KR100629896B1 - Recombinant streptomyces strain having high-producing streptomyces doxorubicin and a dna microarray system for analysis of an expresstion profiles of streptomyces doxorubicin biosynthetic gene cluster - Google Patents

Abstract

The present invention provides a recombinant vector for mass production of doxorubicin, a transformed actinomycetes transformed with the recombinant vector, a DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis including a doxorubicin biosynthesis related gene, and a gene analysis using the DNA microarray chip. It is about a method. The recombinant vector of the present invention and the transformant transformed with the recombinant vector may be utilized for mass production of doxorubicin, and through the DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis of the present invention and a gene analysis method using the same. Genes for mass production of doxorubicin could be selected. The DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis of the present invention and a gene analysis method using the same may be widely used for more efficient doxorubicin production.

Doxorubicin, Actinomycetes, Microarray Chip

Description

RECOMBINANT STREPTOMYCES STRAIN HAVING HIGH-PRODUCING STREPTOMYCES DOXORUBICIN AND A DNA MICROARRAY SYSTEM FOR ANALYSIS OF AN EXPRESSTION PROFILES OF DONE

1A is a schematic diagram showing the biosynthetic pathway of doxorubicin.

1B is a physical functional map of doxorubicin biosynthesis related genes.

2 is a cleavage map of expression vectors pESK403 and pESK404 for high production of doxorubicin.

Figure 3a is S. peucetius / pSE34 transformed with pSE34 (- ◆ -), the S. peucetius / pESK403 transformed with pESK403 (- ■ -) and transformed S. peucetius / pESK404 to pESK404 (- ▲ -) Is a graph showing the degree of production of doxorubicin according to the incubation time in

Figure 3b shows S. peucetius / pSE34 (-VIII) transformed with pSE34, S. peucetius / pESK403 (-VIII) transformed with pESK403 and S. peucetius / pESK404 (-VIII) transformed with pESK404 It is a graph showing the degree of production of danarubisin with the incubation time in.

4 is a photograph of a DNA microarray chip chip in which doxorubicin biosynthesis-related genes have been deposited. (A) Cyto-61 labeled DNA chip; (B) DNA chip comparing gene expression patterns of T ₀ to T ₂ of S. peucetius / pESK404.

5 is a graph showing the growth rate of S. peucetius / pESK403 and S. peucetius / pESK404 in liquid NDYE medium. T ₀ -T ₂ represents the harvest time of the culture for total RNA isolation.

FIG. 6A shows the logarithms of the red fluorescence to green fluorescence intensity ratios of the expression patterns of the doxA gene (-◆-) and the total expression of doxorubicin biosynthesis related genes (-■-) in S. peucetius / pESK403 [log ₂ (red / green)] is a graph of microarray chip analysis results.

FIG. 6B shows the logarithm of the red fluorescence to green fluorescence intensity ratio of the expression patterns of the dnrI gene (-▲-) and the average expression of the total doxorubicin biosynthesis related genes (-■-) in S. peucetius / pESK404 [log ₂ (red / green)] is a graph of microarray chip analysis results.

Figure 7a shows the expression patterns of DnrI dependent genes dnrG (-◆-) , dpsA (-●-) and dpsE (-▲-) in S. peucetius / pESK403 and the average expression patterns of all doxorubicin biosynthesis related genes (-■ This is a graph of microarray chip analysis showing-) as the log value of the red fluorescence to green fluorescence intensity ratio (log ₂ (red / green)).

7b isS. peucetius/ pESK403 in the doxorubicin resistance genedrrA(-▲-), drrB(-●-), drrCThe expression patterns of (-◆-) and the average expression patterns (-■-) of all doxorubicin biosynthesis related genes are calculated as log values of the ratio of red fluorescence to green fluorescence intensity.₂(red / green)] is a graph of microarray chip analysis results.

FIG. 7C shows the logarithm of the red fluorescence to green fluorescence intensity ratio (log ₂ (red) for the expression patterns of dnmL gene (-◆-) and total doxorubicin biosynthesis-related genes in S. peucetius / pESK403 [log ₂ (red) / green)] is a graph of microarray chip analysis results.

Figure 8a shows the expression patterns of DnrI dependent genes dnrG (-◆-) , dpsA (-●-) and dpsE (-▲-) in S. peucetius / pESK404 and the average expression patterns of all doxorubicin biosynthesis related genes (-■ This is a microarray chip analysis graph showing-) as the log value of the red fluorescence to green fluorescence intensity ratio [log ₂ (red / green)].

8b isS. peucetius/ pESK404 is a doxorubicin resistance genedrrA(-▲-), drrB(-●-), drrCThe expression patterns of (-◆-) and the average expression patterns (-■-) of all doxorubicin biosynthesis related genes are calculated as log values of the ratio of red fluorescence to green fluorescence intensity.₂(red / green)] is a graph of microarray chip analysis results.

FIG. 8C shows the logarithms of the red fluorescence to green fluorescence intensity ratios of the expression patterns of dnmL gene (-◆-) and total doxorubicin biosynthesis related genes (-■-) in S. peucetius / pESK404 [log ₂ (red / green)] is a graph of microarray chip analysis results.

The present invention relates to a microarray chip for analyzing the expression pattern of the biosynthetic gene group of doxorubicin and a method for analyzing the expression pattern of the doxorubicin biosynthetic gene group using the same.

