CN106434705B - A kind of acyl-CoA-reductase gene phsR and its application - Google Patents
A kind of acyl-CoA-reductase gene phsR and its application Download PDFInfo
- Publication number
- CN106434705B CN106434705B CN201610528007.XA CN201610528007A CN106434705B CN 106434705 B CN106434705 B CN 106434705B CN 201610528007 A CN201610528007 A CN 201610528007A CN 106434705 B CN106434705 B CN 106434705B
- Authority
- CN
- China
- Prior art keywords
- phsr
- acyl
- coa
- reductase gene
- reductase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/02—Dehydrogenating; Dehydroxylating
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of acyl-CoA-reductase gene phsR and its application, the acyl-CoA-reductase gene phsR has nucleotide sequence shown in SEQ ID No.1.The present invention also provides a kind of acyl-CoA described in missing-reductase gene phsR engineered strains, are named as Mycobacterium neoaurum Δ phsR, and deposit number is CCTCC NO.M 2016321.Engineered strain of the present invention can effectively eliminate the 4-BNA by-product during sterol degradation, the purity and yield dependent on serial steroid products prepared by sterol degradation such as AD, ADD, 9-OH-AD can be significantly improved, reduce the raw materials for production cost and energy consumption of steroidal drug, improve the utilization rate of prodrug, the preparation process of the steroidal drugs such as AD can significantly be simplified, there is industrial application value.
Description
Technical field
The present invention is to be related to a kind of acyl-CoA-reductase gene phsR and its application, belongs to field of biotechnology.
Background technique
Steroid drugs is also known as steroids, clinically occupies an important position.According to its function, steroid drugs can substantially divide
For sex hormone, cortin and protein anabolic hormone.Currently, world market is averaged annual sales amount 40,000,000,000 to steroidal drug
Dollar or more, account for about world's medical product total value 10% (Appl Microbiol Biotechnol, 2012,94,1423-
1447).Wherein, 4-AD (androst-4-end-3,17-dione, AD) is the core of steroid bulk pharmaceutical chemicals
Intermediate, nearly all steroid hormone drug can be produced by starting material of AD, such as: for produce cortin,
Sex hormone, progestational hormone and protein anabolic hormone, and can be used for synthesizing prednisone, progesterone, estrenol, dexamethasone etc. more than 100
Kind drug, is also used directly for production Robust speaker feature mifepristone and all kinds of family planning medications.Currently, global annual sales amount
About 10,000,000,000 dollars, average market growth rate about 10%, we have domestic about 6000 tons of annual requirement, pass through Chinese yam (yellow ginger)
The chemical degradation method of saponin and the microbe transformation method of phytosterol about respectively account for half.Since the former production method is seriously polluted
(having forbidden producing in many provinces), and fluctuations in prices of raw materials is big, therefore is got over the microbial method that phytosterol converts production AD
To be more valued by people.It is contemplated that phytosterol is converted on world market with the further development of production technology
The sales volume of AD is also by rapid growth.Therefore, the market using the large-scale production of phytosterol conversion AD is very huge.
It has been known that there is many bacteriums can degrading plant sterol form AD, such as: new gold mycobacteria, Nocard's bacillus and red ball
Bacterium etc., the bacterial strain that international and China's industry mainly utilizes are new gold mycobacteria.According to existing research shows that: in bacterium
The approach of degrading plant sterol is very similar, has very high conservative；Sterol passes through the beta-oxidation approach in bacterium first and is dropped
Solution forms AD (Int.J.Mol.Sci.2012,13,16514-16543), then under the catalysis of KstD enzyme, is further converted into
ADD.Subsequent some enzymes, such as: 9- hydroxylation multienzyme complex KshAB can be catalyzed 9 hydroxylations of AD or ADD, form new steroidal
Drug 9-OH-AD and 9-OH-ADD (Steroid Biochemistry&Molecular Biology, 91,2004,79-85).
