KR100903485B1 - Combinatorial Biosynthesis of 5-O-desosaminyl erythronolide A as a Precursor of Ketolide Antibiotics and Development of Its Recombinant Strain - Google Patents

Abstract

The present invention is semi-synthesically derived from erythromycin A, a cyclic macrolide of 14 carbons, and is a precursor of a ketolide antibiotic having a 3-keto group instead of the L-cladinos sugar chain of erythromycin A. The present invention relates to a method for combinatorial biosynthesis of O -desosaminyl erythronolide A, which is a erythromycin producing strain, Saccharopolyspora. erythraea) acting on the no-fluoride in erythromycin B to O by a deletion in eryBV gene encodes the glycosyl transferase to attach a sugar chain L- mica LOS-to lead to additional variations in the L- cladinosyl's by methylation 5- O - having no fluoride sosami carbonyl erythromycin B in (DesEB, 2) the accumulation was then deleted Caro Indianapolis Fora EPO la ah eryBV Streptomyces Venezuela deletion mutants in (Streptomyces venezuelae ), which encodes a substrate- thixotropic P450 monooxygenase in the pikromycin biosynthetic pathway. DesEA was successfully prepared by further expression of pikC heterogenes (˜50 μl ⁻¹ ).

Semi-synthesis of ketolides requires a number of processes to protect and deprotect functional groups, resulting in low yields, whereas combinatorial biosynthesis is an effective way to provide more effective precursors for ketolide synthesis. By means; In other words, the use of 5- O -desosaminyl erythronolide A (DesEA, 3) as a precursor during ketolide biosynthesis has been used to eliminate L-cladinos attached to the C-3 position in erythromycin A (1). Can shorten the process.

Ketoride antibiotics; Macrolide system; Erythromycin; Desosaminyl eryronolide A (DesEA); Combinatorial biosynthesis

Description

Combined biosynthesis of 5-O-desosaminyl erythronolide A as a Precursor of Ketolide Antibiotics and Development of Its Recombinant Strain }

The present invention relates to a method for producing 5- O -desosominyl erysonolide A, a precursor of ketolide antibiotics, and to the development of a recombinant strain producing the same.

Macrolides are representative antimicrobial agents that can replace excellent antimicrobial activity and in particular the emergence of multidrug resistant bacteria in recent years. These macrolides exhibit potency when inhibiting protein synthesis in bacteria to bind to peptidyl transferase and adjacent 50s ribosomal subunits.

Clarisithromycin and azithromycin, the second-generation antimicrobials of macrolides, have been developed to address the pharmacokinetic problems associated with acid instability of erythromycin, the first-generation antimicrobial. . In particular, Staphylococcus aureus for these macrolide acids (Staphylococci), Streptococcus (Streptococci), pneumonia, Streptococcus (Pneumococci), enterococci (Enterococci) resistant state appearance, such as was required for the development of antibiotics next generation macrolide flow, and thus Macrolides having a 3-keto group in place of the L-cladinos sugar chain of erythromycin A, and additionally binding aromatic N-substituted carbamate to the C-11 or C-12 position Distinctly, antibiotics with unique structures called ketolides have been developed.

Semi-synthesis of ketorides requires a number of processes to protect and deprotect the functional groups of the precursors, resulting in low yields.

Accordingly, an object of the present invention is the combinatorial synthesis of 5- O -desosaminyl erysonolide A (DesEA, 3), which is a more effective precursor that can be used in the process of ketolide semisynthesis by the combinatorial biosynthesis method. To provide a way.

Another object of the present invention is to provide a recombinant strain for producing 5- O -desosominyl erythronolide A.

The use of 5- O -desosaminyl erythronolide A (DesEA, 3) as a precursor for ketolide synthesis is a process for removing the L-cladinos sugar chain attached to the C-3 position in erythromycin A (1). Can be reduced. Saccharopolyspora , an erythromycin producing strain Deletion mutations in the eryBV gene encoding glycosyltransferase, which acts on erythronoid B in erythraea ) and attaches L-mycarose sugar chains, inducing additional modification to L-cladinos by O -methylation, Accumulate 5- O -Desosaminyl Erysonolide B (DesEB, 2).

Encoding cytochrome P450 monooxygenase in the pikromycin biosynthetic pathway from Streptomyces venezuelae in eryBV deletion strains of Saccharopolis fora erysraeea Further expression of pikC heterogenes successfully produced 5- O -Desosaminyl erylonolide A (DesEA, 3).