Doxorubicin is an anthracycline type II polyketide compound biosynthesized by Streptomyces peucetius , which inhibits the action of DNA topoisomerase II and thus double-strands DNA. It is known to play a role as an anticancer agent through a mechanism of destroying (Arcamone, F., Med. Res. Rev., 4: 153-188, 1984). Doxorubicin produced by S. peucetius is biosynthesized through various enzymatic reactions encoded by about 40 genes clustered in the genome, which is first expanded with a molecule of propionyl coenzyme A (starter unit). Nine malonyl coenzymes A (malonyl CoA), which is an extender unit, are condensed continuously to form polyketide chains. The polyketide skeleton epsilon-rhodomycinone (ε-rhodomycinone) is biosynthesized by cyclization, dehydration and hydroxylation reactions (see FIGS. 1A and 1B), and the biosynthesized epsilon-rhodomycinone The next step is methylation, hydroxylation, decarboxylation, and glycosylation, which is known to be finally biosynthesized into doxorubicin (Grimm, A. et al ., Gene , 151: 1-10, 1994; Hutchinson, CR et al ., J. IND. MICROBIOL. BIOT., 23: 647-652, 1999). In addition to biosynthetic genes, as another important gene family involved in the production of doxorubicin, resistance genes and pathway specific regulatory genes have been reported. The doxorubicin resistance gene product of S. peucetius is known to be composed of DrrA, DrrB, DrrC, and DrrD. Among them, DrrA, an ABC transporter, is paired with DrrB to excrete the synthesized doxorubicin out of cells. (Kaur, P. and Russell, J., J. Biol. Chem., 273: 17933-17939, 1998; Lomovskaya, N. et al., J. Bacteriol., 178: 3238-3245, 1996). The regulatory mechanisms associated with doxorubicin biosynthesis in S. peucetius also consist of cascades of DnrO, DnrN, and DnrI, the last of which is directly linked to the promoter portion of doxorubicin biosynthesis-related genes. It is reported to play a role in promoting the expression of doxorubicin biosynthetic gene (Furuya, K. et al ., J. Bacteriol. 178: 6310-6318, 1996; Otten, SL et al ., Microbiology , 146: 1457-1468 , 2000). However, the regulatory mechanism of doxorubicin is known only to a few genes that are directly related to the biosynthetic process, and as in most actinomycetes, the regulatory mechanisms at higher levels associated with secondary metabolic processes have not been identified.

Strain improvement strategies for improving useful product productivity in actinomycetes, including doxorubicin, which produce industrially useful products, are still being used as mutagens such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or ultraviolet light. Has been relied on for mutagenesis and screening methods. However, since the method is randomized regardless of the understanding of the molecular genetic characteristics of the regulatory genes involved in biosynthesis, it is more systematic and effective by comprehensively comparing the expression of all gene groups involved in doxorubicin biosynthesis. A strain improvement strategy at the molecular level is needed (Rodriguez, E. et al., J. Ind. Microbiol. Biotechnol., 30: 480-488, 2003). In particular, recently, DNA microarray chip (microarray) is erythromycin (erythromycin) and Streptomyces plastic diae (Streptomyces fradiae) to and using the system to produce pneumolysin spokes la Erie tray (Saccharopolyspora erythreae) a saccharide productivity industrial strains Cases of successful analysis of the biosynthetic gene family expression of tylosin produced are reported (Lum, AM et al ., Metabolic Engineering , 6: 186-196, 2003). However, a method of analyzing the expression pattern of doxorubicin biosynthesis genes in actinomycetes using a microarray chip system has not been developed until now.

Accordingly, the present inventors first confirmed that the expression rate of doxorubicin in actinomycetes is greatly improved as a result of transforming a specific biosynthetic pathway gene ( dOx ) involved in pathway specific regulatory gene ( dnrI ) or precursor conversion, thereby finding a highly productive doxorubicin recombinant strain. A DNA microarray chip containing 35 genes involved in doxorubicin biosynthesis of actinomycetes was prepared, and the doxorubicin biosynthetic gene group expression pattern of the doxorubicin high-productive recombinant strain was analyzed using the DNA microarray chip, thereby producing doxorubicin. The present invention was completed by confirming that genes that play a crucial role in improvement can be selected.

The first object of the present invention is to provide an expression vector for actinomycetes comprising a gene associated with doxorubicin biosynthesis.

A second object of the present invention is to provide a doxorushin high productivity transformed strain transformed with the expression vector.

A third object of the present invention is to provide a DNA microarray chip in which genes involved in actinomycetes doxorubicin biosynthesis are integrated.

A fourth object of the present invention is to provide a method for comparatively analyzing the expression patterns of doxorubicin biosynthetic gene groups using the DNA microarray chip.

It is a final object of the present invention to provide a method for selecting genes related to doxorubicin productivity enhancement using the analytical method.

In order to achieve the above object, the present invention provides an expression vector for actinomycetes comprising a gene involved in doxorubicin biosynthesis.

The present invention also provides a doxorubicin high productivity transformed strain transformed with the expression vector.

In addition, the present invention provides a DNA microarray chip in which the doxorubicin biosynthesis related gene of actinomycetes is integrated.

In addition, the present invention provides a method for analyzing the expression of doxorubicin biosynthesis related genes using the DNA microarray chip.

Furthermore, the present invention provides a method for selecting genes related to doxorubicin productivity improvement using the above assay method.

Hereinafter, the present invention will be described in detail.

The expression vector for actinomycetes of the present invention comprises a path specific regulatory gene ( dnrI ) or a precursor conversion biosynthesis gene ( doxA ) of actinomycetes . In this case, it is preferable that the expression vector containing the dnrI pESK404 that the cleavage map as shown in Figure 2 is preferred and, pESK403 having a cleavage map shown expression vectors containing doxA Figure 2.

The transforming strain of the present invention is not particularly limited, but is preferably S. peucetius / pESK404 transformed with pESK404 or S. peucetius / pESK403 transformed with pESK403.

DNA microarray chip for analyzing the expression pattern of the doxorubicin biosynthesis related gene of the present invention includes a solid substrate in which one or more doxorubicin biosynthesis related genes are stored. In this case, the substrate is not particularly limited thereto, but is preferably a glass substrate, and is preferably coated with a cationic polymer for facilitating DNA binding, and the cationic polymer is not particularly limited thereto. Preference is given to -L-lysine. In addition, the DNA microarray chip of the present invention is not particularly limited thereto, but it is preferable to further include a control gene in order to improve the reliability of the analysis result, the control gene is not particularly limited thereto, but a positive control gene and negative It is preferable to include all control genes, and the negative control gene is not particularly limited to genes present in actinomycetes, and the positive control genes are housekeeping genes expressed at a constant level in actinomycetes, but are not particularly limited thereto. It is preferable that it is 16s rRNA of actinomycetes.