Currently, about 16% or so sterol is converted to one kind using the yield of phytosterol conversion AD about 70% or so
Impurity 4-BNA.Since the chemical property of 4-BNA is very similar with AD, it is difficult to separated with AD, industrially need cumbersome step and
Certain economic cost could improve the purity of AD, and other than influencing the purity of AD, impurity composition 4-BNA is also wasted largely
Phytosterol, calculated with annual output 6000 tons of AD, if it is possible to if completely removing 4-BNA, theoretically spend identical
Sterol can produce 1260 tons of AD more.In addition, can be made by transformation sterol degradation approach (being detailed in shown in attached drawing 1)
Bacterial strain directly produces a series of products such as ADD or 9-OH-AD；However due to the presence of 4-BNA by-product, these products are equally deposited
The problem of isolating and purifying.Therefore, meaning of crucial importance will be had to the production of steroid drugs by how removing 4-BNA, but extremely
The present has no related technology reports.
Summary of the invention
For the above problem present in the prior art and deficiency, the present invention is intended to provide a kind of acyl-CoA-reductase
Gene phsR and its application are mentioned with solving the problems, such as how to remove the 4-BNA impurity generated during sterol degradation to reach
The series purity of steroid products such as high AD and the purpose of yield.
For achieving the above object, The technical solution adopted by the invention is as follows:
Acyl-CoA of the present invention-reductase gene phsR has nucleotide sequence shown in SEQ ID No.1.
A kind of engineered strain of acyl-CoA-reductase gene phsR missing, is by genetic engineering means, with homologous
The acyl-CoA-reductase gene phsR seamless missing bacterial strain that recombination form building obtains, is named as
Mycobacterium neoaurum Δ phsR is preserved in China typical culture collection center on June 13rd, 2016, protects
Hiding number is CCTCC NO.M 2016321.
As preferred version, the engineered strain is by knocking out the acyl group on new gold mycobacteria genome
Coacetylase-reductase gene phsR partly or completely nucleotide sequence, causes the acyl-CoA-reductase to lose completely
Bacterial strain living.
Specific gene delection method can be come real by the target practice plasmid of new gold mycobacteria, homologous recombination double crossing over
It is existing, the homology arm of the both wings for the genetic fragment to be lacked can be expanded first, be cloned on knockout carrier by method appropriate, so
Recombinant plasmid is imported in new gold mycobacteria afterwards, is obtained using certain screening, the carrier is in molecular biology
Relatively conventional suicide target practice plasmid preferentially uses anti-screening means.
Preferably, the engineered strain is to make the acyl-CoA-on new gold mycobacteria genome
The the 190th to 1842bp sequence in reductase gene phsR is by an XbaI site sequence: replaced TCTAGA, causing described
Acyl-CoA-reductase complete deactivation bacterial strain.
Preferably, acyl-CoA-reductase of the present invention derives from new gold mycobacteria
M.neoaurum ATCC 25795 is following (a) or protein (b):
(a) protein that the amino acid sequence shown in SEQ ID No.2 forms；
(b) by replacing, missing or adding tool derived from one or several amino acid residues in the amino acid sequence of (a)
There is the protein of same or similar enzymatic activity.
Engineered strain of the present invention can be applied to eliminate the 4-BNA by-product during sterol degradation, improves and relies on
The purity and yield of serial steroid products prepared by sterol degradation, concrete application technique include the following steps:
A) it after carrying out hydrotropy to sterol using surfactant or organic solvent, is obtained using secondary seed culture containing steroid
The conversion culture medium of alcohol；
B) with conversion culture medium of the inoculum concentration switching containing sterol of 5~30wt%, sterol inventory is 5~40g/L, In
It is cultivated 6~8 days under 28~37 DEG C and 100~300rpm.
