CN105087603A - Coenzyme Q10 production method - Google Patents
Coenzyme Q10 production method Download PDFInfo
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- CN105087603A CN105087603A CN201510502517.5A CN201510502517A CN105087603A CN 105087603 A CN105087603 A CN 105087603A CN 201510502517 A CN201510502517 A CN 201510502517A CN 105087603 A CN105087603 A CN 105087603A
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- ddsa
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Abstract
The invention relates to the field of coenzyme Q10 production, in particular to a coenzyme Q10 production method. The coenzyme Q10 production method includes that a ddsA gene with the gene sequence shown in SEQ ID No.1 is transferred into an escherichia coli expression strain for expression. By the coenzyme Q10 production method, coenzyme Q10 can be produced efficiently according to an original coenzyme Q8 production way of the escherichia coli expression strain.
Description
Technical field
The present invention relates to Coenzyme Q10 99.0 production field, in particular to a kind of method of producing Coenzyme Q10 99.0.
Background technology
Coenzyme Q10 99.0 is a kind of antioxidant, is widely used in cardiovascular agent, healthcare products and makeup.The output major part of the Coenzyme Q10 99.0 in the current whole world is at domestic product, and production method from bacterium, extracts product after adopting hydrogenlike silicon ion (Rhodobactersphaeroides) to carry out fermentation culture as bacterial classification.This growth is relatively slower, and the culture medium cost of employing is higher; Improve output narrow with the space reduced costs.
The gene of some protease of coding is proceeded to intestinal bacteria, utilizes large intestine to carry out the production of some product, can be good at utilizing the series of advantages such as Escherichia coli Growth is fast, growth conditions is less demanding, substratum is cheap.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide a kind of method of producing Coenzyme Q10 99.0, by the Coenzyme Q10 99.0 genetically engineered bacterial classification built, production cost significantly reduces, and production technique is simple, and has the space increasing substantially output.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of ddsA gene, its gene order is as shown in SEQIDNo.1.
This gene order clones to obtain from the genome of hydrogenlike silicon ion high-yield strains, and design primer is:
DPF：ATGGGATTGGACGAGGTTTC；
DPR：TCAGGCGATGCGCTCGACCA。
By increasing, the fragment obtained carries out cloning and sequencing, and result is as shown in SEQIDNo.1.
A kind of ubiA gene, its gene order is as shown in SEQIDNo.2.
This gene order clones to obtain, if primer is from the genome of hydrogenlike silicon ion high-yield strains:
UPF：ATGCAGCAGCCGCGCCAAGC；
UPR：TCAGAGGAGCGCGGCTGTGG。
By increasing, the fragment obtained carries out cloning and sequencing, and result is as shown in SEQIDNo.2.
Described ddsA gene and described ubiA gene also by synthetic, to carry out the construction work of follow-up engineering bacteria.
The construction work of engineering bacteria obtains by two kinds of modes: a kind of bacterial classification being construction recombination plasmid and expressing; Another kind is the bacterial classification building genome recombination.
Present invention also offers a kind of containing described ddsA gene or the carrier containing described ddsA gene and described ubiA gene simultaneously.
Namely described ddsA gene can be connected with carrier, obtain the expression plasmid containing ddsA gene; Wherein, carrier can select conventional pET28A etc.Also described ddsA gene and described ubiA gene can be connected on a carrier having multiple multiple clone site.Wherein, carrier can select conventional pETDuet-1 etc.
These two gene orders increase specific restriction enzyme site by design primer, and the product enzyme that pcr amplification obtains is cut, and then carrier adopts identical enzyme to cut, and both connect.
Particularly, described ddsA gene and carrier Connection Step as follows:
Design primer:
DF：ACGGCACGGCATATGGGATTGGACGAGGTTTC
DR：ACGGCACGGCATATGTCAGGCGATGCGCTCGACCA
After carrying out pcr amplification with ddsA gene described in above primer pair, use NdeI digestion products, correspondingly, carrier pET28A also adopts NdeI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, the recombinant plasmid containing ddsA gene can be obtained.
