CN101864441A - A method for knocking out mycobacterial ksdD gene and its application - Google Patents
A method for knocking out mycobacterial ksdD gene and its application Download PDFInfo
- Publication number
- CN101864441A CN101864441A CN201010176347A CN201010176347A CN101864441A CN 101864441 A CN101864441 A CN 101864441A CN 201010176347 A CN201010176347 A CN 201010176347A CN 201010176347 A CN201010176347 A CN 201010176347A CN 101864441 A CN101864441 A CN 101864441A
- Authority
- CN
- China
- Prior art keywords
- ksdd
- gene
- mycobacterium
- transformant
- plasmid
- 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.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种敲除分枝杆菌ksdD基因的方法及其应用，属于生物工程中分子生物学领域。分枝杆菌(Mycobacterium)能将植物甾醇转化为药物中间体4-烯-雄甾-3，17-二酮(AD)和1，4-二烯-雄甾-3，17-二酮(ADD)，其中由ksdD基因编码的3-甾酮-Δ1-脱氢酶(KSDD)是AD转化为ADD的关键酶。本发明以本实验室保藏的一株分枝杆菌为出发菌株，首先扩增出长度约1.7kb编码KSDD的ksdD基因全序列，在基因内部插入一段来源于质粒pET28a的卡那霉素抗性基因(Km)，将得到的基因元件ksdD::Km电转化分枝杆菌，通过PCR验证获得稳定遗传的ksdD基因缺失重组转化子。经检测转化子的比酶活为15.6mu/mg，而原始菌株比酶活为105.4mu/mg，转化子与原始菌株相比，其KSDD酶活性下降了85％。在研究转化子对甾醇的转化情况时，以原始菌株作为对照。两者相比较，转化子AD产量提高了1倍，而ADD产量下降了35.2％。The invention relates to a method for knocking out mycobacterium ksdD gene and its application, belonging to the field of molecular biology in bioengineering. Mycobacterium can convert phytosterols into drug intermediates 4-ene-androst-3,17-dione (AD) and 1,4-diene-androst-3,17-dione (ADD ), wherein 3-sterone-Δ 1 -dehydrogenase (KSDD) encoded by ksdD gene is the key enzyme for converting AD into ADD. In the present invention, a strain of mycobacterium preserved in our laboratory is used as the starting strain. First, the entire sequence of the ksdD gene encoding KSDD with a length of about 1.7 kb is amplified, and a kanamycin resistance gene derived from the plasmid pET28a is inserted into the gene. (Km), the obtained gene element ksdD::Km was electrotransformed into mycobacteria, and a stably inherited ksdD gene-deleted recombinant transformant was obtained through PCR verification. The detected specific enzyme activity of the transformant was 15.6 mu/mg, while that of the original strain was 105.4 mu/mg. Compared with the original strain, the KSDD enzyme activity of the transformant decreased by 85%. When studying the conversion of transformants to sterols, the original strain was used as a control. Comparing the two, the yield of transformant AD increased by 1 times, while the yield of ADD decreased by 35.2%.
Description
Technical field
A kind of method of knocking out mycobacteria ksdD gene and application thereof belong to biology field in the biotechnology.
Background technology
Mycobacterium (Mycobacterium) can be converted into plant sterol pharmaceutical intermediate 4-alkene-androstane-3,17-diketone (AD) and 1, and 4-diene-androstane-3,17-diketone (ADD) is wherein by the 3-sterone-Δ of ksdD genes encoding
1-desaturase (KSDD) is the key enzyme that AD is converted into ADD.
Androstane-4-alkene-3,17-diketone (androst-4-end-3,17-dione, AD) and androstane-1,4-diene-3,17-diketone (androst-1,4-end-3,17-dione ADD) is the important intermediate of preparation steroid drugs, and steroid drugs has pharmacological actions such as very strong anti-infective, antianaphylaxis, antiviral and antishock.In recent years, steroid drugs constantly enlarges in the range of application of medical field, is widely used in treating rheumatosis, cardiovascular, collagen venereal disease disease, leukemic lymphoblastoid, human organ transplant, antitumor, bacterial encephalitis, tetter, endocrine disturbance, geriatric disease etc.AD in the traditional mode of production (D) is mainly synthetic for the prepared using chemical process with the diosgenin, because the numerous length of chemical synthesis process route, " three wastes " are seriously polluted, the sixties in last century, people began to adopt microbial transformation extensively to be present in the intravital sterol of animal and plant to produce AD (D).Microbial method has advantages such as cost is low, environmentally friendly, therefore more and more is subject to people's attention.The research to microbial method production AD (D) abroad starts to walk early, and the 1950's, U.S. Up john company at first utilizes has double-stranded soyasterol to go out AD and ADD for raw material production on the side chain.Early 1970s, the researcher of Japan has horse to open to utilize the microbiological deterioration cholesterol successfully to produce ADD.Dias in 2002 etc. will enrichment from the grease deodorization distillment the plant sterol mixture handle with mycobacterium NRRLB-3805 and obtain AD and ADD, and yield is higher.Domestic research is started late, but has obtained certain achievement.
