CN101864441A

CN101864441A - Method for knocking out mycobacteria ksdD gene and application thereof

Info

Publication number: CN101864441A
Application number: CN201010176347A
Authority: CN
Inventors: 饶志明; 韩冷; 田灵芝; 夏海锋
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-05-19
Filing date: 2010-05-19
Publication date: 2010-10-20

Abstract

The invention provides a method for knocking out a mycobacteria ksdD gene and application thereof, and belongs to the field of molecular biology in biological engineering. The mycobacteria can convert phytosterol into medicinal intermediate 4-alkene-androstane-3,17-diketone (AD) and 1,4-diolefin-androstane-3,17-diketone (ADD), wherein the 3-sterone-delta1-dehydrogenase (KSDD) coded by the ksdD gene is a key enzyme for converting AD into ADD. In the method, a mycobacteria preserved in the laboratory is used as an initial strain, and the method comprises the steps of amplifying a ksdD genetic full sequence with length of about 1.7kb coding KSDD first; inserting a kanamycin resistant gene (Km) from plasmid pET28a inside the gene; and electrotransforming the obtained gene element ksdD::Km to obtain the mycobacteria and performing PCR verification to obtain stably inherited ksdD gene deleted recombinant transformant. The specific enzyme activity of the detected transformant is 15.6mu/mg, while the specific enzyme activity of the original strain is 105.4mu/mg; and compared with that of the original strain, the KSDD enzyme activity of the transformant is reduced by 85 percent. When the condition of converting sterol by the transformant is studied, the original strain is taken as reference. Compared with the original strain, the transformant has the advantages that: the AD yield is increased by one time, and the ADD yield is reduced by 35.2 percent.

Description

A kind of method of knocking out mycobacteria ksdD gene and application thereof

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

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.