Experiments have shown that: acyl-CoA of the present invention-reductase gene phsR missing engineered strain can effectively eliminate
4-BNA by-product during sterol degradation can make AD, ADD, 9-OH-AD etc. depend on the serial steroidal of sterol degradation preparation
The purity and yield of product are improved significantly, such as: with acyl-CoA of the present invention-reductase gene phsR missing
Engineered strain conversion sterol degradation product in the content of 4-BNA therefore can reach 95% less than 1%, AD purity, and adopt
The content of the 4-BNA in sterol degradation product converted with wild strain is that 16%, AD purity only has 80%；Therefore, of the invention
The raw materials for production cost and energy consumption that can obviously reduce steroidal drug, improve the utilization rate of prodrug, can significantly simplify AD
The preparation process of equal steroidal drugs obtains the serial steroid products such as AD of high-purity and yield, has high economic benefit
And industrial application value.
Detailed description of the invention
Fig. 1 is the approach that bacterial degradation phytosterol is converted into the drugs such as AD, ADD, 9-OH-AD and 4-BNA.
Fig. 2 is the acyl-CoA-reductase gene phsR core expanded by new gold mycobacteria M.neoaurum
Sour electrophoretogram.Swimming lane 1 is costing standard molecular weight；Swimming lane 2-5 is that the PhsR gene come is amplified in parallel test.
Fig. 3 is the genotype verifying nucleic acid electrophoresis figure of acyl-CoA-reductase gene phsR missing engineered strain,
In: swimming lane M is DNA standard molecular weight mark；Swimming lane 1-4 is wild-type strain control group (PCR product size is 4.9kb)；Swimming
Road 5-8 is acyl-CoA-reductase gene phsR missing engineered strain (PCR product size is 3.3kb).
Fig. 4 is acyl-CoA-reductase gene phsR missing engineered strain and wild-type strain transformation phytosterin
Result figure, in figure: WT is wild-type strain, and Δ phsR is acyl-CoA-reductase gene phsR missing engineered strain.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Main material source used is tested below:
E.colistraindh5α, nucleic acid toolenzyme are purchased from Takara company, primer synthesis and gene sequencing commission Soviet Union
Zhou Jinwei intelligence Biotechnology Co., Ltd completes, and the source new gold mycobacteria M.neoaurum is American Type Culture Collecti,
Number is ATCC 25795；Sterol substrates used in the present invention are sterol mixture, there is sitosterol, stigmasterol, ergosterol etc.
Phytosterol is bought from industry byproducts such as greases by market.
Gene manipulation techniques of the present invention mainly have gene seamless knockout, gene expression technique, gene expression strong
Promoter replacement, nonconformity formula expression plasmid expression and be integrated into the gene expression singly copied on chromosome, molecule is raw
Object operating method is according to " molecular cloning: laboratory manual ".Other test method without specific conditions, usually according to normal
Rule condition or according to the normal condition proposed by manufacturer.
Embodiment 1: the acquisition of new gold mycobacteria M.neoaurum acyl-CoA-reductase phsR gene, molecule mirror
Fixed and heterogenous expression recombinant plasmid and Escherichia coli recombinant strain building
The present embodiment is only by taking PCR method as an example, by new gold mycobacteria VKM Ac-1815D full-length genome information
Analysis comparison is carried out, obtains complete acyl-CoA-reductase phsR gene, and design pcr amplification product, from new golden branch
The DNA sequence dna that phsR is cloned in bacillus M.neoaurum, as shown in SEQ ID NO.1, corresponding amino acid sequence such as SEQ ID
Shown in NO.2.
By relevant analysis of biological information software Vector NTI of software, BioEdit and biological information website to new
The corresponding conserved sequence and structure of relevant coacetylase-reductase in gold mycobacteria carry out retrieval comparison, obtain VKM
Position and associated sequence information of the phsR in genome, are obtained using the primer amplification that primer5 is designed in Ac-1815D
It obtains the DNA sequence dna of phsR and is sequenced, acyl-CoA-reductase in new gold mycobacteria is found out according to its sequence information
The corresponding complete reading frame of gene phsR.