Because AD is close with ADD character, and separation and purification is very difficult, and it is very high to obtain single AD (D) cost, therefore obtaining per unit area yield AD (D) bacterial strain becomes the target that domestic and international researcher chases in actual production process.Bibliographical information shows that plant sterol passes through at first to obtain AD after the degraded of C17 side chain, further by 3-sterone-Δ
1-desaturase (KSDD) dehydrogenation generates ADD.Along with the announcement successively of the gene ksdD complete sequence of some microorganism encoded K SDD, utilize the metabolic mechanism of the synthetic ADD of genetic engineering means research KSDD zymologic property and AD to become new research direction in recent years.
The present invention at first made up mycobacterium ksdD gene knockout carrier, realized knocking out of ksdD gene in the mycobacterium by electric method for transformation.
Summary of the invention
The objective of the invention is to: to a strain mycobacterium of this laboratory preservation, realize its ksdD gene knockout by making up ksdD gene knockout carrier equimolecular biological means, utilize screening culture medium to screen the transformant that obtains a strain ksdD gene, and the output of and AD (D) alive to its KSDD enzyme is studied.
Technical scheme of the present invention: according to M.neoaurum strain NWIBL-01ksdD gene complete sequence design primers F 1, the R1 that NCBI announces, upstream primer F1 (EcoR I): 5 '-accg
GaattcGtgttctacatgactgcc, downstream primer R1 (Hind III): 5 '-accg
AagcttTcaggcctttccagc.With the mycobacterium chromosomal DNA is template, with F1, R1 is primer, go out the ksdD complete sequence by pcr amplification, PCR product and pMD18-T carrier be connected for 16 ℃ spend the night, thermal shock Transformed E .coli JM109, use amicillin resistance plate screening transformant, and recombinant plasmid is carried out enzyme cut evaluation and sequential analysis.According to the kalamycin resistance gene sequence of carrier pET28a, design primers F 2, R2, upstream primer F2 (Xho I): 5 '-accg
CtcgagGtatctcagttcggtgtag, downstream primer R2 (Xho I): 5 ' accg
CtcgagGgtggcacttttcggggaaatg.With plasmid pET28a is template, and F2, R2 are primer, that resistant gene of pcr amplification card (Km).Plasmid T-ksdD and gene Km are cut with Xho I enzyme respectively, enzyme is cut and is spent the night with 16 ℃ of connections of T4DNA Ligase after product reclaims, Transformed E .coli JM109, use the two resistant panel screening of penbritin and kantlex transformant, and plasmid is carried out enzyme cut evaluation, wherein recombinant plasmid is gene knockout carrier T-ksdD::Km.
Mycobacterium electricity method for transformation: the mycobacterium cell walls contains a large amount of lipid, influences electric transformation efficiency, so need to add a certain amount of penbritin to suppress the growth of cell walls in substratum.Because the mycobacterium growth time is slow, general shake-flask culture 2-3d could arrive logarithmic phase, need get the thalline of OD600=0.6-0.8 during the preparation competence.Need before electricity transforms with 10% glycerine thalline to be washed twice, 8000r/min is centrifugal, at last thalline is resuspended in 200 μ L, 10% glycerine.The electricity conversion condition is respectively voltage 2.0KV, electric transformation time 5ms.The kantlex of finding 15 μ g/mL under study for action is the minimal inhibitory concentration of bacterial strain, so with 15 μ g/mL kalamycin resistance plate screening positive transformants.
