and control pH
Under conditions of, Weilan gum yield reaches 20~30g/L；When preparing gellan gum for fermentation, not under conditions of external source regulation and control pH, knot
Cold glue yield reaches 15~22g/L；The production efficiency of the microbial polysaccharides such as gellan gum, Weilan gum is enhanced, technical process is improved
Economy.
Description of the drawings
Fig. 1 is recombination expression vector establishment schematic diagram；
Fig. 2 is the survival rate recombinated under Sphingol single-cell different temperatures；
Fig. 3 is the survival rate recombinated under Sphingol single-cell difference pH；
Fig. 4 is that stress-resistant Weilan gum produces Sphingol single-cell (recombination Sphingol single-cell) in 42 DEG C, nature pH conditions
Lower fermentation conditional curve；
Fig. 5 is that stress-resistant gellan gum produces Sphingol single-cell (recombination Sphingol single-cell) in 42 DEG C, nature pH conditions
Lower fermentation conditional curve.
Specific implementation mode
The recombination Sphingol single-cell structure of the mutual bacteria GroES genes of the heat containing degeneration-resistant Tengchong of embodiment 1
Step 1: adversity gene expands
Using Tengchong heat, mutually bacteria (CGMCC5161) genome uses software CE Design V1.0 design of primers as template
Software for Design synthetic primer (primer sequence is shown in Table 1) clones degeneration-resistant GroES genes, the nucleotide sequence such as SEQ of GroES genes
ID NO：Shown in 1.The PCR conditions of GroES genes：1) 94 DEG C of denaturation 5min；2) following parameter cyclic is pressed 30 times：94 DEG C of denaturation
1min, 60 DEG C of annealing 60s, 72 DEG C of extension 1min；3) last 72 DEG C of extensions 10min.PCR takes 2 μ L of product after reaction, then
In the Ago-Gel of a concentration of 1g/100mL, electrophoretic analysis is carried out.It is imaged through gel imaging system and is confirming clip size just
After really, the structure that target fragment is used for recombinant expression carrier is recycled using DNA purifying QIAquick Gel Extraction Kits (TaKaRa, 9761).
1 adversity gene primer of table and its nucleotide sequence
Step 2: the recombination bacillus coli structure containing adversity gene
1, pET28a plasmid constructions are recombinated
It is the plasmid that sets out with pET-28a (+) (Novagen, 69864-3), with Nco I (TaKaRa, 1160A)/Not I
(TaKaRa, 1166A) double digestion.The target fragment and linearized vector DNA gel QIAquick Gel Extraction Kit that step 1 obtains
(TaKaRa, 9762) is purified, is recycled.Connect target fragment and carrier by homologous recombination kit (promise is only praised,
C112-1 it) connects, pET-groes recombinant plasmids is obtained with this.
2, pBBR1MCS-5 plasmid constructions are recombinated
Using the pET28a plasmids of recombination as template, design primer (primer sequence is shown in Table 1) amplification clone obtains carrying T7 and opens
The adversity gene segment of mover.Be to set out plasmid with pBBR1MCS-5 (gloomy vast biology, P0309), with Hind III (TaKaRa,
Carrier segments 1060A)/BamH I (TaKaRa, 1010A) are recycled after double digestion, source recombination kit and step are only approved of using promise
The GroEs segments connection of the carrying T7 promoters obtained in rapid 1, structure obtain plasmid name pBBR-groes.By the company of 10 μ L
Object of practicing midwifery is added in the bacillus coli DH 5 alpha competent cell of 100 μ L：1) 30min, 42 DEG C of heat shock 90s are placed on ice；2) on ice
Place 2min；3) the 0.45mL LB culture mediums of preheating, 220rpm, 37 DEG C of stirring 1h are added；4) addition of 200 μ L bacterium solutions is contained
On the LB tablets of the gentamicin of 30 μ g/mL, 37 DEG C are incubated overnight 12~16h, obtain recombinant bacterium E.coli DH5 α (containing recombination
PBBR plasmids), gained Sphingol single-cell is respectively designated as：E.coli GroES, building process are shown in Fig. 1.
