UA84651U - Method for production of landomycine a - Google Patents

Abstract

A method for production of landomycine A is based on introduction of additional copies of activating gene. Gene lanl S. cyanogenus S136 is used as activating gene as part of integrative vector pIJ6902.

Description

A useful model belongs to bacterial genetics and biotechnology and can be used to create strains of bigeriotuse5 suapodepa5 5136 that overproduce the angucycline antibiotic landomycin A and its derivatives.

There is a known method of obtaining superproducers of landomycin, according to which the spore suspension of the producer strain is irradiated with ultraviolet light or treated with a chemical mutagen, it is spread on a nutrient agar medium, and clones with an increased level of biosynthesis of the antibiotic are selected , Rikypaga M,

Vespioia A, MaKatiga T, EedogepKo U. Sepegaiiop oi Bigeriotusez diobizrogiz 5MU622 vigaip my ipsteasei Iapdotusip E rgodisiop apa yg5 ipiya! snagasiegigayop. U. Apiibioi.-2004. - Mine. 57. - R.383-389). The resulting mutants synthesize 2-200 times more landomycin compared to the original strain.

However, the specified method is quite long, includes the use of complex microbiological procedures.

The closest in technical essence - a prototype - is a method of increasing the level of synthesis of landomycin E by strain 5. diorigrogi5 1912 by means of overexpression of the IpaI gene, which encodes a pathway-specific transcriptional activator of the structural genes of the biosynthesis of landomycin E

IFedorenko V.O., Rebets Yu. V, Ostash B.O., Luzhetskyi A.M. The method of obtaining strains of Zigeriotusev diorigrogy5 with an increased level of biosynthesis of landomycins. Declaration patent

of Ukraine for invention. Me200310637 dated 15.12.2003, Bull. Me12). The Ipa!I gene as part of the replicative vector rKS1139 or the integrative vector roET152 was introduced into strain 5. diorigrogiv 1912 by conjugation with EzsPegisnpia soybean ET12567 (ryB307). After incubation at 30 "C for 16 h, each Petri dish with the conjugation mixture was filled with 1 ml of sterile water containing 50 μg/ml of apramycin and 25 μg/ml of nalidixic acid. The incubation was continued until the appearance of transconjugants. Transcon' yugants that inherited the plasmid with the pai gene were selected for apramycin resistance and grown for five days in 50 ml of liquid medium 50. Landomycin E was extracted from the entire fermentation medium with an equal volume of ethyl acetate (50 ml), centrifuged for 10 min at 6000 rpm, the resulting supernatant was evaporated and analyzed by thin-layer and high-performance liquid chromatography.The transconjugants produced 3-5 times more landomycin E than the control

From strain.

However, strain 5. diorizrogy5 1912 does not produce landomycin A, and therefore this strain cannot be used to increase the production of the latter. The protein product of the IpaI gene is not capable of effective transcriptional activation of landomycin A biosynthesis genes in 5. suapodepyv 5136. In addition, in this method, it is necessary to add the antibiotic apramycin to maintain the gene on the replicative plasmid pKSI 139, which complicates the use of the method.

The useful model is based on the task of improving the production method of landomycin A by introducing an additional copy of the Iapi gene into the genome of 5. suapodepych 5136, which will make it possible to increase the level of landomycin A production, simplify and reduce the cost of the process itself.

The task is solved by the fact that in the method of increasing the biosynthesis of landomycin A, which is based on the introduction of additional copies of the activator gene, the gene Iapi 5. suapodepyv 5136 is used as an activator gene as part of the integrative vector riChb902.

Landomycin A is a representative of the largest family of polyketide antibiotics - angucyclines.

Relative to angucyclines are anthracyclines, among which a number of clinically important anticancer compounds, such as doxorubicin, aclacinomycin, nogalamycin, and others, have been identified. Unlike anthracyclines, landomycin A does not intercalate in DNA, but has comparable antitumor activity and effectively destroys cancer cells resistant to anthracyclines (O51ia5p B, KogupeuvKa A,

Zioika AV, Redogepko U. Spetivigu apa Bioiodu oi Iiapdotusipe, ap ehrapaipud Tatu oi roiuKkeiide paishga! rhodisib5 Mipi Vem Med Spet.-2009. - Mine. 9. - R. 1040-1051). This prompts further investigation of landomycin A as a potential basis for a new class of drugs. The world experience in the development of medical preparations based on natural compounds shows that the difficulty of obtaining them in significant quantities, necessary for biological testing, is often the main obstacle on the way to discovering its positive properties. The difficulty of obtaining a pure form of the drug is the main reason for the high cost of drug production, which reduces their attractiveness for patients. The actinomycete culture of bigeriotuse5 suapodepy5 5I3b is the only source of landomycin A, which, however, allows obtaining relatively small amounts of this antibiotic, approximately 60 mg/l of the fermentation medium.