In addition, also can described ddsA gene and described ubiA gene be connected on a carrier, by design primer, different genes is made to carry different restriction enzyme sites, then corresponding with the multiple clone site of carrier, cut by enzyme, connect, transform and verify that Insert Fragment is towards obtaining.
Particularly, described ddsA gene and carrier Connection Step as follows:
Design primer:
DF：ACGGCACGGCATATGGGATTGGACGAGGTTTC
DR：ACGGCACGGCATATGTCAGGCGATGCGCTCGACCA
After carrying out pcr amplification with ddsA gene described in above primer pair, use NdeI digestion products, correspondingly, carrier pETDuet-1 also adopts NdeI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, the recombinant plasmid containing ddsA gene can be obtained.
Described ubiA gene and carrier Connection Step as follows:
Design primer:
UF：CGCGGATCCATGCAGCAGCCGCGCCAAGC
UR：CGCGGATCCTCAGAGGAGCGCGGCTGTGG
After ubiA gene PCR amplification described in above primer pair, use BamHI digestion products, correspondingly, recombinant plasmid containing ddsA gene also adopts BamHI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, the recombinant plasmid containing described ddsA gene and described ubiA gene can be obtained.
Present invention also offers the expression strain containing above-mentioned carrier.Adopt ordinary method to proceed to expression strain by above-mentioned carrier, screening obtains positive strain and get final product.
Preferably, described expression strain is e. coli bl21 (DE3).
Empirical tests, described ddsA gene proceeds to separately, makes expression strain produce Coenzyme Q10 99.0; And described ddsA gene and described ubiA gene are proceeded to simultaneously expression strain, then increase the output of Coenzyme Q10 99.0.
Present invention also offers a kind of method of producing Coenzyme Q10 99.0, the ispB gene that described ddsA gene replaces E. coli expression strains is carried out expressing.
The ispB gene that described ddsA gene provided by the invention replaces E. coli expression strains is expressed, and efficiently original for E. coli expression strains CoQ8 constructive ways can be changed into generation Coenzyme Q10 99.0.
Further, described ddsA gene proceeds to described E. coli expression strains with the form of expression vector or in the mode of homologous recombination.Empirical tests, these two kinds of modes all achieve good effect.
Further, described E. coli expression strains is e. coli bl21 (DE3).
Preferably, the E. coli expression strains of described ddsA gene is proceeded to also containing described ubiA gene.
Further, described ubiA gene proceeds to described E. coli expression strains with the form of expression vector or in the mode of homologous recombination.
Homologous recombination can adopt existing technique means, as red can be adopted to recombinate, but is not limited to this methods of homologous recombination.
Described ddsA gene and described ubiA gene are carried out simultaneously be reassembled as example to illustrate, adopt the mode of red restructuring to carry out, concrete steps are as follows:
1), primer is used
KF:GCGGAATTCGTGTAGGCTGGAGCTGCTTC
And KR:ATGGGAATTAGCCATGGTCC
The resistance fragments Cm of the chloramphenicol resistance in the resistance fragments Km of the anti-kantlex in amplification plasmid pKD4 and amplification plasmid pKD3, then by product cloning in carrier T;
Use EcoRI digested plasmid, and the product dephosphorylation after enzyme is cut.
2), primer is used
DF1:GCGGAATTCATGGGATTGGACGAGGTTTC
And DR1:GCGGAATTCTCAGGCGATGCGCTCGACCA
Increase described ddsA gene;
To increase described ubiA gene with primer UF1:GCGGAATTCATGCAGCAGCCGCGCCAAGC and UR1:GCGGAATTCTCAGAGGAGCGCGGCTGTGG;
Product is cut with EcoRI enzyme respectively;
3), by dephosphorylized containing Km fragment cut with described ddsA gene and described ubiA gene PCR product enzyme respectively with the linearization plasmid of Cm fragment after fragment be connected, then transformation of E. coli DH5a, selects the bacterium colony of terminator codon towards Km or Cm fragment of ddsA gene or ubiA gene by order-checking; Obtain respectively being cloned in the ddsA-Km fragment in carrier T and ubiA-Cm fragment;
4), primer is used
DF2:
TTGCCTTTGTTCACAGTAAGGTAATCGGGGCGAAAAGCCCGGCTTTTGCGATGGGATTGGACGAGGTTTC
And DR2:
TGGAGTTTACTGGAATCATCCCGCGCACTGCGCGGGATGAGGGGAGGGGAATGGGAATTAGCCATGGTCC
Amplification ddsA-Km fragment, obtains the target practice fragment for replacing E. coli ispB gene.
