CN109486738B - Method for producing boldenone by using recombinant escherichia coli whole cell transformation - Google Patents
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Abstract
The invention discloses a method for producing boldenone by utilizing recombinant escherichia coli whole cell transformation, belonging to the field of genetic engineering and enzyme engineering.A 17 β -hydroxysteroid dehydrogenase derived from Bipolaris oryzae is cloned, and excessive co-expression in E.coli B L21 (DE3) is realized.A whole cell transformation system is established, 17 β -HSD derived from Bipolaris oryzae is identified to have the function of 17 β -hydroxysteroid dehydrogenase for the first time, and the function identification of 17 β -HSD derived from Bipolaris oryzae is realized in an escherichia coli expression system, and 1 g/L ADD can be transformed into 872.9 mg/L boldenone by 24h whole cell transformation.
Description
Technical Field
The invention relates to a method for producing boldenone by using whole cell transformation of recombinant escherichia coli, belonging to the field of genetic engineering and enzyme engineering.
Background
With the continuous development of the times, steroid drugs become second class drugs which are only second to antibiotics, and have strong pharmacological actions such as anti-infection, anti-allergy, anti-virus and anti-shock. In 2000, the sales of steroid drugs in the global drug market has broken through $ 200 million, which accounts for about 6% of the total sales of medicine in the world. Steroid hormone drugs are classified into three major classes, adrenocortical hormone, anabolic hormone and sex hormone.
Baodarone is a derivative of testosterone and therefore inherits most of the properties of testosterone, such as androgenic capacity and protein synthesis capacity. The Baodan ketone ensures that the nitrogen balance state of the muscle fiber cells is kept to tend to the positive direction at any time, so that the muscle fiber cells accelerate the synthesis of protein, and the muscle fiber cells expand and swell accordingly, thereby having great effects on strengthening the muscles of physical exercisers and strengthening the endurance. Because the baodan ketone has no obvious tendency of male and female, the baodan ketone has few side effects and is accepted by wide consumers. Currently, the boldenone is mostly synthesized by a chemical method, but the method has the disadvantages of many byproducts, low quality, complex synthetic route, low recovery rate and high cost, and can not meet the production requirement.
17 β -hydroxysteroid dehydrogenase (17 β -hydroxysteroid dehydrogenase, 17 β -HSD) is a catalytic enzyme in the final step of sex hormone synthesis, 17 β -HSD is used as a detection enzyme and can be applied to the field of medical detection, and meanwhile, has wide application value in the fields of food development, medical health care, clinical monitoring, biological pesticides and the like, so that the deep research on 17 β -HSD has far-reaching significance for the development of the whole steroid pharmaceutical industry, and the 17 β -HSD catalyzes the reduction and oxidation reaction between a ketone group and an alcohol group on a C17 position, so that low-biological-activity estrone and androstenedione and high-biological-activity estradiol and testosterone can be mutually converted.
In the aspect of microbial transformation of baodanone, the conversion of AD (androstenedione) to ADD and BD (baodanone) is realized in Pichia pastoris by using aged seedlings and the like at present, the baodanone content in a final product is 41%, a Pseudomonas aeruginosa strain is screened by Mohamed and the like, the Pseudomonas aeruginosa can convert corn oil phytosterol to produce BD L (baodanone), and the baodanone content in the final product is 36.8%.
Disclosure of Invention
The invention aims to solve the technical problem of providing the recombinant escherichia coli for expressing 17 β -hydroxysteroid dehydrogenase, which can produce the pagodane (1(2) -DT) by using Androstenedione (ADD) as a substrate, and the application of the recombinant escherichia coli for catalyzing the ADD to generate the pagodane obtains higher conversion rate and provides beneficial guidance for the industrialization of producing the 1(2) -DT by microbial fermentation.
In one embodiment of the present invention, the nucleotide sequence of the gene encoding the 17 β -hydroxysteroid dehydrogenase is shown in SEQ ID NO. 1.
In one embodiment of the invention, the 17 β -hydroxysteroid dehydrogenase is derived from bipolarisozae.
In one embodiment of the invention, the recombinant Escherichia coli is pET28a (+) as a vector.
In one embodiment of the invention, the recombinant E.coli is E.coli B L21 (DE3) as host.