PhsR primer information is as follows:
Upstream primer 5 ' → 3 ': CGCGGATCCATGGCCCGCATGCATTATGTCG
Downstream primer 5 ' → 3 ': CCCAAGCTTGCCTGCTACCAGTGCACACC
PCR reaction system is as follows:
Take suitable new gold mycobacteria genome as 1 μ L of template, each 2 μ L of 1 μ L, DMSO of primer (20 μm of ol/L),
25 μ L of primeSTAR Max polymerase, moisturizing to 50 μ L of total volume.
Reaction process: 98 DEG C, 10s；56 DEG C, 10s；72 DEG C, 60s, 30 circulations；72 DEG C, 10min.
PMD18-T carrier is connected to after the progress glue recycling (as shown in Figure 2) of the PCR product of acquisition to be sequenced.After sequencing
The genetic fragment of obtained 2004bp size completely encodes 668 amino acid, carries out amino acid consensus sequence analysis, as a result shows
Show with the conservative region in conjunction with NAD (P).
The engineered strain of embodiment 2:phsR gene delection constructs
PhsR gene knockout plasmid in new gold mycobacteria is constructed, which is carried out after corresponding denaturation treatment through electricity
Conversion enters new gold mycobacteria competent cell, carries out first time recombinant screen using resistance, and first round weight will occur
The recon of group carries out the second wheel recombination screening using sucrose plate into after continuous passage, then sharp after the verifying of corresponding resistance
Its recon is verified with PCR.
Specific implementation step is as follows:
1) acquisition of phsR gene upstream and downstream homology arm sequence and the building of recombinant plasmid:
For new gold mycobacteria phsR gene order, the upstream and downstream homology arm primer upstream and downstream for designing gene knockout is same
Source arm, shown in following sequence:
PhsR-L-F:CGGAATTCTGGGGACTTTGTCTTCGATCCAGGCTC
PhsR-L-R:GCTCTAGAGGTGAGGTCACCTACCAGCGCATTCAC
PhsR-R-F:GCTCTAGACTGGGTTACCCGGATTCC
PhsR-R-R:ATAAGCTTTGGGGTTGGAGGCGGTAC
The upstream homology arm DNA of gene is amplified using wild strain DNA as template using primer phsR-L-F and phsR-L-R
Segment (1.6kb), the downstream homology arm DNA fragmentation (1.6kb) amplified with primer phsR-R-F and phsR-R-R；PCR product
After Taq enzyme processing is plus A tail, glue recycles to obtain target DNA fragment, the two homology arms are then cloned into pMD18-T
(Takara) in, sequence verification is errorless；The plasmid of upstream homology arm is had with restriction enzyme EcoRI and XbaI cutting, with
XbaI and HindIII digestion has the plasmid of downstream homology arm, recycles corresponding homology arm segment, and be cloned into EcoRI-
In the pK18mobsacB plasmid of HindIII double digestion, the plasmid for gene knockout is obtained；Recombinant plasmid is denaturalized through NaOH
Freezing is stand-by afterwards.
2) new gold mycobacteria competent cell and plasmid conversion are prepared:
New gold mycobacteria through plate or slant activation is forwarded in primary seed solution and is cultivated, with 1% inoculum concentration
Switching secondary seed culture solution；It is forwarded to according still further to 2% inoculum concentration in the culture medium of competence preparation, culture to OD600 is about
1.5, thalline were collected by centrifugation, is suspended and is rinsed 4 times with 10% glycerol, rear that 10% glycerol suspension thalline is added, and dispenses according to 100 μ L,
It now does current；10 μ L are added into the competent cell of packing in the plasmid handled well, mixes and is added in the electric shock cup of 2mm, ice
Bath stands 10min, and shock by electricity condition: 2500V, 25 μ F, 25ms.
