CN106148263B - Hydrogenlike silicon ion bacterial strain and its preparation method and application - Google Patents
Hydrogenlike silicon ion bacterial strain and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of hydrogenlike silicon ion bacterial strain and its preparation method and application.By the way that hydrogenlike silicon ion bacterial strain will be introduced from the P-hydroxybenzoic acid transport protein pcaK gene of acinetobacter calcoaceticus, ability of the hydrogenlike silicon ion bacterial strain from extracellular intake P-hydroxybenzoic acid is enhanced, and then is improved the yield of hydrogenlike silicon ion bacterial strain synthesizing coenzyme Q 10 intracellular.The present invention solves the disadvantages of Production by Microorganism Fermentation Co-Q10 yield is lower and production cost is higher, can significantly improve the yield of hydrogenlike silicon ion synthesizing coenzyme Q 10 intracellular, reduce the cost of industrialized production Co-Q10.
Description
Technical field
The present invention relates to the production fields of hydrogenlike silicon ion (Rhodobacter sphaeroides) Co-Q10, especially relate to
And hydrogenlike silicon ion bacterial strain and preparation method thereof and using its produce hydrogenlike silicon ion Co-Q10 method.
Background technique
It is quinones that Co-Q10 (Coenzyme Q10), which is a kind of fat-soluble quinones, structure existing for nature,
Derivative is synthesized with multiple isoamyl alkene monomers, and only the number of its side chain iso-amylene unit of the Co-Q10 of separate sources is not
Together, the mankind and mammal are 10 iso-amylene units.Co-Q10 is as the important hydrogen carrier in biological cell respiratory chain, ginseng
With the energetic supersession of cell.In human somatic cell participate in energy manufacture and activation, be prevention of arterial harden to be formed it is most effective
Anti-oxidant composition.In addition to this, Co-Q10 is in antitumor, anti-aging effects and treatment gastrointestinal tract, liver, heart disease and raising
Function of immune system etc. plays an important role, in treatment scurvy, duodenal ulcer, gangrenosum acne periodontitis and rush
Also there is remarkable result into pancreas function and secretion etc., clinical value is high.
Co-Q10 is present in wild plant, in animal body, and the production method of Co-Q10 has animal vegetable tissue extraction at present
Method, chemical synthesis and microbe fermentation method.That there are raw material sources is few for extraction method and chemical synthesis, process is complicated and low output
The problem of, microbe fermentation method has many advantages, such as that from a wealth of sources, lytic activity is good and yield is high, is increasingly becoming Co-Q10 industry
The main method of production.The microbe species for producing Co-Q10 are more, wherein belong to the current work of hydrogenlike silicon ion of photosynthetic bacteria
The critical strain of Co-Q10 is mass produced in industryization, because its synthesizing coenzyme Q 10 content intracellular is higher, extraction step is relatively simple
It is single.However, the principal element for restricting Production by Microorganism Fermentation Co-Q10 at present is that strain hereditary capacity and fermentation control are multiple
Product yield caused by miscellaneous is not high.
Genetic modification and transformation are carried out using biosynthesis pathway of the metabolic engineering means to hydrogenlike silicon ion Co-Q10,
The yield of hydrogenlike silicon ion Co-Q10 can be improved.Chinese patent application CN105441371 A discloses a kind of metabolic engineering
Hydrogenlike silicon ion and its application in production Co-Q10 is transformed in method.By the rate-limiting enzyme base in Co-Q10 biosynthesis pathway
Because UbiE, UbiG, UbiF are converted into hydrogenlike silicon ion genome, it is overexpressed using these three gene tandems to strengthen its own
Co-Q10 synthesis capability, modified engineering bacteria Co-Q10 yield significantly improves.Chinese patent application CN 103509729
A is using the key gene DXS and DDS for strengthening poly- ten isopentenylpyrophosphates synthesis in hydrogenlike silicon ion MEP approach intracellular, to increase
The yield of engineering bacteria Co-Q10 is added.It is still red not over transformation chorismic acid approach, that is, quinone ring synthesis process reinforcing class ball at present
The report of bacterial coenzyme Q10 yield.