Another technical problem to be solved by the present invention is to provide a method for constructing the recombinant Escherichia coli, comprising the steps of:
(1) synthesis of complete sequence of 17 β -HSD gene
According to the 17 β -HSD sequence of Bipolaris oryzae (GeneID:19120956) published by GENBANK website, after codon optimization, enzyme cutting sites BamH I/SacI on pET-28a (+) plasmid are selected, and the synthesized gene is inserted into the corresponding position of the plasmid to obtain recombinant plasmid pET-28a-17 β -HSDBo；
(2) Recombinant plasmid pET-28a-17 β -hsdBoTransformation of Strain E.coli B L21 (DE3)
Recombinant plasmid pET-28a-17 β -hsdBoTransforming into Escherichia coli by chemical transformation method for expression;
(3) recombinant strain E.coli B L21 (DE3)/pET-28a-17 β -hsdBoScreening for Positive transformants
And selecting colonies growing on L B and kanamycin pressure plates, performing shake flask culture, extracting plasmids, and performing single-double enzyme digestion verification to obtain the escherichia coli genetic engineering bacteria.
Another technical problem to be solved by the invention is to provide a method for preparing the boldenone, which takes 17 β -hydroxysteroid dehydrogenase with a nucleotide sequence shown in SEQ ID NO.1 as a catalyst.
In one embodiment of the invention, the 17 β -hydroxysteroid dehydrogenase is derived from bipolarisozae.
The invention aims to solve another technical problem of providing a method for preparing boldenone (1(2) -DT) by using recombinant escherichia coli, wherein the method comprises the steps of resuspending the recombinant escherichia coli by adopting Tris-HCl buffer solution with the concentration of 8-12 m L, the concentration of 45-55 mM and the pH value of 7-8 until the wet weight of cells is 180-220 g/L, adding 8-12 mg of substrate ADD for conversion, wherein the conversion temperature is 25-40 ℃, the conversion pH value is 7-8, the substrate cosolvent is methylated- β -cyclodextrin, the molar ratio of ADD to methylated- β -cyclodextrin is 1:1, and the recombinant escherichia coli expresses 17 β -hydroxysteroid dehydrogenase in a recombinant mode.
In one embodiment of the present invention, the nucleotide sequence of the gene of 17 β -hydroxysteroid dehydrogenase is shown in SEQ ID NO. 1.
In one embodiment of the invention, the 17 β -hydroxysteroid dehydrogenase is derived from bipolarisozae.
In one embodiment of the invention, the recombinant Escherichia coli is pET28a (+) as a vector.
In one embodiment of the invention, the recombinant E.coli is E.coli B L21 (DE3) as host.
Another technical problem to be solved by the invention is to provide a method for culturing recombinant Escherichia coli, namely recombinant bacterium E.coli B L21 (DE3) pET-28a-17 β -hsdBoInoculating the strain in a 5-15 m L L B culture medium, culturing for 10-13 h, then inoculating 8-12% of the strain in a 200-300 m L container filled with a 45-55 m L L B culture medium, culturing at 35-40 ℃ until the late logarithmic growth stage, and then transferring to 22-25 ℃ for induction culture for 8-12 h.
Has the advantages that:
the invention identifies that the 17 β -HSD from the Bipolaris oryzae has the function of 17 β -hydroxysteroid dehydrogenase for the first time, and successfully constructs the recombinant escherichia coli expressing the 17 β -hydroxysteroid dehydrogenase from the fungus, and the whole cell of the recombinant escherichia coli is used as a catalyst, so that 1 g/L ADD can be converted into 872.9 mg/L padanone within 24 h.
The method for producing the boldenone by the whole cells of the microorganism has the advantages of mild reaction conditions, high utilization rate of raw materials, high yield, simple process, easy control and the like, is beneficial to environmental protection, and is easy to popularize and apply.
Drawings
FIG. 1 HP L C detection map of ADD and 1(2) -DT standard;
FIG. 2 measurement of ADD and 1(2) -DT HP L C in recombinant E.coli whole cell transformation medium.
Detailed Description
The examples of the present invention are provided only for further illustration of the present invention and should not be construed as limitations or limitations of the present invention.
The media and detection methods referred to in the following examples are as follows:
l B culture medium, peptone 10 g/L, yeast extract 5 g/L10 g/L (2% agar powder added in solid culture medium).
In HP L C analysis, ADD and 1(2) -DT both have characteristic absorption peaks at 254nm ultraviolet wavelength, so the product concentration is determined by the HP L C method, the chromatographic conditions are that a chromatographic column is Dimosoiil C18(5 mu l,250mm × 4.6.6 mm), a mobile phase is methanol-water (V/V is 70:30), a Detector is UV Detector, the detection wavelength is 254nm, the column temperature is 30 ℃, the sample injection amount is 5 mu L, and the flow rate is 1.0 ml/min.
The main reagents are as follows: ADD and 1(2) -DT were obtained from SIGMA, USA.