The enzyme activity determination method: get the bacterium liquid 100mL that grows to logarithmic phase, the dimethyl sulfoxide (DMSO) that contains 0.1gAD with 2mL is induced.4 ℃, the centrifugal 5min of 8000r/min collects thalline, and the PBS damping fluid washing secondary with 50mL pH 7.2 is resuspended in this damping fluid of 2 times of volumes.50% power ultrasonic fragmentation in the ice bath, work 1s is 3s intermittently, working hour 7min.The centrifugal 30min of 14000r/min obtains supernatant liquor, gets the 0.5mL supernatant liquor, adds to contain 0.1mg PMS, 2mg cytochrome C, 0.02mL and contain the ethanol of 10mg/mLAD, the PBS damping fluid of 0.05mol/L pH 7.2, and the total reaction system reaches 3mL.30 ℃ of reaction 10min detect light absorption value changing conditions under the 550nm wavelength.The required enzyme amount of reduction 1 μ mol cytochrome C in one minute is defined as the enzyme U of unit that lives.
Described substratum is formed: LB substratum (%): peptone 1.0, and yeast powder 0.5, NaCl 1.0; Seed culture medium (%): peptone 1.0, yeast powder 0.3, extractum carnis 0.3, glycerine 1.5; Fermention medium (%): soyasterol 1.0, glucose 1.0, (NH
4)
2HPO
40.35, K
2PO
40.05, MgSO
40.05, MnCl
25 * 10
-4, Tween800.4 regulates pH to 7.0 with 10%NaOH.
Beneficial effect of the present invention: the present invention makes up gene knockout carrier by molecular biology method, and a strain of this laboratory preservation can be transformed the bacterial strain ksdD gene knockout that sterol is AD (D), makes it transform the AD ability and reduces, and AD output has obtained accumulation.This studies confirm that the 3-sterone-Δ of ksdD genes encoding
1-desaturase (KSDD) is the key enzyme that AD transforms ADD, for further research KSDD zymologic property and the metabolic mechanism of illustrating the synthetic ADD of AD lay the foundation.
Description of drawings
The structure of Fig. 1 mycobacterium ksdD gene knockout carrier T-ksdD::Km;
Fig. 2 KsdD complete sequence pcr amplification product
1:DL2000DNA Marker 2:KsdD gene PCR product;
Fig. 3 blocks that resistant gene Km pcr amplification product
1:Km gene PCR product 2:DL2000DNAMarker;
The checking of Fig. 4 T-ksdD::Km gene knockout carrier
1: λ/Hind III DNA marker 2:T-ksdD::Km plasmid EcoR I/Hind III double digestion
3:2000bp DNA ladder 4:T-ksdD::Km plasmid Xho I single endonuclease digestion;
Fig. 5 KsdD genetically deficient transformant PCR checking
M1：λ/Hind?ⅢDNA?marker?M2：DL2000DNA?marker
1: original strain ksdD gene PCR product 2:KsdD disappearance transformant KsdD::Km gene PCR product
3: original strain Km gene PCR product 4:KsdD disappearance transformant Km gene PCR product.
Embodiment
The structure of embodiment 1:ksdD gene knockout carrier T-ksdD::Km
The structure synoptic diagram of carrier as shown in Figure 1, the M.neoaurum strainNWIBL-01ksdD gene complete sequence design primers F 1, the R1 that announce according to NCBI.With the mycobacterium chromosomal DNA is template, is primer with F1, R1, goes out the ksdD complete sequence by pcr amplification, sees Fig. 2.PCR product and pMD18-T carrier be connected for 16 ℃ spend the night, thermal shock Transformed E .coli JM109 uses amicillin resistance plate screening transformant, and recombinant plasmid is carried out enzyme cut and identify and sequential analysis.
According to the kalamycin resistance gene sequence of carrier pET28a, design primers F 2, R2.With plasmid pET28a is template, and F2, R2 are primer, and that resistant gene of pcr amplification card (Km) is seen Fig. 3.Plasmid T-ksdD and gene Km are cut with Xho I enzyme respectively, enzyme is cut and is spent the night with 16 ℃ of connections of T4DNALigase after product reclaims, Transformed E .coli JM109, use the two resistant panel screening of penbritin and kantlex transformant, and plasmid is carried out enzyme cut evaluation, wherein recombinant plasmid is gene knockout carrier T-ksdD::Km.