Summary of the invention
The approach of present inventor's synthesizing coenzyme Q 10 intracellular for hydrogenlike silicon ion has made intensive studies, discovery
The approach of hydrogenlike silicon ion synthesizing coenzyme Q 10 intracellular mainly includes Isoprenoid synthesis (mevalonate pathway), poly- ten
Isoprenoid synthesizes (MEP approach) and the processes such as quinone ring synthesis and modification (chorismic acid approach).Wherein quinone ring synthesis and
Modification is the key rate-limiting step of Co-Q10, and P-hydroxybenzoic acid (pHBA) is the important intermediate of quinone ring synthesis process,
Directly affect Co-Q10 synthetic quantity, the ability of cell itself synthesis P-hydroxybenzoic acid is limited, by strengthen cell from
The ability of extracellular intake P-hydroxybenzoic acid, can be improved the yield of hydrogenlike silicon ion synthesizing coenzyme Q 10.Although in addition, many objects
There is P-hydroxybenzoic acid transport protein (pcaK) gene, but the gene of not all species can be in not jljl in kind
Kind is interior effective or is functioned with high level.Present invention discover that from acinetobacter calcoaceticus (Acinetobacter
Calcoaceticus clone obtains P-hydroxybenzoic acid transport protein (pcaK) gene in), the method combined by bacterium, will
P-hydroxybenzoic acid transporter gene increases the intake of P-hydroxybenzoic acid, Jin Erqiang in hydrogenlike silicon ion intracellular expression
Change the ability of hydrogenlike silicon ion production Co-Q10.
Specifically, the present invention provides the following contents.
In one aspect of the invention, a kind of hydrogenlike silicon ion bacterial strain is provided, it includes pairs from acinetobacter calcoaceticus
Hydroxybenzoic acid transport protein pcaK gene.
In preferred embodiments, in the hydrogenlike silicon ion bacterial strain, P-hydroxybenzoic acid transport protein pcaK gene
Sequence as shown in SEQ ID NO:1.
In other preferred embodiment, hydrogenlike silicon ion bacterial strain is overexpressed active P-hydroxybenzoic acid transhipment
Albumen pcaK.
In other preferred embodiment, ability of the hydrogenlike silicon ion bacterial strain from extracellular intake P-hydroxybenzoic acid
It is strengthened.
In other preferred embodiment, the yield of the hydrogenlike silicon ion bacterial strain synthesizing coenzyme Q 10 intracellular is mentioned
It is high.
Another aspect of the present invention provides a kind of preparation method of hydrogenlike silicon ion bacterial strain comprising by SEQ ID NO:1
Shown in sequence introduce the hydrogenlike silicon ion bacterial strain, so that it is active to hydroxyl to be overexpressed the hydrogenlike silicon ion bacterial strain
Yl benzoic acid transport protein pcaK.
Preferably, hydrogenlike silicon ion bacterial strain preparation method the following steps are included:
(1) the step of constructing pBBR1MCS-2-pcaK recombinant plasmid comprising from the genome of acinetobacter calcoaceticus
Clone obtains sequence shown in SEQ ID NO:1, and the primer pair used when wherein cloning is respectively as shown in SEQ ID NO:2 and 3；
(2) with the recombinant plasmid transformed Escherichia coli S17-1 the step of；
(3) engagement converts the step of recombinant plasmid to hydrogenlike silicon ion bacterial strain.
Other aspects of the present invention provide a kind of method of chorismic acid approach that hydrogenlike silicon ion bacterial strain is transformed comprising
The step of sequence shown in SEQ ID NO:1 is introduced into the hydrogenlike silicon ion bacterial strain.
Another aspect of the invention provides a kind of method for strengthening hydrogenlike silicon ion Co-Q10 yield comprising with coming from
The P-hydroxybenzoic acid transport protein pcaK gene of acinetobacter calcoaceticus is come the step of being transformed chorismic acid approach.
Another aspect of the present invention provides a kind of method for producing hydrogenlike silicon ion Co-Q10 comprising the culture present invention
Described in hydrogenlike silicon ion bacterial strain the step of.
The present invention solves the disadvantages of Production by Microorganism Fermentation Co-Q10 yield is lower and production cost is higher.In addition,
The present invention can significantly improve the yield of hydrogenlike silicon ion synthesizing coenzyme Q 10 intracellular, reduce industrialized production Co-Q10 at
This.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to
This.