Example 1 recombinant E.coli B L21 (DE3) pET-28a-17 β -hsdBoConstruction of the Strain
1. Synthesis and transformation of 17 β -HSD gene complete sequence
According to the sequence of Bipolaris oryzae (GeneID:19120956)17 β -HSD published by GENBANK, codon is optimized to obtain a gene which is shown as SEQ ID NO.1 and is used for coding 17 β -hydroxysteroid dehydrogenase, a target gene and an expression plasmid pET-28a (+) are subjected to enzyme digestion by using restriction enzyme BamH I/SacI, and the target gene is connected with a vector to obtain a recombinant plasmid pET-28a-17 β -HSDBoRecombinant plasmid pET-28a-17 β -hsdBoTransforming into Escherichia coli E.coli B L21 (DE3) by chemical transformation method to obtain recombinant Escherichia coli E.coli B L21 (DE3)/pET-28a-17β-hsdBo。
2. Recombinant Escherichia coli E.coli B L21 (DE3)/pET-28a-17 β -hsdBoScreening for Positive transformants
Colonies growing on L B and kanamycin pressure plates are picked, shake flask culture is carried out, plasmids are extracted for single-double enzyme digestion verification, correct transformants are transferred and induced, cells are collected, whole cell transformation is carried out, the transformant system is 10m L, the temperature is 30 ℃, the transformation pH is 7.5, a substrate cosolvent is methylated- β -cyclodextrin (3 g/L), the substrate concentration is 1 g/L, products 1(2) -DT. in a transformation liquid are detected by using HP L C, and the fact that products 1(2) -DT are detected shows that a recombinant strain with ADD being transformed into 1(2) -DT is successfully constructed.
And a blank control, namely transforming the plasmid pET-28a into escherichia coli E.coli B L21 (DE3) by a transformation method to obtain a recombinant strain E.coli B L21 (DE3)/pET-28a, selecting colonies growing on a L B and kanamycin pressure plate, culturing in a shake flask, transforming and inducing correct transformants, collecting cells, and carrying out whole cell transformation, wherein the transformant is 10m L, the temperature is 30 ℃, the transformation pH is 7.5, a substrate cosolvent is methylated- β -cyclodextrin (3 g/L), the substrate concentration is 1 g/L, and a product 1(2) -DT in a transformation solution is detected by HP L C, so that the product 1(2) -DT is not detected.
Example 2: detection of recombinant strain whole cell transformation performance
The recombinant bacterium E.coli B L21 (DE3) pET-28a-17 β -hsd constructed in example 1 was usedBoInoculating a single colony into a 10m L L B culture medium, culturing for 12h, inoculating into a 250m L container filled with a 50m L0L B culture medium according to the inoculation amount of 10%, culturing at 37 ℃ to the later stage of logarithmic growth, transferring to 24 ℃ for induction culture for 10h, centrifuging to recover the thalli, washing twice, resuspending by using 10m L, 50mM and pH7.5 Tris-HCl buffer solution until the wet weight of the cell is 200 g/L, adding 10mg of substrate ADD for transformation under the conditions that the transformation temperature is 30 ℃, the transformation pH is 7.5, and the substrate cosolvent is methylated- β -cyclodextrin (the molar ratio of ADD: methylated- β -cyclodextrin is 1:1), and performing HP L C determination on ADD and 1- (2) -DT in a recombinant bacterium whole cell transformation solution after 24h of transformation, wherein the results are shown in figure 2, ADD and 1- (2) -DT standard HP L C detection are shown in figure 1.
The results show that 1 g/L ADD can be converted into 872.9 mg/L pagodanone by 24h whole cell transformation.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for producing boldenone by using recombinant escherichia coli whole cell transformation
<160>1
<170>PatentIn version 3.3
<210>1
<211>825
<212>DNA
<213> Artificial Synthesis
<400>1
ggatccatgc cgcacgtgga gagcaccccg tcgacctata tcccgggccg tttagacggc 60
aaagtcgctt tagtcaccgg tagcggccgc ggcatcggcg ccgcggtggc cacccattta 120
ggccgcctcg gcgccaaggt cgtcgtgaac tacgccaact cgaccaagga cgccgaaaag 180
gtggtgtcgg agatcaaggc gctgggctcg gacgcgatcg ccatcaaggc cgacatccgg 240
caagttccgg acatcgtgcg tttattcgat gaggcggtcg cccacttcgg ccatctggac 300
atcgccgtgt cgaactcggg cgtggtgagc ttcggccatt taaaggacgt caccgaggaa 360
gagttcgacc gcgtcttctc gctgaatacc cgcggccagt tcttcgtggc ccgcgaggcc 420
taccgccatt taaccgaggg tggccgcatc attctgactt cttctaacac cagccgcgat 480
ttctcggtcc cgaagcactc gctgtattcg ggctcgaagg gcgccgtcga ctcgttcgtg 540
cgcatcttct cgaaggactg cggtgacaag aagatcaccg tcaacgccgt cgcccccggc 600
ggtaccgtca ccgacatgtt ccacgaggtg tcgcaccact acatcccgaa tggtttagac 660
tacaccgccg agcagcgcca gcagatggcc gcccacgcgt cgcctttaca tcgcaacggc 720
ttcccgcaag atgtggccaa cgtggtgggc tttttagtgt cgaaagaggg cgagtgggtc 780
aacggcaagg tgctgacgct ggacggcggc gcggcctgag agctc 825
Claims (8)
1. A recombinant Escherichia coli is characterized in that 17 β -hydroxysteroid dehydrogenase is expressed, androstenedione is used as a substrate to produce boldenone, and the nucleotide sequence of a gene for coding the 17 β -hydroxysteroid dehydrogenase is shown as SEQ ID No. 1.
