CN1962869B - Regulation protein SnpR and its gene and application - Google Patents

Abstract

The invention discloses a gene nucleotide sequence to code modulating protein SnpR and amino acid sequence of protein and application in the adriablastina preparing course through carrier and gene project bacterium of erythromycin C-14 hydroxylase, which is characterized by the following: cloning SnpR protein gene; adjusting SnpA promoter sequence to plasmid expressive carrier to express coded cerubidin C-14 hydroxylase gene doxA; transmitting cerubidin into adriablastina in the culture liquid directly.

Description

A kind of regulation protein SnpR and its gene and application

technical field

The present invention relates to the field of biological transformation and production of antibiotics, in particular to a novel regulatory protein SnpR and its gene, and regulates the transcription level of the promoter SnpA through SnpR, thereby increasing the level of daunorubicin C-14 hydroxyl The expression of the enzyme, so as to realize the direct conversion of daunorubicin into doxorubicin in the culture medium.

Background technique

Daunorubicin and doxorubicin are clinically important anthracycline antibiotics, which are mainly used in the treatment of various solid tumors and acute leukemia, and are clinically used as first-line anti-tumor drugs. Doxorubicin is a derivative product of C-14 hydroxylation of daunorubicin, which has a wider anti-tumor spectrum and less toxic side effects than daunorubicin. At present, chemical semi-synthesis is used in the industry to transform daunorubicin into doxorubicin, and doxorubicin is obtained from daunorubicin produced by microorganisms through a seven-step reaction. The total yield is up to 40% from daunorubicin, and is generally about 33% in production. Not only the process is complicated, the conversion rate is low, but also the environment is seriously polluted.

Foreign countries have mainly studied the metabolic biosynthetic pathways of daunorubicin and doxorubicin in the two strains of Streptomyces persei (S.peucetics) and Streptomyces (S.sp.) C5. After nearly 20 years of efforts Many research results have been obtained. It is generally believed that the expression product of daunorubicin C-14 hydroxylase gene doxA is the key enzyme that catalyzes the transformation of daunorubicin into doxorubicin. doxA was obtained from the mold S.sp.strain C5.

At present, the metalloprotease gene SnpA promoter has been analyzed in detail and used for the expression of gene doxA in Streptomyces, and it has been found that the promoter is regulated (activated) by its regulatory protein SnpR, but it has not been applied to the biotransformation of Adriamycin It is reported in the production of protein (J. Bacteriol. 1996, 178 (11): 3389, wherein the SnpR sequence is found in Genbank, the accession number is AY072041).

Contents of the invention

One of the technical problems to be solved in the present invention is to provide a new regulatory protein SnpR and its gene, the SnpR protein can regulate the transcription level of promoter SnpA, and improve the expression of daunorubicin C-14 hydroxylase .

The inventor amplified the 1.3Kb-encoded C-14 daunocin containing ribosome binding site from the SIPI-1482 gene of domestic daunorubicin-producing bacteria Streptomycescoeruleorubidus (Streptomycescoeruleorubidus) The doxA gene of mycin hydroxylase and the SnpA promoter regulated by SnpR, after sequencing, it is found that the homology of the doxA gene sequence and the sequence reported in Genbank (accession number is U50973) is 100%; and the SnpR sequence of the present invention and The homology of the sequence reported in Genbank (Accession No. AY072041) is 99.4%, the amino acid homology is 96.5%; the promoter SnpA homology is 100%. Therefore, the regulatory protein SnpR of the present invention is a new protein, and the gene encoding it is one of the following nucleotide sequences:

1) It has the base sequence shown in SEQ ID No.1 in the sequence listing;

2) Encoding a protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence listing.

The regulatory protein SnpR of the present invention is a protein having the amino acid sequence shown in SEQ ID No.2 in the sequence listing.

The second technical problem to be solved by the present invention is to provide a vector expressing daunorubicin C-14 hydroxylase and its genetically engineered bacteria, which include the nucleotide sequence of the gene encoding the regulatory protein SnpR.

In the present invention, the above-mentioned SnpR protein gene, promoter SnpA and doxA gene sequences can be connected in series by using the prior art, and cloned into the vector to construct the expression vector of daunorubicin C-14 hydroxylase. And further use the expression vector to transform the host cell to construct the genetically engineered bacteria expressing the daunorubicin C-14 hydroxylase.

