WO2010127645A2

WO2010127645A2 - The method of biotechnological preparation of lincomycin derivatives and its using

Info

Publication number: WO2010127645A2
Application number: PCT/CZ2010/000057
Authority: WO
Inventors: Dana Ulanova; Jitka Novotna; Yvona Smutna; Zdenek Kamenik; Radek Gazak; Jiri Janata; Jan Kopecky; Jaroslav Spizek
Original assignee: Institute Of Microbiology As Cr, V. V. I.
Priority date: 2009-05-04
Filing date: 2010-05-04
Publication date: 2010-11-11
Also published as: CZ2009277A3; WO2010127645A3; CZ302256B6

Abstract

Lincomycin derivatives are produced by biotechnological cultivation of Streptomyces lincolnensis ATCC 25466 with at least one gene of the 4-L-propylproline biosynthesis coding cluster inactivated. A substitute of the component whose synthesis is blocked by inactivation is added to the medium. In this way new, biologically active lincomycin derivatives are produced biosynthetically.

Description

The method of biotechnological preparation of lincomycin derivatives and its using

Technical Field

The invention involves uses of mutant strains of lincomycin producers defective in lincomycin biosynthesis for the production of hybrid compounds based on lincosamide antibiotics.

Background of the Art

Due to a permanently increasing number of pathogenic microorganisms resistant to known antimicrobial compounds, the interest in the research and development of new, efficient antibiotics also increases. Combinatorial biosynthesis is one of the ways to produce many potentially efficient compounds. By means of genetic modification of already known genes coding biosynthesis of the active compounds or whole clusters of genes it is possible to obtain a microorganism producing compounds with new structures and new properties. Lincomycin and its derivatives are one of those biologically active compounds. Lincomycin is a metabolite produced by different actinomycete species including the strain of Streptomyces lincolnensis used for the treatment of infections caused by grampositive pathogens. Clindamycin is the most common lincomycin derivative and its antimicrobial spectrum has been extended to grampositive anaerobic bacteria and Plasmodium falciparum causing malaria. Other lincomycin derivatives were prepared primarily by chemical synthesis. At the same time, enrichment experiments were performed with the strain Streptomyces lincolnensis based on adding various precursors to the cultivation medium leading to production of new compounds derived from lincomycin. The new compounds were tested, both in vitro and in vivo, against different pathogenic microorganisms.

Biosynthesis of lincomycin includes separate synthesis of two precursors, viz. 4-L-proline (further PPL) and methylthiolincosamide (MTL) that are condensed to give rise to demethyllincomycin (NDL) which is further methylated to yield the resulting lincomycin.

In 1995 the sequence of the lmb cluster of genes coding proteins of the lincomycin biosynthesis in the overproducing strain was described. This cluster contains genes for biosynthesis of both precursors, genes for the condensation reaction, regulatory genes and resistance genes responsible for resistance to the antibiotic. The sequence of the lincomycin cluster of the type strain Streptomyces lincolnensis ATCC 25466 has recently been published. Until now, only the function of proteins LmbBl and LmbB2 responsible for the first steps of biosynthesis of the PPL moiety of the molecule has been demonstrated experimentally. Functions of the other genes are either unknown or were proposed on the basis of homologies with the BLAST database.

Disclosure of the invention

For the preparation of new compounds based on lincosamide antibiotics it is possible to utilize a collection of strains of lincomycin producers defective in one or more steps of biosynthesis. With this aim in mind mutations in some genes of lincomycin biosynthesis were induced in the type strain Streptomyces lincolnensis ATCC 25466. A collection of S. lincolnensis strains defective in PPL biosynthesis (genes imbA, Bl, B2, W, X, Y) and final methylation (gene imbJ) was prepared.

The strains prepared in this way are to be used for a further testing with respect to the production of new compounds depending also on selected cultivation conditions. According to what is coded by a given gene that has been blocked in the designed strain, substituting components are added, namely derivatives of lincomycin precursors; the biosynthesis is thus not blocked any longer and proceeds giving rise to new, biologically active lincomycin derivatives.

In these strains lincomycin biosynthesis is coded by the lmh cluster containing genes ImbA, Bl, B2, W, X, Y coding for proteins involved in the production of the precursor 4-L-propylproline and gene ImbJ coding the final methylation of N-demethyllincomycin. By blocking the 4-L- propylproline production, it is possible to add another proline derivative to the cultivation medium which the microorganism can utilize in the biosynthesis replacing thus 4-L- propylproline. In this way it is possible to prepare a number of new lincomycin derivatives without the use of chemical synthesis.