Doxorubicin biosynthesis-related gene expression analysis method of the present invention comprises the steps of (i) preparing a DNA microarray chip in which one or more doxorubicin biosynthesis-related genes are deposited; (Ii) extracting total RNA from doxorubicin high productivity strains obtained by varying incubation times; (Iii) preparing cDNAs labeled with different fluorescent materials by using reverse transcription from each extracted total RNA; (Iv) competitively hybridizing the cDNAs labeled with the respective fluorescent materials with the DNA microarray chips prepared in step iii; (Iii) detecting each fluorescence after washing and (iii) comparing the intensity of each fluorescence.

In this case, the doxorubicin biosynthesis-related genes are not particularly limited thereto, but dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , dnrF , dnmT , dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC , preferably the doxorubicin high productivity strains Is not particularly limited thereto, but is preferably S. peucetius / pESK404 or S. peucetius / pESK403 of the present invention, the incubation time is not particularly limited thereto, but 24 hours after inoculation before doxorubicin is produced. to T _0, and after the time of inoculation doxorubicin is created and for 48 hours in T _1, it is preferable that 80 hours after inoculation in T _2. On the other hand, the fluorescent material is not particularly limited thereto, but is preferably fluorescein, rhodamine, green fluorescent protein, Cy-3 or Cy-5, and more preferably Cy-3 or Cy-5. In a preferred embodiment of the invention, the total RNA extracted from the doxorubicin high production strain obtained at time point T ₀ is labeled with Cy-3 and the total RNA extracted from the doxorubicin high production strain obtained at time point T ₁ or T ₂ is It is labeled Cy-5. Hybridization methods of step iii are already known in the art. Typically, fluorescently labeled cDNA is mixed with a hybridization solution and left in boiling water to separate DNA double strands, applied to a DNA microarray chip, and then reacted at an appropriate temperature (40 to 70 ° C.) for 8 to 20 hours. . It is then washed several times, dried and the fluorescence is detected with a fluorescence scanner. The step of comparing the fluorescence intensity of step VII may be performed with conventional analysis software of a computer connected to the fluorescence scanner.

In addition, the method of selecting genes related to the productivity improvement of doxorubicin of the present invention comprises the steps of: (i) preparing a DNA microarray chip in which one or more doxorubicin biosynthesis related genes have been deposited; (Ii) extracting total RNA from doxorubicin high productivity strains obtained by varying incubation times; (Iii) preparing cDNAs labeled with different fluorescent materials by using reverse transcription from each extracted total RNA; (Iv) competitively hybridizing the cDNAs labeled with the respective fluorescent materials with the DNA microarray chips prepared in step iii; (Iii) detecting each fluorescence after washing; (Iii) comparing the intensities of each fluorescence and (iii) selecting genes that have a greater fluorescence intensity or no change in expression pattern as compared to the control.

In this case, the doxorubicin biosynthesis-related genes are not particularly limited thereto, but dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , dnrF , dnmT , dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC , preferably the doxorubicin high productivity strains Is not particularly limited thereto, but is preferably S. peucetius / pESK404 or S. peucetius / pESK403 of the present invention, the incubation time is not particularly limited thereto, but 24 hours after inoculation before doxorubicin is produced. to T _0, and after the time of inoculation doxorubicin is created and for 48 hours in T _1, it is preferable that 80 hours after inoculation in T ₂ (see Fig. 5). On the other hand, the fluorescent material is not particularly limited thereto, but is preferably fluorescein, rhodamine, green fluorescent protein, Cy-3 or Cy-5, and more preferably Cy-3 or Cy-5. In a preferred embodiment of the invention, the total RNA extracted from the doxorubicin high production strain obtained at time point T ₀ is labeled with Cy-3 and the total RNA extracted from the doxorubicin high production strain obtained at time point T ₁ or T ₂ is It is labeled Cy-5. Hybridization methods of step iii are already known in the art. Typically, fluorescently labeled cDNA is mixed with a hybridization solution and left in boiling water to separate DNA double strands, applied to a DNA microarray chip, and then reacted at an appropriate temperature (40 to 70 ° C.) for 8 to 20 hours. . It is then washed several times, dried and the fluorescence is detected with a fluorescence scanner. The step of comparing the fluorescence intensity of step VII may be performed with conventional analysis software of a computer connected to the fluorescence scanner.

The present inventors have found that a specific biosynthetic pathway gene (doxA) which is involved in the path specific regulatory gene (dnrI) or conversion of the precursor gene involved in doxorubicin biosynthesis ermE ^* Actinomycetes were transformed with a pESK404 or pESK403 expression vector prepared by linking a promoter to prepare a high productivity strain of doxorubicin. The transformed doxorubicin high productivity strain had a productivity of up to 5.5 times higher than that of the control group (FIG. 6). The transformed actinomycetes strain can be used directly for high efficiency production of doxorubicin, and can be usefully used for analyzing the expression patterns of gene groups related to doxorubicin biosynthesis.

In addition, the inventors of the present invention are dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , which are genes known to be related to doxorubicin biosynthesis. poly-L-lysine coated by PCR amplifying dnrF , dnmT , dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC A DNA microarray chip for analyzing the expression pattern of doxorubicin biosynthesis related genes was prepared by dropping onto a glass substrate. After inoculation of the high productivity strain of doxorubicin, 24 hours after the production of doxorubicin (T ₀ ), it is a time point at which doxorubicin is produced. After 48 hours (T ₁ ) and 80 hours (T ₂ ), the total RNA was extracted from the strain, respectively, and the total RNA extracted at the time point T ₀ was Cy-5, and the total RNA extracted at the time point T ₁ and T ₂ was CDNA was prepared by labeling with Cy-3, and then the DNA. And to the micro array chip competitive hybridization with a fluorescently labeled cDNA produced by varying the time point, after washing were compared with the fluorescence intensity emitted from each fluorophore. As a result, it was possible to select a group of genes whose gene expression is increased above the mean and a group of genes whose gene expression level did not change (see Table 2). In particular, in the case of dnmL , which is a gene whose gene expression does not change, glucose-1-phosphate thymidylyltransferase (glucose-1-phosphate) involved in synthesizing TDP-daunosamine, a deoxysaccharide portion of doxorubicin, thymidylyltrasferase) is a gene encoding a dnrI binding site is known to exist in the promoter region, it was confirmed that there is no change in its expression in the strain overexpressing the actual dnrI (see FIGS. 7c and 8c). This suggests that when an independent expression cassette containing the gene is produced and transformed simultaneously with the dnrI gene, synergistic effects may be exhibited on doxorubicin productivity.