In a preferred example of the present invention, a doxA gene with a ribosome binding site GGAGG sequence is amplified from the SIPI-1482 genome of Streptomyces coeruleorubidus and cloned into the plasmid pUC18. At the same time, the amplified SnpA sequence and the sequence of SnpR activating protein were also placed in pUC18. The doxA, SnpA and SnpR three fragments were subcloned into the plasmid pBluescript IIKS(-)(pBlue2KSM) to construct the vector pYG905, and then the tandem fragment (PstI-HindIII fragment) was combined with the thiostrepton (tsr) resistance gene The vector pYG57 (PstI-HindIII large fragment) was ligated to construct the recombinant plasmid pYG908, which was transformed into Streptomyces lividans TK24 protoplasts and cultured on R ₂ YE medium containing 30 μg/ml thiostrepton (tsr). Positive clones are screened to obtain the expression vector and genetically engineered bacteria of the present invention.

The third technical problem to be solved by the present invention is to provide the application of the above-mentioned gene encoding regulatory protein SnpR in the preparation of doxorubicin by biotransformation of daunorubicin.

In other words, the fourth technical problem to be solved by the present invention is to provide a new method for bioconverting daunorubicin into doxorubicin.

In the method, the above-mentioned expression vector plasmid pYG908 is used to transform the host cell into a transformant, and the transformant is cultivated, and the transformant is fermented to biotransform daunorubicin into doxorubicin.

As we all know, the host cell is usually a Streptomyces cell that does not produce daunorubicin. In the present invention, Streptomyces lividans TK24 is preferred, and the transformant constructed therefrom is the genetically engineered bacterium pYG908/TK24.

The present invention has cloned the metalloprotease gene SnpA promoter activated by the transcriptional regulatory protein SnpR from the disclosed domestic daunorubicin producing bacterium Streptomycescoeruleorubidus SIPI-1482, and proved that the SnpA promoter regulated by SnpR high efficiency. Therefore, the transcription level of the promoter SnpA is regulated by the new regulatory protein SnpR, and the greatly increased transcription level improves the expression of daunorubicin C-14 hydroxylase, thereby realizing the direct transformation of daunorubicin in the culture medium into doxorubicin.

Description of drawings

Figure 1: PCR amplification electrophoresis of SnpR+SnpA, in which 1.2.3 is the PCR map of Streptomyces coelicolor SIPI-1482 genomic DNA as a template, annealing temperature is 62.8 ℃, 65.5 ℃, 68 ℃, 4 is DNA Molecular weight standard DL3000.

Figure 2: Construction map of the recombinant expression plasmid pYG908.

Figure 3: Enzyme digestion verification map of pYG908, wherein 1. λDNA (HindIII), 2. DL3000, 3. pYG908 (EcoRI-PstI), 4. pYG908 (PstI-HindIII).

Figure 4: PCR verification map of the expression plasmid pYG908, wherein 1.2.3 uses pYG908 as a template, respectively uses Sa+da, da+ds, and Sa+Ss as primers, 4.DL3000, 5.λDNA (HindIII), 6.7. 8 is using Streptomyces lividans TK24 as a template, respectively using Sa+da, da+ds, and Sa+da as primers, 9.pYG905 as a template, and Sa+da as primers, wherein S represents SnpR+SnpA, d represents doxA, s stands for sense (forward), and a stands for antisense (reverse).

Figure 5: SDS-PAGE chart of the expression product of engineering bacteria pYG908/TK24 of the present invention, wherein 1. pYG908/TK24, 2. control engineering bacteria pYG57/TK24, 3. TK24, 4. protein standard molecular weight Mark.

Fig. 6: CO binding difference spectrum of engineering bacteria pYG908/TK24 of the present invention.

Figure 7 A-C: HPLC charts of the fermentation products of engineering bacteria pYG908/TK24 of the present invention and control bacteria.

Detailed ways

Further illustrate the present invention with embodiment below.

Materials and methods in the following examples are:

For the molecular cloning technique used, see "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

The tool enzymes used were all purchased from Takala Company, and the specific reaction conditions and methods used were referred to the product instructions.

The gel recovery kit used was purchased from Shanghai Sangon Company, and the usage method refers to the product manual.

The following commercially available plasmids and E. coli strains were used for DNA library construction and gene cloning.

pUC18 (Takala, Japan)

pBluescript II KS(-) (available from Promega)

pYG57 (Chinese Journal of Pharmaceutical Industry, 2004, 35(1): 13)

Streptomyces lividans TK24 (commercially available from ATCC)

The media and reagents not specified are all commercially available products.

