DoxA protein mutant, and coding gene and applications thereof

Abstract

The present invention relates to a DoxA protein mutant having an amino acid sequence set forth in SEQ ID NO: 16, and coding gene thereof. The protein mutant or the coding gene thereof can be used for producing epirubicin. The present invention further relates to a Streptomyces capable of efficiently expressing epirubicin, which is constructed by replacing the dnmV gene of a starting Streptomyces in situ with the avrE gene and mutating the doxA gene of the starting Streptomyces into a gene encoding the protein set forth in SEQ ID NO: 16. The fermentation broth of this Streptomyces has an epirubicin potency of up to 102.0 μg/ml.

Claims

1. A DoxA protein variant comprising the amino acid sequence set forth in SEQ ID NO: 18 with amino acid substitutions Ala133Thr, Ala339Asp, and Cys398Ser, wherein said DoxA protein variant is capable of converting epidaunorubicin to epirubicin.

2. The DoxA protein variant of claim 1, said DoxA protein variant comprising the amino acid sequence set forth in SEQ ID NO: 16.

3. The DoxA protein variant of claim 1, said DoxA protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 16.

4. A nucleic acid molecule encoding the DoxA protein variant of claim 1.

5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 15.

6. An expression cassette comprising the nucleic acid molecule of claim 4.

7. A recombinant vector comprising the nucleic acid molecule of claim 4.

8. A microorganism comprising the recombinant vector of claim 7.

9. A transgenic cell line comprising the recombinant vector of claim 7.

10. A method for constructing an epirubicin-expressing Streptomyces cell, said method comprising the steps of: 1) replacing the dnmV gene of a starting Streptomyces cell in situ with avrE gene, and 2) mutating the doxA gene of the starting Streptomyces cell into a gene sequence encoding the DoxA protein variant set forth in SEQ ID NO: 16.

11. The method according to claim 10, wherein the sequence of the dnmV gene is set forth in SEQ ID NO: 9; the sequence of the avrE gene is set forth in SEQ ID NO: 10; the doxA gene sequence is set forth in SEQ ID NO: 15.

12. A Streptomyces cell produced by the method of claim 10.

13. A method for preparing epirubicin, said method comprising culturing the Streptomyces cell of claim 12 in fermentation medium under conditions wherein the epirubicin is produced.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the chemical structure of epirubicin.

(2) FIG. 2 is a plasmid map of pHY642, the sequence of which is set forth in SEQ ID NO: 1.

(3) FIG. 3 is a plasmid map of pZH5, the sequence of which is set forth in SEQ ID NO: 2.

(4) FIG. 4 is an HPLC detection profile of epirubicin standard.

(5) FIG. 5 is a mass spectrum of epirubicin standard.

(6) FIG. 6 is an HPLC detection spectrum of the ZH98 fermentation broth.

(7) FIG. 7 is an HPLC detection spectrum of the ZH100 fermentation broth.

(8) FIG. 8 is a mass spectrum of epirubicin in the ZH100 fermentation broth.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) The experimental methods used in the following examples are conventional methods unless otherwise specified.

(10) The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

(11) The present invention will now be described in detail in combination with the following examples. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

(12) Streptomyces (CGMCC No. 4827) was purchased from the China General Microbiological Culture Collection Center.

(13) Sucrose-Tris buffer: The solute is sucrose, the solvent is 10 mM Tris-HCl, the mass percentage of sucrose in sucrose-Tris buffer is 10.3%, and the pH of the buffer is 8.0.

(14) The lysozyme solution is a product of Sangon Biotech (Shanghai) Co., Ltd. and its catalog number is A610308.

(15) The saturated phenol solution (pH 8.0) is a product of Sangon Biotech (Shanghai) Co., Ltd. and its catalog number is A504193.

(16) The TSB medium (Bacto™ Tryptic Soy Broth) is a product of BD and its catalog number is 211825.

(17) The pIJ773 is disclosed in the literature “Gust B I, Challis G L, Fowler K, Kieser T, Chater K F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA. 2003 Feb. 18; 100 (4): 1541-6. Epub 2003 Jan. 31”, and can be obtained by the public from Zhejiang Hisun Pharmaceutical Co., Ltd.

(18) The Streptomyces avermitilis is disclosed in the literature “Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol. 2003 May; 21(5): 526-31”, and can be obtained by the public from Zhejiang Hisun Pharmaceutical Co., Ltd.

(19) The Escherichia coli ET12567 (pUZ8002) is disclosed in the literature “Bierman M, Logan R, Obrien K, Seno E T, Rao R N, Schoner B E: Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992, 116(1): 43-49.10.1016/0378-1119 (92) 90627-2”, and can be obtained by the public from Zhejiang Hisun Pharmaceutical Co., Ltd.

(20) The epirubicin standard is a product of Langchem Co. and its catalog number is 10309.

(21) The pSET152 is disclosed in the literature “Bierman M, Logan R, O'Brien K, Seno E T, Rao R N, Schoner B E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992, 116(1): 43-9”, and can be obtained by the public from Zhejiang Hisun Pharmaceutical Co., Ltd.

