KR20090092481A - Biosynthetic method for preparation of antitumor agent epirubicin, novel glycosylated anthracycline derivatives and their preparation methods - Google Patents

Abstract

The present invention is a novel biological system for the production of a semisynthetic improved anthracycline-based anticancer drug epirubicin (epirubicin) while showing a strong anti-cancer activity compared to the anthracycline-based anticancer drug doxorubicin (doxorubicin) A method for producing anthracycline glycosubstituted substance and a novel anthracycline glycosubstituted product produced according to the preparation method thereof. Nucleotide activated sugars biosynthesis protein, glycosyl transferase (glycosyltransferase) - adjuvant protein, for Streptomyces MRS Venezuela expressing the protein acting on the transition to the epirubicin in glycosylation epsilon also Mai during non (glycosylate ε-rhodomycinone) (Streptomyces venezuelae) mutants are prepared and incubated with epsilon rhodomycinone to provide a method for producing epirubicin, including its biosynthetic intermediates and derivatives thereof.

In addition, a novel glycosylated anthracycline is prepared by preparing a Streptomyces S. venezuelae mutant that expresses biosynthetic genes of various nucleotide-activated sugars and their glycosyltransferase-assisted proteins and incubating them with epsilon rhodomycinone. By providing a method and a novel glycosylated anthracycline produced through this, there is an excellent effect of synthesizing an anticancer drug candidate that is more effective than an existing anticancer agent.

Description

Biosynthetic method for preparation of antitumor agent epirubicin, novel glycosylated anthracycline derivatives and their preparation methods

The present invention relates to a biosynthetic method of a semisynthetic improved anthracycline-based anticancer drug epirubicin (Epirubicin) showing a strong anti-cancer action and a method of producing a novel glycosubstituted body. For the synthesis of nucleotide-activated sugars, glycosyltransferase-assisted proteins, proteins that act on the conversion of glycosyllate epsilon rho-mycinone to epirubicin In Streptomyces MRS Venezuela (Streptomyces venezuelae ) mutants and methods for producing epirubicin and epirubicin intermediates by incubating with epsilon rhodomycinone and biosynthetic genes of various nucleotide-activating sugars and their glycosyltransferases (glycosyltransferase) - way to prepare a mutant (S. venezuelae) in Streptomyces MRS Venezuela for the synthesis of the adjuvant protein to produce a novel glycosylated anthracycline derivative by incubation with paddy when my also epsilon and, prepared in this New glycosylated anthracycline derivatives.

Anthracycline-based anticancer substances have a structural feature of having one or more deoxy sugars in an aglycon consisting of four rings. Representative anthracycline anticancer drugs, daunorubicin and doxorubicin, have been used as traditional anticancer chemotherapy agents since their discovery in the 1960s (Hutchinson CR (1997) Chem . Rev. 97: 2525-2535). Natural substances with biological activity have one or more deoxy sugars. In particular, deoxy sugars play a very important role in biological activity, such as anthracycline-based anticancer substances directly act to recognize DNA, a target molecule in the body (Chen KX et al. (1985) Nucleic acids Res ). 14: 2251-2267).

In Streptomyces MRS Venezuela (Streptomyces venezuelae) strain is a recent combination biosynthesis host (as a host), and other species of Streptomyces MRS (Streptomyces) in strains derived from macrolide (macrolide) has been developed as a heterologous expression strain of the family of antibiotics (Yoon YJ et al. (2002 ) Chem Biol 9: 203-214; Hong JSJ et al (2004) FEMS Microbiol Lett 238:...... 291-399; Jung WS et al (2006) Appl Microbiol Biotechnol 72:.. 763-769; yunyeojun, Kwon Seok, Han-Bae "New Olivosyl Tilactone and Its Manufacturing Method" Korean Patent Application No. 10-2007-0063678 (2007.06.29); Method for Producing Mycin A2 and Its Precursor and Recombinant Strain "Patent Application No. 10-2007-68464 (2007.07.09); Kim, Jong-Kyu, Jeon-Gyu Yoon, Debbie Basnet, Park Je-won, Jung Seok, Park Sung-Ryol Combination biosynthesis method of the precursor 5-O-desosaminyl ery noride A and Development of Recombinant Strain "Patent Application No. 10-2007-94101 (September 17, 2007); Yeo, Jun-Joon, Jung-Suk Jung, Soon-Jung Jung, Sung-Ryeol Park, Je-Won Park" Polyketides from Streptomyces venezuelae using expression vectors of pikD regulatory genes ( How to increase the productivity of polyketide) "Korean Patent Application No. 10-2007-0107787 (2007.10.25). In addition, due to the ease of genetic manipulation, the rapid growth rate of S. venezuelae strains compared to other Streptomyces species allows for a short period (3-4 days) of culture in the production of metabolites. (Xue Y. et al. (2001) Metab . Eng . 3: 15-26; Jung WS et al. (2006) Appl . Microbiol Biotechnol 72: 763-769).

In view of the above, the present inventors have considered that the synthesis of nucleotide-activated sugars, glycosyltransferase-adjuvant proteins, and proteins that act on the conversion to epirubicin after glycosylation of epsilon rhodomycinone. S. venezuelae mutants can be prepared and incubated with epsilon rhodomycinone to produce intermediates of epirubicin and epirubicin and through this method the production of novel glycosylated anthracyclines It confirmed and completed this invention.

The present invention provides a method for producing epirubicin using a biological system for overcoming disadvantages such as expensive starting materials of the conventional epirubicin semisynthetic production process, low yield in the stage of separation and purification, and high cost burden during separation and purification. Provide together. For the synthesis of nucleotide-activated sugars, glycosyltransferase-assisted proteins, proteins that act on the conversion of glycosylated epsilon rhodomycinone to epirubicin Streptomyces S. venezuelae mutants were prepared and incubated with epsilon rhodomycinone to prepare intermediates of epirubicin and epirubicin, as well as biosynthetic genes of various nucleotide-activated sugars and their glycosyltransferases A novel glycosylated anthracycline is prepared by preparing a Streptomyces venezuelae mutant for synthesis of ancillary proteins and incubating with epsilon rhodomycinone.

Epirodomycin D represented by Structural Formula 1 of the present invention can be biosynthesized through the following four routes.

Structural Formula 1

First, the full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC ( GeneBank sequence number L76359) in the aknT (S. venezuelae) YJ183 a Streptomyces MRS Venezuela inserted -aknS (GeneBank sequence number AF264025), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank Transformation with a recombinant vector (pYJ371) having a cleavage map as shown in FIG. 13 below, comprising SEQ ID NO: U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) The microorganism obtained by culturing with epsilon rhodomycinone is incubated.

Second, the full-length biosynthetic gene cluster PKS and desamine (des) biosynthetic enzymes encoding pikromycin (Pik) were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC ( GeneBank sequence number L76359) in the stfPII (S. venezuelae) YJ183 a Streptomyces MRS Venezuela -stfG insert (SEQ ID NO: GeneBank AM156932), desIII-desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank Transformation with a recombinant vector (pYJ630) having a cleavage map as shown in FIG. 18 including SEQ ID NO U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) The microorganism obtained by culturing with epsilon rhodomycinone is incubated.

Third, the full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC ( S. venezuelae inserting GeneBank SEQ ID NO: L76359) YJ183 shows snogE ( GeneBank SEQ ID NO: AF187532) , snogN ( GeneBank SEQ ID NO: AF187532), desIII -desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891), dnmZ - dnmU (GeneBank sequence) No. AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237), including the microorganism obtained by transforming with a recombinant vector (pYJ631) having a cleavage map shown below 19 and incubated with epsilon rhodomycinone will be.

Fourth, the full-length biosynthetic gene cluster PKS encoding pikromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC ( GeneBank sequence number L76359) in the aknT (S. venezuelae) YJ183 a Streptomyces MRS Venezuela inserted -aknS (GeneBank sequence number AF264025), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank Transformation with a recombinant vector (pYJ627) having a cleavage map as shown in FIG. 20 including SEQ ID NO: U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), evaE (GeneBank SEQ ID NO: AJ223998), dnmJ (GeneBank SEQ ID NO: M80237) The microorganism obtained by culturing with epsilon rhodomycinone is incubated.

Epi-rhodomycin D to be obtained in the present invention is a culture of the transformed microorganisms YJ183 / pYJ371, YJ183 / pYJ630, YJ183 / pYJ631, YJ183 / pYJ627 and epsilon rhodomycinone in the same amount of methanol Extracted with a mixed solvent, concentrated to methanol and purified.

Epirubicin represented by Structural Formula 2 of the present invention can be biosynthesized through the following route.

Structural Formula 2

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS (GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891) into S. venezuelae YJ183. ), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237), dnrV -doxA (GeneBank SEQ ID NO: U77891), dnrK - dnrP (GeneBank SEQ ID NO: L40425) The microorganism obtained by transformation with a recombinant vector (pYJ546) having a cleavage map as shown in FIG. 14 is incubated with epsilon rhodomycinone.

Epirubicin to be obtained in the present invention is purified by extracting the culture of the transformed microorganisms YJ183 / pYJ546 and epsilon rhodomycinone with the same mixed solvent of methanol and water and concentrated with methanol.

The novel 7- O -D-olibosyl epsilon rhodomycinone (7- O- D-olivosyl ε-rhodomycinone) represented by the following Structural Formula 3 of the present invention can be biosynthesized through the following route.

Structural Formula 3

Depletion of full-length biosynthetic gene cluster PKS and desamine, des biosynthetic enzymes encoding pikromycin (Pik) followed by doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC ( GeneBank sequence number L76359) a streptokinase in MRS Venezuela (S. venezuelae) aknT-aknS ( GeneBank No. AF264025 sequence to YJ183), desIII -desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), oleV (GeneBank insert A microorganism obtained by transforming with a recombinant vector (pYJ613) having a cleavage map as shown in FIG. 15 including oleW (GeneBank SEQ ID NO: AF055579) and urdR (GeneBank SEQ ID NO: AF269227) together with epsilon rhodomycinone It is to culture.

The 7- O -D-olibosyl epsilon rhodomycinone to be obtained in the present invention is isolated from the culture of the transformed microorganisms YJ183 / pYJ613 and epsilon rhodomycinone extracted with the same mixed solvent of methanol and water and concentrated with methanol. Purify.

The novel 7- O -L-3'-N-methyl daunosaminyl epsilon rhodomycinone (7- O- L-3'-N-methyl daunosaminyl ε-rhodomycinone) represented by the following structural formula 4 of the present invention is Biosynthesis is possible through the following route.

Structural Formula 4

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: aknT -aknS (GeneBank sequence number AF264025) to (S. venezuelae) YJ183 a Streptomyces MRS Venezuela insert L76359), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank U77891 SEQ ID NO: ), dnmZ - dnmU , dnmV (GeneBank SEQ ID NO: AF006633), dnmJ (GeneBank SEQ ID NO: M80237) aknX2 (GeneBank SEQ ID NO: AF264025) comprising a transformation vector having a cleavage map as shown in Figure 16 (pYJ656) obtained The microorganism is incubated with epsilon rhodomycinone.

7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone to be obtained in the present invention is a culture of the transformed microorganisms YJ183 / pYJ656 and epsilon rhodomycinone as the same mixed solvent of methanol and water Extract, concentrate with methanol to separate and purify.

The novel 7- O -epi-L-vancosaminyl epsilon rhodomycinone (7- O- epi-L-vancosaminyl ε-rhodomycinone) represented by the following structural formula 5 of the present invention can be biosynthesized through the following route. .

Structural Formula 5

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS ( GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891), evaA (GeneBank SEQ ID NO: AJ223998), inserted into S. venezuelae YJ183. ) , evaB (GeneBank SEQ ID NO: AJ223998) , evaC (GeneBank SEQ ID NO: AJ223998) , evaD (GeneBank SEQ ID NO: AJ223998) , and evaE (GeneBank SEQ ID NO: AJ223998). The microorganism obtained by transformation is incubated with epsilon rhodomycinone.

In the present invention, 7- O -epi-L-vancosaminyl epsilon rhodomycinone is extracted from the culture of the transformed microorganisms YJ183 / pYJ714 and epsilon rhodomycinone with the same mixed solvent of methanol and water, and methanol. Concentrate with to separate and purify.

As described above through the problem solving means, for the synthesis of nucleotide-activated sugars, glycosyltransferase-adjuvant protein, protein acting on the conversion of glycosylated epsilon rhodomycinone to epirubicin By providing a novel glycosylation anthracycline by a method and a way to produce a epirubicin and epi ruby God intermediate by preparing a streptokinase mutant (S. venezuelae) in MRS Venezuela cultured with paddy when my also epsilon Compared to the conventional anti-synthetic method for producing epirubicin, it is easier to separate and purify by-products and to synthesize an effective anticancer substance having higher anticancer activity and lower side effects than conventional anticancer agents.

1 shows Rhodomycin D, Rhodosaminyl aklavinone, Rhamnosyl 8-demethoxy-10-deoxysteffimycinone and Nogalosyl 1-Hyde It shows the biosynthetic pathway of nogalosyl 1-OH-nogalamycinone. a is a reaction scheme of a derivative in which DnmS / DnmQ connects TDP-L-daunosamine to epsilon rhodomycinone, and b is a compound in which AknS / AknT connects TDP-L-rhodosamine to aclabinone. The reaction scheme of the derivative, c is the reaction scheme of the derivative wherein StfG / StfPII linked TDP-L-rhamnose to 8-dimethoxy 10-dioxysteppymycinone, d is TDP-L-nogal The reaction scheme of the derivative linking nogalose to 1-hydroxyl nogalamycinone is shown.

Figure 2 shows the biosynthetic pathways (a) of TDP-epi-L-danosamine, TDP-D-olivose, TDP-L-3'-N-methyl daunosamine and TDP-epi-L-vancosamine Epilodomycin D, epirubicin, 7- O -D-olibosyl epsilon rhodomycinone, 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone, and 7- O -epi The structure (b) of -L-vancosaminyl epsilon rhodomycinone is shown.

Figure 3 is a result of LC / MS analysis of epilodomycin D produced by glycosyltransferase AknS, 4-keto-reductase AvrE.

Figure 4 shows the results of MS / MS analysis of epilodomycin D produced by glycosyltransferase AknS, 4-keto-reductase AvrE.

FIG. 5 shows LC / MS analysis of epirubicin produced by glycosyltransferase AknS, 4-ketoreductase AvrE, epilodomycin D to epirubicin, proteins DnrP, DnrK, DnrV, and DoxA. Results are also shown.

6 shows MS / MS analysis of epirubicin produced by proteins DnrP, DnrK, DnrV, DoxA, which act on glycosyltransferase AknS, 4-ketoreductase AvrE, epilodomycin D to epirubicin conversion. Results and MS / MS analysis of epirubicin standard.

Fig. 7 shows the results of LC / MS analysis of 7- O -D-olibosyl epsilon rhodomycinone.

Fig. 8 shows the results of MS / MS analysis of 7- O -D-olibosyl epsilon rhodomycinone.

Fig. 9 shows the results of LC / MS analysis of 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone.

Fig. 10 shows the results of MS / MS analysis of 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone.

Fig. 11 shows the results of LC / MS analysis of 7- O -epi-L-vancosaminyl rhodomycinone.

Fig. 12 is a diagram showing the results of MS / MS analysis of 7- O -epi-L-vancosaminyl rhodomycinone.

Fig. 13 shows a cleavage map of pYJ371.

14 shows a cleavage map of pYJ546.

15 shows a cleavage map of pYJ613.

16 shows a cleavage map of pYJ656.

17 shows a cleavage map of pYJ714.

18 shows a cleavage map of pYJ630.

19 shows a cleavage map of pYJ631.

20 shows a cleavage map of pYJ627.

The present invention is epi-rhodomycin D (epi-rhodomycin D), epirubicin, 7- O -D-olibosyl epsilon rhodomycinone represented by the following structural formula 1, formula 2, formula 3, formula 4 and formula 5 ( 7- O- D-olivosyl ε-rhodomycinone), 7- O -L-3'-N-methyl daunosaminyl epsilon rhodomycinone (7- O- L-3'-N-methyl daunosaminyl ε-rhodomycinone) , 7- O - epi -L- Banko Sami carbonyl epsilon also when non-Mai (7- O -epi-L-vancosaminyl ε-rhodomycinone) , and provides a preparation method thereof:

Structural Formula 1

Structural Formula 2

Structural Formula 3

Structural Formula 4

Structural Formula 5

Epidomycin D of the present invention can be biosynthesized through the following four pathways.

1) First, the full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank sequence number L76359) in which the Streptomyces MRS Venezuela insert (S. venezuelae) in YJ183 aknT-aknS (GeneBank sequence number AF264025), desIII -desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank SEQ ID NO: U77891), dnmZ-dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) with a recombinant vector (pYJ371) having a cleavage map as shown in FIG. The microorganism obtained by transformation is incubated with epsilon rhodomycinone.

2) second, the full-length biosynthetic gene cluster PKS encoding pikromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and StfPII -stfG ( GeneBank SEQ ID NO: AM156932), desIII -desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT , inserting drrC (GeneBank SEQ ID NO: L76359) into Streptomyces Venezuela ( S. venezuelae ) YJ183 (GeneBank SEQ ID NO: U77891), dnmZ-dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) with a recombinant vector (pYJ630) having a cleavage map as shown in FIG. The microorganism obtained by transformation is incubated with epsilon rhodomycinone.

3) Third, the full-length biosynthetic gene cluster PKS and desamine, des biosynthetic enzymes coding for pykromycin (Pik) were deleted, and then the doxorubicin resistance gene drrA-drrB (GeneBank SEQ ID NO: M73758) and S. venezuelae inserting drrC (GeneBank SEQ ID NO: L76359) YJ183 shows snogE ( GeneBank SEQ ID NO: AF187532) , snogN ( GeneBank SEQ ID NO: AF187532), desIII -desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891), dnmZ - dnmU (GeneBank sequence) No. AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) comprising a microorganism obtained by transforming with a recombinant vector (pYJ631) having a cleavage map as shown in Figure 19 incubated with epsilon rhodomycinone It is.

4) fourth, the full-length biosynthetic gene cluster PKS encoding pikromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank sequence number L76359) in which the Streptomyces MRS Venezuela insert (S. venezuelae) in YJ183 aknT-aknS (GeneBank sequence number AF264025), desIII -desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank SEQ ID NO: U77891), dnmZ-dnmU (GeneBank SEQ ID NO: AF006633), evaE (GeneBank SEQ ID NO: AJ223998), dnmJ (GeneBank SEQ ID NO: M80237), including a recombinant vector having a cleavage map as shown in FIG. 20 (pYJ627) The microorganism obtained by transformation is incubated with epsilon rhodomycinone.

Epiromycin D to be obtained in the present invention is a culture of the transformed microorganisms YJ183 / pYJ371, YJ183 / pYJ630, YJ183 / pYJ631, YJ183 / pYJ627 and epsilon rhodomycinone extracted with the same amount of mixed solvent of methanol and methanol It can be separated and purified by concentration.

In the present invention, S. venezuelae for the synthesis of 4-keto-reductase required for the biosynthesis of nucleotide-activated sugar, epi-L-daunosamine ( S. venezuelae ) Provided are genetically engineered mutants of the expression enzyme, thereby providing a method for preparing epirodomycin D upon injection of an exogenous source of anthracycline and epirodomycin D prepared by the above method.

Streptomyces Epirodomycin D can be produced by culturing a mutant YJ183 / pYJ371 expressing AvrE 4-keto-reductase from avermitilis) in a growth medium containing epsilon rhodomycinone. Epi daunosamine has already been synthesized using AvrE 4-keto-reductase (Madduri K. et al. (1997) Nat. Biotechnol . 16: 69-74).

Ahmiko rapto cis Oriental less (Amycolaptosis mutant YJ183 / pYJ627, expressing one of the epi-L-vancosamine biosynthesis genes derived from orientalis ) , can be produced by culturing YJ183 / pYJ627 in a growth medium containing epsilon rhodomycinone. have. The above results, which can produce epi-L- vanosamine as well as epi-L-Danosamine, confirm the substrate flexibility of EvaE and select useful proteins for biosynthesis of Epi-L-Danosamine. .

The present invention provides genetically engineered mutants of Streptomyces S. venezuelae expressing enzymes for the synthesis of glycosyltransferase-assisted protein pairs as well as nucleotide activated sugars, thereby exogenous of anthracyclines When the source) is injected, the present invention provides a method for preparing Epirodomycin D and Epirodomycin D prepared by the above method.

In the present invention, Streptomyces galileae ( Streptomyces galilaeus) confirms the flexibility of the deoxy sugar (deoxy sugar AknS glycosyl transferase of the origin) and aglycone (aglycon). Epsilon rhodomycinone was added to the mutant YJ183 / pYJ371 (TDP-Epi-L-Danosamine) expressing the AknS / AknT glycosyltransferase-assisted protein pair to produce epilodomycin D. Based on previous research, AknS has been shown to be the native substrate of TDP-L-rhodosamine, TDP-L-daunosamine, and TDP-2-deoxy-L-fucose (2-deoxy-L). -fucose) (Leimkuhler C. et al. (2007) J. Am. Chem . Soc . 129: 10546-10550). The results of the present invention confirmed that AknS can also accept TDP-epi-L-danosamine.

In the present invention, the flexibility of the deoxy sugar and aglycon of StfG glycosyltransferase derived from Streptomyces steffisburgensis is confirmed. Epsilon rhodomycinone was added to the mutant YJ183 / pYJ630 (TDP-Epi-L-Danosamine) expressing the StfG / StfPII glycosyltransferase-assisted protein pair to produce epilodomycin D. Based on previous research, StfG is a natural substrate of NDP-L-rhamnose, NDP-L-olivose, NDP-L-digitoxose and NDP-L-Olandose. oleandrose, NDP-L-mycarose, NDP-L-amicetose, NDP-D-boivinose, NDP-D-olivose, NDP-D-Digi It is known to attach digitoxose (Olano C. et al. (2008) Chem . Bio. Chem . 9: 1-11). The results of the present invention confirmed that StfG can accept TDP-epi-L-danosamine in addition to the existing known deoxy sugar (deoxy sugar).

In the present invention, Streptomyces MRS furnace divided emitter (Streptomyces nogalater ) SongE glycosyltransferase from the deoxy sugar (aglycon) and the deg (sugar) is confirmed. Epsilon rhodomycinone was added to the mutant YJ183 / pYJ631 (TDP-epi-L-daunosamine) expressing a SnogE / SnogN glycosyltransferase-assisted protein pair to produce epilodomycin D. Previous studies have predicted that SnogE can attach NDP-L-nogalose, the original substrate (Torkkell S. et al. (2001) Mol . Genet. Genomics 266: 276-288). The results of the present invention confirmed that SnogE can accept TDP-epi-L-danosamine as a substrate.

In the present invention, epirubicin can be biosynthesized through the following pathway.

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS (GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891) into S. venezuelae YJ183. ), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237), dnrV -doxA (GeneBank SEQ ID NO: U77891), dnrK - dnrP (GeneBank SEQ ID NO: L40425) The microorganism obtained by transformation with a recombinant vector (pYJ546) having a cleavage map as shown in FIG. 14 is incubated with epsilon rhodomycinone.

Epirubicin to be obtained in the present invention can be separated and purified by extracting the culture of the transformed microorganisms YJ183 / pYJ546 and epsilon rhodomycinone with the same mixed solvent of methanol and water and concentrated with methanol.

The 7- O -D-olibosyl epsilon rhodomycinone of the present invention can be biosynthesized through the following route.

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) into S. venezuelae YJ183 has aknT- aknS (GeneBank SEQ ID NO: AF264025), desIII-desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891), oleV (GeneBank SEQ ID NO: AF055579) , oleW (GeneBank SEQ ID NO: AF055579), urdR (GeneBank) A microorganism obtained by transformation with a recombinant vector (pYJ613) having a cleavage map as shown in FIG. 15 including No. AF269227) is incubated with epsilon rhodomycinone.

The 7- O -D-olibosyl epsilon rhodomycinone to be obtained in the present invention is isolated by extracting a culture of the transformed microorganisms YJ183 / pYJ613 and epsilon rhodomycinone with the same mixed solvent of methanol and water and concentrating with methanol. It can be purified.

7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone of the present invention can be biosynthesized through the following route.

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: aknT -aknS (GeneBank sequence number AF264025) to (S. venezuelae) YJ183 a Streptomyces MRS Venezuela insert L76359), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank U77891 SEQ ID NO: ), dnmZ - dnmU , dnmV (GeneBank SEQ ID NO: AF006633), dnmJ (GeneBank SEQ ID NO: M80237) aknX2 (GeneBank SEQ ID NO: AF264025) comprising a transformation vector having a cleavage map as shown in Figure 16 (pYJ656) obtained The microorganism is incubated with epsilon rhodomycinone.

7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone to be obtained in the present invention is a culture of the transformed microorganisms YJ183 / pYJ656 and epsilon rhodomycinone as the same mixed solvent of methanol and water It can be separated and purified by extraction and concentration with methanol.

The 7- O -epi-L-vancosaminyl epsilon rhodomycinone of the present invention can be biosynthesized through the following route.

The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS ( GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891), evaA (GeneBank SEQ ID NO: AJ223998), inserted into S. venezuelae YJ183. ) , evaB (GeneBank SEQ ID NO: AJ223998) , evaC (GeneBank SEQ ID NO: AJ223998) , evaD (GeneBank SEQ ID NO: AJ223998) , and evaE (GeneBank SEQ ID NO: AJ223998). The microorganism obtained by transformation is incubated with epsilon rhodomycinone.

In the present invention, 7- O -epi-L-vancosaminyl epsilon rhodomycinone is extracted from the culture of the transformed microorganisms YJ183 / pYJ714 and epsilon rhodomycinone with the same mixed solvent of methanol and water and The concentration can be separated and purified.

The present invention confirms the flexibility of the AknS glycosyltransferase to the deoxy sugar. Epsilon rhodomycinone to YJ183 / pYJ613 (TDP-D-Olivos), YJ183 / pYJ656 (TDP-L-3-N-methyl-Danosamine), YJ183 / pYJ714 (TDP-Epi-L-Vancosamine) Each addition produced a new glycosylated anthracycline. These results indicate that AknS has broad substrate flexibility with respect to deoxy sugars.

One disadvantage of combinatorial biosynthesis techniques for the development of new antibiotics is the significantly lower yield of hybrid compounds (Tang L. et al. (2001) Chem . Biol . 8: 547-555; Yoon YJ et al. (2002) Chem . Biol . 9: 203-214). Epilodomycin D and YJ183 / pYJ613 (TDP-D-olibos), YJ183 / pYJ656 (TDP-L-) prepared from YJ183 / pYJ371 (TDP-Epi-L-Daunosamine) injected with epsilon rhodomycinone 3-N-methyl daunosamine), new anthracycline 7- O -D-olibosyl epsilon rhodomycinone, 7- O- L-, prepared from YJ183 / pYJ714 (TDP-epi-L-vancosamine). 3'-N-methyl daunosaminyl epsilon rhodomycinone, 7- O -epi-L-vancosaminyl epsilon rhodomycinone is deoxy sugar due to the catalytic specificity of AknS, StfG and SnogE. It was judged that this was introduced into oxygen at the C-7 position of epsilon rhodomycinone.

The newly obtained S. venezuelae mutant provides a simple system for the biosynthesis of glycosylated anthracyclines of various structures of aglycons. The present invention further examined the flexibility of glycosyltransferases for aglycon and deoxy sugars. In addition, (S. venezuelae) in Streptomyces MRS MRS in Venezuela other Streptomyces (Streptomyces genus ) has the advantage of growing faster than the strain, which is advantageous for the biosynthesis of novel glycosylated anthracyclines.

Hereinafter, an Example demonstrates this invention in detail. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1: Bacterial Lineage, Culture Conditions and Genetic Manipulation

In Streptomyces MRS Venezuela (Streptomyces venezuelae ) ATCC 15439 was used for mutant preparation. S. venezuelae mutants were cultured in R2YE medium containing the appropriate antibiotic, Thiostrepton (0.025 mg / mL). It was cultured in MRS Streptomyces ISP2 medium page dominant T mouse (Streptomyces peucetius) ATCC 29050, Uz (S, galilaeus) in Streptomyces MRS galril La ATCC 31615, Streptomyces Mrs. Bruno Gala emitter (S. nogalater) ATCC 27451, Streptomyces MRS stacking piece beoljen system (S. steffisburgensis) NRRL 3193, Mrs. Arbor streptomycin US Antilles (S. avermitilis) ATCC 31267, ahmiko rapto cis Oriental less (A. orientalis) NRRL 18098 was used for genomic DNA preparation. Genetic manipulations were performed in E. coli DH5α according to a recognized procedure (Sambrook J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3 ^rd Edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Litmus 28, New England Biolabs, T-easy vector (Promega) was used for subcloning.

Example  2: Epirubicin  Including New Glycosylation Anthracyclines  For manufacturing To doxorubicin  Manufacture of microorganisms resistant to

Pik PKS and des gene cluster-deleting mutant YJ028 (Jung WS et al. (2007) Appl . Microbiol . Biotechnol . 76 of Streptomyces venezuelae for the production of novel glycosylated anthracyclines, including epirubicin . : 1373-81) should confer self-resistance to doxorubicin to ensure stable production of glycosylated anthracyclines. To prepare the expression vector pSABC containing the drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) doxorubicin resistance genes from S. peucetius , the following method was performed. . drrA - drrB (GeneBank SEQ ID NO: M73758) genes were amplified from genomic DNA of Streptomyces Peustius by polymerase chain reaction (PCR) using primers of SEQ ID NOs: 1 and 2: 5 '-CCC AGATCT CCATGTGACTACTGGGGGCGTTAG-3' and 5'-GGG AAGCTT TCCGGTACCTGTGTCAGTGGGCGT-3 '(underlined parts are restricted). The drrC (GeneBank SEQ ID NO: L76359) gene was amplified from the genomic DNA of S. peucetius by Polymerase chain reaction (PCR) using the primers of SEQ ID NOs: 3 and 4. 5 '-CCC AAGCTT CCGTCAATGTGAGGATGCTCATGC-3' and 5'-GGG TCTAGA GGGTTAATTAACTTAGCACCCGGGGTCAAGGATCA-3 '(underlined parts are restricted). The PCR product of drrA-drrB was cut into Bgl II and Hind III and cloned into the same site of Litmus28 . The PCR product of drrC (GeneBank SEQ ID NO: L76359) was cut into Hind III and Xba I and cloned into the same site of Litmus28 cloned with drrA - drrB (GeneBank SEQ ID NO: M73758). DrrA- drrB (GeneBank SEQ ID NO: M73758) Litmus28 cloned with drrC (GeneBank SEQ ID NO: L76359) was cloned into pSET152, which was cut with Bgl II and Xba I and with BamH I and Xba I. PSABC prepared as above was integrated into the chromosomal DNA of mutant YJ028 to produce YJ183. Mutant YJ183 prepared as described above grows very well when cultured in SGGP of 15 µg / ml doxorubicin, whereas mutant YJ028 without pSABC does not grow when cultured in SGGP of 15 µg / ml doxorubicin. It was confirmed. It was confirmed that the doxorubicin resistance genes drrA -drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) integrated in the chromosomal DNA of YJ183 were performing the resistance function.

Example  3: Preparation of Expression Plasmids and Mutants

A basic expression vector for biosynthesis of TDP-epi-L-danosamine and transformation by glycosyltransferase was prepared as follows. dnmQ -dnmS (SEQ ID NO: GeneBank L47164) gene was amplified from the genomic DNA of Streptomyces MRS page dominant T-house (S. peucetius) by PCR using the primers of SEQ ID NO: 5 and 6: 5'- AGATCTTTAATTAAGATATC ACAGTGTGAGGAGCACGA-3 'and 5'- TCTAGACATATGGCTACG ACAAGACAGCGA-3 '(underlined parts are restricted). desIII -desIV (GeneBank SEQ ID NO: AF079762) Gene sequence was amplified from the genomic DNA of (S.venezuelae) in Streptomyces MRS Venezuela by PCR using primers 7 and 8: 5'- TTAATTAAACTAGT TAACTCGCCACGCCGACCGTT-3 'and 5'- TCTAGA GAGCTCCTCGTAGGCGGCCTT-3' (the underlined Part is a restricted part). dnmW (GeneBank sequence number U77891) gene was amplified from the genomic DNA of Streptomyces MRS page dominant T-house (S. peucetius) by PCR using the primers of SEQ ID NO: 9 and 10: 5'- TTAATTAAACTAGTATCGAT CTCGCAGCCTCTCGTTGACGAAGA-3 'and 5' TCTAGA GTCAGTCCAGCCCGTCCCGGGTGA-3 '(underlined parts are restricted). dnmT (GeneBank sequence number U77891) gene was amplified from the genomic DNA of Streptomyces MRS page dominant T-house (S. peucetius) by PCR using the primers of SEQ ID NO: 11 and 12: 5'- TTAATTAAACTAGTGCTAGC TCAAACACCGCACCGTCACCCGGG-3 'and 5' TCTAGA AACAGGACGCGCACGGGGAACTCA-3 '(underlined parts are restricted). The dnmZ- dnmU (GeneBank SEQ ID NO: AF006633) gene was amplified from genomic DNA of Streptomyces Peustius by PCR using the primers of SEQ ID NOs: 13 and 14: 5'- TTAATTAAACTAGT CTCCGATCCTTACGAGGAGTC-3 'and 5'- TCTAGA ATGTCCGCCTCACCCGTCTCC-3 '(underlined parts are restricted). avrE (GeneBank sequence number AB032523) gene SEQ ID NO: 15, and by PCR using the primers of 16 was amplified from the genomic DNA of Streptomyces Mrs. Arbor US subtilis (S. avermitilis): 5'- TTAATTAAACTAGTTACGTA GCACCAGGAATTCGAGCGCCGCTA -3 ' and 5' TCTAGACCTAGG TCGACGAGAAGCTGTGACGGCCGA-3 '(underlined parts are restricted). The dnmJ (Genebank SEQ ID NO: M80237) gene was amplified from genomic DNA of Streptomyces Peustius by PCR using the primers of SEQ ID NOs: 17 and 18: 5'- TTAATTAAACTAGT CCGACAAGGAGTCACGTGTCC-3 'and 5'. TCTAGAGAATTC CCGGCCTAGCTCACAGGCTTC-3 '(underlined parts are restricted). PCR products of dnmJ (Genebank SEQ ID NO: M80237) were cut into Pac I and Xba I and cloned into the same site of Litmus28. The PCR product of avrE (GeneBank SEQ ID NO: AB032523) was cut to Pac I and Xba I so that Litmus28 cloned with dnmJ (Genebank SEQ ID NO: M80237) was cloned to the site cut to Pac I and Spe I. DnmZ-dnmU (GeneBank SEQ ID NO: AF006633) , dnmT (GeneBank SEQ ID NO: U77891) , dnmW (GeneBank SEQ ID NO: U77891) , desIII - desIV (GeneBank SEQ ID NO: AF079762) , dnmQ-dnmS (GeneBank SEQ ID NO: L47164) Are cloned in turn. Consequently dnmQ (GeneBank sequence number L47164), dnmS (GeneBank sequence number L47164), desIII (GeneBank sequence number AF079762), desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank sequence number U77891), pYJ351 is generated in which dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , avrE (GeneBank SEQ ID NO: AB032523) , and dnmJ (Genebank SEQ ID NO: M80237) are included in Litmus28. First, the biosynthesis of TDP-epi-L-danosamine and transformation by AknS / AknT were made as follows. The aknT - aknS ( GeneBank SEQ ID NO: AF264025) gene was amplified from genomic DNA of Streptomyces S. galilaeus by PCR using primers of SEQ ID NOs: 19 and 20: 5'- GATATC CACGTCCCGAAGGAGACTGCCGTG- 3 'and 5'- AAGCTT TCACGCGGAGGTGGTCAGGGGGAA-3' (underlined portions are restricted). The PCR product of aknT - aknS ( GenenBank SEQ ID NO: AF264025) was cut with EcoR V and Nde I and cloned into the same site of pYJ351 to make pYJ355. aknT obtained cut pYJ355 with Pac I and Xba I (GeneBank sequence number AF264025), aknS (GeneBank sequence number AF264025), desIII (GeneBank sequence number AF079762), desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), Fragment containing dnmT (GeneBank SEQ ID NO: U77891) , dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , avrE (GeneBank SEQ ID NO: AB032523) , dnmJ (Gen eBank SEQ ID NO: M80237) Cloning to the same site to prepare pYJ371. In addition, an expression vector for biosynthesis of TDP-epi-L-daunosamine and transformation by StfG / StfPII was prepared as follows. The stfPII- stfG ( GeneBank SEQ ID NO: AM156932) gene was amplified from genomic DNA of Streptomyces staphyzalgenes ( S.steffisburg ensis) by PCR using the primers of SEQ ID NOs: 21 and 22: 5'- TTAATTAAGATAT CGCTCGACCGCCACGCTCTGTTCGA-3 ' And 5′- TCTAGACATATG CCCATGGATAGGTGCGGGGCTTAC-3 ′ (underlined portions are restriction portions). The PCR product of stfPII-stfG ( GenenBank SEQ ID NO: AM156932) was cut with EcoR V and Nde I and cloned into the same site of pYJ351 to make pYJ628. pYJ628 the stfG obtained cut with Pac I and Xba I (GeneBank SEQ AM156932), stfPII (GeneBank SEQ AM156932), desIII (GeneBank sequence number AF079762, desIV (GeneBank sequence number AF079762, dnmW (GeneBank sequence number U77891), dnmT ( A fragment containing GeneBank SEQ ID NO: U77891) , dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , avrE (GeneBank SEQ ID NO: AB032523) , dnmJ (GeneBank SEQ ID NO: M80237) was placed in the same site of pSE34. Cloning was carried out to prepare pYJ630.In addition, the plasmid was prepared as follows for biosynthesis of TDP-epi-L-daunosamine and transformation by SnogE / SnogN.songE (GeneBank SEQ ID NO: AF187532) Genes were amplified from genomic DNA of Streptomyces nogalater by PCR using primers SEQ ID NOs: 23 and 24: 5'- TTAATTAAACTAGT ACCACCGAGCGGACTTCCGGTGAC-3 'and 5'- TCTAGACATATG TCCGGGCCCTCGGTGTGTCACGCG-3' (underlined) Hits are restricted). The songN (GeneBank SEQ ID NO: AF187532) gene was amplified from genomic DNA of Streptomyces nogalater by PCR using the primers of SEQ ID NOs: 25 and 26: 5'- TTAATTAAGATATC CTTGACGTGGCCTGGAAGCTGGTT-3 'and 5'- TCTAGA GTCCATGGAGAGTCCCTTCCGTTG-3 '(underlined parts are restricted). PCR products of the snogE (GeneBank SEQ ID NO: AF187532) gene were cut into Pac I and Xba I and cloned into the same site of Litmus28. snogN (GeneBank sequence number AF187532) PCR product was Pac I, and cut off with Xba I songE (GeneBank sequence number AF187532) is cloned in the cut area with Pac I and Spe I in the embedded Litmus28 then cut with EcoR V and Nde I snogN of (GeneBank SEQ ID NO: AF187532) and A fragment containing snogE (GeneBank SEQ ID NO: AF187532) was cloned into the same site of pYJ351 to make pYJ629. snogN obtained cut pYJ629 with Pac I and Xba I (GeneBank sequence number AF187532), snogE (GeneBank sequence number AF187532), desIII (GeneBank sequence number AF079762), desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), The fragments containing dnmT (GeneBank SEQ ID NO: U77891) , dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , avrE (GeneBank SEQ ID NO: AB032523) , dnmJ (GeneBank SEQ ID NO: M80237) are the same as pSE34. Cloning to the site produced pYJ631. In addition, in the biosynthesis of TDP-epi-L-daunosamine, the plasmid was prepared as follows using EvaE as a 4-keto-reductase and transformation of TDP-epi-L-danosamine by AknS / AknT. It became. The evaE (GeneBank SEQ ID NO: AJ223998) gene was amplified from the genomic DNA of A. orientalis by PCR using the primers of SEQ ID NOs: 27 and 28: 5'-CGG TTAATTAAACTAGTTACGTA CTCGCACGCTTCTCGCGTGCATCA-3 'and 5' -CGG TCTAGACCTAGG ACACGCGTGGTCATGCGCGAGCCT-3 '(underlined parts are restricted). evaE (GeneBank sequence number AJ223998) PCR product of the gene is truncated in pYJ355 with SnaB I and Avr II is cloned in the same site to prepare a pYJ620. pYJ620 the aknS obtained cut with Pac I and Xba I (GeneBank sequence number AF264025), aknT (GeneBank sequence number AF264025), desIII (GeneBank sequence number AF079762), desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), The fragments containing dnmT (GeneBank SEQ ID NO: U77891) , dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , evaE (GeneBank SEQ ID NO: AJ223998) , dnmJ (GeneBank SEQ ID NO: M80237) are the same as pSE34. Cloning to the site produced pYJ627.

Biosynthesis of TDP-Epi-L-Daunosamine for the production of Epirubicin and the transformation of TDP-Epi-L-Danosamine by AknS / AknT, expression of proteins required for the conversion of Epirodomycin D to Epirubicin A plasmid pYJ546 was prepared. dnrV -doxA (GeneBank SEQ ID NO: U77891) The gene was amplified from genomic DNA of S. peucetius by PCR using the primers of SEQ ID NOs: 29 and 30: 5'- TTAATTAAACTAGT CGTGTGCCGATCCATCGAAA-3 'and 5'- TCTAGA ATCAGCGCAGCCAGACGGGCAGT-3' Underlined parts are restricted). dnrK -dnrP (GeneBank SEQ ID NO: L40425) The gene was amplified from genomic DNA of Streptomyces Peustius by PCR using primers of SEQ ID NOs: 31 and 32: 5'- TTAATTAAACTAGT CTACCTCGGCACCTACCGCCAGCT-3 'and 5'- TCTAGA TCACACTGTCGCGGCCCGGGTGTG-3' Underlined parts are restricted). The ermE * promoter was amplified from pSE34 containing the ermE * promoter by PCR using the primers of SEQ ID NOs: 33 and 34: 5'- TTAATTAAACTAGT GAGCTCGGTACCAGCCCG-3 'and 5'- TCTAGA TACCAACCGGCACGATTG-3' (underlined) Part is a restricted part). PCR product of dnrK -dnrP (GeneBank SEQ ID NO: L40425) gene was cut with Pac I and Xba I, cloned into the same site of Litmus28, and PCR product of dnrV - doxA (GeneBank SEQ ID NO: U77891) gene was cut with Pac I and Xba I Cloning into sites cut with Pac I and Spe I of Litmus28 containing dnrK (GeneBank SEQ ID NO: L40425) and dnrP (GeneBank SEQ ID NO: L40425) genes. This strategy further clones dnrV (GeneBank SEQ ID NO: U77891) , doxA (GeneBank SEQ ID NO: U77891), ermE * promoters. As a result, the ermE * promoter, dnrV (GeneBank SEQ ID NO: U77891) , doxA (GeneBank SEQ ID NO: U77891) , dnrV (GeneBank SEQ ID NO: U77891) , doxA (GeneBank SEQ ID NO: U77891) , dnrK (GeneBank SEQ ID NO: L40425) And Litmus28 containing the dnrP (GeneBank SEQ ID NO: L40425) gene was constructed. ErmE * promoter obtained by cutting this plasmid with Pac I and Xba I, dnrV (GeneBank SEQ ID NO: U77891) , doxA (GeneBank SEQ ID NO: U77891) , dnrV (GeneBank SEQ ID NO: U77891) , doxA (GeneBank SEQ ID NO: U77891) , dnrK (GeneBank) SEQ ID NO: L40425) And dnrP (GeneBank SEQ ID NO: L40425) The fragment is cloned into the same site of pSE34. Then Pac I and Spe aknT cut into I obtained cut pYJ355 with Pac I and Xba I (GeneBank sequence number AF264025), aknS (GeneBank sequence number AF264025), desIII (GeneBank sequence number AF079762), desIV (GeneBank sequence number AF079762) , dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891) , dnmZ (GeneBank SEQ ID NO: AF006633) , dnmU (GeneBank SEQ ID NO: AF006633) , avrE (GeneBank SEQ ID NO: AB032523) , dnmJ (GeneBank SEQ ID NO: M80237) Cloning with the prepared fragments yielded pYJ546.

Plasmids were prepared as follows for transformation by TDP-D-olivose and AknS / AknT. pYJ146 (Hong et al. (2004) FEMS . Microbiol . Lett . 238: 391-399) was prepared by oleV (GeneBank SEQ ID NO: AF055579) , oleW (GeneBank SEQ ID NO: AF055579) cut with Xba I and Hind III. And A fragment containing urdR (GeneBank SEQ ID NO: AF269227) was cloned into the same site of pSE34. pYJ355 was cut into Pac I and Nhe I so that aknT (GeneBank SEQ ID NO: AF264025) , aknS (GeneBank SEQ ID NO: AF264025) , desIII (GeneBank SEQ ID NO: AF079762) , desIV (GeneBank SEQ ID NO: AF079762) , dnmW (GeneBank SEQ ID NO: U77891) Fragments included were oleV (GeneBank SEQ ID NO: AF055579) , oleW (GeneBank SEQ ID NO: AF055579) And cloned into the sites cut with Pac I and Xba I of pSE34 containing urdR (GeneBank SEQ ID NO: AF269227) to make pYJ613.

A plasmid was prepared as follows for transformation by TDP-L-3'-N-methyl daunosamine and AknS / AknT. The aknX2 (GeneBank SEQ ID NO: AF264025) gene was amplified from genomic DNA of S. galilaeus by PCR using the primers of SEQ ID NOs: 35 and 36: 5'- TTAATTAAACTAGTCAATTG CGCCGACGGAGGGAACAGACATGT-3 'and 5' TCTAGA CTCAGCGCTGTTCGCGAACACCGA-3 '(underlined parts are restricted). The dnmV gene was amplified from genomic DNA of S. peucetius by PCR using the primers of SEQ ID NOs: 37 and 38: 5'- TTAATTAAACTAGTTACGTA TCACGCGGAGACGGGTGAGGCGGA-3 'and 5'- TCTAGACCTAGG TCGTCGGAAGCCTGTGAGCTAGGC-3' (The underlined portion is a restriction). dnmV (GeneBank No. AF006633 sequence) PCR product of the gene is cut off so as SnaB I and Avr II is pYJ346 was produced are cloned into the same sites of pYJ355. aknX2 (GeneBank No. AF264025 sequence) PCR product of the gene is cut off so as Mfe I and Xba I is cloned pYJ656 was produced in the cut area with EcoR I and Xba I of pYJ346.

Plasmids were prepared as follows for transformation by TDP-epi-L-vancosamine and AknS / AknT. The evaA (GeneBank SEQ ID NO: AJ223998) gene was amplified from genomic DNA of A. orientalis by PCR using primers of SEQ ID NOs: 39 and 40: 5'- TTAATTAAACTAGTGCTAGC CGCGGCTCGCCGGCTGAACACGAA-3 'and 5'- TCTAGA CTCATGCACCTCCCCGAGGCTGGG-3 '(underlined parts are restricted). The evaB (GeneBank SEQ ID NO: AJ223998) gene was amplified from genomic DNA of A. aorientalis by PCR using primers of SEQ ID NOs: 41 and 42: 5'- TTAATTAAACTAGT GACATCAGTTCATGGAAAGGC-3 'and 5'- TCTAGA GTTGTCAGAGGGTCGACAGCACTT-3 '(underlined parts are restricted). The evaC (GeneBank SEQ ID NO: AJ223998) gene was amplified from genomic DNA of A. aorientalis by PCR using primers of SEQ ID NOs: 43 and 44: 5'- TTAATTAAACTAGT TCCCCACAATTTCGTCGACGACAAG-3 'and 5'- TCTAGA TTTTGCGACATGGCGATCACGTCA-3 '(underlined parts are restricted). The evaD (GeneBank SEQ ID NO: AJ223998) gene was amplified from genomic DNA of A. aorientalis by PCR using primers of SEQ ID NOs: 45 and 46: 5'- TTAATTAAACTAGT GACCCTCTGACAACTCCCACTACA-3 'and 5'- TCTAGA TGTGGGCAAGCAGTCAGGTCGAGA-3 '(underlined parts are restricted). The PCR product of evaE (GeneBank SEQ ID NO: AJ223998) gene was cut into Pac I and Xba I and cloned into the same site of Litmus28. The PCR product of the evaD (GeneBank SEQ ID NO: AJ223998) gene was cut into Pac I and Xba I and cloned into the site where Litmus28 containing evaE (GeneBank SEQ ID NO: AJ223998) was cut into Pac I and Spe I. In this strategy, evaC (GeneBank SEQ ID NO: AJ223998) , evaB (GeneBank SEQ ID NO: AJ223998) , and evaA (GeneBank SEQ ID NO: AJ223998) were cloned in this order. As a result, the evaA hereafter (GeneBank sequence number AJ223998), evaB (GeneBank sequence number AJ223998), evaC (GeneBank sequence number AJ223998), evaD (GeneBank sequence number AJ223998), Litmus28 containing the evaE (GeneBank sequence number AJ223998) was Pac I And Xba I and cloned into the same site of pSE34. Pac I- Nhe I fragment with aknT (GeneBank SEQ ID NO: AF264025) , aknS (GeneBank SEQ ID NO: AF264025) , desIII (GeneBank SEQ ID NO: AF079762) , desIV (GeneBank SEQ ID NO: AF079762) , dnmW (GeneBank SEQ ID NO: U77891) of pYJ355 (fragment) is evaA (GeneBank sequence number AJ223998), evaB (GeneBank sequence number AJ223998), evaC (GeneBank sequence number AJ223998), evaD (GeneBank sequence number AJ223998), evaE (GeneBank sequence number AJ223998) of pSE34 contains a Pac I Cloned to / Spe I site produced pYJ714.

Table 1

Strain or plasmid Characteristic references Strain S. venezuelae ATCC 15439 Wild type YJ028 pikA and pikB ( des ) deletion mutant Jung WS et al. (2007) Appl. Microbiol . Biotechnol.76 : 1373-1381 YJ183 YJ028 / pSABC The present invention Plasmid pSET152 Shuttle vector for E. coli and Streptomyces Bierman M. et al. (1992) Gene 116: 43-49 pSE34 Shuttle vector for E. coli and Streptomyces Yoon YJ et al (2002) Chem . Biol . 9: 203-214 pSABC drrA , drrB , drrC The present invention pYJ146 desVII , desVIII , oleV , oleW, urdR Hong JSJ et al. (2004) FEMS . Microbiol . Lett . 238: 391-399 pYJ366 dnmS , dnmQ , desIII , desIV , dnmW, dnmT , dnmZ , dnmU , avrE, dnmJ The present invention pYJ371 aknS , aknT , desIII , desIV , dnmW, dnmT , dnmZ , dnmU , avrE, dnmJ The present invention pYJ630 stfG , stfPII , desIII , desIV, dnmW , dnmT , dnmZ , dnmU, avrE , dnmJ The present invention pYJ631 snogE , snogN , desIII , desIV, dnmW , dnmT , dnmZ , dnmU, avrE , dnmJ The present invention pYJ627 aknS , aknT , desIII , desIV , dnmW , dnmT , dnmZ , dnmU , evaE , dnmJ The present invention pYJ613 aknS , aknT , desIII , desIV , dnmW , oleV , oleW , urdR The present invention pYJ656 aknS , aknT , desIII , desIV , dnmW , dnmT , dnmZ , dnmU , dnmV , dnmJ , aknX2 The present invention pYJ714 aknS , aknT , desIII , desIV , dnmW , evaA , evaB , evaC , evaD, evaE The present invention

Table 2 Primer List

Gene Sequence (5'-3 ') Restriction site drrA - drrB forward CCC AGATCT CCATGTGACTACTGGGGGCGTTAG BglII reverse GGG AAGCTT TCCGGTACCTGTGTCAGTGGGCGT HindIII drrC forward CCC AAGCTT CCGTCAATGTGAGGATGCTCATGC HindIII reverse GGG TCTAGA GGGTTAATTAACTTAGCACCCGGGGTCAAGGATCA XbaI dnmQ - dnmS forward AGATCTTTAATTAAGATATC ACAGTGTGAGGAGCACGA BglII, PacI, EcoRV reverse TCTAGACATATGGCTACG ACAAGACAGCGA XbaI, NdeI desIII - desIV forward TTAATTAAACTAGT TAACTCGCCACGCCGACCGTT PacI, SpeI reverse TCTAGA GAGCTCCTCGTAGGCGGCCTT XbaI dnmW forward TTAATTAAACTAGTATCGAT CTCGCAGCCTCTCGTTGACGAAGA PacI, SpeI, ClaI reverse TCTAGA GTCAGTCCAGCCCGTCCCGGGTGA XbaI dnmT forward TTAATTAAACTAGTGCTAGC TCAAACACCGCACCGTCACCCGGG PacI, SpeI, NheI reverse TCTAGA AACAGGACGCGCACGGGGAACTCA XbaI dnmZ - dnmU forward TTAATTAAACTAGT CTCCGATCCTTACGAGGAGTC PacI, SpeI reverse TCTAGA ATGTCCGCCTCACCCGTCTCC XbaI avrE forward TTAATTAAACTAGTTACGTA GCACCAGGAATTCGAGCGCCGCTA PacI, SpeI, SnaBI reverse TCTAGACCTAGG TCGACGAGAAGCTGTGACGGCCGA XbaI, AvrII dnmJ forward TTAATTAAACTAGT CCGACAAGGAGTCACGTGTCC PacI, SpeI reverse TCTAGAGAATTC CCGGCCTAGCTCACAGGCTTC XbaI, EcoRI aknT - aknS forward GATATC CACGTCCCGAAGGAGACTGCCGTG EcoRV reverse AAGCTT TCACGCGGAGGTGGTCAGGGGGAA NdeI stfPII - stfG forward TTAATTAAGATAT CGCTCGACCGCCACGCTCTGTTCGA PacI, EcoRV reverse TCTAGACATATG CCCATGGATAGGTGCGGGGCTTAC XbaI, NdeI snogE forward TTAATTAAACTAGT ACCACCGAGCGGACTTCCGGTGAC PacI, SpeI reverse TCTAGACATATG TCCGGGCCCTCGGTGTGTCACGCG XbaI, NdeI snogN forward TTAATTAAGATATC CTTGACGTGGCCTGGAAGCTGGTT PacI, EcoRV reverse TCTAGA GTCCATGGAGAGTCCCTTCCGTTG XbaI evaE forward CGG TTAATTAAACTAGTTACGTA CTCGCACGCTTCTCGCGTGCATCA PacI, SpeI, SnaBI reverse CGG TCTAGACCTAGG ACACGCGTGGTCATGCGCGAGCCT XbaI, AvrII dnrV - doxA forward TTAATTAAACTAGT CGTGTGCCGATCCATCGAAA PacI, SpeI reverse TCTAGA ATCAGCGCAGCCAGACGGGCAGT XbaI dnrK - dnrP forward TTAATTAAACTAGT CTACCTCGGCACCTACCGCCAGCT PacI, SpeI reverse TCTAGA TCACACTGTCGCGGCCCGGGTGTG XbaI ermE * forward TTAATTAAACTAGT GAGCTCGGTACCAGCCCG PacI, SpeI reverse TCTAGA TACCAACCGGCACGATTG XbaI aknX2 forward TTAATTAAACTAGTCAATTG CGCCGACGGAGGGAACAGACATGT PacI, SpeI, MfeI reverse TCTAGA CTCAGCGCTGTTCGCGAACACCGA XbaI dnmV forward TTAATTAAACTAGTTACGTA TCACGCGGAGACGGGTGAGGCGGA PacI, SpeI, SnaBI reverse TCTAGACCTAGG TCGTCGGAAGCCTGTGAGCTAGGC XbaI, AvrII evaA forward TTAATTAAACTAGTGCTAGC CGCGGCTCGCCGGCTGAACACGAA PacI, SpeI, NheI reverse TCTAGA CTCATGCACCTCCCCGAGGCTGGG XbaI evaB forward TTAATTAAACTAGT GACATCAGTTCATGGAAAGGC PacI, SpeI reverse TCTAGA GTTGTCAGAGGGTCGACAGCACTT XbaI evaC forward TTAATTAAACTAGT TCCCCACAATTTCGTCGACGACAAG PacI, SpeI reverse TCTAGA TTTTGCGACATGGCGATCACGTCA XbaI evaD forward TTAATTAAACTAGT GACCCTCTGACAACTCCCACTACA PacI, SpeI reverse TCTAGA TGTGGGCAAGCAGTCAGGTCGAGA XbaI

Biosynthesis and transformation of plasmids pYJ371, pYJ630, pYJ631, pYJ627, TDP-D-olivose for the biosynthesis and transformation of TDP-Epi-L-Danosamine plasmids pYJ613, TDP-L-3'-N-methyl The plasmid pYJ656 for biosynthesis and transformation of daunosamine and the plasmid pYJ714 for biosynthesis and transformation of TDP-epi-L-vancosamine were transformed into mutant YJ183 to YJ183 / pYJ371, YJ183 / pYJ630, YJ183 / pYJ631, YJ183 / pYJ627, YJ183 / pYJ613, YJ183 / pYJ656 and YJ183 / pYJ714 were prepared, respectively.

Example  4: mutant Streptomyces In Venezuela ( S. venezuelae Anthracycline Biosynthesis and Analysis in

(S. venezuelae) to each of the mutant Streptococcus MRS Venezuela i.e., YJ183 / pYJ371, YJ183 / pYJ630 , YJ183 / pYJ631, YJ183 / pYJ627, YJ183 / pYJ613 ,, YJ183 / pYJ656 or YJ183 / pYJ714 also in the 0.6 mg epsilon Mai Synon was incubated in 60 mL of a R2YE solid medium containing 0.025 mg / ml at 30 ° C. for 5 days. The resulting culture was extracted with the same mixed solvent of water and methanol and then concentrated with methanol. The obtained product was analyzed using a combination of LC (Liquid Chromatography) / MS (Mass spectrometry) and MS / MS. LCs were double mobile phase [solvent A: 5 mM ammonium acetate, 0.05% glacial acetic acid; Solvent B: 5 mM ammonium acetate, acetonitrile / methanol 4: 1 mixed solvent in 0.05% glacial acetic acid; A: Phenomenex Synergi Polar with 80-30%, 25 minutes, 30-20%, 15 minutes, 20%, 9 minutes, 20-80%, 1 minute, 80%, 10 minutes]. -RP) column ((150 mm × 4.6 mm, 4 μm) was run with a Waters, Milford, MA Model 2690 separation module at a flow rate of 0.25 mL / min. The resulting effluent was a micromass quattro LC triple tandem. After injection into a Micromass Quattro LC triple tandem quadrupole mass spectrometer (Baverly, Mass.), The mass spectroscopic data were collected in positive-ion mode. Comparison was made using the peak intensities obtained from the MC chromatogram.

First, epsilon rhodomycinone was added to the growth medium of YJ183 / pYJ371 to investigate what epilodomycin D (FIG. 2B) was synthesized by the action of AknS glycosyltransferase, AvrE 4-keto-reductase. . Analysis of the organic extract of YJ183 / pYJ371 infused with epsilon rhodomycinone by LC / MS showed a peak of m / z = 558 corresponding to epidomycin D (Fig. 3) at a retention time of 38.5 minutes. lost. In the MS / MS analysis, m / z = 558 peak to as epi erythromycin D is the ion of m / z = 558 corresponding to erythromycin also epi D peak of the epi -L- Dow representing labor seeds lose m / z = 428 Peak, and a peak of m / z = 130 corresponding to epi-L-danosamine (FIG. 4). It was confirmed that about 30% of the injected epsilon rhodomycinone was converted to epilodomycin D.

Further, epsilon rhodomycinone was injected into the growth medium of YJ183 / pYJ630, YJ183 / pYJ631 or YJ183 / pYJ627. Through LC / MS analysis, peaks corresponding to epilodomycin D were obtained by YJ183 / pYJ371, in which AknS / AknT was expressed as glycosyltransferase, an auxiliary protein pair, and AvrE as a 4-ketoreductase. Each of these compounds produced could be observed at the same retention time. This proves the presence of epilodomycin D in the growth medium of YJ183 / pYJ630, YJ183 / pYJ631 and YJ183 / pYJ627 respectively, and 30%, 10% and 25% of the injected epsilon rhodomycinone, respectively It was confirmed that it is converted to mycin D.

Meanwhile, epirubicin (FIG. 2B) was synthesized by the action of proteins DnrP, DnrK, DnrV, and DoxA, which act on AknS glycosyltransferase, AvrE 4-keto-reductase, and epirodomycin D to epirubicin. In order to examine whether or not, epsilon rhodomycinone was added to the growth medium of YJ183 / pYJ546. Analysis of the organic extract of YJ183 / pYJ546 infused with epsilon rhodomycinone by LC / MS showed a small peak of m / z = 544 corresponding to epirubicin (Fig. 5) at a retention time of 26.9 minutes. lost. As a result of MS / MS analysis, epirubicin of m / z = 544 peak showed an ionic peak of m / z = 544 corresponding to epirubicin and a peak of m / z = 130 corresponding to epidaunosamine (FIG. 6). This MS / MS fragmentation pattern was consistent with the MS / MS fragmentation pattern of standard epirubicin (FIG. 6).

Epsilon rhodomycinone was added to the growth medium of YJ183 / pYJ613 to investigate whether 7- O -D-olibosyl epsilon rhodomycinone (FIG. 2B) was synthesized by the action of AknS glycosyltransferase. Analysis of the organic extract of YJ183 / pYJ613 infused with epsilon rhodomycinone by LC / MS showed m / z = 559 of 7- O -D-olibosyl epsilon rhodomycinone (FIG. 7). Peaks were observed at retention time 40 minutes 6 seconds. As a result of MS / MS analysis, m / z = 559 peak of 7- O -D-olibosyl epsilon rhodomycinone has an ion of m / z = 559 corresponding to 7- O -D-olibosyl epsilon rhodomycinone. Peak, m / z = 428 indicating loss of D-olivose was shown (FIG. 8). About 2% of the injected epsilon rhodomycinone was converted to 7- O -D-olibosyl epsilon rhodomycinone.

In order to investigate whether 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone (FIG. 2B) was synthesized by the action of AknS glycosyltransferase, epsilon rhodomycinone was added to YJ183 / pYJ656. Was added to the growth medium. Analysis of the organic extract of YJ183 / pYJ656 injected with epsilon rhodomycinone by LC / MS, corresponds to 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone (FIG. 9) A peak of m / z = 572 was observed at retention time 38 minutes 12 seconds. As a result of MS / MS analysis, 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone with m / z = 572 peak was 7- O- L-3'-N-methyl daunosaminyl epsilon M / z = 572 ion peak corresponding to rhodomycinone, m / z = 428 peak representing loss of L-3'-N-methyl daunosamine, and L-3'-N-methyl daunosamine The corresponding m / z = 144 is shown (FIG. 10). It was confirmed that about 85% of the injected epsilon rhodomycinone was converted to 7- O- L-3'-N-methyl daunosaminyl epsilon rhodomycinone.

In order to investigate whether 7- O -epi-L-vancosaminyl epsilon rhodomycinone (FIG. 2B) was synthesized by the action of AknS glycosyltransferase, epsilon rhodomycinone was added to the growth medium of YJ183 / pYJ714. Added. Analysis of the organic extract of YJ183 / pYJ714 injected with epsilon rhodomycinone by LC / MS showed m / z corresponding to 7- O -epi-L-vancosaminyl epsilon rhodomycinone (FIG. 11). A peak of = 572 was observed at retention time 40 minutes 48 seconds. As a result of MS / MS analysis, m / z = 572 peak of 7- O -epi-L-vancosaminyl epsilon rhodomycinone is m corresponding to 7- O -epi-L-vancosaminyl epsilon rhodomycinone. The ion peak at / z = 572, the peak at m / z = 428 indicating loss of epi-L-vancosamine and the m / z = 144 corresponding to epi-L-vancosamine (FIG. 12). It was confirmed that about 45% of the injected epsilon rhodomycinone was converted to 7- O -epi-L-vancosaminyl epsilon rhodomycinone.

<110> Ewha University-Industry Collaboration Foundation <120> Biosynthetic Method for preparion of antitumor agent epirubicin, novel glycosylated anthracycline derivatives and their preparaion methods <130> 20080222-ewha <160> 46 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (drrA-drrB) (forward) <400> 1 cccagatctc catgtgacta ctgggggcgt tag 33 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (drrA-drrB) (reverse) <400> 2 gggaagcttt ccggtacctg tgtcagtggg cgt 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (drrC) (forward) <400> 3 cccaagcttc cgtcaatgtg aggatgctca tgc 33 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (drrC) (reverse) <400> 4 gggtctagag ggttaattaa cttagcaccc ggggtcaagg atca 44 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmQ-dnmS) (forward) <400> 5 agatctttaa ttaagatatc acagtgtgag gagcacga 38 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmQ-dnmS) (reverse) <400> 6 tctagacata tggctacgac aagacagcga 30 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR Primer (desIII-desIV) (forward) <400> 7 ttaattaaac tagttaactc gccacgccga ccgtt 35 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer (desIII-desIV) (reverse) <400> 8 tctagagagc tcctcgtagg cggcctt 27 <210> 9 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmW) (forward) <400> 9 ttaattaaac tagtatcgat ctcgcagcct ctcgttgacg aaga 44 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmW) (reverse) <400> 10 tctagagtca gtccagcccg tcccgggtga 30 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmT) (forward) <400> 11 ttaattaaac tagtgctagc tcaaacaccg caccgtcacc cggg 44 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmT) (reverse) <400> 12 tctagaaaca ggacgcgcac ggggaactca 30 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmZ-dnmU) (forward) <400> 13 ttaattaaac tagtctccga tccttacgag gagtc 35 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmZ-dnmU) (reverse) <400> 14 tctagaatgt ccgcctcacc cgtctcc 27 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (avrE) (forward) <400> 15 ttaattaaac tagttacgta gcaccaggaa ttcgagcgcc gcta 44 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> PCR Primer (avrE) (reverse) <400> 16 tctagaccta ggtcgacgag aagctgtgac ggccga 36 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmJ) (forward) <400> 17 ttaattaaac tagtccgaca aggagtcacg tgtcc 35 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmJ) (reverse) <400> 18 tctagagaat tcccggccta gctcacaggc ttc 33 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (aknT-aknS) (forward) <400> 19 gatatccacg tcccgaagga gactgccgtg 30 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (aknT-aknS) (reverse) <400> 20 aagctttcac gcggaggtgg tcagggggaa 30 <210> 21 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (stfPII-stfG) (forward) <400> 21 ttaattaaga tatcgctcga ccgccacgct ctgttcga 38 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> PCR Primer (stfPII-stfG) (reverse) <400> 22 tctagacata tgcccatgga taggtgcggg gcttac 36 <210> 23 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (snogE) (forward) <400> 23 ttaattaaac tagtaccacc gagcggactt ccggtgac 38 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> PCR Primer (snogE) (reverse) <400> 24 tctagacata tgtccgggcc ctcggtgtgt cacgcg 36 <210> 25 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (snogN) (forward) <400> 25 ttaattaaga tatccttgac gtggcctgga agctggtt 38 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (snogN) (reverse) <400> 26 tctagagtcc atggagagtc ccttccgttg 30 <210> 27 <211> 47 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaE) (forward) <400> 27 cggttaatta aactagttac gtactcgcac gcttctcgcg tgcatca 47 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaE) (reverse) <400> 28 cggtctagac ctaggacacg cgtggtcatg cgcgagcct 39 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnrV-doxA) (forward) <400> 29 ttaattaaac tagtcgtgtg ccgatccatc gaaa 34 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnrV-doxA) (reverse) <400> 30 tctagaatca gcgcagccag acgggcagt 29 <210> 31 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnrK-dnrP) (forward) <400> 31 ttaattaaac tagtctacct cggcacctac cgccagct 38 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnrK-dnrP) (reverse) <400> 32 tctagatcac actgtcgcgg cccgggtgtg 30 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> PCR Primer (ermE *) (forward) <400> 33 ttaattaaac tagtgagctc ggtaccagcc cg 32 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR Primer (ermE *) (reverse) <400> 34 tctagatacc aaccggcacg attg 24 <210> 35 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (aknX2) (forward) <400> 35 ttaattaaac tagtcaattg cgccgacgga gggaacagac atgt 44 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (aknX2) (reverse) <400> 36 tctagactca gcgctgttcg cgaacaccga 30 <210> 37 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmV) (forward) <400> 37 ttaattaaac tagttacgta tcacgcggag acgggtgagg cgga 44 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <220> PCR Primer (dnmV) (reverse) <400> 38 tctagaccta ggtcgtcgga agcctgtgag ctaggc 36 <210> 39 <211> 44 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaA) (forward) <400> 39 ttaattaaac tagtgctagc cgcggctcgc cggctgaaca cgaa 44 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaA) (reverse) <400> 40 tctagactca tgcacctccc cgaggctggg 30 <210> 41 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaB) (forward) <400> 41 ttaattaaac tagtgacatc agttcatgga aaggc 35 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaB) (reverse) <400> 42 tctagagttg tcagagggtc gacagcactt 30 <210> 43 <211> 39 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaC) (forward) <400> 43 ttaattaaac tagttcccca caatttcgtc gacgacaag 39 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaC) (reverse) <400> 44 tctagatttt gcgacatggc gatcacgtca 30 <210> 45 <211> 38 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaD) (forward) <400> 45 ttaattaaac tagtgaccct ctgacaactc ccactaca 38 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (evaD) (reverse) <400> 46 tctagatgtg ggcaagcagt caggtcgaga 30