Example 1 PCR amplification and sequencing results of doxA gene and snpR+snpA

Using the published Streptomyces coelicolor SIPI-1482 genome as a template, the doxA gene was amplified by 97°C for 5min, 95°C for 30s, 72°C for 5min, 30 cycles, and 72°C for 10min to amplify a single target The stripe is about 1.3Kb in size. The amplification of snpR+snpA uses gradient PCR, and the designed primers are:

Forward primer: CG GAATTC GCGCATCGATGACTCCTCGAG (underlined as EcoRI),

Reverse primer: AA CTGCAG GTACCGCCGACCCGCTGCAT (PstI is underlined). The annealing temperatures were 62.8°C, 65.5°C, and 68°C, respectively. It can be seen from Figure 1 that no target band was amplified at 62.8°C. To enhance the specificity of amplification, annealing at 68°C was used.

The above-mentioned doxA gene containing ribosome binding sites was sequenced, and it was found that its sequence was 100% homologous to the sequence reported in Genbank (accession number U50973), while the snpR sequence derived from SIPI-1482 and Genbank The reported sequence (Accession No. AY072041) has a homology of 99.4% (SEQ ID No.1), an amino acid homology of 96.5% (SEQ ID No.2), and a promoter snpA homology of 100%.

Example 2 Recombinant plasmid construction, enzyme digestion verification and PCR inspection

The above doxA, snpA and SnpR gene sequences were cloned into pUC18. The doxA, snpA and SnpR fragments excised from it were subcloned into the plasmid pBluescript II KS(-), and then the tandem fragment (PstI-HindIII fragment) was combined with the vector pYG57 containing the thiostrepton (tsr) resistance gene (PstI-HindIII large fragment) connection constructs recombinant plasmid pYG908, transforms TK24 protoplasts, and screens positive clones on the R ₂ YE medium containing 30 μg/ml thiostrepton (tsr), obtains the expression vector of the present invention and Refer to Figure 2 for the construction process of the genetically engineered bacteria pYG908/TK24 and the recombinant plasmid pYG908.

The fragments of snpR+snpA and doxA, as well as the tandem fragments of the two, can be excised with corresponding enzymes for the recombinant plasmid pYG908 (Fig. 3). By diluting the recombinant plasmid as a template, the sequences of different combinations of primers Sa, da, ds, and Ss are:

Ss: CG GAATTC GCGCATCGATGACTCCTCGAG (EcoRI),

Sa: AA CTGCAG GTACCGCCGACCCGCTGCAT (PstI),

ds: G GAATTC GGAGG GGTGCCTCATGAGCGGCGAG (EcoRI),

da: CCC AAGCTT CCATCAACGCAGCCAGACGG (HindIII),

The corresponding target fragment (1.3Kb in size) and tandem fragment (2.6Kb in size) could also be amplified, while the control TK24 could not be amplified (Figure 4). Thus, it was judged that the expression plasmid of Streptomyces was constructed correctly.

Expression of embodiment 3 recombinant protein

Insert the genetically engineered bacteria pYG908/TK24 into YMB medium (see the Streptomyces operation manual for the formula) 20ml (+tsr 30μg/ml), 30°C, 250r/min shaker culture for 2d as seeds, and then 10 (v/ v) After the % inoculum is inserted into the above-mentioned medium, continue to cultivate for 2 days, collect the mycelium by centrifugation (10000r/min, 10min), wash the mycelium with 100mmol/L phosphate buffer (pH7.5), and then sonicate Break up (work for 5s, stop for 5s, cycle 80 times), collect supernatant after centrifugation, and use for SDS-PAGE protein electrophoresis analysis.

As shown in Figure 5, from the analysis of the results of mycelium protein electrophoresis, it can be known that the genetically engineered bacteria pYG908/TK24 produced a band of about 47kD, the size and the theoretical daunorubicin C-14 hydroxylase gene encoding 423 amino acids The size of the control engineering bacteria pYG57/TK24 (obtained by transforming the TK24 strain with the expression plasmid pYG57) is an engineering bacteria in which the doxA gene cooperates with Dnrv and is controlled by the ermE promoter. There is no obvious difference between the band of about 45KD and the control TK24 , indicating that daunorubicin C-14 hydroxylase is not produced.