3) recombinant screen and verifying:
After recovery medium culture 2-3h is added in electrotransformation product, it is coated on the resistance containing 30 μ g/mL kanamycins
On plate, 3-5d is cultivated, the single colonie grown is chosen and is cultivated into fluid nutrient medium, the correctness of single-swap is verified by PCR；
It carries out after passing on twice, culture is subjected to appropriate dilution spread on the sucrose plate containing 10%；Picking Dan Ke after culture
It is grand to carry out verifying the experiment of sucrose lethal efficiency in resistance containing kanamycin and the not plate of kalamycin resistance respectively；It will
The monoclonal that cannot be grown on kanamycins plate is seeded to fluid nutrient medium, selects through PCR and distinguishes deletion mutant
With back mutation bacterial strain；As a result as shown in Figure 3: using phsR-L-F and phsR-R-R as primer after gene knockout, with above-mentioned
Non-resistant bacterium solution genome as template, expanded, gene reversion bacterial strain, that is, wild-type strain amplified production is big
Small about 4.9kb, and the amplified fragments size of deletion mutant bacterial strain is about 3.3kb, it may be assumed that phsR gene delection is fallen intermediate
1.6kb, remaining size about 400bp destroy the structure and function of enzyme.
Prepared acyl-CoA-reductase gene phsR missing engineered strain, name are as follows: Mycobacterium
Neoaurum Δ phsR is preserved in China typical culture collection center (address: Wuhan City, Hubei Province on June 13rd, 2016
City Wuchang Luo Jia Shan Wuhan University, postcode: 430072), deposit number is CCTCC NO.M 2016321.
The engineered strain of embodiment 3:phsR gene delection prepares the application in AD in phytosterol degradation
Hydrotropy is carried out to sterol using surfactant or organic solvent etc., using secondary seed culture；With 5-30wt%
Inoculum concentration transfer the conversion culture medium containing sterol, sterol inventory is 5-40g/L, 28-37 DEG C, is converted under high dissolved oxygen condition
6-8d；Conversion fluid is extracted in two times with the ethyl acetate of two volumes after conversion, and combining extraction liquid is spin-dried for rear methanol or second
Nitrile, which redissolves, carries out HPLC analysis for use.
The present embodiment carries out hydrotropy to sterol using 1% Tween80, using secondary seed culture；Convert culture medium dress
Liquid measure is 50mL/250mL, and inoculum concentration 10wt%, converts 6d by 30 DEG C, 200rpm；Fermentation by C18 reversed-phase column to preparation
Product carries out HPLC detection and analysis, analysis method are as follows:
Flow velocity: 1ml/min；
Mobile phase:
B (acetonitrile): 50-90%, 20min to 90%, 5min；A(H2O)；
Sample volume: 10 μ L；
Detector: UV 240nm.
The relative amount of converted product is as shown in figure 4, as seen from Figure 4: the sterol degradation product converted by wild-type strain
In AD purity be 80%, 4-BNA content be 16%, separately have 4% other impurity；And by phsR gene of the present invention
AD purity in the sterol degradation product of the engineered strain Mycobacterium neoaurum Δ phsR conversion of missing is 95%,
Content < 1% of 4-BNA separately has 4% other impurity.Illustrate the engineered strain using phsR gene delection of the present invention
Mycobacterium neoaurum Δ phsR, can obviously eliminate the by-product 4-BNA generated in degrading plant sterol process
Accumulation so that the serial steroid products such as AD of high-purity can be obtained, in sterol conversion process so as to make the serial steroid such as AD
The yield of body product has promotion by a relatively large margin, in addition, the content because of 4-BNA is significantly reduced, therefore makes the series such as AD
The preparation process of steroid products is obviously simplified, and is greatly saved because of sterol starting material spent by 4-BNA, can obviously reduce
The raw materials for production cost and energy consumption of steroidal drug, improve the utilization rate of prodrug, have high economic benefit and industrial
Application value.
Finally need described herein be: phsR gene of the present invention, including homology be more than 80% or more it is same
Source gene can be in other sterol degradation bacterial strains, be also possible to the gene order as shown in SEQ ID NO.1 by dashing forward
Become, there is very wide universality, can be used to that all sterol degradations including new gold mycobacteria are transformed
Bacterial strain, to improve the purity and production of steroid drugs produced for eliminating the 4-BNA by-product generated during sterol degradation
Rate.