Unless specifically stated, " % " of the invention indicates weight percent wt%.Heretofore described term tool
There is normally understood meaning in this field, unless clearly stating in the present invention.Heretofore described cell culture, engagement turn
Change, genetic engineering means etc. be in the art by using means, for details, reference can be made to such as " Molecular Cloning:A Laboratory guides "
(the 4th edition), Cold Spring Harbor Laboratory are published, Science Press's Imported；" cell culture " Si Tuzhenqiang, Wu Junzheng, generation
Boundary's bibliogony etc..
In the present invention, " P-hydroxybenzoic acid transport protein pcaK gene " is to refer to encode active para hydroxybenzene
The full-length gene of formate transporter pcaK or its function fragment or their artificial reconstructed form.In the present invention, " the people
Work transformation " includes, but are not limited to the gene mutation of insertion, missing, displacement, replacement of more than one nucleic acid etc. or gene lures
Become." artificial reconstructed " can occur at least one in any position at the 3 ' ends in gene, 5 ' ends and gene internal, as long as above-mentioned
" artificial reconstructed " does not reduce, reduces or loses the function that gene encodes active P-hydroxybenzoic acid transport protein pcaK
?.Preferably, heretofore described P-hydroxybenzoic acid transport protein pcaK gene can be full-length gene.For example, SEQ
Gene shown in ID NO:1.
In the present invention, " active " refer to make P-hydroxybenzoic acid from it is extracellular absorb, absorb, enter to it is intracellular
Function." active P-hydroxybenzoic acid transport protein pcaK " includes full length protein, its functional fragment or natural
The protein of structural transformation is carried out on the basis of protein structure.
In the present invention, the expression preferably by P-hydroxybenzoic acid transport protein pcaK gene in bacterial strain is strengthened from extracellular
The ability for absorbing P-hydroxybenzoic acid, further preferably can be improved the yield of hydrogenlike silicon ion bacterial strain synthesizing coenzyme Q 10 intracellular.
In the present invention, " reinforcing " for absorbing ability refers to the raising of cellular uptake amount, the raising including single transport protein transfer efficiency
And/or the increase of protein quantity is transported in cell.In the present invention, preferably described " reinforcing " not only includes transporting egg in cell
The increase of white quantity, it is often more important that the raising of single rotary protein transfer efficiency.
In the present invention, it is preferable that from acinetobacter calcoaceticus (Acinetobacter calcoaceticus) genome gram
Grand P-hydroxybenzoic acid transport protein (pcaK) gene.The genome for extracting acinetobacter calcoaceticus is preferably included, PCR is passed through
Method clones pcaK gene.It preferably, may include restriction enzyme site in PCR primer pair.Restriction enzyme site usually requires to be carried according to plasmid
Have restriction enzyme site in body and suitably selects.In the present invention, preferably select for example, EcoRI restriction enzyme site, BamHI restriction enzyme site.
It is particularly preferred that primer pcaK-F (EcoRI restriction enzyme site) and pcaK-R (BamHI restriction enzyme site) are used in the present invention, they
Sequence be respectively SEQ ID NO:2 and SEQ ID NO:3.
In the present invention, for linker because the preferable plasmid of carrier.It can be used as plasmid any existing or existing
The plasmid or plasmid class carrier being transformed on plasmid basic.However, discovery has when selecting plasmid pBBR1MCS-2 in the present invention
More good changing effect.
In the present invention, method that gene is connect with carrier can include:
1) distinguished with EcoRI enzyme and BamHI enzyme, or cut gene simultaneously with the two；
2) distinguished with EcoRI enzyme and BamHI enzyme, or with both at the same cut vector or plasmid；
3) gene after cutting is connected with carrier or plasmid with ligase.
It should be noted that for above-mentioned steps 1) and sequence 2) be not particularly limited, can preferentially carry out step 1), so
Carry out step 2) again afterwards, vice versa.Optionally, step 1) and 2) can be carried out simultaneously.For step 3), it is preferable to use T4DNA
Ligase.The condition for connecting reaction is preferred: temperature is between 10~20 DEG C, and preferably 16 DEG C；Reaction time preferably 1 to 24 hour,
More preferable 3~8 hours, such as 3 hours.
In the present invention, by plasmid or channel genes donor bacterium.As the method for plasmid or channel genes, this field can be used
The methods of interior commonly used approach, including but not limited to electrization, particle bombardment, microinjection.Selecting plasmid
In the case where pBBR1MCS-2, it is preferred to use heat shock method is converted.
In the present invention, usually used any method in the art is can be used in the verifying of the recon as plasmid conversion,
Including but not limited to, PCR amplification proof method, digestion verification method, immune proof method and enzymatic activity proof method etc..It is accurate from verifying
Property angle, preferably sequence verification method.Specifically, positive recombinant is gone out by resistance screening in post-conversion, extracts plasmid, and be sequenced
Plasmid gene sequence is verified.
In the present invention, gene is preferably introduced by hydrogenlike silicon ion by the method for engagement conversion.Preferably, by using big
Enterobacteria (for example, E.coli S17-1) is converted as the engagement of donor bacterium and the hydrogenlike silicon ion as recipient bacterium and is led
Enter, has many advantages, such as to import high-efficient.In the present invention, the mixed proportion of donor bacterium and recipient bacterium can be 1:2 to 1:50, preferably
1:2.5 to 1:25, more preferable 1:3 to 1:20, further preferred 1:4 to 1:15, still more preferably 1:6 to 1:10.For example, 1:
3,1:6,1:10 etc..
In the present invention, the culture of donor bacterium and recipient bacterium is preferably cultivated in same culture medium.It is preferred that LB is cultivated
Fluid nutrient medium, solid medium etc. can be used as the form of culture medium in base.In the present invention, preferred liquid culture medium.Separately
Outside, it is not limited as training method, occasional drive mode (batch process), continuous training method, shaking flask training can be used
The mode of supporting etc..
In the present invention, hydrogenlike silicon ion is preferably cultivated or is engaged with engagement special culture media (solution 1), ingredient,
Content or property are as follows:
Every liter of culture medium contains 2.0g succinic acid, 0.8g (NH4)2SO4, 0.10g sodium glutamate, 0.04g aspartic acid,
1.0mg niacin, 0.50mg vitamin B1,0.010mg biotin, 2.44g MgCl2·6H2O or 3.0gMgSO4·7H2O,
0.344g CaCl2·H2O, 0.02g FeSO4·7H2O, 0.2ml (NH4)6MO7O24(1%Solution), pH is adjusted to 4.5~
4.9。
In the present invention, for donor bacterium, the concentration of logarithmic growth phase is OD600=0.4~0.6)；For recipient bacterium,
The concentration of logarithmic growth phase is OD600: 0.3~0.6.Cultivation temperature for donor bacterium and/or recipient bacterium is usually 32 DEG C to 38
DEG C, preferably 35 DEG C to 36 DEG C, more preferable 37 DEG C.
Hydrogenlike silicon ion bacterial strain of the invention can pass through low temperature such as liquid nitrogen cryogenics preservation.When in use, it such as is producing
The bacterial strain of preservation is activated when hydrogenlike silicon ion Co-Q10.The method of production hydrogenlike silicon ion Co-Q10 of the invention includes culture
The step of hydrogenlike silicon ion bacterial strain of the invention.Wherein, described to cultivate the bacterial strain that the state of activation can be directly used, it can also be by protecting
The bacterial strain of hiding state activates.In the method for production hydrogenlike silicon ion Co-Q10, preferably it is added in the medium to hydroxyl
Benzoic acid, concentration range preferably 0.1 to 2.0mol/L, more preferable 0.2 to 1.8mol/L, further preferred 0.3 to 1.5mol/
L, most preferably 0.5 to 1mol/L.
In the present invention, hydrogenlike silicon ion bacterial strain is preferably further collected, for example, can carry out by following steps: 7000
~9000rpm centrifugation, collect thallus, with concentration be 15~25mM, the phosphate buffer washing thalline of pH=6.5~7, again
Thalline were collected by centrifugation.In a preferred embodiment of the present invention, it is centrifuged with 8000rpm, thallus is collected, with 20mM, pH=
6.8 phosphate buffer washing thalline, thalline were collected by centrifugation again.
Embodiment
1. strain and experimental material
All strains and material in the present embodiment are sold to obtain by market purchase.For example, hydrogenlike silicon ion
Rhodobacter sphaeroides 2.4.1 is purchased from ATCC (bacterium numbering ATCC-17023), acinetobacter calcoaceticus
(Acinetobacter calcoaceticus) is purchased from ATCC (bacterium numbering ATCC-31926).Plasmid, genome extract reagent
Box is purchased from TIANGEN company.PCR product QIAquick Gel Extraction Kit is purchased from U.S. Omega company.T4Ligase is purchased from U.S. NEB company.
Chemical reagent is purchased from Sinopharm Chemical Reagent Co., Ltd., is that analysis is pure.
The building of 2.pBBR1MCS-2-pcaK carrier:
Clone obtains P-hydroxybenzoic acid transport protein (pcaK) gene from the genome of acinetobacter calcoaceticus:
Acinetobacter calcoaceticus is seeded in the 50mL triangular flask of the culture medium containing LB and is incubated overnight 8h, bacterium is collected by centrifugation
Body extracts the genome of acinetobacter calcoaceticus using genome extraction kit (TIANGEN company, Beijing)；Use primer
PcaK-F (EcoRI restriction enzyme site) (SEQ ID NO:2) and pcaK-R (BamHI restriction enzyme site) (SEQ ID NO:3) is from acetic acid
Clone obtains pcaK gene (SEQ ID NO:1) in the genome of calcium acinetobacter calcoaceticus.The condition of PCR are as follows: 94 DEG C of initial denaturation 5min,
94 DEG C of denaturation 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 recycle.
It is recycled using the pcaK gene that PCR product QIAquick Gel Extraction Kit (Omega company, the U.S.) obtains clone, uses EcoRI
Double digestion pcaK gene and plasmid pBBR1MCS-2 are distinguished with BamHI, and T4 ligase (NEB company, the U.S.) 16 DEG C of companies are used after digestion
3h is met, heat shock method converts Escherichia coli T1 cell, is sieved on the LB plate for receiving chloramphenicol resistance containing concentration for 50mg/ml card
Choosing, picks out culture for the bacterial strain with Kan resistance, extracts sun using plasmid extraction kit (TIANGEN company, Beijing)
Property cloned plasmids pBBR1MCS-2 is spare.
PBBR1MCS-2-pcak converts E. coli S17-1 (donor bacterium):
1 pipe Escherichia coli S17-1 competence is taken from -80 DEG C of refrigerators, 0.5 μ l recombinant plasmid is added after ice bath 20min
PBBR1MCS-2-pcaK, again common ice bath 10min, 42 DEG C of heat shock 60s, ice bath 10min, is added 650 μ l LB liquid again
Culture medium, 5000r/min is centrifuged 5min after 37 DEG C of culture 45min, abandons 300 μ l supernatants, remaining liq is applied to 50mg/ml
In Kan resistant panel, the positive recombinant that picking can be grown extracts plasmid order-checking verifying.
Engagement converts pBBR1MCS-2-pcaK to hydrogenlike silicon ion (recipient bacterium):
(1) hydrogenlike silicon ion 2.4.1 is purchased from ATCC (bacterium numbering ATCC17023), and donor bacterium S17-1 (contains plasmid
PBBR1MCS-2-pcaK) 37 DEG C of shaking flasks are incubated overnight in the LB liquid medium containing antibiotic, and next day is with the switching of 1:10
Amount is forwarded to fresh LB liquid medium, continues to cultivate about 1~2h or so, guarantees that logarithmic growth phase is fully achieved in donor bacterium
(OD600: 0.4~0.6)；
(2) recipient bacterium hydrogenlike silicon ion in LB liquid medium 30 DEG C culture be then forwarded to fresh LB culture medium after
Continuous culture, until recipient bacterium is completely in logarithmic growth phase state (OD600: 0.3~0.6).Take the bacterium of 2~5 μ l in sterile respectively
In EP pipe, 8000rpm is centrifuged 3~5min, is then washed 2 times with fresh LB culture medium, is distinguished afterwards with donor bacterium and recipient bacterium
It takes 50 μ l and 100 μ l LB culture mediums to dilute to be resuspended.Donor bacterium and recipient bacterium mixed proportion are 1:3,1:6,1:10；
(3) bacterium of mixing is applied on solid non-resistant LB plate, preculture 20~for 24 hours, by the bacterium on plate in advance
Cold 1ml solution 1 (mating culture medium) culture medium is eluted in EP pipe；
(4) 4 DEG C, 8000rpm centrifugation 2min, abandon supernatant, pre- 1 culture medium of solution of 500 μ l ice are added, and pressure-vaccum mixes,
Step is repeated to wash 1 time；
(5) it is coated on after bacterium being resuspended with the solution of 0.1ml 1 containing final concentration of 200mg/L K2TeO3And 50mg/ml
On 1 plate of solution of Kan resistance, 32 DEG C of 3~5d of culture grow black joint element, and as pcaK recombinates hydrogenlike silicon ion.