Example 4 CO binding difference spectrum analysis (Chinese Journal of Pharmaceutical Industry, 2004, 35 (1): 13)

The pYG908/TK24 engineering strain was cultured in the above YMB for 2 days, then transferred to GMS medium and cultured at 30°C for two days, then the hyphae were collected and homogenized. TK24 was used as a control to carry out the CO binding difference spectrum analysis experiment. The experimental samples, controls and references were first reduced with sodium dithionite (Na ₂ S ₂ O ₄ ), and then the samples and controls were ventilated with CO for 3 minutes (the references were not ventilated), and scanned at 400-500 nm.

From the CO binding difference spectrum (Figure 6), it can be seen that pYG908/TK24 has a small peak at 450nm, which proves from another angle that the C-14 daunorubicin hydroxylase gene is expressed. Because the C-14 daunorubicin hydroxylase gene is a P450 oxidase, it has a smaller absorption peak at 450nm after being reduced with sodium dithionite and then reduced with CO.

Embodiment 5 Genetic Engineering Bacteria Fermentation Transformation Daunorubicin is Doxorubicin

Inoculate the fresh spores of the genetically engineered bacterium pYG908/TK24 obtained in Example 2 and the TK24 strain into 20ml of YMB (5 μg/ml tsr is also added when the engineered strain is cultivated) culture medium from the slant, and culture with shaking at 30°C and 250r/min 2d later as liquid seeds. After above-mentioned liquid seed is inserted into GMS culture medium with 10% (v/v) inoculum amount (5 μ g/ml tsr also will be added when engineering bacterial strain is cultivated), add daunorubicin to final concentration after culturing 3d under the same condition. 2 μg/ml, and then stop the fermentation after continuing to culture for 3 days. Then adjust the pH of the culture solution to 8.5 with 5mol/L NaOH, add an equal volume of extractant (chloroform:methanol=9:1) and extract at 30°C and 220r/min for 30min. Centrifuge (Beckman AvantiJ-25, rotor JA17, 8,000r/min, 10min) to separate the above-mentioned extraction mixture, discard the upper aqueous phase and bacteria, collect the obtained organic phase solution and drain it on a rotary evaporator, the red product Dissolve in an appropriate volume of methanol, and then subject the product to HPLC analysis.

Daunorubicin is added to the fermentation medium as a substrate, and it can be seen that the contrast TK24 bacterium does not produce the peak (Fig. 7B) with the same retention time as doxorubicin (English abbreviation dxr) by the HPLC figure (see Figure 7), but Can still see the peak of residual daunorubicin (English abbreviation dnr), and also see that there is a peak with a retention time close to it before the daunorubicin peak. The product produced by the element. However, the genetically engineered bacterium of the present invention can produce a peak (see FIG. 7C ) with the same retention time as the doxorubicin standard sample ( FIG. 7A ), which can be preliminarily considered to be doxorubicin. Simultaneously the peak of daunorubicin still exists, and explanation also has part and does not transform, also has other by-products to generate simultaneously.

Sequence Listing

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Claims (8)

1. A gene derived from the coding regulatory protein SnpR of Streptomyces coeruleorubidus, which is one of the following nucleotide sequences: 1) It is the base sequence shown in SEQ ID No.1 in the sequence listing; 2) Encoding a protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence listing. 2. A regulatory protein SnpR derived from Streptomyces coeruleorubidus (Streptomyces coeruleorubidus), which is a protein composed of the amino acid sequence shown in SEQ ID No.2 in the sequence listing. 3. A carrier expressing daunorubicin C-14 hydroxylase, comprising the gene nucleotide sequence of the coding regulatory protein SnpR according to claim 1. 4. carrier as claimed in claim 3, is characterized in that, the promotor of the gene expressing daunorubicin C-14 hydroxylase in this carrier is SnpA promotor, and carrier is plasmid pBluescript IIKS (-). 5. A genetically engineered bacterial strain for expressing daunorubicin C-14 hydroxylase, which is obtained by transforming the expression vector described in claim 3 or 4 into Streptomyces lividans TK24 Genetically engineered strains. 6. The application of the gene encoding regulatory protein SnpR as claimed in claim 1 in the preparation of doxorubicin by daunorubicin biotransformation. 7. A method that daunorubicin is biotransformed into doxorubicin, is characterized in that utilizing the expression vector described in claim 4 to transform host cells, cultivating transformants, and daunorubicin is biotransformed into by the transformant fermentation Adriamycin. 8. The method according to claim 7, characterized in that the host cell is Streptomyces lividans TK24.

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