LB culture mediums：NaCl 10g/L, peptone 10g/L, yeast powder 5g/L, solvent are water.
Step 3: the recombination Sphingol single-cell structure containing adversity gene
By three parents engage method, with help plasmid pRK2013 be mediate, realize recombination pBBR1MCS-5 plasmids with
Recombinate Sphingol single-cell (Sphingomonas sp.；S.sp. the engagement transfer between).
1, thalline culture：Donor bacterium E.coli GroES (plasmids of the pBBR1MCS-5 containing recombination), help bacterium E.coli
HB101 (gloomy vast biology, P1446)/pRK2013 (gloomy vast biology, P0469), is inoculated into the LB culture mediums added with corresponding antibiotic
In 37 DEG C culture 8h, recipient bacterium S.sp. in Sphingol single-cell seed culture medium 30 DEG C culture 16h, incubation time about its
Mid log phase, each Sphingol single-cell vital movement is vigorous at this time, compares and is appropriate for mating experiment；
Wherein, the reference of bacterium E.coli HB101/pRK2013 construction methods is helped《Molecular Cloning:A Laboratory guide》(third edition,
Science Press, in January, 2016,93-97 pages) in Escherichia coli method for transformation part.
Wherein, a concentration of 50 μ g/mL of gentamicin in the LB culture mediums of donor bacterium E.coli GroES, culture side are cultivated
Help a concentration of 100 μ g/mL of the LB culture medium streptomycins of bacterium E.coli HB101/pRK2013.
Sphingol single-cell seed culture medium：Glucose 20g/L, yeast extract 1g/L, peptone 3g/L, K2HPO4·3H2O
2g/L, MgSO40.1g/L, pH 7.2~7.4.
Wherein, recipient bacterium Sphingol single-cell is Alcaligenes sp.CGMCC2428 or Sphingomonas
sp.CGMCC31461。
2, three parents engage：Above-mentioned receptor parent bacterium solution 2mL is taken, thalline were collected by centrifugation, brine 2~3 times,
Fall supernatant, the help bacterium of 2.0mL is added, centrifuges the supernatant that inclines after mixing, 2mL donor bacterium is added, LB culture mediums are clear after centrifugation
Mixing after washing 3 times takes a part of liquid to be applied to non-resistant Sphingol single-cell tablet/slant medium, and 30 DEG C are incubated overnight.
Sphingol single-cell tablet/slant medium：Glucose 10g/L, peptone 10g/L, beef extract 3g/L,
NaCl5g/L, agar 20g/L adjust pH to 7.2~7.4.
3, joint element screens：The combination thalline of overnight incubation is washed with sterile water from solid medium, is applied to
It on Double screening and culturing medium containing gentamicin and streptomysin, cultivates 2~4 days, screening recon obtains expression bacterium
S.sp.GroEs。
Screening and culturing based formulas is as follows：Glucose 10g/L, peptone 10g/L, beef extract 3g/L, NaCl 5g/L, agar
20g/L, 50 μ g/mL of gentamicin, 100 μ g/mL of streptomysin adjust pH to 7.2~7.4.
Recombination Sphingol single-cell structure of the embodiment 2 containing degeneration-resistant GroEL genes
Construction method is with embodiment 1, the difference is that adversity gene is GroEL genes, derives from thermus thermophilus
(CGMCC15059), nucleotide sequence such as SEQ ID NO：Shown in 2, primer sequence table is shown in Table 1, obtains S.sp.GroEL
Recombination Sphingol single-cell structure of the embodiment 3 containing degeneration-resistant IrrE genes
Construction method is with embodiment 1, the difference is that adversity gene is IrrE genes, derives from the unusual coccus of radiation hardness
(CGMCC633), nucleotide sequence such as SEQ ID NO：Shown in 5, primer sequence table is shown in Table 1, obtains S.sp.IrrE.
Recombination Sphingol single-cell structure of the embodiment 4 containing degeneration-resistant DnaK genes
Construction method is with embodiment 1, the difference is that adversity gene is DnaK genes, nucleotide sequence such as SEQ ID NO：3
It is shown.