The checking of embodiment 2:ksdD gene knockout carrier T-ksdD::Km
The structure synoptic diagram of carrier is a template with the mycobacterium chromosomal DNA as shown in Figure 1, is primer with F1, R1 respectively, by pcr amplification ksdD gene complete sequence.Goal gene is connected with the pMD18-T carrier, makes up plasmid T-ksdD.With plasmid pET28a is template, and F2, R2 are primer, that resistant gene of pcr amplification card (Km).The size that amplifies is about 1.4kb kalamycin resistance gene Km inserts plasmid T-ksdD in Xho I site.Recombinant plasmid is cut through Xho I enzyme and is discharged 4.4kb and 1.4kb size fragment, and corresponding gene fragment Km and plasmid T-ksdD size are seen Fig. 4 respectively.Recombinant plasmid is cut release 3.1kb and 2.7kb fragment through EcoR I and Hind III enzyme, and corresponding gene fragment ksdD::Km and T carrier size are seen Fig. 4 respectively.Above presentation of results kalamycin resistance gene successfully inserts in the T-ksdD plasmid.
Embodiment 3: the mycobacterium electricity transforms with transformant and identifies
By electric method for transformation gene element ksdD::Km is imported mycobacterium, on that resistant panel of card, screen positive transformant.At first positive transformant is carried out the phenotype checking, on the kalamycin resistance flat board, original strain can't be grown, and transformant is well-grown then.Secondly it being carried out the PCR checking, is primer with F1, R1, and transformant karyomit(e) is template, obtain the 3.1kb fragment of corresponding ksdD::Km size by pcr amplification, and original strain can only amplify the 1.7kb fragment of corresponding gene ksdD size; Be primer with F2, R2 simultaneously, positive transformant can amplify the Km gene fragment of big or small 1.4kb, and original strain can't amplify this fragment (see figure 5), proves that it is that gene ksdD lacks that gene element ksdD::Km successfully has been incorporated on the mycobacterium karyomit(e).
Embodiment 4: transformant KSDD enzyme activity determination and sterol transformation experiment
Get 0.5mL transformant cytoclasis liquid supernatant and be used for the enzyme biopsy and survey, with the reaction system that do not add substrate A D as blank, simultaneously in contrast with original strain.The ratio enzyme of transformant is lived and to be 15.6mu/mg after testing, and original strain alives than enzyme be 105.4mu/mg, transformant is compared with original strain, its KSDD enzymic activity has descended 85%.At the research transformant during to the conversion situation of sterol, in contrast with original strain.Behind the fermentation 72h, control group A D output reaches the highest, is 0.167g/L, and transformant AD output behind the 98h just reaches maximum 0.336g/L.Behind the fermentation 168h, ADD output all reaches maximum, and ADD output is 1.095g/L in the control group at this moment, and ADD output is 0.709g/L in the transformant.Both compare, and transformant AD output has improved 1 times, and ADD output has descended 35.2%.
Claims (2)
1. made up mycobacterium ksdD gene knockout carrier T-ksdD::Km, it is characterized in that the mycobacterium karyomit(e) can transform sterol generation AD (D) is that the template pcr amplification goes out the ksdD gene, obtaining the T-ksdD plasmid after connecting the PMD18-T carrier, will be that resistant gene of card Km that template amplification goes out inserts and asks in the ksdD gene and obtain gene knockout plasmid T-ksdD::Km with the PET28a plasmid.
2. knocking out plasmid T-ksdD::Km according to the reorganization that makes up in the claim 1 utilizes electric method for transformation to transform mycobacterium.The mycobacterium cell walls contains a large amount of lipid, influences electric transformation efficiency, so need to add a certain amount of penbritin to suppress the growth of cell walls in substratum.It is characterized in that needing with 10% glycerine thalline to be washed twice, 8000r/min is centrifugal, at last thalline is resuspended in 200 μ L, 10% glycerine.The electricity conversion condition is respectively voltage 2.0KV, electric transformation time 5ms.With 15 μ g/mL kalamycin resistance plate screening positive transformants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010176347A CN101864441A (en) | 2010-05-19 | 2010-05-19 | A method for knocking out mycobacterial ksdD gene and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010176347A CN101864441A (en) | 2010-05-19 | 2010-05-19 | A method for knocking out mycobacterial ksdD gene and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101864441A true CN101864441A (en) | 2010-10-20 |
Family
ID=42956363