Saccharopolyspora erythraea ) NRRL 2338 strain is known to produce erythromycin A (1) (see FIGS. 1 and 2). An entire gene cluster ( ery ) responsible for the biosynthesis of erythromycin A1 was cloned and sequenced, and then the characteristics of each open reading frame were predicted. 6- deoxyerysonolide B (6-dEB), a macrolactone with a 14 carbon cyclic structure, is a modular polyketide consisting of three separate polypeptides encoded by eryA I , eryA II and eryAIII It is synthesized by a synthetic polyketide synthase (PKS). The addition of two sugars and two hydroxyl groups then produces erythromycin A (1) (see FIG. 2).

Streptomyces Among the post-PKS customized enzymes present in the pikromycin biosynthesis gene of the venezuelae strain, DesVII, a desosaminyl transferase that flexibly acts on substrates such as various macrolides and sugar chains, and various sun macrolides PikC's ability to hydrate species is of great value for combinatorial biosynthesis performance. In addition, the discovery of the PikC crystal structure is known that the combination of two different desosamin binding pockets with a single macrolactone binding site shows the substrate flexibility of the enzyme.

Synthesis of the ketoride begins with the hydrolysis of the 6- O -methyl erythromycin A derived L-cladinos sugar chain chemically synthesized in erythromycin A (1). These synthesis steps can be shortened by preparing 3-decladinosyl derivatives of erythromycin A directly from erythromycin producing strains by combinatorial synthesis methods.

In the present invention, a combinatorial synthesis method was used to produce 5- O -desosaminyl erysonolide A (DesEA, 3) as a precursor for ketolide synthesis. First, eryBV deletion mutant strains of Zaccaropolyphora erythraea were prepared to produce 5- O -desosaminyl erysonolide B (DesEB, 2), and then the substrate-flexible mono in Streptomyces venezuela. Successful conversion of DesEB (2) to DesEA (3) using a PikC-expressing recombinant strain obtained by inserting the heterologous gene pikC encoding an oxygenase into the chromosomal DNA of the eryBV deletion mutant strain under the control of the actI promoter.

In the present invention, by producing 5- O -desosominyl erythronolide A using a recombinant strain, not only can the ketoride precursor, which is difficult to obtain by the conventional chemical process, be obtained by a combination biosynthetic method, but also in a simple process with high yield. There is an advantage that can synthesize ketorides.

[ Experimental Example ]

1. Experimental Materials and Methods.

1.1. Microbial strains, culture conditions and DNA manipulation.

The microbial strains and plasmids used in the present invention are listed in Table 1 below.

[Table 1] Plasmid and strain list

Zaccaropolisa erythraea NRRL 2338 (red variant) microorganism is J. M. Provided by Weber (Pharma Logic Inc., USA), R2T2 (peptone agar R2T; sucrose 103.0 gl ^-1 , K ₂ SO ₄ 0.25 gl ^-1 , yeast extract 6.5 gl ^-1 , tryptome 5.0 gl ^-1 , Bacto agar 22.0 gl ^-1 ). Tryptic Soy Broth; pancreatic digest of casein 17.0 gl ^-1 , enzymatic digest of soybean meal 3.0 gl ^-1 , sucrose 2.5 gl ^-1 , NaCl 5.0 gl ^-1 , K ₂ HPO ₄ 2.5 gl ⁻¹ ) was used. Glycine SGGP liquid medium without ^{(peptone 4.0 gl -1, yeast extract} 4.0 gl -1, casamino acid 4.0 gl -1, MgSO 4 and ^{_{7H 2 O 0.5 gl -1, glucose}} 10.0 gl -1, KH 2 PO 4 1.3 gl - ¹ ) Zaccaropolis Fora Erisraea It was used for the cultivation of the protoplast of the strain. Zaccaropolis Fora Erisraea In the selection and maintenance of transformants, thiostrepton was used at a concentration of ²⁵ μg ⁻¹ . Escherichia coli (Escheria coli ) XL1-Blue (Stratagene) was used as a cloning host for DNA manipulation and E. coli JM110 was used as demethylation host. E. coli microbial strains were routinely cultured in Luria-Bertani (LB; tryptone 10.0 gl ^-1 , yeast extract 5.0 gl ^-1 , NaCl 10.0 gl ^-1 , Bacto agar 15.0 gl ^-1 ) agar, To maintain the plasmids were grown in LB cultures containing appropriate concentrations of antibiotics (100 μg ml ^{−1 of} ampicillin). DNA manipulations in E. coli were performed in Litmus 28 (New England Biolabs) and pGEM Easy T-vector (Promega) according to standard procedures. On the other hand, shuttle vector pWHM3 (pYJ585) was used for the production of eryB V deletion mutants of Zaccarpolyporia erysraea . Plasmids isolated from E. coli cells were isolated according to the use of Plasmid Midi Kit (Qiagen) or Sambrook et al. Cleavage of DNA via restriction enzymes was performed as recommended by the enzyme supplier (New England Biolabs and Roche).