Claims (7)
1. a kind of acyl-CoA-reductase gene phsR missing engineered strain, the acyl-CoA-reductase gene
PhsR has nucleotide sequence shown in SEQ ID No.1, it is characterised in that: the engineered strain is by genetic engineering hand
Section, the acyl-CoA-reductase gene phsR seamless missing bacterial strain of acquisition is constructed in a manner of homologous recombination, is named as
Mycobacterium neoaurum Δ phsR is preserved in China typical culture collection center on June 13rd, 2016, protects
Hiding number is CCTCC NO.M 2016321.
2. acyl-CoA according to claim 1-reductase gene phsR missing engineered strain, it is characterised in that: institute
The engineered strain stated is by knocking out the portion the acyl-CoA-reductase gene phsR on new gold mycobacteria genome
Point or whole nucleotide sequences, cause the acyl-CoA-reductase complete deactivation to obtain.
3. acyl-CoA according to claim 2-reductase gene phsR missing engineered strain, it is characterised in that: institute
The engineered strain stated is by making on new gold mycobacteria genome in the acyl-CoA-reductase gene phsR
190 to 1842bp sequence is by an XbaI site sequence: replaced TCTAGA, causing the acyl-CoA-reductase complete
Inactivation obtains.
4. acyl-CoA according to claim 2 or 3-reductase gene phsR missing engineered strain, feature exist
In the protein that acyl-CoA-reductase amino acid sequence shown in SEQ ID No.2 forms.
5. acyl-CoA according to claim 4-reductase gene phsR missing engineered strain, it is characterised in that: institute
Acyl-CoA-the reductase stated derives from new gold mycobacteria M.neoaurum ATCC 25795.
6. a kind of acyl-CoA described in any one of claims 1 to 3-reductase gene phsR missing engineered strain
Using, it is characterised in that: it is used to eliminate the 4-BNA by-product during sterol degradation with the engineered strain.
7. application according to claim 6, which is characterized in that concrete application technique includes the following steps:
A) it after carrying out hydrotropy to sterol using surfactant or organic solvent, is obtained using secondary seed culture containing sterol
Convert culture medium；
B) it is transferred the conversion culture medium containing sterol with the inoculum concentration of 5~30wt%, sterol inventory is 5~40g/L, 28~
It is cultivated 6~8 days under 37 DEG C and 100~300rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528007.XA CN106434705B (en) | 2016-07-01 | 2016-07-01 | A kind of acyl-CoA-reductase gene phsR and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528007.XA CN106434705B (en) | 2016-07-01 | 2016-07-01 | A kind of acyl-CoA-reductase gene phsR and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106434705A CN106434705A (en) | 2017-02-22 |
| CN106434705B true CN106434705B (en) | 2019-11-26 |
Family
ID=58184014
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610528007.XA Active CN106434705B (en) | 2016-07-01 | 2016-07-01 | A kind of acyl-CoA-reductase gene phsR and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106434705B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280758B (en) * | 2019-11-18 | 2024-07-19 | 沈阳博泰生物制药有限公司 | Steroid 5 beta reductase variant and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2592138A2 (en) * | 2010-06-10 | 2013-05-15 | Korea Advanced Institute Of Science And Technology | Mutant microorganism having a hydrocarbon-generating ability and method for producing hydrocarbon using same |
| CN104781411A (en) * | 2012-11-09 | 2015-07-15 | 凯利斯塔公司 | Compositions and methods for biological production of fatty acid derivatives |
| CN105154380A (en) * | 2008-10-28 | 2015-12-16 | Reg生命科学有限责任公司 | Methods and compositions for producing fatty alcohol |
| CN106459985A (en) * | 2014-12-12 | 2017-02-22 | 华东理工大学 | Hydroxyacyl-coenzyme a dehydrogenase gene and acyl-coenzyme a thiolase gene, and genetically-engineered bacteria and use thereof |
| CN108138126A (en) * | 2016-06-16 | 2018-06-08 | 华东理工大学 | A kind of Mycobacterium tuberculosis genes engineering bacteria and its application in steroidal compounds are prepared |
-
2016