Recombination Sphingol single-cell structure of the embodiment 5 containing degeneration-resistant DnaJ genes
Construction method is with embodiment 1, the difference is that adversity gene is DnaJ genes, nucleotide sequence such as SEQ ID NO：4
It is shown.
Embodiment 6 recombinates Sphingol single-cell different temperatures survival rate and investigates
Sphingol single-cell (recipient bacterium is Alcaligenes sp.CGMCC2428) S.sp.GroES will be recombinated,
S.sp.GroEL, S.sp.IrrE are research object, collect 2mL bacterium solutions respectively, with brine 2~3 times, outwell supernatant
Liquid, is added certain volume physiological saline, and gradient dilution takes 200uL dilutions to apply the celebrating that respective concentration is added at identical OD values
Big mycin and streptomysin plating medium (formula is with reference to " screening and culturing medium " in embodiment 1) place incubator culture 72h, and set
Set control group (i.e. Alcaligenes sp.CGMCC2428), respectively record 30 DEG C, 33 DEG C, 36 DEG C, 39 DEG C, 42 DEG C, 45 DEG C not
Synthermal lower Sphingol single-cell viable count.As a result see Fig. 2, when temperature is less than 36 DEG C, the expression of adversity gene is to sphingol list
Born of the same parents' bacterium survival rate has not significant impact；After temperature is higher than 39 DEG C, seriously reduction is even all dead for control group viable count, and table
The Sphingol single-cell survival rate for having reached adversity gene is significantly higher than control group, shows apparent growth vigor.In addition, just warm
It spends for tolerance effects, the Sphingol single-cell S.sp.IrrE effects for expressing global transcription factor are the most apparent, can be by sphingol
Monad tolerable temperature is improved to 42 DEG C.
Embodiment 7 recombinates Sphingol single-cell difference pH survival rates and investigates
Method distinguishes dilution spread on the tablet of pH4.0,7.0,10.0 with embodiment 6, by bacterium solution, is placed on 30 DEG C of trainings
Case culture 72h is supported, viable count is recorded.As a result see Fig. 3, control group is under the conditions of more high or low pH, Sphingol single-cell survival rate
It is substantially reduced, and the pH tolerance ranges of Sphingol single-cell can be expanded to pH4.0~10.0 by the expression of adversity gene.
8 stress-resistant of embodiment recombinates Sphingol single-cell and is used for Weilan gum fermenting and producing
Sphingol single-cell (recipient bacterium is Alcaligenes sp.CGMCC2428) S.sp.GroES will be recombinated respectively,
S.sp.GroEL, S.sp.IrrE and control group A lcaligenes sp.CGMCC2428 be inoculated in Sphingol single-cell tablet/tiltedly
Face culture medium (formula is with reference to embodiment 1), 30 DEG C of cultures are for 24 hours.A ring bacterium is connect in equipped with (the formula reference of 135mL seed culture mediums
Embodiment 1) 1000mL triangular flasks in, rotating speed 200r/min, 30 DEG C of culture 16h.Cultured seed liquor is pressed into volume basis
Than in 7.5L fermentation tanks of the inoculum concentration access equipped with 4L fermentation mediums for 3%, 72h is cultivated under 42 DEG C, nature pH.Pass through
Weilan gum synthesis capability under the conditions of high temperature, nature pH is investigated and is found, compares original Sphingol single-cell under these conditions not
Weilan gum can be largely synthesized, and the Weilan gum yield of the recombination Sphingol single-cell through genetic modification reaches 20g/L or more,
The Weilan gum yield highest of middle S.sp.IrrE, reaches 25.7 ± 0.25g/L (Fig. 4).It is most ideal in actual industrial fermentation process
State be Sphingol single-cell optimum growth temperature range to high temperature extend, and have wider growth pH ranges, because
This recombination Sphingol single-cell can better meet industrial production demand.