DNA fragments were separated by agar electrophoresis using a QIAquick Gel extraction kit (Qiagen). DNA ligation was performed according to standard protocol with T4 DNA ligase (New England Biolabs). The total genomic DNA of Zaccaropolyphora erythraea was isolated using the Genomic DNA Separation Kit (G-Spin, Qiagen). Polymerase chain reaction (PCR) was performed with DNA-polymerase (New England Biolabs) and pfu polymerase (Stratagene) using the conditions recommended by the manufacturer. PCR reaction was carried out according to the procedures and methods commonly used in the art, and PCR conditions are not particularly important for the invention.

1.2 Zaccaropolis Fora Erisraea eryBⅤ  Fruitful Mutantism Design.

Primers used for PCR amplification of genes according to the invention are listed in Table 2 below. Since pWHM3 is not cloned from Zaccaropophora erythroa derived from pIJ101 and is easy to prepare a deletion mutant strain by homologous recombination method, pWHM3 derivative is inserted into the chromosomal DNA of a Saccaropophora eryraea strain. I was. At another site in the eryB V region two fragments of DNA were amplified by PCR using primers. LA5F and LA5R upstream of the eryB V gene and Nco I / at the 5'- and 3'- ends of the primers to introduce Bam HI and Spe I restriction sites at the 5'- and 3'- ends of the primers, respectively. In order to introduce Nhe I and Xba I restriction sites, RA5F and RA5R were used in the downstream region of the eryB V gene (see Table 2). Each of the PCR-fabricated DNA fragments was cloned in turn into Litmus 28; A 968 bp Nco I- Nhe I fragment downstream of the eryB V gene was cloned into litmus 28, followed by a clone of 1 kb Bam HI- Spe I fragment, and finally 1.9 kb finally prepared in litmus 28 The Bam HI- Xba I fragment of was cloned in pWHM3 to obtain pYJ584, a deletion plasmid to transform Zaccaropolyphora erythraea.

Plasmid pYJ584 was transformed into Saccharopolyphora erythraea by protoplast transformation. Introduced into. Thiostrepton resistant colonies appeared after 4-5 days, and colonies were passaged twice on R2T2 plates containing thiostrepton. Double crossover mutants were screened from thiostrepton sensitive phenotypes, and deletion mutants were selected by confirming genotype by Southern hybridization. Southern hybrids were performed according to the protocol and hybridizing probes were labeled with DIG (Roche Molecular Biochemicals).

Finally obtained eryB V deletion mutant Zaccaropolis Fora Erisraea Named YJ584.

[Table 2] Primer List

1.3 Design of Expression Plasmids and Mutant Strains.

The plasmid pYJ585 was designed by cloning a DNA fragment containing the act I promoter containing the cognate regulator gene act II-ORF4 in pWHM3.

DNA fragments were amplified from the template pHGF7505 using a set of primers; Each is in the 5'-and 3'-end of the primer Mfe Ⅰ and Bgl Ⅱ- Sbf Ⅰ- Kpn Ⅰ- Avr actⅠF and actⅠR Ⅱ the introduction of restriction sites (see Table 2).

The DNA sequence of the homologous region for recombination of the plasmid at the erm E-terminus, ie, chromosomal DNA, was used as the oligonucleotide and amplified from the genomic DNA of Saccharophora erythraea as a template; DisF and disR with Eco R I and Nsil I restriction sites at the fragment 5'- and 3'- ends, respectively. 1.5 kb Eco RI- Nsil I fragments were cloned into litmus 28.

Streptomyces as a template using primers from a 1.2 kb DNA fragment containing the pikC gene Amplified from genomic DNA of Venezuelaa ; PikCF and pikCR which introduced Eco RI / Avr II and Eco RI restriction sites at the 5'- and 3'- ends of the primers, respectively. PCR-manufactured pikC containing DNA fragments were transferred to litmus 28 containing homologous DNA regions for recombination. Finally, it was cloned into Avr Ⅱ- Ⅰ Nsil fragment was digested with Avr pYJ585 Ⅱ / sbf Ⅰ screen including pikC to obtain an expression plasmid pikC pYJ586. Zaccaropolis Fora Erisraea pYJ586 plasmid was transformed into eryB V deletion strain YJ584 to obtain pikC expression variant strain YJ584 / pYJ586.

fragments comprising eryB V were amplified using primers; EryBVF and eryBVR with Avr II and Eco RI restriction sites introduced at the 5'- and 3'- ends of the primers, respectively. Avr II- Eco Ri fragments, including eryB V , were cloned into litmus 28 as homologous DNA regions for recombination. Finally, the Avr II- Nsi I fragment was cloned into pYJ585 digested with Avr II / sbf I to obtain eryB V expression plasmid pYJ591. Isaac Caro Paul less Fora EPO la ah transformed pYJ591 deletion in eryB Ⅴ main YJ584 by the eryB Ⅴ return obtain a YJ584 / pYJ591 mutant strain.

Each plasmid is called Zaccaropolisa The EPO la Oh that inserted into the chromosomal DNA of YJ584 strain was confirmed by Southern hybridization method (Southern hybridization).

1.4. Analysis of macrolide antibiotics

That at 32 ℃ 5 ilgan cultured in TSB medium Isaac Caro Indianapolis Fora Erisraea The whole culture (50 mL) of each of the wild and mutant strains was transferred to a separatory funnel, and then the pH was adjusted to ˜9 (or 10) with concentrated NaOH, followed by extraction with the same volume of ethyl acetate. The organic solvent layer was dried over Na ₂ SO ₄ , evaporated to dryness on a rotary evaporator and placed in a refrigerator until analysis. The extract was dissolved in 60 μl of methanol, and a portion of this solution was analyzed by HPLC-ESI-MS / MS analyzer.

Zaccaropolis Fora Macrolide flow analysis obtained by the O strain culture solution on EPO la was carried out using a Waters / Micromass Quattro micro / MS interface on Micromass Quattro micro MS. Separation was performed using a Nova-Pak C ₁₈ (4.0 μm, water) reversed phase column of 150 × 3.9 mm. Analytes were added to water (A) consisting of 5 mM (w / v) ammonium acetate, 0.05% acetic acid (v / v) and 80% (v / v) acetonitrile in the same concentration (B). ) The liquid gradient was eluted at a flow rate of 180 μL / min so that the liquid B became 20% to 70% for 25 minutes and the liquid B became 90% for 20 minutes. To rebalance the column, B was maintained at 90% for 10 minutes and B was maintained at 20% for 10 minutes. The column effluent was experimented in cationic mode by connecting to ESI-MS. The experimental instrument was calibrated by injecting erythromycin (Sigma, 50 μg ⁻¹ ) standard solution directly into the ion source (60 mL min ⁻¹ ). The optimal parameters of the ESI-MS / MS system were based on maximum generation, first based on protonated molecular ions and then on corresponding segmented ions. The calibration parameters of the HPLC-ESI-MS detector were optimized as follows: capillary and cone voltages were 4 kV and 35 V, respectively, and the source and desolvation temperatures were 130 and 250 ° C, respectively. Desolvent gas flow rate and cone gas flow rate were 500 and 50 Lhr ^−1, respectively. The scan range was m / z 100 to 800 Da. The aaa impact energy in MS / MS mode was 1.5 V in parallel with all argon-induced cleavage of molecular ions.

2. Results

2.1. DesEB To produce Zaccaropolis Fora Erisraea eryB Ⅴ  Fruitful mutations.

pYJ584, an eryB V deletion plasmid, was prepared and transformed into wild saccharopolypora Oh by the EPO transformed into LA, Isaac Caro Police Fora PYJ584 that is inserted into the chromosomal DNA of the EPO Oh la was confirmed by PCR and Southern hybridization method. The expected size of DNA fragments PCR prepared from genomic DNA of wild strain (2.5 kb) and eryB V deletion mutant strain (1.3 kb) indicated that pYJ584 was successfully inserted into chromosomal DNA (data not shown). Double cross mutants were selected as single cross mutants by selecting a number of thiostrepton resistant colonies from thiostrepton sensitive colonies. Genomic DNA digested in Apa I was analyzed by Southern hybridization using a DNA fragment containing the downstream proximal region of eryB V as a probe. As a result, a single band of 3.3 kb was found in wild species and eryB V deletion in wild species. A reduced size single band of 2.1 kb was observed (see FIG. 3). Analysis of the extract of the eryB V mutant strain of the present invention by HPLC-ESI-MS / MS revealed the loss of major macrolides such as erythromycin A (1) and erythromycin B (4) detected in wild species (Fig. 5a, b). Whereas erythromycin is intermediate, DesEB (2) of erythromycin A (1) grinding Caro Indianapolis Fora It was detected as the main macrolide in the extract of Erysraeea YJ584 mutant strain (see FIG. 5B).

Molecular ions [DesEB] was detected at m / z 560.1 consistent with the H ^+, fragmentation sun m / in _{z 542.0 [DesEB-H 2 O} ] H +, m / z 384.8 In [DesEB-desosamine-H ₂ O ] H ⁺ , m / z 158.0 shows corresponding fragmentation ions with [desosamine] H ⁺ , which can be interpreted as 5- O -desosaminyl erythronolide B (2) (see FIG. 6C).

2.2. eryBⅤ  Recovery of erythromycin production by self-return in mutant strains.

eryBⅤ the deletion mutant plasmid pYJ591 for EryBⅤ expressed under the promoter control actⅠ Isaac Caro Indianapolis Fora Erisraea Transformed with YJ584. Hybridization was a thio streptavidin gene tone cover the genomic DNA cut with Eco RⅠ with DIG as a probe bar, grinding Caro Indianapolis Fora The expression plasmid was inserted into the chromosomal DNA of Erisraea YJ584 / pYJ591. Southern hybridization was confirmed. That is, a single band appeared at 11.5 kb to confirm the genotype of pYJ591 inserted into the chromosomal DNA (see FIG. 4).

Compared with wild Isaac Caro Police Fora It was confirmed that erythromycin production in the extract of the erythroa YJ584 / pYJ591 mutant strain was restored to about 80% level compared to the wild species, demonstrating that the self-reversion of eryB V was successfully expressed in the mutant strain (FIG. 5C). ). It was confirmed that there are two major macrolides showing the same retention time and MS / MS fragmentation pattern of erythromycin A (1) and B (4) produced in wild strains; Molecular ion m / z 734.3 of erythromycin A (ErA, 1) [ErA ] consistent with H ^+, fragmentation pattern in the ^{H +, m / z 576.0 [} ErA-H 2 O] at m / z 716.0 [ ErA-cladinose] H ⁺ , and m / z 158.2 showed corresponding fragmentation ions with [desosamine] H ⁺ ; Erythromycin B is consistent with the molecular ions in the m / z 718.2 [ErB] H + of (ErB, 4), the fragmentation pattern m / z 560.1 In [ErB-mycarose] H ^+, and m / z 158.2 In [desosamine ] H ⁺ and corresponding fragmentation ions were shown (see FIGS. 5C and 6A, B).

2.3. eryBⅤ Within fruit mutants pikC  Production of DesEA by Expression.

Delete the plasmid pYJ586 Caro Indianapolis Fora EPO la transgenic ah YJ584 to and confirmed the insertion of the claw mosom DNA eryBⅤ deletion mutants by Southern hybridization method. In other words, the genomic DNA digested with Eco R I was conjugated with a TIG-labeled thiostrepton gene with a probe. As a result, the genotype confirmed that the pikC expression plasmid pYJ586 was successfully inserted into the chromosomal DNA at 11.6 kb. (See Figure 4). Streptomyces The pikC gene in the Pyclomycin biosynthetic gene family of Venezuela is known to encode cytochrome P450 monooxygenase with excellent substrate flexibility, acts on cyclic monolactones of various sizes, and has a hydroxyl functional attachment site. According to various patterns, Zaccaropolis Pora The EPO called Oh My PikC YJ584 expression strains are, Isaac Caro Police Fora The production of the hydroxide derivative was expected of DesEB (2) it made in the O strain YJ584 the EPO la. As expected, the mutant expression PikC, Isaac Caro Indianapolis Fora Erisraea YJ584 / pYJ586 converted the produced DesEB to DesEA (3), a hydroxylated derivative, and the extract of the culture was confirmed by HPLC-ESI-MS / MS (see FIG. 5D). This compound [DesEA] showed a molecular ion at m / z 575.5 corresponding to the H ^+, fragmentation pattern m / in _{z 558.0 [DesEA-H 2 O} ] H +, m / z 540.1 In [DesEA-2H ₂ O] H ⁺ , and m / z showed fragmentation ions consistent with [DesEA-desosamine-H ₂ O] H ⁺ , and m / z 158.1 with [desosamine] H ⁺ at m / z 158.1, which derivative was 5-O- It can be interpreted as desosaminyl eryronolide A (3) (see FIG. 6D).

3. Conclusion

The Saccharopolyphora erythraea NRRL 2338 strain is known to produce erythromycin A (1). The commercial importance of erythromycin has led to an in-depth study of biosynthesis and combinatorial biosynthesis, which has stimulated the production of various homologues of related antibiotics.

Erythromycin A (1) is used as a starting material for the synthesis of ketorides. Methods of preparing non-natural biosynthetic intermediates that can replace erythromycin A (1) by a combinatorial biosynthesis approach can serve as a useful resource for ketolide precursors. DesEA (3) is synthesized by hydroxylation according to the results of PikC's catalysis at C-12 position of DesEB (2), and DesEB is obtained by deleting eryB V in Zaccaropolyphora erythraea , ketoride. It can be a good candidate as starting material in synthesis.

Zaccaropolis Fora Erisraea The production of DesEB (2) in YJ584 is a result of the flexibility of the desosaminyl transferase EryCIII, which has been reported to be 6-degree using 3-α-mycarosyl-6-deoxyeryslorinoid B as substrate. were obtained oxy erythromycin a, Isaac containing EryCⅢ methyl person nosil erythromycin no fluoride precursor Caro Indianapolis Fora Erisraea It has been reported that the strain SGT2 can be used to convert to methyllamnosyl erythromycin.

On the other hand, even though EryK, which catalyzes the hydroxylation of the erythromycin D at the C-12 position in its natural substrate, is not expected to be affected by the genetic manipulation introduced in the present invention, the saccharopolypora Erisraea The absence of DesEA (3) in the YJ584 and YJ584 / pYJ591 mutants was probably due to the relatively stringent substrate specificity of EryK, which is consistent with previous reports. In other words, hydroxylation of the C-12 position was not observed among the 3-rhamnosyl derivatives of erythromycin that appeared when OleG2 was replaced with EryBV in the eryB V deletion mutant.

C-12 hydroxylation chain DesEA (3) of DesEB (2) has been detected in a small amount of the derivative base concentrate commercial crystals of erythromycin, as previously reported, Isaac Caro Indianapolis Fora Erisraea There were no reported papers confirming the presence of DesEB (2) in the culture of wild strains. Also, in this study, DesEB (2) used wild saccharopolis fora even though it adopted high sensitivity ESI-MS / MS. Erisraea Or eryB V autologous was not detected from the return mutant strain . PikC has a similar function to oxidation of macrolides, like EryK, but more flexible substrate specificity than EryK upon introduction of C-12 hydrosyl functional groups in DesEB (2).

In summary, combinatorial biosynthetic methods using post-PKS custom enzymes that are flexible to a variety of substrates, such as PikC, are useful for the development of various derivatives through chemical modifications and may be non-natural precursors for the production of polyketide antimicrobials. It is considered a means to biosynthesize derivatives.

[references]

Figure 1 shows the structure of the macrolide antibiotics, erythromycin A (1); 5- O -Desosaminyl erythronolide B (2); And 5- O -Desosaminyl eryronolide A (3).

FIG. 2 shows the biosynthetic pathway of erythromycin A (1), 6-deoxyerythronolide B is 3 having 6 modules using furopionyl CoA as extension and 6 methylmalonyl CoA as extension Synthesized into PKS genes. Hydroxylation at the C-6 position yields erythronolide B, followed by a two-step process of glycosylation by EryBV and EryCIII, respectively, to obtain erythromycin D.