2. The recombinant Escherichia coli of claim 1, wherein pET series is used as a vector and Escherichia coli E.coli B L21 (DE3) is used as a host.
3. A method for constructing the recombinant Escherichia coli of any one of claims 1 to 2, comprising the steps of:
(1) the Bipolaris oryzae 17 β -HSD sequence from GeneID 19120956 is synthesized into the gene shown in SEQ ID NO.1 after codon optimization;
(2) the gene is connected to pET28a (+) to obtain a recombinant plasmid pET28a-17 β -hsd;
(3) after linearization, the recombinant vector is electrically transferred into E.coli B L21 (DE3), and positive clones are screened and verified to obtain the recombinant Escherichia coli E.coli B L21 (DE3)/pET-28a-17 β -hsdBo。
4. A method for preparing boldenone is characterized in that 17 β -hydroxysteroid dehydrogenase with a nucleotide sequence shown as SEQ ID NO.1 is used as a catalyst.
5. The method for preparing boldenone by using the recombinant escherichia coli as claimed in any one of claims 1-2, wherein the recombinant escherichia coli is used as a biocatalyst, and androsta-1, 4-diene-3, 17-dione ADD is used as a substrate.
6. The method of claim 5, wherein the recombinant Escherichia coli is resuspended in 8-12 m L, 45-55 mM Tris-HCl buffer solution with pH of 7-8 until the wet weight of the cell is 180-220 g/L, 8-12 mg of substrate ADD is added for conversion, the substrate cosolvent is methylated- β -cyclodextrin, the conversion temperature is 25-40 ℃, the conversion pH is 7-8, and the molar ratio of ADD to methylated- β -cyclodextrin is 1: 1.
7. A method for culturing the recombinant Escherichia coli as claimed in any one of claims 1 to 2, wherein the recombinant Escherichia coli is inoculated into 5-15 m L L B culture medium, cultured for 10-13 h, then inoculated into 200-300 m L container filled with 45-55 m L L B culture medium with an inoculum size of 8-12%, cultured at 35-40 ℃ until late logarithmic growth, and then transferred to 22-25 ℃ for induction culture for 8-12 h.
8. The use of the recombinant Escherichia coli of any one of claims 1 to 2 in the preparation of food, medical care, and biopesticide.
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Citations (5)
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|---|---|---|---|---|
| CN102168099A (en) * | 2011-01-21 | 2011-08-31 | 华东理工大学 | 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof |
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN106367465A (en) * | 2016-09-26 | 2017-02-01 | 上海中医药大学 | Method for producing artificial bear gall powder through engineering bacteria fermentation |
| CN107099516A (en) * | 2017-06-05 | 2017-08-29 | 华东理工大学 | 7 β hydroxy sterols dehydrogenase mutants and its application in ursodesoxycholic acid synthesis |
| CN108707553A (en) * | 2018-05-10 | 2018-10-26 | 上海师范大学 | It is capable of bacterial strain and its application of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD |
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| US20030027805A1 (en) * | 2000-08-21 | 2003-02-06 | Llewellyn William Charles | Use of 1,4-androstadienedione as a method of increasing levels of the anabolic/androgenic steroid boldenone in humans |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168099A (en) * | 2011-01-21 | 2011-08-31 | 华东理工大学 | 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof |
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN106367465A (en) * | 2016-09-26 | 2017-02-01 | 上海中医药大学 | Method for producing artificial bear gall powder through engineering bacteria fermentation |
| CN107099516A (en) * | 2017-06-05 | 2017-08-29 | 华东理工大学 | 7 β hydroxy sterols dehydrogenase mutants and its application in ursodesoxycholic acid synthesis |
| CN108707553A (en) * | 2018-05-10 | 2018-10-26 | 上海师范大学 | It is capable of bacterial strain and its application of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD |
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