- 2016-07-01 CN CN201610528007.XA patent/CN106434705B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154380A (en) * | 2008-10-28 | 2015-12-16 | Reg生命科学有限责任公司 | Methods and compositions for producing fatty alcohol |
| EP2592138A2 (en) * | 2010-06-10 | 2013-05-15 | Korea Advanced Institute Of Science And Technology | Mutant microorganism having a hydrocarbon-generating ability and method for producing hydrocarbon using same |
| CN104781411A (en) * | 2012-11-09 | 2015-07-15 | 凯利斯塔公司 | Compositions and methods for biological production of fatty acid derivatives |
| CN106459985A (en) * | 2014-12-12 | 2017-02-22 | 华东理工大学 | Hydroxyacyl-coenzyme a dehydrogenase gene and acyl-coenzyme a thiolase gene, and genetically-engineered bacteria and use thereof |
| CN108138126A (en) * | 2016-06-16 | 2018-06-08 | 华东理工大学 | A kind of Mycobacterium tuberculosis genes engineering bacteria and its application in steroidal compounds are prepared |
Non-Patent Citations (1)
| Title |
|---|
| Mycobacterium sp. NRRL B-3805, complete genome,GenBank: CP011022.1;Rodriguez-Garcia,A等;《GenBank》;20160418;gene complement(1570350..1572353）所在段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106434705A (en) | 2017-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110423732B (en) | Enzyme expressed in saccharomyces cerevisiae, genetic engineering bacteria for high yield of alpha-and gamma-tocotrienols and construction method thereof | |
| CN101565709B (en) | 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof | |
| CN105779444B (en) | A kind of Gene expression improving bacillus protein expression quantity | |
| CN106459985A (en) | Hydroxyacyl-coenzyme a dehydrogenase gene and acyl-coenzyme a thiolase gene, and genetically-engineered bacteria and use thereof | |
| CN109988722A (en) | A kind of method of recombinant Saccharomyces cerevisiae bacterial strain and its application and production tyrosol and/or rhodioside | |
| CN101565710B (en) | 3-sterone-Delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof | |
| CN110982722A (en) | Construction method of saccharomyces cerevisiae for efficiently synthesizing α -amyrin | |
| CN102168099A (en) | 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof | |
| CN111763677A (en) | Promoter for heterologous expression of keratinase | |
| CN106434705B (en) | A kind of acyl-CoA-reductase gene phsR and its application | |
| CN108138126A (en) | A kind of Mycobacterium tuberculosis genes engineering bacteria and its application in steroidal compounds are prepared | |
| CN111154665B (en) | Recombinant yarrowia lipolytica and construction method and application thereof | |
| CN105886415B (en) | A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid | |
| CN109554378B (en) | Xanthan gum yield related gene and application thereof in constructing high-yield xanthan gum engineering bacteria | |
| CN108220216B (en) | Ammonium-resistant nitrogen-fixing microorganism for over-expression of glnR gene and construction method and application thereof | |
| CN110964649A (en) | Engineering strain for accumulating emodin-8-methyl ether and construction method and application thereof | |
| CN110540943B (en) | Gene engineering strain for producing emodin and construction method and application thereof | |
| CN109762814A (en) | A kind of inducible promoter and application thereof | |
| CN109504699A (en) | A kind of method and application reducing the accumulation of yellow wine yeast urea | |
| Borne et al. | Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design | |
| CN107083375B (en) | Medium-temperature alpha-amylase and gene and application thereof | |
| CN110564630A (en) | aspergillus ochraceus mutant strain with ochratoxin A gene knockout function and construction and application thereof | |
| CN110527638B (en) | Gene engineering strain for accumulating emodin and construction method and application thereof | |
| CN112899172B (en) | Homologous recombination efficient gene engineering bacterium of phoma sphingomyelina and construction method and application thereof | |
| CN104293758A (en) | Rhizoma panacis majoris beta-amyrin synthase gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |