KR101197026B1 - Tacrolimus producing strain and Tacrolimus biosynthetic genes obtained by the same - Google Patents

Abstract

The present invention provides a novel Streptomyces sp. Which can produce tacrolimus FK506, which is known to have immunosuppressive activity, with high efficiency. MJM7001 strain and tacrolimus biosynthetic gene obtained from the strain, the tacrolimus producing strain according to the present invention is capable of mass production of tacrolimus in high efficiency, compared to the conventional tacrolimus producing strain, high purity Since it can be isolated, it can be useful for the production of immunosuppressive agents used for the prevention and treatment of rejection by organ transplantation, graft-versus-host disease, autoimmune disease, infectious disease, etc. Tacrolimus biosynthesis genes isolated from them may suggest new possibilities for industrial strain improvement methods suitable for the optimization and mass production of industrially and medically useful bioactive substances.

Description

Tacrolimus producing strain and Tacrolimus biosynthetic genes obtained by the same

The present invention relates to a tacrolimus producing strain and a tacrolimus biosynthetic gene obtained therefrom, and more specifically, tacrolimus (hereinafter, 'tacrolimus FK506' or 'known as having a conventional immunosuppressive activity. New Streptomyces sp. It relates to the MJM7001 strain and tacrolimus biosynthesis gene obtained from the strain.

In order to treat organ transplantation or autoimmune disease, undesirable immune response should be suppressed. In this case, immunosuppressive agents are essential. To date, the most used substance as an immunosuppressive agent is cyclosporin, which is mainly composed of cyclosporin A. However, the cyclosporin A has a problem that can cause side effects such as kidney toxicity, hypertension, diabetes mellitus and post-transplant lymph cell proliferative diseases. Due to this problem, tacrolimus is currently the most popular material.

Tacrolimus was found in fermentation broth of Streptomyces tsukubaensis, a microorganism obtained from soil samples collected in the Tsukuba region of northern Japan, and is an antibiotic substance belonging to the macrolide system having an immunosuppressive effect. to be. Tacrolimus also inhibits the activity of T-cell and interleukin-2 in the human immune system, lowers immunity and shows excellent efficacy without adverse effects on atopic dermatitis, while suppressing rejection after organ transplantation with excellent immunosuppressive effects. It is also attracting attention as a possible immunosuppressive agent, and is expected to replace steroidal drugs causing side effects when taken for a long time.

Known microorganisms producing tacrolimus are known Streptomyces tsukubaensis No.9993, Streptomyces sp. MA 6858, Streptomyces clavuligerus CKD 1119, and the like. However, the production of tacrolimus by such microorganisms had a problem that the yield is low and uneconomical.

Accordingly, the present inventors have screened and improved strains that produce tacrolimus using molecular biological techniques, create high-yield production strains of tacrolimus through mutations of key genes, and increase molecular yield of production yield in mutant strains. As a result of intensive research to present the scientific evidence, the present invention has been reached.

It is therefore an object of the present invention to provide novel strains that produce Tacrolimus compounds with high efficiency.

Another object of the present invention is to provide a tacrolimus biosynthesis gene derived from the novel tacrolimus producing strain.

In addition, another object of the present invention is Streptomyces sp. It is to provide an isolated nucleic acid which is a recombinant vector comprising a nucleic acid encoding a protein domain involved in the synthesis of allylmalonyl-epi required to synthesize tacrolimus from the MJM7001 strain.

It is another object of the present invention to provide a recombinant host cell comprising a recombinant gene coding of the enzyme involved in the synthesis of the allylmalonyl- recipe.

It is another object of the present invention to provide an isolated nucleic acid which is a recombinant vector capable of self-replicating or fusion to a chromosome of a host cell.

In addition, another object of the present invention is Streptomyces sp. Transform host cells using a recombinant vector comprising a nucleic acid encoding a protein domain involved in the synthesis of allylmalonyl-epi required to synthesize tacrolimus from the MJM7001 strain, and allylmalonyl-epi synthase It is to provide a method for producing a polyketide, characterized in that the host cell is cultured under conditions that are produced to catalyze the synthesis of the polyketide.

The object of the present invention as described above is to screen for novel strains that produce tacrolimus, improve the selected strains by mutagenesis and investigate their morphological properties, while optimizing fermentation medium conditions, solid culture and preservation methods and liquids. This was achieved by establishing culture methods, optimizing fermenter conditions, studying tacrolimus biosynthesis due to the addition of precursors, building a genetic library in tacrolimus producing strains, and searching for FK506 biosynthetic gene populations.

The present invention is Streptomyces sp. To produce a tacrolimus compound of the formula (1). Provides the MJM7001 strain.

In addition, the present invention is the Streptomyces sp. Provided is a tacrolimus biosynthesis gene of SEQ ID NO: 1 from MJM7001 strain.

In addition, the present invention Streptomyces sp. An isolated nucleic acid is provided that is a recombinant vector comprising a nucleic acid encoding a protein domain involved in the synthesis of allylmalonyl-epi required to synthesize tacrolimus from a MJM7001 strain.

The present invention also provides a recombinant host cell comprising a recombinant gene coding of the enzyme involved in the synthesis of the allylmalonyl- recipe.

The present invention also provides an isolated nucleic acid which is a recombinant vector capable of self-replicating or fusion to a chromosome of a host cell.

In addition, the present invention Streptomyces sp. Transform host cells using a recombinant vector comprising a nucleic acid encoding a protein domain involved in the synthesis of allylmalonyl-epi required to synthesize tacrolimus from the MJM7001 strain, and allylmalonyl-epi synthase Provided is a method for producing a polyketide, characterized in that the host cell is cultured under conditions that are produced to catalyze the synthesis of the polyketide.

Tacrolimus producing strain according to the present invention can produce a large amount of tacrolimus with high efficiency compared to the conventional tacrolimus producing strain, and can separate tacrolimus with high purity, rejection by organ transplantation, bone marrow transplantation Can be useful for the production of immunosuppressive agents used in the prevention and treatment of graft-versus-host disease, autoimmune diseases, infectious diseases, etc., and the tacrolimus biosynthesis gene isolated therefrom is a bioactive substance useful industrially and medically. It is possible to suggest new possibilities of industrial strain improvement methods suitable for the optimization of productivity and mass production.

1 is a photograph comparing the present invention with the test strain strain.
Figure 2 is an electron micrograph of the strain of the present invention.
3 is a photograph showing colonies having three different colors.
Figure 4 is a photograph comparing the form of colonies in the FK506-containing solid medium.
5 shows the starting strain and high producing strain Streptomyces sp. MJM7001 fermentation concentration is a graph comparing.
6 is a diagram showing the effect of the feeding of Proline and Lysine on FK506 productivity.
7 shows the structure of FK506 and FK520.
8 shows the sequence diagram of the FK506 biosynthesis gene.
FIG. 9 shows a map of recombinant plasmids with genes fkbV , fkbW , fkbX , fkbY inserted that are predicted to be involved in allylmalonyl- epi biosynthesis.
10 is Streptomyces sp. All expressed fkbV, fkbW, fkbX, fkbY. MJM7001 is a graph confirming the increase in tacrolimus productivity.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Screening of Tacrolimus Producing Strains

In this example, a plate dilution technique was used to isolate the microorganisms producing tacrolimus FK506. About 1 g of the soil collected by the rare microorganism bank possessed by the present inventors was put in a sterile test tube, mixed by adding sterilized water, and left for about 10 minutes to obtain a supernatant. After about 10 ⁵ times diluted with sterile water and the resultant supernatant, the yeast in the dilution plate 0.1㎖-malt agar (yeast extract 4 g / ℓ, wort 10 g / ℓ, glucose 4 g / ℓ, Agar 20 g / L, 1 L of distilled water, medium prepared at pH 6.8 to 7.0). Thereafter, after culturing for about 21 days at 28, colonies grown on yeast-malt agar medium were passaged at regular intervals on fresh yeast-malt agar medium using sterile needles.

The colonies grown by passage in the above culture were again incubated for 28 to 10 days, and then, aspergillus fumigatus on the medium where colonies were grown. Aspergillus 0.5 ml of spore suspension of fumigatus KCCM 60027) was applied. At this time, Aspergillus fumigatus KCCM 6002 was used as a candidate strain that inhibited growth by immunosuppressive substances. After applying these strains, colonies were further cultured for 28 days for 2 days to select 350 colonies whose candidates inhibited the growth of Aspergillus fumigators.

As a result, after selecting 108 strains of actinomycetes excluding fungi and bacteria from the selected strains, strains showing immunosuppressive activity were secondarily selected through an immunosuppressive activity test of a BNC company which is a partner company. It was confirmed whether the strain selected primarily from the above produces a material having immunosuppressive activity. Concentration of 0.5x10 ⁶ cells / ml of T-cells in rat spleens in 96-well plates containing RPMI 1640 medium (GIBCO, GrandIsland, NY) containing 10% fetal calf serum inactive by heat treatment Each 200 μL was subdivided. Then, after the allocation of the methanol extract concentrate of the primary starter strain 108 weeks culture solution in the well, 250 ng / ㎖ of Ino azithromycin [EO No pore Ca ^{2 +} ion-conducting material as a kind of (ionophore)) and 10 ng / ml PMA (Peptonized Milk Agar; 1 g of peptonized milk, 1 L of distilled water) was added.

Thereafter, the mixture was reacted with radioisotope 3H-TdR (Thimidine deoxyribose) (S.A. (Specific activity) 5 Ci / mmol; concentration 1 μCi / ml) for about 5 hours, and then cultured for 48 hours at 37 ° C. The culture solution was filtered through glass wool to harvest the cells, and then subjected to radioactivity analysis using a spectrophotometer. At this time, the radioactivity of the control group without the culture solution was set to 100% and compared with the results of the experimental group, and the experiment was repeated three times to increase the accuracy.

As a result, in the experimental group to which the culture medium sample was added, it was confirmed that strain 1 strain showed a result of reducing the radioactivity to 100% or less. That is, it was confirmed that specific components of the strain inhibited the proliferation of T cells, and that the culture sample contained a substance having an immunosuppressive function. Production strain Streptomyces sp. MJM7001 was used as starting strain.
The strain was deposited with the Korean microbiological conservation center with accession number KCCM11116P as of October 18, 2010.

Example 2 improvement by random mutagenesis of tacrolimus producing strain

Starting strain Streptomyces sp. Was cultured in yeast-malt agar medium at 28 ° C. for 7-14 days. Spore suspension was made using sterile water to collect spores of MJM7001.

Specifically, 3 mL of physiological saline was added to the culture plate and spores were collected by platinum. Physiological saline containing spores was put in a Erlenmeyer flask containing glass beads and shaken for 30 minutes. It was then passed through cotton wool to obtain a single spore suspension. To determine the spore concentration, 0.1 mL of the spore suspension was serially diluted in 0.9 mL physiological saline, and 0.1 mL was taken and plated on a plate. Spore concentration was adjusted to 10 ⁶ per ml and induced mutation by UV, HNO _{3 and} other mutation methods, and then plated on yeast-malt agar medium and incubated at 28 ° C. for 10 days. UV transition conditions were irradiated for 30 seconds at a distance of 30cm with 15W UV having a wavelength of 253nm. HNO ₃ Mutation condition is 0.1 mol / L of NaNO ₂ The solution was treated for 10 to 60 minutes. The single colony thus obtained was inoculated again in yeast-malt agar medium, incubated at 28 ° C. for 7-14 days, and then inoculated in a Erlenmeyer flask to carry out liquid culture to confirm final potency. The high production strain MJM7001 with good genetic performance was obtained by assaying the fermentation potency and stability through passage of the high-producing strain obtained several times in slope medium.

Example 3 Investigation of Morphological Characteristics of Production Strains

Streptomyces sp. Although the MJM7001 producing strains were actinomycetes, the culture, morphological, physiological and carbon source availability were examined. As a result, the known strain of FK506 production, Streptomyces tsukubaensis No.9993 and Streptomyces sp .) This is very different from ATCC 55098.

Specifically, the strains isolated on yeast-malt agar medium, potato agar medium, oatmeal agar medium, zappe glucose agar medium, glucose-asparagine agar medium, peptone-glucose agar medium and skim milk powder agar medium, respectively, and then, at 28 ℃ After culturing for 7-14 days, growth, spore formation, colony color and water-soluble pigment were observed. As a result, the growth and spore formation of yeast-malt agar medium were relatively strong, and Streptomyces sp. ATCC55098 (MA6858) strains were tan to white, while Streptomyces sp. The MJM7001 producing strain had an orange color, and the known strain did not produce a pigment, whereas the producing strain of the present invention secreted a dark orange water-soluble pigment (see FIG. 1).

In order to examine the morphological characteristics of the production strain, MJM7001 strains were cultured in yeast-malt agar medium for 28 to 14 days, and observed through optical and electron microscopy (see FIG. 2). On the other hand, the cell wall composition of all three strains, type I, LL-type diaminopimelic acid (diaminopimelic acid) was observed the same point, but the DNA hybridization test, a molecular biological technique for measuring genetic similarity was used. MJM7001 strains belong to the same genus as the known strains of tacrolimus production, but they correspond to other species because their sequence homology is 70% or less. It can be seen.

DNA-DNA Hybridization of MJM7001 with Test Strains Strain % Recombination with Marked DNA Strains of the Invention Streptomyces Tsukuba Ensis No.9993 Streptomyces in ATCC 55098 Strains of the Invention 100 60 50 Streptomyces tsukubaensis No.9993
65
100
55 Streptomyces sp. ATCC 55098
48
53
100

Example 4 Stabilization and Screening of Tacrolimus Producing Strains

Development of high tacrolimus strains and optimization of fermentation conditions were performed. Streptomyces sp. Tacrolimus FK506 productivity by MJM7001 strain is greatly influenced by fermentation conditions. In the selection of good strains, each time mutation was performed, multiple passages were used to reduce the frequency of different types of colonies in solid medium and to obtain more stable strains. In addition, the fermentation culture was performed by selecting colonies grown in a solid medium to which FK506 (200 mg / l) was added (see FIG. 3). The colony forms three main colors, light yellow, gray and orange. In liquid culture, the initial growth rate is faster than the yellow colony, but not as much as the orange colony in FK506 productivity (see Table 2). Three colonies confirmed resistance to FK506, orange orange colonies showed the strongest resistance consistent with the productivity pattern (see Fig. 4).

FK506 Productivity Comparison of Three Different Color Colonies in Liquid Cultures Sample name PMV (%) Final pH Volume activity (mg / l) Red colony-1 38 6.8 220 Red colony-2 33 6.8 225 Red colony-3 33 6.8 250 Yellow colony-1 31 6.7 180 Yellow colony-2 33 6.8 200 Yellow colony-3 35 6.8 195 Gray colony-1 22 6.7 170 Gray colony-2 17 6.7 65 Gray colony-3 23 6.7 145

Example 5: Optimization of Fermentation Medium Condition of Tacrolimus Producing Strains

Tacrolimus producing strain Streptomyces sp. Substituted carbon source experiments of the MJM7001 strain confirmed that the use of a carbon source other than starch significantly reduced tacrolimus FK506 production. However, when soluble starch is used as a carbon source, a high viscosity phenomenon of the culture medium is required, and a higher stirring speed is required to maintain the oxygen supply. Dextrin was used as a carbon source to reduce the viscosity of the cultures and was found to produce more FK506 than soluble starch under the same fermentation conditions. So soluble starch was used in the preculture and dextrin was used in the main culture. As a nitrogen source, yeast extract and corn steep powder were used in preculture instead of yeast extract in this culture. In addition, the regulation of FK506 biosynthesis by glucose, ammonium, phosphate, etc. was observed. In medium containing 2% or more glucose as a carbon source, it was confirmed that FK506 production was inhibited by metabolic inhibition. High concentrations of phosphate medium are generally disadvantageous for the production of antibiotics. It is known that many antibiotics do not produce in medium rich in inorganic phosphate for the growth of production strains. However, it was found that the productivity of FK506 was increased by using a phosphate added medium (phosphate concentration of 0.5%). K ₂ HPO ₄ (0.2% for control medium composition and 0.5% for high phosphate medium composition) was added to a medium containing a suitable carbon source and nitrogen source to increase FK506 biosynthesis by 50% compared to the control.

Example 6 Establishment of Solid Culture and Preservation Methods of Production Strains

Specifically, the production strain 30 yeast-malt agar medium (starch 10 g / l, yeast extract 4 g / l, malt juice 10 g / l, agar 20 g / l, distilled water 1 l, after sterilization pH 6.8 ~ 7.0 ) Was incubated at 28 ℃ in a medium containing a single colony (monocolony) for 7 to 14 days. Thereafter, single colonies incubated in 1.5 ml of saline were suspended, then plated onto several plates containing the solid medium, and incubated at 28 ° C. for 7-14 days. 5 ml of sterilized 15% (w / v) skim milk solution was added to each of the cultured plates, and then, spores and some mycelium suspensions were harvested by scraping with a flame sterilized loop. Thereafter, the spore suspension was passed through the glass wool using a sterile syringe to prepare a spore suspension from which the mycelium was removed. In case of short-term storage, store in freezer (-70 ℃). In the case of long-term preservation, it is desirable to make ampoule by lyophilization method. 0.2 ml of the suspension was put into an ampoule with a pasture pipette, and the inlet was closed with the original sterilized cotton, and vacuum dried in a freeze dryer (-70 ° C.) for 30 to 40 minutes.

Example 7 Establishment of Liquid Culture of Production Strains

For spawn culture, 25 ml of spawn culture medium (Glucose 1.0%, Soluble starch 1.0%, Yeast extract 1%, Corn steep powder 0.5%, CaCO ₃ 0.1%, pH 6.6) was added to a 100 ml Erlenmeyer flask at 121 ° C. Sterilized for 20 minutes. The prepared spore suspension was inoculated in yeast-malt agar medium and incubated at 28 ° C. until a single colony (monocolony) was formed for 4-5 days. Subsequently, one of the colonies selected empirically by observing the pigment or colony form was inoculated with a flame sterilized loop and inoculated in the seed culture medium, followed by culturing at 28 ° C. at 220 rpm for 48 hours to prepare a seed culture. Subsequently, the main culture medium (250 glucose 1.0%, Dextrin 10%, Dried Yeast 1%, Corn steep powder 0.5%, K ₂ HPO ₄ 2%, CaCO ₃ 0.1%, pH 6.8) in a 250 ml Erlenmeyer flask for the main culture 50 ml was added and sterilized at 121 ° C. for 20 minutes. Thereafter, the prepared seed culture solution (2% (v / v)) was inoculated in the main culture medium and cultured for 6-9 days at 25 ℃ 220rpm to prepare a main culture culture. In consideration of industrial properties, low-cost industrial raw materials were mainly used. Fermentation was relatively stabilized by adding anti forming regent such as Neorin, a synthetic polymer (concentration was 0.1% (v / v)).

Example 8 Optimization of Fermenter Condition Using Production Strains

Streptomyces sp. FK506 productivity in the fermenter was observed using MJM7001. Specifically, 50 ml of preculture medium was added to a 500 ml round flask and sterilized at 121 ° C. for 40 minutes, and then the spore suspension was grown to 1% (v / v). Thereafter, the cultured strains were incubated for 48 hours at 28 ° C. and 120 round trips to obtain a single stage culture medium. The first stage preculture was inoculated in a 2.5 L fermenter containing 1 L of the preculture to 2.0% (v / v). Then, two-stage preculture at 28 ° C., 400 rpm, and 1 vmv for 24 hours, and then the two-stage pre-culture solution was added to 10% (v / v) in a 7.5-l fermentation tank containing 4 L of the main culture solution. Phagocytosis was incubated for 7-8 days at 28 ℃, 300 ~ 500rpm, 1vvm conditions. The culture solution thus obtained was analyzed by HPLC using the extraction method described above, and it was confirmed that about 700 mg / L of FK506 was produced (see FIG. 5).

In particular, it is important to provide sufficient oxygen during the growing season up to the third day. In antibiotic production of actinomycetes, some strains have been reported to increase productivity by changing from aerobic to anaerobic during fermentation. Actinomycetes achieve optimal growth by inhibiting most of the unnecessary biosynthetic mechanisms when nutrients such as carbon sources, phosphorus and nitrogen are readily available. Thus, most antibiotic production occurs during stagnant periods after major nutrient depletion and the inhibition of antibiotic biosynthetic mechanisms by major metabolites is eliminated. Streptomyces sp. For the MJM7001 strain, it is growing until the 3rd day of culture, after which the production of FK506 begins. In view of these growth characteristics, FK506 productivity under aerobic (control) and semi-anaerobic conditions (sealing the flask inlet with paraffin after 3 days of aerobic culture) was investigated. Streptomyces sp. The growth of MJM7001 was significantly suppressed, and thus the production of FK506, the secondary metabolite, was reduced by 50% compared to the control, but there was no significant difference in specific production (ug / ml mycelium) of FK506 production. Therefore, by adjusting the oxygen concentration in the production period at the end of the growing fermenter will increase productivity.

Example 9 FK506 Biosynthesis Study by Addition of Precursor

Antibiotic biosynthesis can also be caused by imbalances in the major metabolic mechanisms of actinomycetes. Biosynthesis of the red pigment undecylprodigiosin produced by the actinomycetes model strain S. coelicolor is partially derived from the amino acid proline. S. coelicolor mutants, which have abnormalities in proline transport and proline catabolism, produce large amounts of undecylprodigiosin, consuming extra proline accumulated in cells. Although these findings do not yet indicate how proline accumulation accelerates the timing of undecylprodigiosin biosynthesis and which gene regulation is involved, the imbalance of major metabolic mechanisms may also lead to antibiotic biosynthesis regulation. Is showing.

Therefore, the effects of FK506 production were investigated by adding two amino acids (Lysine, Proline) which are expected to be precursors in FK506 biosynthesis. Streptomyces sp. Cultured under the fermentation conditions described above. As a result of confirming the biosynthesis of FK506 in MJM7001 cell extract, Proline increased the biosynthesis up to 30% than the control at the concentration of 1g / ℓ, but reduced the biosynthesis by 50% at the concentration of 2g / ℓ. Lysine showed similar biosynthesis to the control at the concentration of 1 g / l, but increased biosynthesis by 20% at the concentration of 2 g / l (see FIG. 6). Of course, in order to see the effect on the production medium due to the addition of these amino acids, the concentration of the amino acid contained in the medium components must be calculated first to determine the appropriate amino acid concentration. Several other amino acid addition experiments are also in progress.

Example 10 Gene Library Construction and Tapping of FK506 Biosynthetic Gene Group in Tacrolimus Producing Strains

FK520 (Ascomycin) is distinguished from FK506 in that it lacks an allyl group at C-21 of FK506 and instead has an ethyl group at that position, and has similar activity to FK506, although it is a reduced immunosuppressive activity (see FIG. 7).

In fact, a small amount of FK520 is produced from the FK506 production strain. In order to overcome the shortcomings of strains that showed a limitation in productivity due to the improvement of FK506 production strains by repeated mutation methods without considering the molecular biological characteristics of the strain genome, the expression control system of actinomycetes regulatory gene was developed. . However, this method also creates structurally similar FK520s, which can lead to separation difficulties. Therefore, in order to further optimize the production-separation process, creative molecular genetic strain improvement method using the FK506 production strain genome utilization technology is urgently required.

Therefore, the present invention is to find the FK506 whole biosynthetic gene sequence in the production strain. To date, wild strains of FK506 production, Streptomyces sp. The FK506 biosynthetic gene family of MA6548 was only partially identified. Biosynthesis of FK506 involves several enzymatic actions. The FK506 polyketide biosynthesis (PKS) enzyme, consisting of the fkbA , fkbB , fkbC and fkbP gene products, synthesizes the core structure of the molecule. Also takes place not be oxidized by the fkbO gene product leading to the formation of the cake in earthenware C-9 and C-9 of the hydroxylation fkbD gene product, P450 hydroxylase parameters. Methylation at C-31 is also mediated by O-methytransferase, the fkbM gene product. But usually the regulatory genes at both ends are still unknown.

Against this background, FK506 biosynthesis genes were isolated by the following method. As described by Practical Streptomyces Genetics, Streptomyces sp. Was isolated using lysozyme / proteinase K protocal. It was isolated from MJM7001. The average size of DNA was estimated to be 80-120 kb by electrophoresis on 0.3% agarose gel.

A commercial SuperCos 1 Cosmid Vector Kit (Stratagene) was used for library preparation. About 100ug of genomic DNA and Sau3AI enzyme were added at 1000mU to 1mU, and partially pulverized at 37 ° C for 5 minutes, followed by electrophoresis on a 0.7% agrarose gel to collect only 30-50kb of DNA, and then ligation to SuperCos vector arms.

The resulting ligation mixture is mixed with the packaging extract of the Gigapack III Plus Packaging Extract Kit (Stratagene) and then infected with the host E. coli XL1-Blue MR cells. From this, a library of approximately 10,000 independent cosmid clones was obtained. In order to find gene groups involved in FK506 biosynthesis among the constructed genomic libraries, primers for primary screening were prepared using fkbM gene located on the right side based on the published FK506 biosynthetic gene sequence. [ Forward ] 5'-gagtgacgtggtggagacct-3 '[ Reverse ] 5'-cttctccacgtcgatcttcag-3'. Streptomyces sp. Using a PCR reaction with the above primer. A probe for fkbM was isolated from MJM7001 . Southern hybridization was performed using this probe. Two cosmids were separated from the library by Southern hybridization. Both ends of these Cosmids were sequenced. It was also mapped by several restriction enzymes.

The results showed that these two cosmids contained DNA inserts that overlap each other. Pointing out that these two Cosmid sequence data were obtained on the FK506 PKS gene cluster, sequences similar to those of the FK520 and Rapamycin biosynthetic clusters were generated. In order to obtain Cosmid containing a broader sequence, probes representing the middle fkbP gene and the left fkbC gene were isolated based on sequences on the middle and left side of the published FK506 biosynthetic sequence region. Three additional cosmids were obtained from the DNA fragments of adjacent chromosomes by screening additional libraries. Finally, a total of five Cosmids were selected to decode the entire sequence and predict gene function (see FIG. 8 and Table 3).

Example 11 Cloning and Overexpressing Transformants of Allyl-Moiety Biosynthetic Gene Fragments Isolated from Example 10

The allyl group in C-21 of FK506 plays a very important role in the immunosuppressive activity. To date, FK506 is the only polyketide with an allyl group. The FK506 biosynthetic genes so far identified are only partially known. Since FK520 has an ethyl group in C-21, the immunosuppressive activity is markedly reduced, and it is produced as a by-product during FK506 fermentation, which causes great difficulty in the separation process. Thus, four genes ( fkbV , fkbW, fkbX , fkbY ) with no homology compared to the FK520 biosynthetic gene group were newly discovered in the FK506 biosynthetic gene group. These are expected to be genes encoding acyl transferase, crotonyl-CoA reductase, ketosynthase and dehydrogenese, respectively. By overexpressing the genes involved in allylmoty biosynthesis, the allylmotiful pool in the FK506 biosynthesis process was increased, resulting in increased FK506 productivity and a decrease in the proportion of analogues such as FK520. In this Example, the gene involved in the biosynthesis of allylmalonyl- epi is obtained by PCR (Forward 5'-gag gaattc cggcccgagctgt-3; Reverse 5'-cac aagctt cctgacgcgggact-3 ') to clone the gene involved in the biosynthesis of allylmalonyl- epi into the overexpression vector pWHM3. FkbV , fkbW , fkbX , fkbY using genomic DNA A fragment containing the gene was obtained, and the ends of the DNA fragment contained restriction enzymes EcoR1 and HindIII recognition sequences.

fkbV , fkbW , fkbX , fkbY Was cloned DNA fragment (6,353 bp) containing the gene for Escherichia coli cyano (E. coli) Streptomyces shuttle vector pWHM3 which, over-expression vector that is the other by conventional gene Instruction introduced in Crawley mousse strain to obtain a transformant. 9 is a map of the overexpression vector inserted DNA fragment.

Example 12 Transformed Streptomyces sp. Increased tacrolimus production at MJM7001

The overexpression vector prepared in the above Example was Streptomyces sp. It was introduced into MJM7001 and transformed. In addition, a transformant having a pWHM3 empty vector was prepared as a control. Thereafter, tacrolimus production of Streptomyces Tsukubaensis wild type, empty vector control, and gene overexpressing transformants was measured (see FIG. 10).

FK506 Biosynthesis Gene Size and Function DNA coordinates amino acid Proposed function fkbA
fkbB
fkbC
fkbO
fkbP
fkbD
fkbM
fkbN
fkbQ
fkbR
fkbL
fkbK
fkbJ
fkbI
fkbH
fkbG
fkbF
fkbE
fkbS
fkbT
fkbU
fkbV
fkbW
fkbX
fkbY 63347-82600
34568-57535
23770-34584
57715-58746
58746-63341
82600-83766
83763-84545
84560-87331
87386-88165
89085-90029
22629-23666
21757-22632
21500-21760
20407-21507
19308-20396
18662-19330
17738-18490
16538-17722
15124-16350
14553-15020
13339-14493
12166-13326
10832-12169
8455-10835
7142-8437 6418
7655
3604
343
1531
388
260
923
259
314
345
291
86
366
362
222
250
394
408
155
384
386
445
793
431 FK506 polyketide synthase
FK506 polyketide synthase
FK506 polyketide synthase
C9 hydroxyl oxidase
Pipecolate-incorporating peptide synthetase
C9 hydroxylase
31-O-Methyl transferase
Transcription regulator
Type II thioesterase
LysR family transcription regulator
Lysine cyclodeaminase
Hydroxyacyl-CoA dehydrogenase
Acyl Carrier protein
Formation of glyceryl-ACP
Acyl-ACP dehydrogenase
O-Methyl transferase
3-oxoacyl- [acyl-carrier protein] reductase
Acyl-CoA dehydrogenase
Cytochrome P450
AsnC family transcriptional regulator
Methionine gamma-lyase
Acyl-CoA dehydrogenase
Crotonyl-CoA reductase
Polyketide synthase domain
Acyl transferase domain

Therefore, molecular genetic and biochemical basic studies on the biosynthetic genes of these production strains are expected to present new possibilities of industrial strain improvement methods suitable for the optimization of productivity and mass production of bioactive substances which are industrially and medically useful.

Korea Microorganism Conservation Center (overseas) KCCM11116P 20101018

Attach an electronic file to a sequence list

Claims (6)

Streptomyces sp. To produce a tacrolimus compound of Formula 1 MJM7001 (KCCM11116P)
Formula 1

Streptomyces sp according to claim 1. Tacrolimus biosynthesis gene derived from strain MJM7001 (KCCM11116P) and having SEQ ID NO: 1.
delete delete delete delete

Images