(ingredient of solution 1: every liter of culture medium contains 2.0g succinic acid, 0.8g (NH4)2SO4, 0.10g sodium glutamate,
0.04g aspartic acid, 1.0mg niacin, 0.50mg vitamin B1,0.010mg biotin, 2.44g MgCl2·6H2O or
3.0gMgSO4·7H2O, 0.344g CaCl2·H2O, 0.02g FeSO4·7H2O, 0.2ml (NH4)6MO7O24(1%
Solution), pH is adjusted to 4.5~4.9)
The red bacterial fermentation culture of pcaK recombination classes ball:
Hydrogenlike silicon ion positive colony plasmid intracellular is extracted using plasmid extraction kit (TIANGEN company, Beijing),
Sequence verification positive colony obtains more plants of pcaK recombinant bacterial strains.It takes and is wherein carried out as recombinant bacterial strain of the invention for two plants at random
Following experiment.
Recombinant bacterial strain of the invention is taken to the LB seed culture medium of 500 μ l to 25ml from conservation pipe, 32 DEG C, 220rpm is trained
It supports for 24 hours.Bacterium solution after culture is forwarded to the hydrogenlike silicon ion fermentation medium containing 45ml by 20% inoculum concentration, 32 DEG C,
220rpm culture culture 48h.
Hydrogenlike silicon ion fermentation medium group is divided into every 1 liter of culture medium and contains: glucose 7g, peptone 3.2g, glutamic acid
Sodium 1.2g, NH4Cl 3.8g, FeCl30.25g/L, MgSO46g/L, corn pulp 1.2g, Na2HPO41.8g, NaH2PO42g,
ZnCl20.0015g, KCl2.4g, CuSO40.06g, Ca (HCO3) 27.2g, thiamine hydrochloride 20g, riboflavin 3g, folic acid 1g, niacin
Amine 25g.The red bacterial transport P-hydroxybenzoic acid of recombination classes ball and conjunction are detected by the P-hydroxybenzoic acid of additional various concentration
At the ability of Co-Q10.
The extraction and analysis of Co-Q10:
1ml fermentation culture is taken, the hydrochloric acid of the 6mol/L of 200 μ l is added, concussion mixes；2ml acetone is added, concussion is mixed
It is even；30% hydrogen peroxide of 100 μ l is added, concussion mixes；It is settled to 10mL with dehydrated alcohol, concussion mixes；After mixing well
Ultrasonic extraction 45min (overall process temperature is controlled at 30 DEG C or less) shakes up the solution after ultrasound, stands 10min, upper layer is taken to have
Machine mutually removes bacterial debris through 0.22 μm of organic filtering with microporous membrane, is analyzed with efficient liquid phase HPLC.The analysis condition of HPLC
Are as follows: liquid phase analysis instrument is the high performance liquid chromatograph DAD3000 of Thermo company, and column model: SunFireTM, C18 are anti-
Xiang Zhu (4.6 × 150mm, 1.7 μm), mobile phase is methanol: ethyl alcohol=3:7,30 DEG C of flow velocity 1ml/min of column oven, Detection wavelength
275nm, 10 μ l of sample volume.
Table 1pcaK recombinant bacterial strain is compared with original strain
Table 1 shows original strain and the bacterial strain by pcaK modified recombinant, and in the P-hydroxybenzoic acid of various concentration, there are items
The yield of Co-Q10 under part, original strain addition P-hydroxybenzoic acid not can increase the production of Co-Q10 as can be seen from the results
Amount, and pcaK recombinant bacterial strain yield of Co-Q10 under conditions of adding P-hydroxybenzoic acid outside increases 8.5%~12.6%,
Illustrate that pcaK recombination hydrogenlike silicon ion can use P-hydroxybenzoic acid to increase the yield of Co-Q10.And work as para hydroxybenzene
The additive amount of formic acid is excessive (2mol/L), can inhibit the synthesis of Co-Q10 and the eubolism growth of thallus instead.
Present invention is not limited to the embodiments described above, without departing from the essence of the present invention, this field skill
Any deformation, improvement, the replacement that art personnel are contemplated that each fall within the scope of the present invention.