EXAMPLES

Example 1—Construction of Plasmid pZH11 for the dnmV Gene Knockout

(22) 1. Extraction of Genomic DNA of Streptomyces (CGMCC No. 4827)

(23) 50 μl cell suspension of Streptomyces (CGMCC No. 4827) was inoculated into 30 ml of TSB medium, cultured at 28° C., 220 rpm for 48 hr, centrifuged in a 50 ml centrifuge tube at 4000 rpm for 10 min, and the supernatant was removed. The obtained precipitate was washed with 30 ml sucrose-Tris buffer twice and then suspended in 5 ml sucrose-Tris buffer. 20 μl lysozyme solution (100 mg/ml) was added and the resulting mixture was kept in 37° C. water bath for 2 hr. 500 μl of 10% SDS solution was added and the mixture was gently inverted until essentially clear. 5 ml of a saturated phenol solution (pH 8.0) was added, and after gently inverting several times, the mixture was centrifuged at 4000 rpm for 10 min. 4 ml of the upper layer solution was taken, and 4 ml of a phenol-chloroform-isoamyl alcohol (pH 8.0) solution was added, and the mixture was gently inverted several times, and then centrifuged at 4000 rpm for 10 minutes. 3 ml of the upper layer was taken, 300 μl of 3 M HAc/NaAc buffer (pH 5.3) and 3 ml of isopropanol were added, and after gently inverting several times, the pellet of the agglomeration was picked up to a 1.5 ml centrifuge tube with a pipette tip. The precipitate was washed twice with aqueous ethanol (70% by volume) and dried at room temperature. The mixture was dissolved by adding 500 μl of Tris-HCl (pH 8.0) to obtain genomic DNA of Streptomyces (CGMCC No. 4827).

(24) 2. Using the genomic DNA of Streptomyces (CGMCC No. 4827) obtained in step 1 as a template, the Dnm VLF/Dnm VLR primer pair and the Dnm VRF/Dnm VRR primer pair were used respectively to amplify and obtain the upstream DNA fragment of the dnmV gene and the downstream DNA fragment of the dnmV gene.

(25) The sequences of the primers were as follows:

(26) TABLE-US-00001 Dnm VLF: (SEQ ID No. 3) 5′-CCCAAGCTTCCACTCTGCCCGTCCACCTCTT-3′, (The underlined sequence is the recognition site of restriction endonuclease HindIII) Dnm VLR: (SEQ ID No. 4) 5′-TGCTCTAGACTCACCCGTCTCCGCGTG-3′, (The underlined sequence is the recognition site of restriction endonuclease XbaI) Dnm VRF: (SEQ ID No. 5) 5′-TGCTCTAGACGGGCTGGTCGTCAACATCG-3′, (The underlined sequence is the recognition site of the restriction endonuclease XbaI) Dnm VRR: (SEQ ID No. 6) 5′-CCGGAATTCGCTCCTTCCTGGGCTTCCTG-3′. (The underlined sequence is the recognition site of the restriction endonuclease EcoRI)

(27) (The underlined sequence is the recognition site of the restriction endonuclease EcoRI)

(28) According to instruction of the PrimeSTAR kit (TaKaRa, catalog number: R044A), the PCR amplification system was prepared according to the following ratio:

(29) TABLE-US-00002 2 × PrimeSTAR GC buffer 40 μl 2.5 mM dNTP 6.4 μl Dnm VLF(Dnm VRF) 0.8 μl Dnm VLR(Dnm VRR) 0.8 μl template 0.8 μl H.sub.2O 30.5 μl PrimeSTAR polymerase 0.8 μl

(30) According to the different prime pairs, the PCR was carried out in two different tubes. The PCR procedure was: 95° C. for 5 min; 30 cycles of 95° C. for 30 sec, 55° C. for 30 sec and 72° C. for 3 min; 72° C. for 5 min; 16° C. for 1 min.

(31) 3. The upstream DNA fragment of dnmV gene obtained by PCR amplification was digested with HindIII and XbaI to obtain upstream fragment 1; vector pHY642 (FIG. 2) was digested with HindIII and XbaI to obtain vector fragment 1; the upstream fragment 1 was ligated with the vector fragment 1 to obtain a recombinant plasmid, which was named pZH9. The pZH9 was sent for sequencing and the results were in line with expectations.

(32) The downstream DNA fragment of dnmV gene obtained by PCR amplification was digested with EcoRI and XbaI to obtain the downstream fragment 2; pZH9 was digested with EcoRI and XbaI to obtain vector fragment 2; the downstream fragment 2 was ligated with vector fragment 2 to obtain a recombinant plasmid, which was named pZH10. The pZH10 was sent for sequencing and the results were in line with expectations.

(33) The pIJ773 was digested with XbaI to obtain an apramycin-resistant gene fragment; pZH10 was digested with XbaI to obtain vector fragment 3; and the apramycin-resistant gene fragment was ligated with the vector fragment 3 to obtain a recombinant plasmid, which was named pZH11. The pZH11 was sent for sequencing and the results were in line with the expected sequence.

(34) pZH11 is the finally constructed vector for the dnmV gene knockout.

Example 2—Construction of Plasmid pZH12 for In Situ Replacement of dnmV Gene with avrE Gene

(35) 1. Using the genomic DNA of Streptomyces avermitilis as a template, avrEF/avrER primer pair was used to amplify and obtain the avrE gene fragment.

(36) The PCR amplification system and procedure were the same as in Example 1 except that the template and the primers were different.

(37) The sequences of the primers were as follows:

(38) avrEF:

(39) TABLE-US-00003 avrEF: (SEQ ID No. 7) 5′-ACGCGGAGACGGGTGAGGCGGACATGGGGCGGTTTTCGGTGTGC- 3′; avrER: (SEQ ID No. 8) 5′-GTCGTCGGAAGCCTGTGAGCTACACGTAAGCCGCCACCATG-3′.

(40) 2. The avrE gene fragment obtained in step 1 was digested with XbaI to obtain avrE fragment 3; pZH10 was digested with XbaI to obtain vector fragment 3; the avrE fragment 3 was ligated with the vector fragment 3 to obtain a recombinant plasmid, which was named pZH12. The pZH12 was sent for sequencing and the results were in line with the expected sequence.

(41) pZH12 was used to replace the dnmV gene in situ with the avrE gene.

Example 3—Construction of Epirubicin-Producing Bacteria

(42) 1. Construction of recombinant E. coli ET12567 (pUZ8002, pZH11) and ET12567 (pUZ8002, pZH12)

(43) pZH11 and pZH12 were transformed into E. coli ET12567 (pUZ8002), respectively, as follows:

(44) 1 μl of the plasmid pZH11 prepared in Example 1 and 1 μl of the plasmid pZH12 prepared in Example 2 were separately added to 100 μl of E. coli ET12567 (pUZ8002) competent cells, placed on ice for 30 min, and then heat-shocked at 42° C. for 90 sec, and then quickly placed on ice cooling for 1 min. 900 μl of liquid LB medium was added, and kept in 37° C. water bath for 50 min. 100 μl of each was plated on solid LB medium containing 25 μg/ml chloramphenicol (Cm), 50 μg/ml kanamycin (Km), and 50 μg/ml ampicillin (Amp), and cultured overnight at 37° C. The transformants were grown, i.e. recombinant E. coli ET12567 (pUZ8002, pZH11) and ET12567 (pUZ8002, pZH12).

(45) 2. Cultivation of recombinant Escherichia coli ET12567 (pUZ8002, pZH11) and ET12567 (pUZ8002, pZH12)

(46) The single colonies of recombinant Escherichia coli ET12567 (pUZ8002, pZH11) and ET12567 (pUZ8002, pZH12) were inoculated into 3 ml of liquid LB medium containing 25 μg/ml Cm, 50 μg/ml Km and 50 μg/ml Amp. After cultured overnight at 37° C., 250 rpm, 300 μl of each was inoculated into 30 ml of liquid LB medium containing 25 μg/ml Cm, 50 μg/ml Km and 50 μg/ml Amp, and cultured at 37° C., 250 rpm for 4-6 h to an OD600 of 0.4-0.6. After centrifugation, the cells were respectively collected, washed twice with liquid LB medium, and finally, 500 μl of liquid LB medium was added to suspend the cells for use.

(47) 3. Conjugal transfer and screening of dnmV gene knockout strain ZH11

(48) (1) Conjugal Transfer

(49) 50 μl of Streptomyces (CGMCC No. 4827) cell suspension was inoculated into 30 ml of TSB medium, and cultured at 28° C., 220 rpm for 48 hr. Then, 500 μl of the culture was added to 500 μl of ET12567 (pUZ8002, pZH11) cultured in step 2. 800 μl of the supernatant was removed by centrifugation. The cells were suspended in the remaining supernatant and plated on MS solid medium plates, and cultured at 28° C. for 16-20 h. Then the surface of the medium was covered with 1 ml of sterilized water containing 500 μg of apramycin (Am) and 500 μg of nalidixic acid (NaI), cultured at 28° C. for 4-8 days, and the conjugant was grown.

(50) (2) Screening

(51) One conjugant obtained in step (1) was inoculated by streaking on MS solid medium (20.0 g of agar, 20.0 g of mannitol, 20.0 g of soybean cake powder, and tap water was added until the final volume was 1000 ml) containing 25 μg/ml of NaI and cultured at 28° C. for 4-6 d. Each of the grown single colonies was simultaneously transferred to the following two solid media: MS solid medium containing 25 μg/ml thiostrepton (Tsr) and 25 μg/ml apramycin (Am), respectively. After cultivation at 28° C. for 5 days, the growth was observed. The colony which was grown on the MS solid medium containing apramycin (Am) while not grown on the MS solid medium containing thiostrepton was the dnmV gene knockout strain, which was named ZH11.

(52) 4. Construction of Epirubicin-Producing Strain

(53) (1) Conjugal Transfer

(54) 50 μl cell suspension of ZH11 obtained in step 3 was inoculated into 30 ml TSB medium, and cultured at 28° C., 220 rpm for 48 hr. Then, 500 μl of the culture was added to 500 μl of ET12567 (pUZ8002, pZH12) cultured in step 2. 800 μl of the supernatant was removed by centrifugation. The cells were suspended in the remaining supernatant and plated on MS solid medium plates, and cultured at 28° C. for 16-20 h. The surface of the medium was covered with 1 ml of sterilized water containing 500 μg of Tsr and 500 μg of NaI, cultured at 28° C. for 4-8 days, and the conjugant was grown.

(55) (2) Screening

(56) One conjugant obtained in step (1) was inoculated by streaking on MS solid medium containing 25 μg/ml of NaI and cultured at 28° C. for 4-6 d. Each of the grown single colonies was simultaneously transferred to the following two MS solid media: one contained 25 μg/ml Am, and the other contained neither Am nor Tsr. After cultivation at 28° C. for 5 days, the growth was observed. The colony, which was grown on the MS solid medium containing no Am and Tsr while not grown on the MS solid medium containing Am (i.e., indicating that dnmV was replaced with avrE), was an epirubicin-producing bacterium, which was named ZH12.

(57) The sequence of the dnmV gene is set forth in SEQ ID NO: 9.

(58) The sequence of the avrE gene is set forth in SEQ ID NO: 10.

Example 4—Fermentation Test of Epirubicin

(59) 1. Fermentation of Epirubicin

(60) A loop of mycelia of ZH12 prepared in Example 3 was inoculated on the solid slant medium, and after cultivation at 28° C. for 10 days, a mass of about 1×1 cm was dug from the slant, inoculated into the seed culture medium and incubated at 28° C., 250 rpm for 45 hr to obtain seed culture. Then, 2.5 ml of the seed culture was inoculated into the fermentation medium, and cultured at 28° C., 250 rpm for 7 days to obtain a fermentation broth.

(61) The formulations of each of the above media are as follows:

(62) Solid slant medium (g/L): yeast extract 4.0, malt extract 10.0, glucose 4.0, agar 20.0, the balance was water, the pH was adjusted to 6.80, and the mixture was sterilized, slanted and cooled.

(63) Seed culture medium (g/L): soluble starch 30.0, glucose 10.0, soybean cake powder 20.0, CaCO.sub.3 2.0, NaCl 3.0, the balance was water, and the pH was adjusted to 6.8.

(64) Fermentation medium (g/L): corn starch 80.0, yeast powder 30.0, CaCO.sub.3 3.0, NaCl 3.0, the balance was water, and the pH was adjusted to 6.80.

(65) The sterilization methods of the above media were all as follows: sterilized at 121° C. for minutes.

(66) 2. HPLC Detection

(67) After the fermentation, the fermentation broth was adjusted to pH 1.5 with HCl, and ethanol with 3 times the volume of the mixture was added. After standing for 1 hr, the resulting mixture was centrifuged at 4000 rpm. The supernatant sample was taken for HPLC detection, and the epirubicin standard was used as a control. The epirubicin potency of the fermentation broth was calculated by multiplying the target peak area ratio of the sample and the standard by the concentration of the standard.

(68) The HPLC detection method is as follows:

(69) Column: C18 column, 5 μm, 4.6×250 mm;

(70) Buffer: prepared by dissolving 1.44 g of sodium dodecyl sulfate and 0.68 ml of phosphoric acid in 500 ml of ultrapure water;

(71) Mobile phase: buffer:acetonitrile:methanol is 500:500:60 (volume ratio);

(72) Flow rate: 1.35 ml/min;

(73) Detection wavelength: 254 nm;

(74) Injection volume: 10 μl.

(75) The HPLC detection profile of the epirubicin standard is shown in FIG. 4, wherein the retention time of epirubicin is 10.316 min.

(76) The mass spectrum of the epirubicin standard is shown in FIG. 5.

(77) The results showed that the epirubicin potency of the ZH12 fermentation broth was 0.65 μg/ml.

Example 5—Mutation and Cloning of the doxA Gene

(78) 1. The genomic DNA of Streptomyces (CGMCC No. 4827) was used as a template, DoxAF/DoxAR was used as a primer pair, and MnCl.sub.2 with a final concentration of 0.5 μM was added for error-prone PCR to amplify a doxA mutant gene fragment, thereby conducting random mutation of the doxA gene.

(79) The sequences of the primers were as follows:

(80) DoxAF: 5′-ACAGAGCTCGTGGCCGTCGACCCGTTC-3′ (SEQ ID NO: 11) (The underlined sequence is the recognition site of the restriction endonuclease SacI);

(81) DoxAR: 5′-GGAAGATCTTCAGCGCAGCCAGACGGG-3′ (SEQ ID NO: 12) (The underlined sequence is the recognition site of the restriction endonuclease BglII).

(82) According to the instruction of Taq Polymerase kit (Sangon Biotech (Shanghai) Co., Ltd., catalog number: B500010), the PCR amplification system was prepared according to the following ratio:

(83) TABLE-US-00004 10 × Taq buffer 10 μl 2.5 mM dNTP 10 μl DoxAF 2 μl DoxAR 2 μl template 1 μl 50 μm MnCl.sub.2 1 μl H.sub.2O 73 μl Taq polymerase 1 μl

(84) The PCR was carried out in 4 tubes, and the PCR procedure was: 94° C. for 5 min; 30 cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 2 min; 72° C. for 5 min; 16° C. for 1 min.

(85) 2. The doxA mutant gene fragment obtained in step 1 was digested with SacI and BglII to obtain the doxA mutant fragment 4; the vector pZH5 (FIG. 3) was digested with SacI and BglII to obtain the vector fragment 4; the doxA mutant fragment 4 was ligated with the vector fragment 4 to obtain a recombinant plasmid, which was named pZH99. In this process, the doxA mutant gene was cloned downstream of the promoter ermE*.

(86) 3. The pZH99 was digested with XbaI and BglII to obtain ermE*+doxA mutant gene fragment; the vector pSET152 was digested with XbaI and BamHI to obtain vector fragment 5; the ermE*+doxA mutant gene fragment was ligated with the vector fragment 5 to obtain a recombinant plasmid, which was named pZH100.

(87) The sequence of the ermE* was set forth in positions 46 to 325 of SEQ ID NO: 2. pZH100 was a DoxA protein mutant-expressing plasmid.

(88) 4. Using the genomic DNA of Streptomyces (CGMCC No. 4827) as a template and DoxAF/DoxAR as a primer pair, the doxA gene was amplified.

(89) The PCR amplification system and procedure were the same as in Example 1 except that the primers were different.

(90) Using the amplified doxA gene, recombinant plasmid pZH98 was obtained through steps 2 to 3. The pZH98 was sent for sequencing and the results were in line with expectations. pZH98 was a DoxA protein-expressing plasmid, used as a control for pZH100.

Example 6—Screening of doxA Mutants

(91) 1. Construction of recombinant E. coli ET12567 (pUZ8002, pZH98) and ET12567 (pUZ8002, pZH100)

(92) pZH98 and pZH100 were transformed into E. coli ET12567 (pUZ8002), respectively, as follows:

(93) 1 μl of the plasmid pZH98 and pZH100 prepared in Example 5 were added to 100 μl of E. coli ET12567 (pUZ8002) competent cells respectively, placed on ice for 30 min, heat-shocked at 42° C. for 90 sec, then rapidly placed on ice cooling for 1 min. 900 μl of liquid LB medium was added and the resulting mixture was kept in 37° C. water bath for 50 min. 100 μl of each was plated on solid LB medium containing 25 μg/ml chloramphenicol (Cm), 50 μg/ml kanamycin (Km) and 50 μg/ml Apramycin (Am), and cultured overnight at 37° C. Transformants were grown respectively, namely recombinant E. coli ET12567 (pUZ8002, pZH98) and ET12567 (pUZ8002, pZH100).

(94) 2. Conjugal Transfer

(95) (1) One single colony of the ET12567 (pUZ8002, pZH98) transformants obtained in step 1 is selected and then inoculated into 3 ml of liquid LB medium containing 25 μg/ml Cm, 50 μg/ml Km and 50 μg/ml Am, and cultured overnight at 37° C., 250 rpm. After that, 300 μl of the culture was inoculated into 30 ml of liquid LB medium.

(96) (2) All the transformants of ET12567 (pUZ8002, pZH100) obtained in step 1 were washed with 1 ml of liquid LB medium and all inoculated into 30 ml of liquid LB medium containing 25 μg/ml Cm, 50 μg/ml Km and 25 μg/ml Am. The transformants were cultured at 37° C., 250 rpm for 4-6 h to an OD600 of 0.4-0.6. After centrifugation, the cells were collected, washed twice with liquid LB medium, and finally, 500 μl of liquid LB medium was added to suspend the cells for use.

(97) (3) 50 μl of the cell suspension of ZH12 prepared in Example 3 was inoculated into 30 ml of TSB medium, and cultured at 28° C., 220 rpm for 48 hr. Then 500 μl of the culture was added to 500 μl of the ET12567 (pUZ8002, pZH98) obtained in the step (1) and the ET12567 (pUZ8002, pZH100) obtained in the step (2) respectively. 800 μl of the supernatant was removed by centrifugation. The cells were suspended in the remaining supernatant and plated on MS solid medium plates, and cultured at 28° C. for 16-20 h. Then the surface of the medium was covered with 1 ml of sterilized water containing 500 μg of apramycin (Am) and 500 μg of nalidixic acid (NaI), cultured at 28° C. for 4-8 days, and the conjugants was grown. They were named ZH98 and ZH100, respectively. The conjugants ZH98 and ZH100 were spotted onto the solid slant medium in Example 4.

(98) 3. Fermentation Test

(99) Fermentation and HPLC detection of epirubicin of ZH98 and ZH100 was carried out in accordance with the method of Example 4.

(100) The HPLC detection spectrum of the ZH98 fermentation broth was shown in FIG. 6.

(101) The HPLC detection spectrum of the ZH100 fermentation broth was shown in FIG. 7, wherein the retention time of epirubicin was 10.316 min.

(102) The mass spectrum of epirubicin in the ZH100 fermentation broth was shown in FIG. 8.

(103) Using the fermentation results of ZH98 as a control, epirubicin high-producing strains of doxA mutant ZH100 were screened. One strain, whose epirubicin potency was significantly increased, was obtained. The epirubicin potency of its fermentation broth reached 102.0 μg/ml, while the control ZH98 was 0.72 μg/ml.

(104) 4. DoxA Mutation Sites Detection

(105) The genomic DNA of the high-producing strain of ZH100 obtained in step 3 was extracted according to step 1 in Example 1. ermEF/DoxAR were used as a primer pair to carry out a PCR reaction to obtain PCR amplification products.

(106) The sequences of the primers were as follows:

(107) TABLE-US-00005 ermEF: (SEQ ID No. 13) 5′-AGCCCGACCCGAGCACGC-3′ DoxAR: (SEQ ID No. 14) 5′-GGAAGATCTTCAGCGCAGCCAGACGGG-3′

(108) The PCR amplification products were sent for sequencing.

(109) The sequencing results showed that the sequence of the doxA mutant gene of the strain was as set forth in SEQ ID NO: 15.

(110) The amino acid sequence of the protein encoded by the doxA mutant gene was set forth in SEQ ID NO: 16.

(111) The sequence of the doxA gene of Streptomyces (CGMCC No. 4827) was set forth in SEQ ID NO: 17.

(112) The amino acid sequence of the DoxA protein encoded by the doxA gene of Streptomyces (CGMCC No. 4827) was set forth in SEQ ID NO: 18.

(113) Sequencing revealed that the base mutations of the doxA gene in this strain were 397G>A, 399C>T, 1016C>A, and 1193G>C, and the changes of amino acid codon were 5′-GCC-3′ at positions 397-399 to 5′-ACT-3′, 5′-GCC-3′ at positions 1015-1017 to 5′-GAC-3′, and 5′-TGC-3′ at positions 1192-1194 to 5′-TCC-3. Correspondingly, amino acids changed at three sites of the DoxA protein: the alanine at position 133 of DoxA protein was changed to threonine (A133T), the alanine at position 339 was changed to aspartic acid (A339D) and the cysteine at position 398 was changed to serine (C398S).

(114) TABLE-US-00006 SEQ ID NO: 1: 5′-agcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcatcgatgatcagat caaggcgaatacttcatatgcggggatcgaccgcgcgggtcccggacggggaagagcggggagcttgccagaga gcgacgacttccccttgcgttggtgattgccggtcagggcagccatccgccatcgtcgcgtagggtgtcacaccccag gaatcgcgtcactgaacacagcagccggtaggacgaccatgactgagttggacaccatcgcaaatccgtccgatccc gcggtgcagcggatcatcgatgtcaccaagccgtcgcgatccaacataaagacaacgttgatcgaggacgtcgagcc cctcatgcacagcatcgcggccggggtggagttcatcgaggtctacggcagcgacagcagtccttttccatctgagttg ctggatctgtgcgggcggcagaacataccggtccgcctcatcgactcctcgatcgtcaaccagttgttcaagggggag cggaaggccaagacattcggcatcgcccgcgtccctcgcccggccaggttcggcgatatcgcgagccggcgtggg gacgtcgtcgttctcgacggggtgaagatcgtcgggaacatcggcgcgatagtacgcacgtcgctcgcgctcggagc gtcggggatcatcctggtcgacagtgacatcaccagcatcgcggaccggcgtctccaaagggccagccgaggttacg tcttctcccttcccgtcgttctctccggtcgcgaggaggccatcgccttcattcgggacagcggtatgcagctgatgacg ctcaaggcggatggcgacatttccgtgaaggaactcggggacaatccggatcggctggccttgctgttcggcagcga aaagggtgggccttccgacctgttcgaggaggcgtcttccgcctcggtttccatccccatgatgagccagaccgagtct ctcaacgtttccgtttccctcggaatcgcgctgcacgagaggatcgacaggaatctcgcggccaaccgataagcgcct ctgttcctcggacgctcggttcctcgacctcgattcgtcagtgatgatctgccggtctccctatagtgagtcgtattaatttc gataagccaggttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat ttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatctgacgggtgcgcatgatcgtg ctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcga acgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaag tctggaaacgcggaagtcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagc ggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa aaggccagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcc cccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggc gtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcg ggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtg cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgact tatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagt ggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaaga gttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga aaaaaaggatctcaagaagatcdttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggatt ttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatg agtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttg cctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgaga cccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgca actttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgt tgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaagg cgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggcc gcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggt gagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataata ccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtg agcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcc tttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaata ggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaat aggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggaga cggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtg tcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatggacatattgtcgttagaacgcg gctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactatagaactcgacctgcaggtccccggg gatcggtcttgccttgctcgtcggtgatgtacttcaccagctccgcgaagtcgctcttcttgatggagcgcatggggacgt gcttggcaatcacgcgcaccccccggccgttttagcggctaaaaaagtcatggctctgccctcgggcggaccacgcc catcatgaccttgccaagctcgtcctgcttctcttcgatcttcgccagcagggcgaggatcgtggcatcaccgaaccgc gccgtgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggtcgccattgatgcgggcca gctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacggccagcaggtaggccgacagg ctcatgccggccgccgccgcatttcctcaatcgctcttcgttcgtctggaaggcagtacaccttgataggtgggctgcc cttcctggttggcttggtttcatcagccatccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagag caggattcccgttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcgggtgggcctac ttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacgaaccattggcaaaatcctgtatatc gtgcgaaaaaggatggatataccgaaaaaatcgctataatgaccccgaagcagggttatgcagcggaaaagatccgt cgagcagctga-3′ SEQ ID NO: 2: 5′-gaactcgagcagctgaagcttgcatgcctgcaggtcgactctagaagcccgacccgagcacgcgccggc acgcctggtcgatgtcggaccggagttcgaggtacgcggcttgcaggtccaggaaggggacgtccatgcgagtgtcc gttcgagtggcggcttgcgcccgatgctagtcgcggttgatcggcgatcgcaggtgcacgcggtcgatcttgacggct ggcgagaggtgcggggaggatctgaccgacgcggtccacacgtggcaccgcgatgctgttgtgggctggacaatcg tgccggttggtaggatccagcggtgagcgagctcgaattcatcgatgatatcagatctgccggtctccctatagtgagtc gtattaatttcgataagccaggttaacctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggc gctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggc ggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagg aaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaa gtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgt tccgaccctgccgcttaccggatacctgtccgcattctcccttcgggaagcgtggcgattctcatagctcacgctgtag gtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcc ttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggat tagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttac cttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccg ctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatatttct acggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcaccta gatcatttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatc agtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatac gggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaa taaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgc cgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacg ctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaa gcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgt atgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctca tcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtg cacccaactgatcttcagcatatttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaa aagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca cctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccattcgtctcgcgcgtt tcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccggga gcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagca gattgtactgagagtgcaccatatggacatattgtcgttagaacgcggctacaattaatacataaccttatgtatcatacac atacgatttaggtgacactata-3′ SEQ ID NO: 9: 5′-atgcgggtcgtggttctgggggcgacgggcagcgtcggtcggcaggtgtgtgcggcgtaccaggcgca cgggtgggacgtgcacggggtggcccgccgcccggcgccgcacctgagcgggtgcgggttcacggagctggacc tcgcggccgccgcgcctgggcggatcgccacggtgctgggtgatcttccggcggacgtcgtggtcaacgcggcggg cggctggggcgacaccgaggaggagatgacgtactcgcatctgcgactggtgcgacgcctggtggaggcgctcgc gctgctcccgttccggccccggctggtccatctggggtcggtgcacgagtacggtcccgtgccggccggcacgctgc tgcacgaggacctgctgccggagccggtcacgccgtacgcgcgcgtcaaactggagacctcgtcggccgtcctgac cgcagcgcgggccggtgtcctggacgcggtggtgctgcgcgcggcgaacatgtcgggcccgcatccgccgcagga gagtttcctggccgccctgatggcgcgtatcagcacggcattcgcgcacggtgggcggctggagttgagcgtcgcgg acgcacggcgggacttcatcgacgtgcgggacgtcgcacaggcggtggtgcgtgccgggcgggctccggcggtcg gcgggctggtcgtcaacatcgggcgcggggacgccgtgccgatcggtgatctggtcggctggctgctggaggccgc cgccttcccggaggaccgggtcgaccgccgggaggcgccggtgcggagcaagggcggcgactggacccggctg gacatcgggcgggcccggcggttgctgtcctgggcgccgcgcatcggcctgcgggactccgtccacagcatgtggc ggaccgcgcacggcgccccggcctag-3′ SEQ ID NO: 10: 5′-atggggcggttttcggtgtgcccgccccggccgaccggaatactgaagagcatgctgacgactgggatgt gcgaccgaccgctggtcgtcgtactcggagcctccggctatatcgggtcggccgtcgcggcggaactcgcccggtg gccggtcctgttgcggctggtggcccggcgaccgggcgtcgttccgccgggcggcgccgcggagaccgagacgc gtacggccgacctgacggcggcgagcgaggtcgccctcgccgtgacggacgccgacgtggtgatccacctggtcg cgcgcctcacccagggagcggcatggcgggcggcggagagcgatccggtggccgagcgggtgaacgtcggggt gatgcacgacgtcgtcgcggccctgcggtccgggcgccgcgccgggccgcccccggtggtggtgttcgccgggtc ggtctaccaggtgggccgcccgggtcgggtcgacggcagtgagccggacgagcccgtgacggcctatgcccgtca gaaactcgacgccgaacggacgttgaagtccgccacggtcgagggtgtcctgcgggggatctcgctgcggctgccc accgtctacggcgcggggccgggcccgcagggcaacggcgtcgtgcaggcgatggtgctccgggcgctcgccga cgaggccctcaccgtgtggaacggaagcgtggtggagcgtgacctggtgcatgtggaggatgtcgcgcaggccttc gtgagctgcctggcgcacgcggatgcgctcgccgggcggcactggctgctcggcagcggtcgtcctgtgaccgtcc cgcacctcttcggtgccatcgccgccggcgtgtccgcccgcaccgggcgccccgcggtgcccgtgaccgcggtgga ccctccggcgatggcgacggcggcggacttccacgggaccgtcgtcgactcctcggcgttccgcgcggtcaccggg tggcggccgcggctgtcgcttcaggagggcctggaccacatggtggcggcttacgtgtag-3′ SEQ ID NO: 15: 5′-gtggccgtcgacccgttcgcgtgtcccatgatgaccatgcagcgcaagcccgaggtgcacgacgccttcc gggaggcgggcccggtcgtcgaggtgaacgcccccgcgggcggacccgcctgggtcatcaccgatgacgccctc gcccgcgaggtgctggccgatccccggttcgtgaaggaccccgacctcgcccccgccgcctggcggggggtggac gacggtctcgacatccccgttccggagctgcgtccgttcacgctcatcgccgtggacggcgaggcccaccggcgcct gcgccgcatccacgcacctgcgttcaacccgcgccggctggccgagcggacggatcgcatcgccgcgatcgccgg ccggctgctcaccgaactcgccgacacttccggccggtcgggcaaaccggccgagctgatcggcggcttcgcgtac cacttcccgctgttggtcatctgcgagctgctcggtgtgccggtcaccgatccggcgatggcccgcgaggccgtcagc gttctcaaggcactcggcctcggcggcccgcagagcggcgggggtgacggcacggaccctgccgggggcgtgcc ggacacctcggccctggagagcctgctcctcgaagccgtgcactcagcccggcggaacgacaccccgaccatgacc cgcgtgctgtacgagcgcgcgcaggccgagttcggctcggtctccgacgaccagctcgtctacatgatcaccgggct catcttcgccggccacgacaccaccggctccttcctgggcttcctgctcgcggaggtcctggcgggccgcctcgcgg cggatgccgacgaggacgccgtctcccggttcgtggaggaggcgctgcgctaccacccgccggtgccctacacgtt gtggaggttcgctgccacggaggtgaccatcggcggcgtccggctgccccgcggagcgccggtgctggtggacatc gagggcaccaacaccgacggccgccatcacgacgacccgcacgccttccacccggaccgtccctcgtggcggcgg ctcaccttcggcgacgggccgcactactgcatcggggagcagctcgcccagctggagtcgcgcacgatgatcggcg tactgcgcagcaggttccccgaggcccgactggccgtgccgtacgacgagttgcggtggtcccggaagggggccca gacggcgcggctcaccgaactgcccgtctggctgcgctga-3′ SEQ ID NO: 16: VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALA REVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLR RIHAPAFNPRRLAERTDRIAAIAGRLLTELADTSGRSGKPAELIGGFAYHFPL LVICELLGVPVTDPAMAREAVSVLKALGLGGPQSGGGDGTDPAGGVPDTS ALESLLLEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIFA GHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLWR FAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDDPHAFHPDRPSWRRLTF GDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWSRKGAQTA RLTELPVWLR SEQ ID NO: 17: 5′-gtggccgtcgacccgttcgcgtgtcccatgatgaccatgcagcgcaagcccgaggtgcacgacgccttcc gggaggcgggcccggtcgtcgaggtgaacgcccccgcgggcggacccgcctgggtcatcaccgatgacgccctc gcccgcgaggtgctggccgatccccggttcgtgaaggaccccgacctcgcccccgccgcctggcggggggtggac gacggtctcgacatccccgttccggagctgcgtccgttcacgctcatcgccgtggacggcgaggcccaccggcgcct gcgccgcatccacgcacctgcgttcaacccgcgccggctggccgagcggacggatcgcatcgccgcgatcgccgg ccggctgctcaccgaactcgccgacgcctccggccggtcgggcaaaccggccgagctgatcggcggcttcgcgtac cacttcccgctgttggtcatctgcgagctgctcggtgtgccggtcaccgatccggcgatggcccgcgaggccgtcagc gttctcaaggcactcggcctcggcggcccgcagagcggcgggggtgacggcacggaccctgccgggggcgtgcc ggacacctcggccctggagagcctgctcctcgaagccgtgcactcagcccggcggaacgacaccccgaccatgacc cgcgtgctgtacgagcgcgcgcaggccgagttcggctcggtctccgacgaccagctcgtctacatgatcaccgggct catcttcgccggccacgacaccaccggctccttcctgggcttcctgctcgcggaggtcctggcgggccgcctcgcgg cggatgccgacgaggacgccgtctcccggttcgtggaggaggcgctgcgctaccacccgccggtgccctacacgtt gtggaggttcgctgccacggaggtgaccatcggcggcgtccggctgccccgcggagcgccggtgctggtggacatc gagggcaccaacaccgacggccgccatcacgacgccccgcacgccttccacccggaccgtccctcgtggcggcgg ctcaccttcggcgacgggccgcactactgcatcggggagcagctcgcccagctggagtcgcgcacgatgatcggcg tactgcgcagcaggttccccgaggcccgactggccgtgccgtacgacgagttgcggtggtgccggaagggggccca gacggcgcggctcaccgaactgcccgtctggctgcgctga-3′ SEQ ID NO: 18: VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALA REVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLR RIHAPAFNPRRLAERTDRIAAIAGRLLTELADASGRSGKPAELIGGFAYHFP LLVICELLGVPVTDPAMAREAVSVLKALGLGGPQSGGGDGTDPAGGVPDT SALESLLLEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIF AGHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLW RFAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDAPHAFHPDRPSWRRLT FGDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWCRKGAQT ARLTELPVWLR