Use primer
UF2:
TAACAGAACTGTTTTTACCGGCGTCACCGTTGTACTAAGAGGAAAAAAATATGCAGCAGCCGCGCCAAGC
And UR2:
ATCAGGCAACCCAGAAGAAAGCCGGATGATCATCCGGCTTTTTTACATCAATGGGAATTAGCCATGGTCC
Amplification ubiA-Cm fragment, obtains the target practice fragment for replacing intestinal bacteria ubiA gene.
5), use the ispB gene of ddsA-Km target practice fragment to e. coli bl21 (DE3) replace and use the ubiA gene of ubiA-Cm target practice fragment to e. coli bl21 (DE3) to replace, screening positive bacteria comes to the engineering bacteria of the production Coenzyme Q10 99.0 built.
In addition, the engineering bacteria obtained can also carry out the optimization of bacterial strain, to improve the output of Coenzyme Q10 99.0.As the bacterial strain obtained carried out replacing and the screening of promotor and ribosome bind site, to obtain the bacterial strain of higher output yield, for the production of Coenzyme Q10 99.0.
Compared with prior art, beneficial effect of the present invention is:
(1) the invention provides a kind of new ddsA gene order, this gene is used for the production of Coenzyme Q10 99.0, has extraordinary effect.
(2) present invention also offers a kind of new ubiA gene order, this gene is used for the production of Coenzyme Q10 99.0, has the effect of extraordinary increase Coenzyme Q10 99.0 output.
(3) present invention also offers containing described ddsA gene and the carrier containing described ddsA and described ubiA gene simultaneously, and the expression strain containing this carrier.
(4) present invention also offers the method for producing Coenzyme Q10 99.0, efficiently original for E. coli expression strains CoQ8 constructive ways can be changed into generation Coenzyme Q10 99.0.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercially available acquisition.
Embodiment 1
The ddsA gene of synthetic as shown in SEQIDNo.1;
The recombinant plasmid of preparation containing described ddsA gene:
Design primer:
DF：ACGGCACGGCATATGGGATTGGACGAGGTTTC
DR：ACGGCACGGCATATGTCAGGCGATGCGCTCGACCA
After carrying out pcr amplification with ddsA gene described in DF and DR primer pair, use NdeI digestion products, correspondingly, carrier pET28A also adopts NdeI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, namely obtain the recombinant plasmid containing described ddsA gene;
Recombinant plasmid containing described ddsA gene is proceeded to e. coli bl21 (DE3), and screening obtains positive strain;
Embodiment 2
The ddsA gene of synthetic as shown in SEQIDNo.1;
The ubiA gene of synthetic as shown in SEQIDNo.2;
The recombinant plasmid of preparation containing described ddsA gene:
Particularly, described ddsA gene and carrier Connection Step as follows:
Design primer:
DF：ACGGCACGGCATATGGGATTGGACGAGGTTTC
DR：ACGGCACGGCATATGTCAGGCGATGCGCTCGACCA
After carrying out pcr amplification with ddsA gene described in above primer pair, use NdeI digestion products, correspondingly, carrier pETDuet-1 also adopts NdeI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, the recombinant plasmid containing ddsA gene can be obtained.
Described ubiA gene and carrier Connection Step as follows:
Design primer:
UF：CGCGGATCCATGCAGCAGCCGCGCCAAGC
UR：CGCGGATCCTCAGAGGAGCGCGGCTGTGG
After ubiA gene PCR amplification described in above primer pair, use BamHI digestion products, correspondingly, recombinant plasmid containing ddsA gene also adopts BamHI enzyme to cut, both products by ligase enzyme connect transform after checking Insert Fragment towards, the recombinant plasmid containing described ddsA gene and described ubiA gene can be obtained.Recombinant plasmid simultaneously containing described ddsA gene and described ubiA gene is proceeded to e. coli bl21 (DE3), screening obtains positive strain, is the bacterial strain simultaneously containing the recombinant plasmid of described ddsA gene and the recombinant plasmid of described ubiA gene.