Claims (8)
1. a kind of hydrogenlike silicon ion bacterial strain, it includes the P-hydroxybenzoic acid transport protein pcaK bases from acinetobacter calcoaceticus
Cause；Wherein, the sequence of the P-hydroxybenzoic acid transport protein pcaK gene is as shown in SEQ ID NO:1；The class ball is red thin
Bacteria strain is the hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1 that bacterium numbering is ATCC-17023.
2. hydrogenlike silicon ion bacterial strain according to claim 1 is overexpressed active P-hydroxybenzoic acid transhipment egg
White pcaK.
3. hydrogenlike silicon ion bacterial strain according to claim 1, wherein the hydrogenlike silicon ion bacterial strain is from extracellular intake to hydroxyl
The ability of yl benzoic acid is strengthened.
4. hydrogenlike silicon ion bacterial strain according to claim 1, wherein the hydrogenlike silicon ion bacterial strain synthesizing coenzyme Q 10 intracellular
Yield be improved.
5. a kind of preparation method of hydrogenlike silicon ion bacterial strain comprising sequence shown in SEQ ID NO:1 is introduced the class ball
Rhodobacter strain, so that the hydrogenlike silicon ion bacterial strain be made to be overexpressed active P-hydroxybenzoic acid transport protein pcaK；
The hydrogenlike silicon ion bacterial strain is the hydrogenlike silicon ion Rhodobacter that bacterium numbering is ATCC-17023
sphaeroides2.4.1。
6. according to the method described in claim 5, comprising:
(1) the step of constructing pBBR1MCS-2-pcaK recombinant plasmid comprising cloned from the genome of acinetobacter calcoaceticus
Sequence shown in SEQ ID NO:1 is obtained, wherein the primer pair used when clone is respectively such as SEQ ID NO:2 and SEQ ID NO:
Shown in 3；
(2) with the recombinant plasmid transformed Escherichia coli S17-1 the step of；
(3) engagement converts the step of recombinant plasmid to hydrogenlike silicon ion bacterial strain.
7. a kind of by improving cellular uptake amount the method that improves the Co-Q10 yield of hydrogenlike silicon ion comprising with coming from
The P-hydroxybenzoic acid transport protein pcaK gene of acinetobacter calcoaceticus is come the step of being transformed chorismic acid approach；Wherein, described
The sequence of P-hydroxybenzoic acid transport protein pcaK gene is as shown in SEQ ID NO:1；The hydrogenlike silicon ion is bacterium numbering
For the hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1 of ATCC-17023.
8. a kind of method for producing hydrogenlike silicon ion Co-Q10 comprising culture is according to any one of claims 1 to 4
The step of hydrogenlike silicon ion bacterial strain；The step includes being added P-hydroxybenzoic acid in the medium, and concentration range is 0.1~
2.0mol/L。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610535089.0A CN106148263B (en) | 2016-07-08 | 2016-07-08 | Hydrogenlike silicon ion bacterial strain and its preparation method and application |
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| CN201610535089.0A CN106148263B (en) | 2016-07-08 | 2016-07-08 | Hydrogenlike silicon ion bacterial strain and its preparation method and application |
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| CN107119001B (en) * | 2017-04-26 | 2019-07-19 | 福建师范大学 | The production method of genetically engineered hydrogenlike silicon ion and preparation method thereof and farnesol |
| CN108795968A (en) * | 2017-05-03 | 2018-11-13 | 华东理工大学 | A kind of genetic transforming method of hydrogenlike silicon ion superior strain |
| JP2020532307A (en) | 2017-09-05 | 2020-11-12 | インメド ファーマシューティカルズ インコーポレイティド | E. for biosynthesis of cannabinoid products. Metabolic engineering of E. coli |
| SG11202109724QA (en) * | 2019-03-06 | 2021-10-28 | Inmed Pharmaceuticals Inc | Compositions and methods for biosynthesis of terpenoids or cannabinoids in a heterologous system |
| CN110358700B (en) * | 2019-05-24 | 2020-10-13 | 湖南省植物保护研究所 | Photosynthetic bacterium strain, biocontrol microbial inoculum, biocontrol fermentation liquor, preparation method and application |
| CN115181753B (en) * | 2021-12-31 | 2024-07-26 | 杭州恩和生物科技有限公司 | Rhodobacter sphaeroides conjugation transformation method |
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