As described above, the DNA microarray chip including the doxorubicin biosynthesis-related genes of the present invention and the gene expression pattern analysis method using the same allow more systematic analysis of the expression patterns of genes related to the biosynthesis of doxorubicin, and these genes. It can be usefully used to determine the functional relationship between the liver, and furthermore, it can be useful in establishing a strategy for high efficiency production of doxorubicin.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1> Preparation of Doxorubicin High Productivity Strains

The introduction of the dnrI gene, which specifically regulates the doxorubicin biosynthesis process, has already been reported to increase doxorubicin productivity (Park, H.-S. et al., J. Microbiol. Biotechnol., In press, Stutzman-Engwall , KJ et al., J. Bacteriol., 174: 144-154, 1992). However, since this report is simply the result obtained by introducing the dnrI gene in a multicopy form, the inventors have found that ermE ^* By cloning and introducing the dnrI gene into pSE34 (Pfizer Inc, USA), an actinomycete expression vector containing a promoter, it was intended to induce dnrI expression in cells stably and efficiently. In addition, the doxA gene, which is involved in the bioconversion of doxorubicin from the precursor of doxorubicin, was also cloned into pSE34 to enhance the bioconversion efficiency of danarubicin to doxorubicin (Lomovskaya, N. et al., J. Bacteriol. , 181: 305-318, 1999).

To this end, pathway specific regulatory genes ( dnrI ) and precursor converting biosynthetic genes ( doxA ) were cloned from the S. peucetius ATCC29050 strain in the following manner:

First, the S. peucetius ATCC29050 strain was incubated in R2YE medium for 5 days to isolate genomic DNA by Hopwood et al. (Hopwood, DA et al., Genetic Manipulation of Streptomyces , A Laboratory Manual, Norwich, UK, John Innes Foundation, 1985). 5'-GGATCCGGTCGAACTGCGGGAGGGG-3-3 '(forward primer doxA- 1, SEQ ID NO: 1) and 5' for doxA as a primer for amplifying doxA and dnrI from the genomic DNA, respectively, by polymerase chain reaction (PCR). -AAGCTTGCGGATCAGCGCAGCCAGAC-3 '(reverse primer doxA -2, SEQ ID NO: 2) was used, with respect to dnrI 5'-GGATCCGGAAATATGCCACCTGCTGA-3' (forward primer dnrI -1, SEQ ID NO: 3) and 5'-AAGCTTGTGGACACGTGACTCCTTGT-3 '( Reverse primer dnrI- 2, SEQ ID NO: 4) was used. Polymerase chain reaction was repeated 30 times using Rapid Thermocycler ^™ (Idaho Technology, USA) for 30 seconds at 96 ° C., slow cooling at 40 ° C. for 30 seconds, and elongation at 35 ° C. at 68 ° C. (Kim, E. -S. et al., J. Bacteriol ., 177: 1202-1207, 1995). The PCR product amplified by the reaction was cloned into pGEM T-easy (Promega, USA) vector, and then transformed into E. coli DH5α. Plasmid DNA was isolated from the transformed E. coli DH5α, digested with Bam H I and Hin d III present in the multicloning site of the pGEM T-easy vector to extract dnrI and doxA gene segments, and then, respectively, pSE34 ( Pfizer Inc, USA) (Figure 2). The plasmid vectors prepared as described above were named as pESK403 and pESK404, respectively, and were transformed into S. lividans TK21 strain by PEG transformation method through protoplasm (Kim, E.-S. et al., J. Bacteriol ., 177: 1202-1207, 1995). Transformed strains were selected using 0.5 mg / ml thiostrepton. On the other hand, in order to prepare a highly productive strain of doxorubicin, strains of S. peucetius ATCC27952, which do not produce doxorubicin, were transformed with pESK403 and pESK404, respectively, isolated from the transformed S. lividans TK21 strains. It was named S. peucetius / pESK403 and S. peucetius / pESK404. The strain was stored at −20 ° C. as a stock containing 20% glycerol, and the stock was stored in 25 ml of NDYE medium (4.5% maltose, 0.5% yeast extract, 0.48% HEPES, 0.43). NaNO ₃ , 0.023% K ₂ HPO ₄ , 0.012% MgSO ₄ , 0.2% 10X trace element solution pH7.4) and incubated at 30 ℃, 200rpm was used for subsequent analysis.

Example 2 Quantitative Analysis of Doxorubicin Production of Doxorubicin High Productive Strains According to Culture Time

S. peucetiusATCC27952 and the high productivity strains prepared in Example 1S. peucetius/ pESK403 andS. peucetiusDoxorubicin produced by / pESK404) was quantified by high-performance liquid chromatography (HPLC) using a C-18 reversed phase (250 x 4.6 mm) column (Waters, Ireland) in the following manner: First, a predetermined amount of the culture solution was taken, a 1-volume extract (Iso-propyl alcohol: 30% HCl = 50: 1) was mixed, stirred for 30 minutes, and centrifuged at 15000 rpm for 10 minutes to prepare an extract. Subsequently, as a mobile phase for HPLC analysis, 1.327 g / l of sodium dodecyl sulfate and 0.68 ml / l of phosphoric acid were mixed in an acetonitrile: distilled water (1: 1 v / v) mixture. The flow rate was 1 ml / min and the column temperature was maintained at room temperature to quantify the production of doxorubicin at a wavelength of 254 nm with a UV detector (FIGS. 3A and 3B).