Sphingosine unit cell strain fermentation culture medium prescription is as follows：Glucose 50g/L, yeast extract 8g/L, K2HPO4·3H2O
3g/L, MgSO4 0.4g/L。
9 stress-resistant of embodiment recombinates Sphingol single-cell and is produced for gellan gum fermentation
Method is with embodiment 8, the difference is that recombination Sphingol single-cell (recipient bacterium S.sp.CGMCC31461)
S.sp.GroES, S.sp.GroEL, S.sp.IrrE, control group S.sp.CGMCC31461.42 DEG C are investigated, natural pH fermentations item
Under part, the case where sphingosine unit cell strain fermentation produces gellan gum is recombinated.As a result see Fig. 5, the original Sphingol single-cell of control group is not
Gellan gum can be largely synthesized, and the recombination Sphingol single-cell for expressing adversity gene shows good production performance, knot is cold
Glue yield reaches as high as 18.1 ± 0.16g/L, is significantly better than control group.
Wherein, sphingosine unit cell strain fermentation culture medium is：K2PO41.5g/L, KH2PO41g/L, MgSO40.6g/L, yeast extract
0.2g/L, soyabean protein powder 2g/L, sucrose 30g/L, bubble enemy 0.1% (v/v).
Sequence table
<110>Nanjing University of Technology
<120>A kind of Sphingol single-cell genetic engineering bacterium and its construction method and application
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ttaatcatgc gtgaggctga cattttagca atcgtataa 279
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atcgctgaag caatggaaaa agtaggtaaa gatggtgtaa tcactgttga agaagcaaaa 540
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ttggtagtca acaaaatccg tggtgcatta aaagttgcag ctgttaaagc tccaggtttt 840
ggtgaccgtc gtaaggcaat gttacaagac atcgctatct taactggtgg tactgtaatc 900
tctgacgaga cgggtatgaa gctagaagat gcgactatcg acatgcttgg tactgctgag 960
aaagtaatca tcgacaaaga caatactgtt gttgtaaatg gtgctggttc tgctgaagct 1020
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aaagcaaatc aagataagaa aatcgcagta tttgaccttg gtggtggtac tttcgatatc 600
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ttaggtggtg atgacttcga taaagtaatc attgactggt tagcggaaga atttatgaaa 720
gatcacccaa caatcgatct tcgtaaagac ccaatggcat tacaacgttt gaaagaggca 780
gctgaaaaag caaaaatcga gctttctgca ggtgctaaaa cagacatcaa cttgccatac 840
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gatgctgact tatctacttc agatatcgat gaagtaatct tagtaggtgg ttctactcgt 1020
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ccaagaggtg tacctcaaat cgaagtaact tttgatattg atgcgaacgg tatcgtaagt 1440
gtatcagcaa aagataaagc aactggtaaa gagcagtcta tcaaaattga ggcttcttca 1500
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gaggcagcat tgaaagactt gaaagaagct cacgctgctc aagatttgga taaaatccaa 1740
cctgctttag acgcattaaa tgcggcatgg caagcggctt ctcaagagat gtaccaagct 1800
acaggtggtg acgctgcagg tgcagctggt gctgatccta acgctgctca aggtggtgca 1860
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ttaggtgaaa aatttaaaga ggtcaacgaa gcctatgaga ccttgagtga tgatagtaag 180
aaatttcact atgatcatgg cttaacttcg ttcactacat tttcgaatac ccctactcca 240
acagaggtgg ctgaggaaga aattcgaaca caaagaaaat ctgaagtaaa agaagagatt 300
tttaaagaaa aagctaagga gaaaaaaagt aaaaagtttt tcgttccttt tactgtaggt 360