Embodiment 3
1), primer is used
KF:GCGGAATTCGTGTAGGCTGGAGCTGCTTC
And KR:ATGGGAATTAGCCATGGTCC
The resistance fragments Cm of the chloramphenicol resistance in the resistance fragments Km of the anti-kantlex in amplification plasmid pKD4 (GeneBridges company) and amplification plasmid pKD3 (GeneBridges company), then by product cloning in the carrier T of promega company;
Use EcoRI digested plasmid, enzyme cut after the TSAP enzyme dephosphorylation of product promega company;
2), primer is used
DF1:GCGGAATTCATGGGATTGGACGAGGTTTC
And DR1:GCGGAATTCTCAGGCGATGCGCTCGACCA
Increase described ddsA gene;
To increase described ubiA gene with primer UF1:GCGGAATTCATGCAGCAGCCGCGCCAAGC and UR1:GCGGAATTCTCAGAGGAGCGCGGCTGTGG;
Respectively product is cut with EcoRI enzyme respectively;
3), by dephosphorylized containing Km fragment cut with described ddsA gene and described ubiA gene PCR product enzyme respectively with the linearization plasmid of Cm fragment after fragment be connected, then transformation of E. coli DH5a, selects the bacterium colony of terminator codon towards Km or Cm fragment of ddsA gene or ubiA gene by order-checking;
Obtain respectively being cloned in the ddsA-Km fragment in carrier T and ubiA-Cm fragment;
4), primer is used
DF2:
TTGCCTTTGTTCACAGTAAGGTAATCGGGGCGAAAAGCCCGGCTTTTGCGATGGGATTGGACGAGGTTTC
And DR2:
TGGAGTTTACTGGAATCATCCCGCGCACTGCGCGGGATGAGGGGAGGGGAATGGGAATTAGCCATGGTCC
Amplification ddsA-Km segments-segment, obtains the target practice fragment for replacing E. coli ispB gene;
Use primer
UF2:
TAACAGAACTGTTTTTACCGGCGTCACCGTTGTACTAAGAGGAAAAAAATATGCAGCAGCCGCGCCAAGC
And UR2:
ATCAGGCAACCCAGAAGAAAGCCGGATGATCATCCGGCTTTTTTACATCAATGGGAATTAGCCATGGTCC
Amplification ubiA-Cm fragment, obtains the target practice fragment for replacing intestinal bacteria ubiA gene.5), use the ispB gene of ddsA-Km target practice fragment to e. coli bl21 (DE3) replace and use the ubiA gene of ubiA-Km target practice fragment to e. coli bl21 (DE3) to replace, screening obtains the bacterial strain 1 only containing described ddsA and the positive strain 2 containing described ddsA and described ubiA respectively.
Experimental example 1
The bacterial strain obtained by embodiment 1-3 is expressed under the same conditions, and substratum used is common LB liquid nutrient medium, and inoculum size is 3%, and actual conditions is: 37 DEG C, 220r/min cultivates 16 hours, then carries out the detection of Coenzyme Q10 99.0 content.
Coenzyme Q10 99.0 detects as follows: lucifuge operates, get the bacterium liquid 5ml mixing and let cool to room temperature, move in the brown volumetric flask of 50ml (not allowing to blow out with rubber suction bulb), add the hydrochloric acid 0.5ml of 1mol/L, then add the hydrogen peroxide 0.5ml of 30%, add appropriate dehydrated alcohol, put and all drive out of to bubble in ultrasonic cleaner ultrasonic 1 ~ 2 minute, fermented liquid, without till conglomeration, after placing room temperature, adds dehydrated alcohol constant volume.Control water temperature 30 ~ 35 DEG C, more ultrasonic 60 minutes, take out, place room temperature, filter with 0.45 μm of organic system filtering head, discard just filtrate, collect subsequent filtrate, for high performance liquid chromatograph analysis.Each sample does 3 Duplicate Samples.