Figure 3a is S. peucetius / pSE34 transformed with pSE34 (- ◆ -), the S. peucetius / pESK403 transformed with pESK403 (- ■ -) and transformed S. peucetius / pESK404 to pESK404 (- ▲ -) Figure 3b is a graph showing the degree of production of doxorubicin according to the incubation time in, S. peucetius / pSE34 (-▤-) transformed with pSE34, S. peucetius / pESK403 (-▥-) transformed with pESK403 and It is a graph showing the production level of danarubicin according to the incubation time in S. peucetius / pESK404 (-▨-) transformed with pESK404.

As shown in FIGS. 3A and 3B, the S. peucetius / pESK404 to which the pathway-specific regulatory gene dnrI was introduced was found to have a productivity increase of about 5.5 times compared to S. peucetius / pSE34 containing only a vector as a control. In the case of S. peucetius / pESK403 in which doxA , a precursor conversion biosynthesis gene, was introduced, the productivity of doxorubicin was increased by about 2.5 times compared to the control group. As a result of measuring the productivity of dolanrubicin, a precursor of doxorubicin, it was confirmed that trace amounts of danarurubicin are produced by S. peucetius / pESK403 until the 3rd day. Is believed to have been bioconverted to doxorubicin. On the other hand, Danarubicin in S. peucetius / pESK404 was found to be produced at high concentrations until day 3, which suggests that doxorubicin biosynthesis is continued until late cell growth by DnrI. However, S. peucetius / pESK403 For that reason, unlike New Ruby Dana continued productivity in the S. peucetius / pESK404 is because the expression of my most DoxA cells extremely low Dana Ruby God does not remain switched production to doxorubicin I think it is. These results suggest the possibility of improving the productivity of useful physiologically active substances by smoothing the metabolic flux of regulatory genes or biosynthesis.

<Example 3> doxorubicin biosynthesis gene group PCR amplification and DNA microarray chip production

Doxorubicin biosynthetic gene sequences for DNA microarray chip fabrication were obtained from the Entrez Nucleotides Database at the National Center for Biotechnoloy Information (NCBI), and primers for PCR amplification. Was constructed to include all ORFs of each gene (Table 1). PCR conditions for amplifying each gene, No. 1: separation for 30 seconds at 96 ° C, slow cooling for 30 seconds at 40 ° C, elongation for 35 seconds at 68 ° C; No. 2: separation at 96 ° C. for 30 seconds, slow cooling at 55 ° C. for 30 seconds, elongation at 68 ° C. for 35 seconds; No. 3: three seconds of separation at 96 ° C for 30 seconds, slow cooling at 50 ° C for 30 seconds, and elongation at 68 ° C for 60 seconds. Primer sequences and PCR conditions for each gene are summarized in Table 1 (* purchased commercially available products (Stratagene, USA)). Meanwhile, the DNA chip contained 35 doxorubicin biosynthesis genes and three positive controls. Two positive genes from plants and 16S rDNA from S. peucetius were used as positive controls. Each gene product amplified by PCR was cloned into a pGEM T-easy (Promega, USA) vector and sequenced to confirm the DNA sequence. Two genes, dnrU and dumQ, were identified as different genes during sequencing and were excluded from the DNA microarray chip fabrication process. Each gene cloned into the pGEM T-easy vector was amplified by PCR using a universal primer included in the vector and used as a sample for DNA microarray chip fabrication. The DNA microarray chip was a slide glass coated with poly-L-lysine, and Pixsys 5500 ^TM (Cartesian Technologies Inc., USA) was used for the drip of the gene. Genepix Personal 4100A ^™ (Axon Instruments Inc., USA) was used for scanning.