gtatttacct tagtagttgg tgggatgatc tcttttttcg ttcaacgcca gagtaaactg 420
gctgcatcta cctttgaaga tgcaacaaca aaccttgaat tgaaagattt taatgctgtt 480
aaaggtaata ttgatgtctt atatcactta gaatctcctc ttcttgcaga tattattgag 540
gctggtttat acgtccaact caaccaaagt aatgaagcta ttgatctatt agaaccaaaa 600
ttatatatga ttaatgagga acctaaaaat attgtttctc aagcatatct ctatttaggt 660
attgcctact atcaaagaaa attagggaat aaggcagttg attatttaga gaaatcaatt 720
tcttacaata acaaaaacaa acaagtttat tattggttag gagtcactta ctcagaactt 780
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gaaaaccaat ctattttaag tttaggaata gcatatcaac gttccggaca atataatgct 900
gcccaagata attttgagct tttattattc aaccctgatt ttaaaaagga agccaactat 960
tatatgggtt ggaattattt cctaagtaac aaaaacacta ataaagcttg ccttttatgg 1020
gagaaagctg ctaaaatggg ttctcaagaa gctagatatc aagtgcagag gcattgtggt 1080
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gtgaccgccc ccgacgccct cgccgccgcc aaagccagga tgcgcgacct ggcggcggcc 180
tacgtggcgg ccctgcccgg acgcgacacc cacagcctga tggcgggggt gcccggcgta 240
gacctcaaat tcatgccgct cggctggcgc gacggggcgt tcgaccccga gcacaacgtc 300
atcctcatca actcggcggc ccgccccgaa cgccagcgct tcaccctcgc ccacgaaatc 360
gggcacgcga ttttactcgg cgacgacgac ctgctctccg acatccacga cgcctacgag 420
ggcgagcggc tcgaacaggt catcgaaacg ctgtgcaacg tggcggcggc ggcgattttg 480
atgcccgaac ccgtcatcgc ggaaatgctg gaacgcttcg gccccaccgg gcgcgccctc 540
gccgaactcg ccaagcgggc cgaagtcagc gcgtcgtcgg cgctctacgc cctgaccgag 600
cagaccccgg tgcccgtcat ctacgcggtc tgtgcgccgg gcaagcctcc gcgtgagcag 660
gccgcaagcg acgaggacgc tggcccaagc acagaaaaag tcctgacggt ccgcgccagc 720
agctcgacgc ggggcgtcaa gtacaccctg gcgagcggca cgccggtacc cgccgaccac 780
ccggcggcgc ttgccctcgc cacgggcatg gaagtgcgcg aggaaagcta cgtgcccttt 840
cgctcgggcc ggaaaatgaa ggcggaggtg gacgcctacc cgtcgcgcgg catcgtggcc 900
gtcagtttcg agttcgaccc cgcccgcctg ggccgcaagg acagcgagca ggccgaccgg 960
gacgagccgc aggacgctgc acagtga 987
Claims (10)
1. a kind of Sphingol single-cell genetic engineering bacterium, which is characterized in that the genetic engineering bacterium is to have imported adversity gene
Sphingol single-cell, the adversity gene come from extreme microorganism；Wherein, the adversity gene is encoding heat shock proteins
The gene of gene or encoding global transcription regulatory factor.
2. Sphingol single-cell genetic engineering bacterium according to claim 1, which is characterized in that the extreme microorganism is to rise
Punching heat mutual bacteria (Thermoanaerobacter tengcongensis), thermus thermophilus (Thermus
Thermophilus), sulfolobus solfataricus (Sulfolobus solfataricus), heat color bacillus (Flammeovirga sp.) or
The unusual coccus of radiation hardness (Deinococcus radiodurans).
3. Sphingol single-cell genetic engineering bacterium according to claim 1, which is characterized in that the adversity gene is as follows
(1) to any gene in (5)：
(1) the GroES genes of encoding heat shock proteins GroES；
(2) the GroEL genes of encoding heat shock proteins GroEL；
(3) the DnaK genes of encoding heat shock proteins DnaK；
(4) the DnaJ genes of encoding heat shock proteins DnaJ；
(5) the IrrE genes of encoding global transcription regulatory factor IrrE.