Experimental group: experimental group 1 bacterial strain used is in embodiment 1, described ddsA gene is proceeded to e. coli bl21 (DE3) with plasmid to obtain; Experimental group 2 bacterial strain used is that described ddsA gene and described ubiA gene are proceeded to e. coli bl21 (DE3) with same plasmid and obtain by embodiment 2; To be embodiment 3 with described ddsA replace the ispB gene of e. coli bl21 (DE3) and obtain with the ubiA gene that described ubiA gene replaces e. coli bl21 (DE3) experimental group group 3 bacterial strain used.Arrange standard substance group, standard concentration is 100 μ g/ml simultaneously; Also arrange control group, bacterial strain used is e. coli bl21 (DE3).
High performance liquid chromatograph Parameter Conditions:
Chromatographic system: chromatographic column: C18 reversed-phase column 4.6mm × 150mm5 μm; Determined wavelength: 275nm;
Moving phase: ethanol: methyl alcohol (chromatographic grade)=35:65; Flow velocity: 1.0ml/min, is advisable with Coenzyme Q10 99.0 main peak appearance time 10min; Column temperature: 35 DEG C;
Sample size: 10 μ l; Working time: 20min.
According to the result of HPLC, calculate the output of Coenzyme Q10 99.0 in each sample, result is as shown in table 1.
The output of Coenzyme Q10 99.0 under the common LB culture medium condition of table 1
| Group | Coenzyme Q10 99.0 output (μ g/ml) |
| Control group | 0 |
| Experimental group 1 | 0.5 |
| Experimental group 2 | 0.7 |
| Experimental group 3 | 2.0 |
As can be seen from Table 1, ddsA gene order provided by the invention has the effect changed into by original for E. coli expression strains CoQ8 constructive ways efficiently and generate Coenzyme Q10 99.0, and, ubiA gene order provided by the invention can improve the output of Coenzyme Q10 99.0 further, and can by the transformation to expression strain, promote the output of Coenzyme Q10 99.0 further, the Coenzyme Q10 99.0 genetically engineered bacterial classification of structure has the space increasing substantially output; In addition, production cost significantly reduces, and production technique is simple, significant to the production of Coenzyme Q10 99.0.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. a ddsA gene, is characterized in that, its gene order is as shown in SEQIDNo.1.
2. a ubiA gene, is characterized in that, its gene order is as shown in SEQIDNo.2.
3. the carrier containing ddsA gene according to claim 1 or contain ddsA gene according to claim 1 and ubiA gene according to claim 2 simultaneously.
4. the expression strain containing carrier according to claim 3.
5. expression strain according to claim 4, is characterized in that, described expression strain is e. coli bl21 (DE3).
6. produce a method for Coenzyme Q10 99.0, it is characterized in that, ddsA gene according to claim 1 is proceeded to E. coli expression strains and carries out expressing.
7. method according to claim 6, is characterized in that, described ddsA gene proceeds to described E. coli expression strains with the form of expression vector or in the mode of homologous recombination.
8. method according to claim 7, is characterized in that, described E. coli expression strains is e. coli bl21 (DE3).
9. method according to claim 6, is characterized in that, proceeds to the E. coli expression strains of described ddsA gene also containing ubiA gene according to claim 2.
10. method according to claim 9, is characterized in that, described ubiA gene proceeds to described E. coli expression strains with the form of expression vector or in the mode of homologous recombination.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886562A (en) * | 2016-05-31 | 2016-08-24 | 兰州天和生物催化技术有限公司 | Method for preparing coenzyme Q 10 through microbiological fermentation method |
-
2015
- 2015-08-14 CN CN201510502517.5A patent/CN105087603A/en active Pending
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| COPELAND,A.等: "CP000143.2", 《GENBANK》 * |
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| 孔璐等: "大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q10合成能力的影响", 《食品与药品》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105886562A (en) * | 2016-05-31 | 2016-08-24 | 兰州天和生物催化技术有限公司 | Method for preparing coenzyme Q 10 through microbiological fermentation method |
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