Doxorubicin biosynthesis related genes and primers for their amplification gene Drop location Gene products PCR primers SEQ ID NO: PCR conditions line Heat dnmZ One One Potential Flavoproteins Forward: catatgacaaagccatctgtgcacg Reverse: tcatcggcccaacgccacttcgctc 5 6 One dnrD One 2 Methylaclanonic acid cyclase Forward: catatgagcacgcagatcgatctgg Reverse: tcaggggcgccagccctcgggggtg 7 8 One dnmJ One 3 Potential Protein Kinases Forward: catatgtccacctacgtctggcaat reverse: tcacaggcttccgacgacctcccgc 9 10 One dnmL One 4 Glucose-1 Phosphate Transferase Forward: catatgaaggctctcgtcctgtccg Reverse: tcatgaggagatctccaccctgctg 11 12 One dnrN One 5 Regulatory protein Forward: catatgaccatccgagtcgtgattg Reverse: tcagatccagccggacatactggcg 13 14 One dpsG One 6 Danarubicin acyl carrier protein Forward: catatggctgagctcagcctggcgg Reverse: tcatgccgccgctcgcagcctctcg 15 16 One dpsH One 7 Danarubicin biosynthetic enzyme Forward: catatggaccgacggttgtccgctc Reverse: tcagtccagcccgtcccgggtgacg 17 18 One dnrE One 8 Function unknown Forward: catatggagaacacacagcgatccg Reverse: tcacgtcaccgagagacccccgtcc 19 20 One drrB One 9 Danarubisin resistant protein Forward: catatgacgacgtcccccggcaccg Reverse: tcagtgggcgttcttgttgcggtac 21 22 One dpsY One 10 Polyketide cyclase Forward: catatgtgctgtggtggcccgagag Reverse: tcattcgtcgacgaaggcgaccgcg 23 24 One drrA 2 One Danarubisin resistant protein Forward: catatgaacacgcagccgacacggg Reverse: tcatgccaccttctcctcttccgcc 25 26 One drrD 2 2 Danarubisin resistant protein Forward: catatgagcggcgcacctactgtcc Reverse: tcacgaaggcgcccccgtagccgcg 27 28 One dpsF 2 3 Danarubicin-doxorubicin polyketide synthase Forward: catatgtccgagttgccgcttcagc Reverse: tcaggcgctggactcggcgtactgc 29 30 One dpsD 2 4 Danarubicin-doxorubicin polyketide synthase Forward: catatggatgcgcccgcgattctcc Reverse: tcaccgcccgggtcgcccactgagc 31 32 One dnrI 2 5 Potential transcriptional regulators Forward: ggatccggaaatatgccacctgctga Reverse: aagcttgtggacacgtgactccttgt 33 34 One doxA 2 6 Danarubicin C-14 hydroxylase Forward: ggatccggtcgaactgcgggaggggt Reverse: aagcttgcggatcagcgcagccagac 35 36 One dnrM 2 7 TDP-D-glucose-4,6-dehydratase Forward: catatgaccatgaagatcctggtga Reverse: tcaggatccggtgagcggtgcccac 37 38 2 dnrO 2 8 Potential suppressor Forward: catatgaccgagacgccatcccgca Reverse: tcacccggctgctggcccgctgacc 39 40 2 dnrF 2 9 Aclavinone C-11 hydroxylase Forward: catatggccttgacgaagccggatg Reverse: tcagttcgccggagggggcggcgac 41 42 2 dnmT 2 10 Doxorubicin biosynthetic protein DnmT Forward: catatgaccgcccagatcgcccggt Reverse: gaactcacctccggggcggggcgga 43 44 2 dnmV 3 One Thymidine Diphosphate-4-keto-2,3,6-tridioxy-hexelosose reductase Forward: catatgcgggtcgtggttctggggg Reverse: ctaggccggggcgccgtgcgcggtc 45 46 2 dnrG 3 2 Anthraquinol monooxygenase Forward: catatgccacagccagaaccgaacg Reverse: ttcacgtcgtccgccctccgggccc 47 48 2 dpsA 3 3 Danarubicin-doxorubicin polyketide synthase Forward: catatgaaccggcggatcgtcatca Reverse: tcacctctcgagacggctcagcagc 49 50 2 dpsB 3 4 Danarubicin-doxorubicin polyketide synthase Forward: catatgacaggtaccgcggcacgga Reverse: tcacgccgccccccggaccaccaga 51 52 2 dnrV 3 5 Doxorubicin biosynthetic protein DnrV Forward: catatgaccaggttcgcgcccggcg Reverse: tcgccgctcacgaggcaccgcccgc 53 54 2 dpsE 3 6 Danarubicin-doxorubicin polyketide synthase Forward: catatgtccgaggcagccgaccggg Reverse: tcagtagctgccgaggccaccacag 55 56 2 dnrH 3 7 Potential Baumycin Biosynthetic Protein Forward: catatgcgcgtcctgttcgccacca Reverse: ctaggcaccgccggtggaaggcgac 57 58 2 dnrK 3 8 Caminomycin O-Methyltransferase Forward: catatgacagccgaaccgacggtcg Reverse: tcgtgtgtcaggcgccggtggccgc 59 60 2 dnrP 3 9 10-carbomethoxy-13-dioxycarminomycin esterase Forward: catatgccgacacgcatgatcacca Reverse: tcacactgtcgcggcccgggtgtgg 61 62 2 dnmS 3 10 Glycosyl Transferase Forward: catatgaaggtgctcgtgacggcgt Reverse: aaagcccctagtgccggacgccctg 63 64 2 dpsC 4 One Danarubicin-doxorubicin polyketide synthase Forward: catatgagcgtgccgcagggggcgc Reverse: atgacgggctcctcaccgggcggcc 65 66 2 dnrX 4 2 Danarubicin / doxorubicin biosynthetic enzyme Forward: catatggagccgaacgaatcgacat Reverse: tcaccctgagcggatccagcggccc 67 68 3 dnmU 4 3 Potential epimerase Forward: catatgaaggcgcgggaactggcgg Reverse: tcacccgtctccgcgtgacccggcc 69 70 3 dnrC 4 4 Function unknown Forward: catatgcaggattcgtcctacaaag Reverse: tcagtgggcgacccgggcggtgacg 71 72 3 drrC 4 5 Danarubicin resistant protein (UvrA-like protein) Forward: catatgcagcagcaggctattcaga reverse: aaggatcagcgccgggcccggtggt 73 74 3 dnrU - - Tanarubicin C-13 ketoriductase Forward: catatgacggcctccaccccgcacc Reverse: tcagtgcgcggtgtcgccgacggcg 75 76 2 dnmQ - - Function unknown Forward: catatgcccacacccacgtccgcgc Reverse: tcacttctgggccagccgcagcgag 77 78 3 Control group 1 4 6 S. peucetius 16S rDNA (positive control) Forward: agagtttgatcmtggctcag Reverse: ggctaccttgttacgactt 79 80 2 Control 2 4 7 Plant genes (Arabidopsis thaliana system I chlorophyll a / b-binding protein (X56062)) (positive control)                _  _ * Control group 3 4 8 Plant genes (Arabidopsis Thaliana RUBISCO Activase (X14212)) (positive control)                _  _ *

Example 4 Gene Expression Pattern Analysis Using DNA Microarray System

The high productivity strain of doxorubicin prepared in Example 1 was cultured, and total RNA was extracted using a total RNA separation kit (RNeasy ^TM Mini Kit, QIAGEN, Germany) according to a specific incubation time, and then used as a template for cDNA synthesis. . 24 hours after the inoculation before doxorubicin was produced as the T ₀ time point, cDNA was prepared by labeling the extracted total RNA with a fluorescent substance Cy-3. That point subsequent were positive for the 48 hours after inoculation of the time the doxorubicin production starting T ₁ time, doxorubicin productivity up to the inoculation After 80 hours of the T ₂ the time, T ₁ and T ₂ total extracted at the time of RNA Were each labeled with Cy-5 to prepare cDNA (FIG. 5).

5 is a graph showing the growth rate of S. peucetius / pESK403 and S. peucetius / pESK404 in liquid NDYE medium. At this time, T ₀ -T ₂ represents the harvest time of the culture for total RNA separation.

The labeled cDNA at the T ₀ time point and the cDNA at the T ₁ or T ₂ time point were mixed in a 1: 1 manner and hybridized on the DNA chip in the following manner: First, the total used for cDNA synthesis. RNA was 15 μg each and a random hexamer with 70% GC-content was used as a primer for reverse transcription. For fluorescent labeling, total RNA was first separated by treatment for 10 minutes at 70 ° C., and then immediately stored on ice, and the random primer and reaction solution [5X first stand buffer, 0.1M DTT, dNTP (4mM dATP, 4mM dTTP, 10mM). dGTP, 0.5 mM dCTP), Cy-3 (or Cy-5) -dCTP (PerkinElmer, USA) and reverse transcriptase (SuperScript II, Invitrogen, USA)]. Then, cDNA was synthesized by reacting at 42 ° C. for 2 hours, a stop solution was added, and then left at 65 ° C. for 10 minutes. 3 M sodium-acetate (pH 5.2) and 100% ethanol were added thereto, precipitated in a -20 ° C. freezer for 30 minutes, and cDNA was isolated by centrifugation. Hybridization was performed by mixing the labeled cDNA and the hybridization solution, and then allowed to stand at 65 ° C. for 10 minutes, mixing the cDNA labeled with Cy-3 and Cy-5, respectively, 1: 1, then leaving it in boiling water for 2 minutes and immediately 45 It was left to stand at 20 degreeC for 20 minutes. The cDNA sample was loaded on the DNA chip onto the DNA dripping portion, covered with cover glass, and hybridized at 65 ° C. for about 12 to 16 hours (Huang, J. et al., Genes Dev., 15: 3183-3192, 2001). ). To wash the hybridized DNA chip, it was soaked in 50 ml of 2X SSC buffer containing 0.1% SDS, shaken for 2 minutes, immediately transferred to 50 ml of 1X SSC buffer, and shaken for 3 minutes. Finally, after transferring to 0.2X SSC buffer, shake again for 2 minutes, discard the wash solution, and centrifuged to remove the wash solution components from the hybridized DNA chip. After washing the dried DNA, the fluorescent signal was detected using a DNA chip scanner (Genepix Personal 4100A, Axon Instruments Inc., USA) (Fig. 4).

4 is a photograph of a DNA microarray chip in which doxorubicin biosynthesis related genes have been deposited. At this time, A represents a Cyto-61-labeled DNA microarray chip, and B represents a DNA microarray chip comparing gene expression patterns of T ₀ to T ₂ of S. peucetius / pESK404.

Results analysis was analyzed by grouping the expression patterns of the entire genes by means of hierachical clustering based on the value of the fluorescence intensity of each point analyzed through scanning (Table 2 and FIGS. 6 to 8).

Expression Patterns of Doxorubicin Biosynthesis Related Genes in S. peucetius / pESK403 and S. peucetius / pESK403 gene S. peucetius / pESK403 S. peucetius / pESK404 Ratio of fluorescence intensity Ratio of fluorescence intensity T ₁ / T ₀ T ₂ / T ₀ T ₁ / T ₀ T ₂ / T ₀ dnmZ F ^* 3.053 0.521 2.103 dnrD 1.412 6.638 1.093 3.924 dnmJ F 3.454 1.043 3.584 dnmL F 1.485 0.32 1.329 dnrN F 3.887 0.925 4.848 dpsG F 7.992 0.75 5.674 dpsH F F 0.607 2.334 dnrE F 1.26 F 1.71 drrB F 5.478 0.611 4.4 dpsY F 5.513 0.478 2.417 drrA F 11.05 0.879 8.53 drrD F F F F dpsF F 2.304 F 2.996 dpsD F F F F dnrI 1.361 7.311 1.847 3.808 doxA 1.932 23.951 0.113 3.606 dnrM F 3.029 0.435 2.278 dnrO F 6.606 0.864 6.207 dnrF F F 0.363 1.328 dnmT F 5.447 F 2.191 dnmV F 1.814 F 2.042 dnrG F 10.486 F 99.765 dpsA F 3.054 F 8.578 dpsB F F F F dnrV F F F F dpsE F 5.635 0.575 7.65 dnrH F F F F dnrK 0.025 2.863 1.134 2.203 dnrP F 4.58 0.393 4.082 dnmS F F F 2.704 dpsC F 3.51 0.151 4.992 dnrX F 4.523 0.521 5.062 dnmU F 5.426 0.594 11.619 dnrC F 4.945 0.687 10.942 drrC F 17.99 2.696 35.153

As shown in Table 2, as a result of analysis using a DNA microarray chip, both strains of S. peucetius / pESK403 and S. peucetius / pESK404 showed a similar pattern of expression pattern of the doxorubicin biosynthetic gene group as doxorubicin productivity increased. Seemed. However, at some points no signal was commonly observed (indicated by F ^* in the table: Flagging), which was either at extremely low levels of expression of the genes or randomly used for cDNA synthesis, even though the expression of the genes occurred. It is speculated that enemy hexamers were not suitable for synthesizing cDNAs of all biosynthetic related genes. Both doxA gene of S. peucetius / pESK403 and dnrI gene of S. peucetius / pESK404 induced higher expression than those of other doxorubicin biosynthetic genes. This is the ermE ^* of pSE34 Promoting the gene expression from the early growth of the cell by the promoter has been demonstrated (Fig. 6a and 6b).

In addition,S. peucetiusThe expression pattern of the doxorubicin-related gene group of / pESK403 significantly increased the expression of the resistance gene as well as the expression of the doxorubicin biosynthetic gene as a whole (Fig. 7b).S. peucetiusSimilar results were found for / pESK404 (FIG. 8B). Therefore, the present results suggest the possibility of inducing an overexpression of the resistance gene, leading to an increase in doxorubicin productivity (Dairi, T. et al.,Biosci. Biotechnol. Biochem. 59 (10): 1835-1841, 1995).

Doxorubicin biosynthesis genes regulated by DnrI are known as drrAB, dnrG-dpsABCD, dpsEF, dnrDKPQS, dnmLM operon and dnrC, drrC genes (Otten, SL et al., J. Bacteriol., 177: 1216-1224, 1995; Sheldon, PJ et al., Mol. Microbiol ., 44 (2): 449-460, 2002). In the case of S. peucetius / pESK403, the expression of genes regulated by DnrI was observed, and the expression was similar to that of other doxorubicin biosynthesis genes (FIG. 7A). On the other hand, S. peucetius / pESK404 overexpressing the dnrI gene from the relatively early, it can be seen that most of the genes regulated by DnrI maintain a higher expression level than other biosynthetic genes (Fig. 8a). However, in the case of S. peucetius / pESK404, the dnmL gene, a glucose-1 phosphate thymidylyltransferase involved in synthesizing TDP-daunosamine, the deoxysaccharide portion of doxorubicin, despite overexpressing the dnrI gene Compared to other DnrI dependent genes, it was confirmed that the expression level is significantly low (Fig. 8c). The results were similarly confirmed for S. peucetius / pESK403 (FIG. 7C). As a result of confirming the nucleotide sequence of the promoter portion of dnmL , it was confirmed that the DnrI-binding region was intactly preserved like other dnrI -dependent genes. The results suggest that maximizing the expression of dnmL in S. peucetius biosynthesis process can facilitate the supply of TDP-danosamine , and consequently increase the doxorubicin productivity.

The present inventors have analyzed the doxorubicin biosynthetic gene expression pattern through the DNA microarray chip system as described above, as well as the dnrI gene, which is a path-specific gene that promotes the expression of all biosynthetic genes, as well as the deoxysaccharide synthesis gene regulated by DnrI It was confirmed that stable and steady expression of dnmL and doxorubicin resistance genes drrA, drrB, and drrC are major factors for increasing doxorubicin productivity.

The present invention provides a recombinant vector for mass production of doxorubicin, a transformed actinomycetes transformed with the recombinant vector, a DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis including a doxorubicin biosynthesis related gene, and a gene analysis using the DNA microarray. It is about a method. As claimed and demonstrated above, the recombinant vector of the present invention and transformants transformed with the recombinant vector were capable of mass production of doxorubicin. In addition, the gene for mass production of doxorubicin could be selected through the DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis of the present invention and a gene analysis method using the same. The DNA microarray chip for analyzing gene expression patterns related to doxorubicin biosynthesis of the present invention and a gene analysis method using the same may be widely used for more efficient doxorubicin production.

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Claims (15)

In the expression vector for doxorubicin production, it is represented by the cleavage map of FIG. 2 and includes an expression vector for production of doxorubicin (pESK404) or a precursor conversion biosynthesis gene ( dox A) including a path specific regulatory gene ( dnr I) of actinomycetes. Expression vector for doxorubicin production (pESK403). delete Actinomycetes Streptomyces fusetius transformed with the expression vector for doxorubicin production of claim 1. 4. The transformed actinomycete Streptomyces fusethius according to claim 3, which is S. peucetius / pESK404 or S. peucetius / pESK403. DNA microarray chip for analyzing the expression pattern of doxorubicin biosynthesis related genes comprising one or more doxorubicin biosynthesis related genes. The method according to claim 5, wherein the doxorubicin biosynthesis related genes are dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , dnrF , dnmT for analyzing expression patterns of doxorubicin biosynthesis related genes, characterized in that selected from the group consisting of dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC DNA microarray chips. The DNA microarray chip according to claim 5, further comprising a control gene, for analyzing the expression pattern of doxorubicin biosynthesis related genes. (Iii) preparing a DNA microarray chip in which one or more doxorubicin biosynthesis related genes have been deposited; (Ii) extracting total RNA from doxorubicin high productivity strains obtained by varying incubation times; (Iii) preparing cDNAs labeled with different fluorescent materials by using reverse transcription from each extracted total RNA; (Iv) competitively hybridizing the cDNAs labeled with the respective fluorescent materials with the DNA microarray chips prepared in step iii; (Iii) detecting each fluorescence after washing, and (Iii) Doxorubicin biosynthesis-related gene expression analysis method comprising the step of comparing the intensity of each fluorescence. The method according to claim 8, wherein the doxorubicin biosynthesis related genes are dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , dnrF , Doxorubicin biosynthesis related gene expression pattern analysis method characterized in that it is selected from the group consisting of dnmT , dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC . The method of claim 8, wherein the doxorubicin high productivity strainS. peucetius/ pESK404 orS. peucetius/ pESK403 Doxorubicin biosynthesis-related gene expression analysis method characterized in that. The method of claim 8, wherein the incubation time is T ₀ 24 hours after inoculation before doxorubicin is produced, 48 hours after inoculation, which is the time when doxorubicin is produced, is T ₁ , and 80 hours after inoculation is T _2. Doxorubicin biosynthesis-related gene expression analysis method characterized in that the. The method of claim 8, wherein the fluorescent material is fluorescein, rhodamine, green fluorescent protein, Cy-3 or Cy-5. (Iii) preparing a DNA microarray chip in which one or more doxorubicin biosynthesis related genes have been deposited; (Ii) extracting total RNA from doxorubicin high productivity strains obtained by varying incubation times; (Iii) preparing cDNAs labeled with different fluorescent materials by using reverse transcription from each extracted total RNA; (Iv) competitively hybridizing the cDNAs labeled with the respective fluorescent materials with the DNA microarray chips prepared in step iii; (Iii) detecting each fluorescence after washing; (Iii) comparing the intensity of each fluorescence; and (Iii) A method for selecting genes related to doxorubicin productivity improvement, comprising selecting a gene having a greater fluorescence intensity or no change in expression pattern compared to a control group. The method according to claim 13, wherein the doxorubicin biosynthesis related genes are dnmZ , dnrD , dnmJ , dnmL , dnrN , dpsG , dpsH , dnrE , drrB , dpsY , drrA , drrD , dpsF , dpsD , dnrI , doxA , dnrM , dnrO , dnrF , Screening for genes related to doxorubicin productivity enhancement, characterized in that it is selected from the group consisting of dnmT , dnmV , dnrG , dpsA , dpsB , dnrV , dpsE , dnrH , dnrK , dnrP , dnmS , dpsC , dnrX, dnmU, dnrC and drrC Way. The method of claim 13, wherein the doxorubicin high-producing strain is S. peucetius / pESK404 or S. peucetius / pESK403.

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