Claims (9)

Novel 7- O -D-olibosyl epsilon rhodomycinone represented by Structural Formula 3 (7- O- D-olivosyl ε-rhodomycinone). Structural Formula 3 Novel 7- O -L-3'-N-methyl daunosaminyl epsilon rhodomycinone represented by the following structural formula 4 (7- O- L-3'-N-methyl daunosaminyl ε-rhodomycinone). Structural Formula 4 Novel 7- O -epi-L-vancosaminyl epsilon rhodomycinone represented by Structural Formula 5 (7- O- epi-L-vancosaminyl ε-rhodomycinone). Structural Formula 5 In the method of biosynthesis of epilodomycin D, 1) First, the full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank sequence number L76359) in which the Streptomyces MRS Venezuela insert (S. venezuelae) in YJ183 aknT -aknS (GeneBank sequence number AF264025), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank SEQ ID NO: U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237), including a recombinant vector having a cleavage map as shown in FIG. 13 below (pYJ371) The microorganism obtained by transformation is incubated with epsilon rhodomycinone, 2) second, the full-length biosynthetic gene cluster PKS encoding pikromycin (Pik) and desamine (des) biosynthetic enzymes were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank sequence number L76359) in which the Streptomyces MRS Venezuela insert (S. venezuelae) in YJ183 stfPII-stfG (GeneBank SEQ AM156932), desIII -desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank SEQ ID NO: U77891), dnmZ-dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) with a recombinant vector (pYJ630) having a cleavage map as shown in FIG. The microorganism obtained by transformation is incubated with epsilon rhodomycinone, 3) Third, the full-length biosynthetic gene cluster PKS and desamine, des biosynthetic enzymes coding for pykromycin (Pik) were deleted, and then the doxorubicin resistance gene drrA-drrB (GeneBank SEQ ID NO: M73758) and S. venezuelae inserting drrC (GeneBank SEQ ID NO: L76359) YJ183 shows snogE ( GeneBank SEQ ID NO: AF187532) , snogN ( GeneBank SEQ ID NO: AF187532), desIII -desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891), dnmZ-dnmU (GeneBank sequence) No. AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237) comprising a microorganism obtained by transforming with a recombinant vector (pYJ631) having a cleavage map as shown in Figure 19 incubated with epsilon rhodomycinone do or, 4) fourth, the full-length biosynthetic gene cluster PKS encoding pikromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank sequence number L76359) in which the Streptomyces MRS Venezuela insert (S. venezuelae) in YJ183 aknT -aknS (GeneBank sequence number AF264025), desIII - desIV (GeneBank sequence number AF079762), dnmW (GeneBank sequence number U77891), dnmT (GeneBank SEQ ID NO: U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), evaE (GeneBank SEQ ID NO: AJ223998), dnmJ (GeneBank SEQ ID NO: M80237), including a recombinant vector having a cleavage map as shown in FIG. 20 (pYJ627) The microorganism obtained by transformation was incubated with epsilon rhodomycinone, 5) The cultures of the transformed microorganisms YJ183 / pYJ371, YJ183 / pYJ630, YJ183 / pYJ631, YJ183 / pYJ627 and epsilon rhodomycinone prepared according to the above method were extracted with the same mixed solvent of methanol and water and concentrated with methanol. A process for preparing epidomycin D represented by the following structural formula (1). Structural Formula 1 The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS (GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891) into S. venezuelae YJ183. ), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633), avrE (GeneBank SEQ ID NO: AB032523), dnmJ (GeneBank SEQ ID NO: M80237), dnrV -doxA (GeneBank SEQ ID NO: U77891), dnrK - dnrP (GeneBank SEQ ID NO: L40425) The microorganism obtained by transforming with a recombinant vector (pYJ546) having a cleavage map as shown in FIG. 14 was then incubated with epsilon rhodomycinone, Method for producing epirubicin represented by the following structural formula 2 characterized in that the culture of the transformed microorganisms YJ183 / pYJ546 and epsilon rhodomycinone is extracted with a mixed solvent of methanol and water in the same amount and concentrated with methanol. Structural Formula 2 The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS (GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891), oleV (GeneB79 SEQ ID: AF0555) into S. venezuelae YJ183. ) , oleW (GeneBank SEQ ID NO: AF055579), urdR (GeneBank SEQ ID NO: AF269227) containing a recombinant vector (pYJ613) having a cleavage map as shown in Figure 15 and then incubated with epsilon rhodomycinone , A novel 7- O -D-olibosyl epsilon represented by the following structural formula 3 is characterized by extracting the culture of the transformed microorganism YJ183 / pYJ613 and epsilon rhodomycinone with the same mixed solvent of methanol and water and concentrating with methanol. Process for preparing rhodomycinone: Structural Formula 3 The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) into S. venezuelae YJ183 shows aknT -aknS ( GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891) , dnmT (GeneBank SEQ ID NO: U77891), dnmZ -dnmU (GeneBank SEQ ID NO: AF006633) , dnmV ( A microorganism obtained by transforming the recombinant microorganism (pYJ656) having a cleavage map as shown in FIG. 16 including GeneBank SEQ ID NO AF006633), dnmJ (GeneBank SEQ ID NO M80237) aknX2 (GeneBank SEQ ID NO AF264025), together with epsilon rhodomycinone After incubation The culture of the transformed microorganisms YJ183 / pYJ656 and epsilon rhodomycinone is extracted with the same mixed solvent of methanol and water and concentrated with methanol, a novel 7- O -L-3 ' Method for preparing -N-methyl daunosaminyl epsilon rhodomycinone. Structural Formula 4 The full-length biosynthetic gene cluster PKS coding for pykromycin (Pik) and the desamine (des) biosynthetic enzyme were deleted, followed by the doxorubicin resistance genes drrA - drrB (GeneBank SEQ ID NO: M73758) and drrC (GeneBank SEQ ID NO: L76359) inserts aknT- aknS ( GeneBank SEQ ID NO: AF264025), desIII - desIV (GeneBank SEQ ID NO: AF079762), dnmW (GeneBank SEQ ID NO: U77891), evaA (GeneBank SEQ ID NO: AJ223998), inserted into S. venezuelae YJ183. ) , evaB (GeneBank SEQ ID NO: AJ223998) , evaC (GeneBank SEQ ID NO: AJ223998) , evaD (GeneBank SEQ ID NO: AJ223998) , evaE (GeneBank SEQ ID NO: AJ223998), including a recombinant vector having a cleavage map as shown in FIG. 17 (pYJ714) Microorganisms obtained by transformation with incubated with epsilon rhodomycinone, The transformed microorganism YJ183 / pYJ656 and epsilon to as Mai when discussing extracting the culture with an equal volume of methanol and a mixed solvent of water and to, characterized in that the concentration of methanol novel 7- O represented by the formula 5-epi -L- Method for preparing vancosaminil epsilon rhodomycinone. Structural Formula 5 Biosynthesis of deoxysugar through genetic modification, synthesis of glycosyltransferase which can attach deoxysugar to epsilon rhodomycinone and novel glycosylated epsilon rhodomycinone Incubated Streptomyces venezuelae with epsilon rhodomycinone capable of synthesizing a protein which is responsible for the conversion of a novel epirubicin to a derivative of epirubicin, and then cultured the same amount of a mixed solvent of methanol and water. A method for producing a novel anthracycline sugar substituent, characterized in that the extraction with and concentrated with methanol.