4. Sphingol single-cell genetic engineering bacterium according to claim 3, which is characterized in that the core of the GroES genes
Nucleotide sequence such as SEQ ID NO：Shown in 1, the nucleotide sequence such as SEQ ID NO of the GroEL genes：Shown in 2, the DnaK
The nucleotide sequence of gene such as SEQ ID NO：Shown in 3, the nucleotide sequence such as SEQ ID NO of the DnaJ genes：Shown in 4,
The nucleotide sequence of the IrrE genes such as SEQ ID NO：Shown in 5.
5. the construction method of Sphingol single-cell genetic engineering bacterium described in claim 1-4 any one, which is characterized in that including
Following steps：
(1) it is the plasmid that sets out with pET28a, PCR amplification adversity gene builds the recombination pET28a plasmids containing adversity gene；
(2) for the recombination pET28a plasmids obtained using step (1) as template, PCR amplification obtains carrying the adversity gene of T7 promoters
Segment；
(3) be to set out plasmid with pBBR1MCS-5, the adversity gene segment for the carrying T7 promoters that step (2) is obtained and
PBBR1MCS-5 plasmids connect, structure recombination pBBR1MCS-5 plasmids；
(4) recombination pBBR1MCS-5 plasmids are imported in E.coli DH5 α competent cells, obtains recombination E.coliDH5 α；
(5) using Sphingol single-cell as recipient bacterium, the recombination E.coli DH5 α that step (4) obtains are donor bacterium, E.coli
HB101/pRK2013 is to assist bacterium, and the method combined by three parents, structure obtains Sphingol single-cell genetic engineering bacterium.
6. according to the method described in claim 5, it is characterized in that, Sphingol single-cell described in step (5) is Alcaligen
es sp.CGMCC2428。
7. according to the method described in claim 5, it is characterized in that, Sphingol single-cell described in step (5) is Sphingomo
nas sp.CGMCC31461。
8. application of the Sphingol single-cell genetic engineering bacterium in fermentation prepares polysaccharide described in claim 1-4 any one.
9. application according to claim 8, which is characterized in that polysaccharide prepared by the fermentation is Weilan gum, the fermentation
Stage fermentation temperature is 30~42 DEG C, and fermentation pH is 4.0~10.0.
10. application according to claim 8, which is characterized in that polysaccharide prepared by the fermentation is gellan gum, the fermentation
Stage fermentation temperature is 30~42 DEG C, and fermentation pH is 4.0~10.0.
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| CN109294950A (en) * | 2018-10-11 | 2019-02-01 | 浙江理工大学 | High activity gellan gum oligosaccharides producing strains and its application |
| CN109504618A (en) * | 2018-10-25 | 2019-03-22 | 上海交通大学 | Can efficient-decomposition estrogen and polycyclic aromatic hydrocarbon the unusual coccus of radiation hardness and application thereof |
| CN111057711A (en) * | 2019-12-25 | 2020-04-24 | 廊坊梅花生物技术开发有限公司 | Sphingomonas engineering bacteria and construction method and application thereof |
| CN112094877A (en) * | 2020-08-17 | 2020-12-18 | 广西大学 | Method for increasing yield of welan gum produced by sphingosine monad |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109294950A (en) * | 2018-10-11 | 2019-02-01 | 浙江理工大学 | High activity gellan gum oligosaccharides producing strains and its application |
| CN109294950B (en) * | 2018-10-11 | 2021-03-30 | 浙江理工大学 | High-activity gellan gum oligosaccharide producing strain and application thereof |
| CN109504618A (en) * | 2018-10-25 | 2019-03-22 | 上海交通大学 | Can efficient-decomposition estrogen and polycyclic aromatic hydrocarbon the unusual coccus of radiation hardness and application thereof |
| CN111057711A (en) * | 2019-12-25 | 2020-04-24 | 廊坊梅花生物技术开发有限公司 | Sphingomonas engineering bacteria and construction method and application thereof |
| CN111057711B (en) * | 2019-12-25 | 2022-01-18 | 廊坊梅花生物技术开发有限公司 | Sphingomonas engineering bacteria and construction method and application thereof |
| CN112094877A (en) * | 2020-08-17 | 2020-12-18 | 广西大学 | Method for increasing yield of welan gum produced by sphingosine monad |
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