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010176347A Pending CN101864441A (en) | 2010-05-19 | 2010-05-19 | A method for knocking out mycobacterial ksdD gene and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101864441A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531746A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Method for realizing whole-cell transformation from AD (androst-4-end-3,17-dione) to ADD (androst-1,4-end-3,17-dione) by utilizing recombinant corynebacterium crenatum |
| CN106544298A (en) * | 2016-10-27 | 2017-03-29 | 广东省微生物研究所 | A kind of preparation method of bacilluss competent cell |
| CN111662858A (en) * | 2020-07-17 | 2020-09-15 | 湖北工业大学 | Application of mutant of HGMS2 strain in preparation of 4-androstene-3, 17-dione (4-AD) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718732A (en) * | 2004-07-07 | 2006-01-11 | 新疆农业科学院微生物应用研究所 | A kind of electric method for transformation that shuttle plasmid is imported brevibacterium flavum |
| CN101565710A (en) * | 2009-05-20 | 2009-10-28 | 华东理工大学 | 3-sterone-Delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof |
-
2010
- 2010-05-19 CN CN201010176347A patent/CN101864441A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718732A (en) * | 2004-07-07 | 2006-01-11 | 新疆农业科学院微生物应用研究所 | A kind of electric method for transformation that shuttle plasmid is imported brevibacterium flavum |
| CN101565710A (en) * | 2009-05-20 | 2009-10-28 | 华东理工大学 | 3-sterone-Delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李玉等: "微生物 3-甾酮-Δ1-脱氢酶的研究进展", 《药物生物技术》 * |
| 田琳等: "分枝杆菌甾酮C1，2位脱氢酶基因敲除的研究", 《中国生物工程杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531746A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Method for realizing whole-cell transformation from AD (androst-4-end-3,17-dione) to ADD (androst-1,4-end-3,17-dione) by utilizing recombinant corynebacterium crenatum |
| CN104531746B (en) * | 2014-12-09 | 2019-09-03 | 江南大学 | A method for transforming AD into ADD by utilizing recombinant Corynebacterium crenatum whole cells |
| CN106544298A (en) * | 2016-10-27 | 2017-03-29 | 广东省微生物研究所 | A kind of preparation method of bacilluss competent cell |
| CN106544298B (en) * | 2016-10-27 | 2020-03-24 | 广东省微生物研究所 | Preparation method of bacillus subtilis competent cells |
| CN111662858A (en) * | 2020-07-17 | 2020-09-15 | 湖北工业大学 | Application of mutant of HGMS2 strain in preparation of 4-androstene-3, 17-dione (4-AD) |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Woolston et al. | Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi) | |
| He et al. | Transcriptome profiling of Zymomonas mobilis under furfural stress | |
| ES2651076T3 (en) | Suitable cell for the fermentation of a mixed sugar composition | |
| Dezam et al. | Microbial production of organic acids by endophytic fungi | |
| US20110053216A1 (en) | Modified Cyanobacteria | |
| US9315832B2 (en) | Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures | |
| Cao et al. | A genetic toolbox for metabolic engineering of Issatchenkia orientalis | |
| Liu et al. | Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination | |
| CN102421890A (en) | Recombinant bacterium for producing ethanol and application thereof | |
| EP3620528B1 (en) | Use of a mutant lacking the acyl-coenzyme a thiolase gene | |
| CN101948794A (en) | Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof | |
| Lee et al. | Analysis of Clostridium beijerinckii NCIMB 8052’s transcriptional response to ferulic acid and its application to enhance the strain tolerance | |
| Dai et al. | Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species | |
| US9476067B2 (en) | Shuttle vector capable of transforming multiple genera of cyanobacteria | |
| CN101864441A (en) | A method for knocking out mycobacterial ksdD gene and its application | |
| CN103635577A (en) | Microbial syntheses of aldehydes and corresponding alcohols | |
| Sheng et al. | Generation of tetramycin B derivative with improved pharmacological property based on pathway engineering | |
| Diallo et al. | Transcriptomic and phenotypic analysis of a spoIIE mutant in Clostridium beijerinckii | |
| Joseph et al. | Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference | |
| CN107937297B (en) | A strain of multi-inhibitor stress-tolerant Saccharomyces cerevisiae and preparation method and application | |
| Ujor et al. | Ribozyme-mediated downregulation uncovers DNA integrity scanning protein A (DisA) as a solventogenesis determinant in Clostridium beijerinckii | |
| CN107119001A (en) | The production method of genetically engineered hydrogenlike silicon ion and preparation method thereof and farnesol | |
| Speer | Development of a genetically modified silage inoculant for the biological pretreatment of lignocellulosic biomass | |
| WO2018124984A1 (en) | A method for improving the biogas potential of anaerobic digestions with rumen fungi | |
| DE102008064249A1 (en) | Improved acid and solvent production in microorganisms |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20101020 |