The authors proposed for the first time to use stable and inducible gene overexpression

Iapi for increasing the production yield of landomycin A by strain 5. suapodepis 51 36. The Iapi gene encodes a pathway-specific transcriptional activator of the structural genes of the biosynthesis of landomycin A. By testing various gene expression systems in 5. suapodepis 5136, the optimal system for increased expression of Iapi was selected. It is based on the integrative vector rizhb902 INiapa, 9., Zi, u., Moye, M. Stoz55-gedshianyop atopu divragaiye apiirioiiis Biozupipeiis raSshulauz oi bieriotuse5 soviiisoYog. My. MystobrioI.-2005. - Mine. 58. - R. 1276-1287), in which the transcription of the Iapi gene will be under the control of the thiostrepton-inducible promoter iirAr.

Since the overexpression of pathway-specific regulator genes makes it possible to quickly obtain strains that produce an increased amount of only a certain class of antibiotics, this will lead to an increase in the yield of landomycin A production.

The drawing explains the essence of a useful model.

Fig. 1 Chemical structure of landomycin A, the main representative of antibiotics of the landomycin series.

Fig. 2 Results of the analysis of the production of landomycin A by strains using spectrophotometry, where: 1 - the level of production of landomycin A by strain 5. suapodepyv 5136; 2 - the level of production of landomycin A by strain 5. suapodepyv 5136 (rMO15). The method can be illustrated by examples:

Plasmid RMO15 is constructed. First, to construct the plasmid RMO15, a DNA fragment from chromosome 5. suapodepib5 5136, which contains the Iati gene, is amplified with the help of the primers Iapimaeishr o (5-AAASATATAasISTSAatTtttoaAS(cS-3) and Iapimaeitr ((5-

AAAASAATTatSsASTastTTASSO,АСаСоСО-3). The obtained fragment with a size of 0.8 t.p.n. treated with restriction endonucleases Mavei! and Mines are further purified according to commonly used techniques |Glover D. Molecular cloning of DNA. Methods. M., Myr-1989-t.1-374s)|.

The vector riShb902 INiapa, 5., ZP, 9.,

Moie, M. Sgov5-gedshayop atopu daizragaiye apiibioiis Biozupieiiis raiyulauz oi Bngeriotusev5 soeiiisoYog. My. MisgobioI.--2005. - Mine. 58. - R. 1276-1287). The linearized vector and the amplified fragment are ligated. The ligase mixture is used to transform the strain of E. soii ONba, and clones carrying recombinant plasmids are selected on HA medium with 25 μg/ml of apramycin. Plasmid

DNA from transformants is analyzed by restriction mapping with Mabeyi enzymes! and Mye and using plasmid sequencing | Lover D. Molecular cloning of DNA. Method! M.,

Mir-1989-t.i-374s|. The resulting plasmid is designated pRMO15.

The BE strain is transformed with Plasmid RMO15. soybean ET12567 (ryv307), which provides conjugative transfer of co-resident plasmids by TsGriei E. due to the iga genes of the rOV307 plasmid,

Megzipiaz M., Ztiy S.R. Nidn eyisiepsu ipiegdepegis sopishda! іShapviei oi riaztiy OMA iot

EzsePetisnia soii tyu teinuI-OMA-teziysiipa Zigeriotuseiez II REM5 Mistobio!. And hey.-1997. - my I.155. -

R.223-229)|. One colony of E. soya culture ET12567 (ryv307), grown at night for 12 hours, is sown in 10 ml of IV medium with 50 μg/ml of kanamycin. The culture is grown to an optical density of 0.1 IU, transferred to microtubes with a volume of 1.5 ml and precipitated by centrifugation at 8,000 rpm for 1 min. Drain off the supernatant and resuspend the cells in 50 μl of medium IV. Cool the resulting cell suspension in ice for 5 min, add 1 ml of 0.1 M SasSig solution and incubate in ice for 1 h. Cells are pelleted by centrifugation at 8,000 rpm for 1 min, the supernatant is drained and resuspended in 100 μl of 0.1 M SasSig solution - Incubate for 1 h. in ice and add the plasmid DNA solution. Incubate for 1 hour. in ice, after which the cells are subjected to heat shock for 1 min at 40 "C, cooled and 1 ml of medium is added | B. Incubate for 2 h at 37 "C to induce the expression of resistance genes and sow on dishes with HA medium with 25 μg/ ml of apramycin and 50 μg/ml of kanamycin. Cups are incubated at 37 "C for 16 hours, after which the transformants are transplanted onto fresh HA medium with 25 μg/ml of apramycin and 50 μg/ml of kanamycin.

Plasmid rMO15 in 5. suapodepy5 5136 is transferred by intergeneric conjugation with the corresponding BE strain. soybean ET12567 (pOV307; pMO115). The mycelium suspension of strain 5. dpapayepviv is sown on medium OM g/l: oatmeal - 30, agar - 18, tap water - up to 1 l, pH before sterilization - 7.0 and grown for 7 days at 28 "С to obtain a lawn of cells for The strain E. soya ET12567 (ryB307; pMO15) is sown on a plate with medium IA with 25 μg/ml of apramycin and 50 μg/ml of kanamycin and grown for 18 hours at 37 "С.

Prepare a suspension of cells in 1 ml of medium II and B. At the same time, prepare a suspension of cells of strain 5. suapodepis in 1 ml of sterile water. Cells are subjected to heat shock for 5 min at 50 °C.

The suspension of Escherichia coli and streptomycetes cells is precipitated by centrifugation for 2 min at 8,000 rpm, the supernatant liquid is drained off, dissolved in 100 μl of IV medium, mixed with each other, and inoculated onto plates with OM medium. Cups are incubated for 20 hours. at 28C and pour 1 ml of an aqueous solution of 25 μg of apramycin and 50 μg of nalidixic acid.

The resulting strain 5. suapodepy5 5I 36 (rMO15) is checked for the presence of a plasmid.

They are grown in 15 ml of T5B liquid medium and total DNA is isolated, samples of which are used to transform E. soy strain OH5a |Kiezeg T., VIrB M.9., Vypeg M.)., Spayeg K.E., Norzhosa 0.A.

Rgasiisa! Bigeriotusev depeiis5 // doip Ippe5 Eoshipaaiyop, Mogmisn, Opiei Kipdaot.-2000.-613 radez). Plasmid DNA from transformants is analyzed by sequencing.

The levels of landomycin A production by the original and recombinant strain 5. suapodepyv 5136 are compared. The strains are sown in flasks with a volume of 0.3 l containing 30 ml of T5B medium and grown for 2 days at 30 "C on an orbital shaker (200 rpm. ).1 ml of the previous culture is inoculated into flasks with a volume of 0.5 l containing 200 ml of medium 52, g/l: soy peptone -10, glucose - 20, calcium carbonate anhydrous - 2, cobalt chloride - 0.001, and grown during

For days at 30 "C on an orbital shaker (200 rpm). Then the pH of the fermentation medium is brought to -7, and extracted with an equal volume of ethyl acetate at 25 "C for 6 hours. on a reciprocating rocker (170 oscillations/min.) The organic phase is separated from the fermentation medium by centrifugation (10 min, 6000 rpm) and evaporated to a dry residue in a rotary evaporator.

When conducting the experiment, the following are used: to determine the temperature - a mercury thermometer with an error of readings x 0.5 "C; for the volume of liquids - measuring cylinders with an error of readings x 0.5 ml and laboratory dispensers with an error of readings of 0.05-0.5 μl; masses of substances - the analytical weight of "Bapogiyib" with an error of readings - 0.00005 g and the laboratory weight of AKI5 with an error of readings of the same 0.05 g; time - with an electronic clock with an error of readings x 0.5 sec. Spectrophotometric analysis of landomycin products is performed on the MapoOhor 2 device, with a measurement error of 0.01 9o.

The dry residue is dissolved in methanol. The concentration of landomycin A in an alcoholic solution is determined by determining the optical density of the solution at a wavelength of A-445 nm (optimum absorption of landomycin A in the visible part of the spectrum) on a spectrophotometer

MapOhor. The obtained data are normalized relative to the same amount of biomass - dry weight and converted into production percentages, where 100 95 is the production level of the original strain. The experiment is repeated three times and the average value, error and standard deviation are determined.

The results of the experiments (Fig. 2) indicate that the recombinant strain produces

Zo is approximately 5 times more than landomycin A, which confirms the expected result.

Claims (1)

USEFUL MODEL FORMULA The method of production of landomycin A, which is based on the introduction of additional copies of the activator gene, which differs in that the Iapi 5. suapodepi5 5136 gene is used as an activator gene as part of the integrative vector riShShb902. OM she a her Bosh t g not this and I wk need to p A PAH dn et M and Seya which I znzok ele YOU na se WE USOU che NE la VAH vena, in I ko Y Sho t Ken neo cho she ey chtrig.