Figure 3 (a) is a Zaccaropolis Pora Erisraea Wild Strains and Zaccaropolis Fora Erisraea Southern hybridization method of genomic DNA derived from pYJ584 ( eryB V deletion mutant). Genomic DNA digested with Apa I was used for Southern hybridization and the DNA fragment downstream to eryB V was used as a probe. Lane 1. wild strain (3.3 kb); 2. The Zaccaropolis Fora Erisraea pYJ584 (2.1 kb); 3. relates to a single cross-derived return (3.3 kb).

(b) the Zaccaropolis fora Erisraea Wild type strain (top) and Ⅴ eryB in the wild strain shows that the gene is deleted deleted Caro Indianapolis Fora Erisraea The restriction map of genomic DNA of pYJ584 (bottom) is shown.

Figure 4 is deleted Caro Indianapolis Fora for pikC and eryB Ⅴ insertion mutant Erisraea It is a related photograph of the hybrid binding Southern genomic DNA from the strain. Genomic DNA digested with Eco RI was used for Southern hybridization and thiostrepton gene was used as probe. Lane 1.Saccharopolis Fora Erisraea YJ584 / pYJ591 (11.5 kb); 2-4.The Zaccaropolis Pora Erisraea YJ584 / pYJ586 (11.6 kb).

FIG. 5 relates to HPLC-ESI-MS / MS analytical chromatogram, wherein (a) is Zaccaropolyphora Erisraea Of wild-derived extracts, (b) relates to YJ584, eryB V deletion mutants, (c) relates to YJ584 / pYJ591, eryB V autologous return mutants, and (d) to YJ584 / pYJ586, pikC It relates to inserted variant strains.

FIG. 6 relates to mass spectra, in which (a) and (b) each represent a saccharopolyphora. Erythromycin A (1) and erythromycin B (4) obtained from extracts of wild erythroaea strains, and (c) is a saccharopolis fora It relates to EPO la DesEB (2) obtained from the Oh YJ584, extract the eryB Ⅴ deletion mutant on, and (d) is deleted Caro Indianapolis Fora EPO O YJ584 / pYJ586, eryB La Ⅴ relates to a fruit and DesEA (3) pikC is obtained from the extract of the insertion mutants.

In the description, ErA stands for erythromycin A; ErB stands for erythromycin B; cla is cladinosyl's stands for (cladinose) and; des is an abbreviation for dessamine.

<110> Geno Tech Corp. <120> Combinatorial Biosynthesis of 5-O-desosaminyl erythronolide A as          a Precursor of Ketolide Antibiotics and Development of Its          Recombinant Strain <130> 2007905ewha <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (disF) <400> 1 cgagacgaat tcggcggaat attaacggtt aaa 33 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> PCR Primer (disR) <400> 2 gatgttatgc atcgaatgca cgacgaagaa ctg 33 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (LA5F) <400> 3 gcccgtggat ccgacctgga aagcgagcaa 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (LA5R) <400> 4 acccgcacta gtgctcctcg gtgggggtca 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (RA5F) <400> 5 gccaccatgg ctagcggttt ccgaccgaca 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR Primer (RA5R) <400> 6 tctgcgtcta gaccggaatc gtccggtcgc 30 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer (eryBVF) <400> 7 accctaggcg gacggtggcc gccctga 27 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR Primer (eryBVR) <400> 8 gtggatccgg aaaccgctag ccggcgtg 28 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR Primer (actlF) <400> 9 aaacaattgc tcgagtttaa acagctcgg 29 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> PCR Primer (actlR) <400> 10 aaagatctcc tgcaggtacc taggttaatt aatcgatgcg ttc 43 <210> 11 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR Primer (pikCF) <400> 11 aagaattcct aggtttccgt tccgcttccg gcccggt 37 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> PCR Primer (pikCR) <400> 12 aagaattcgt aatgggtgac gtgcgggttc aa 32  

Claims (1)

Saccharopolyspora erythraea in Saccharopolyspora erythraea acts on erythronolide B and deletes the eryBV gene encoding the glycosyltransferase that attaches the L-mycarose sugar chain. Encoding cytochrome P450 monooxygenase in the pikromycin biosynthetic pathway from Streptomyces venezuelae in eryBV deletion strains A method for combinatorial synthesis of 5-O-desosaminyl erysonolide A, characterized by extracting from a culture medium a strain further expressing pikC heterogenes.

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