Figures

Fig. 1: Primers for synthesis of inactivation cassettes

Fig. 2: Chromatogram UPLC analysis, peaks of 4' - butyl- 4'depropyllincomycin (further

BuLIN) and 4' - pentyl- 4'- depropyllincomycin (further PeLIN) are detected

Fig. 3: Chemical structure of lincomycin Fig. 4: The list of the used E. coli strains including the contained plasmid and antibiotic present in the cultivation medium and, eventually, the required cultivation temperature.

Fig. 5: The list of the used antibiotics with their abbreviations and concentrations in the cultivation media.

Fig. 6: The list of the used cultivation media with their composition

Examples

Example 1

To obtain a strain Streptomyces lincolnensis ATCC 25466 defective in lincomycin biosynthesis one of the genes involved in 4-L-propylproline biosynthesis was inactivated using a kit for directed mutagenesis in streptomycetes (REDIRECT PCR targeting system, Proc Natl Acad Sci

USA.100(4). 1541-1546. 2003).

Cosmid LK6 maintained in E. coli BW25113/pIJ790, carries the whole lmb cluster from

Streptomyces lincolnensis. In this cosmid the gene imbXwas replaced with inactivation cassette aac(3)IV/oriT coding apramycin resistance. This inactivation cassette was formed by PCR reaction with the aid of primers Xf and Xr containing homologous flanking regions of gene imbX. A 1383 pb fragment produced by cleaving of plasmid pIJ773 with restriction enzymes

EcoRl and HindIII served as template. E. coli BW25113 containing cosmid LK6 and plasmid pIJ790 with genes required for recombination was transformed with the inactivation cassette by electroporation and selected in LB medium for apramycin (50 mg/L), kanamycin (50 mg/L) and carbamycin (100 mg/L) resistance. The cassette was by recombination integrated into cosmid

LK6 by exchange for gene imhX.

The cosmid prepared in this way was by conjugation transferred to S¹. lincolnensis ATCC

25466 according to the protocol of Kieser T., Bibb M.J., Chater K.F., Hopwood D.A.: Practical

Streptomyces genetics, John Innes Centre, Norwich Research Park, Colney, Norwich, England.

(2000)

Transformation of S. lincolnensis with cosmid DNA by interspecies conjugation with E. coli. By means of electroporation E. coli ET1267/pUZ8002 cells were transformed with cosmid LK6 carrying the inactivation cassette and selected on LB medium with apramycin (50μg/ml) and carbenicillin (100 μg/ml). One of the colonies was inoculated in 10 ml of medium LB containing apramycin (50 μg/ml), chloramphenicol (25 μg/ml) and kanamycin (50μg/ml) and cultivated on a shaker to OD₆₀₀ = 0.4 (about 4 h). The antibiotics were removed from the culture by washing the cells twice with 10 ml of medium LB (1000 g, 10 min, 20 ° C) and the cells were then resuspended in 1 ml of medium LB.

To 10 μl of spore suspension of Streptomyces Hncolnensis ATCC 25466 (10^s cfu) 500 μl of 2 x YT medium were added and the spores were heat-shocked at 50 C and then cooled down to laboratory temperature.

0.5 ml suspension of E. coli ET12567/pUZ8002 cells was mixed with 0.5 ml of spore suspension of Streptomyces Hncolnensis ATCC 25466 and centrifuged (10 000 g, 5 s, 20 ⁰C). The sediment was resuspended in 50 μl of the residual medium. A dilution series from 10^"1 to 10^"4 was prepared (suspension was diluted with sterile distilled water). One-hundred μl of each diluted suspension were spread on MS agar with 10 mmol/1 MgCl₂ without selection and incubated at 30 ⁰C. After 16 - 2O h each 8 cm Petri dish was evenly overlaid with 1 ml sterile distilled water containing 0.5 mg of nalidixic acid and 1.25 mg of apramycin and further incubated at 30 ° C for 3 - 5 days.

S. Hncolnensis clones containing the inactivation cassette in their DNA were selected for apramycin (50 mg/L) resistance, first on MS agar and then on DNA agar. To eliminate the effect of the inactivation cassette on transcription of genes in the same transcription unit, in LK6 cosmid bearing E. coli DH5α the cassette was cleaved off with the aid of FLP recombinase coded by a gene carried on plasmid pBT340. FLP recombinase removed the central part of the inactivation cassette and left an 81 pb DNA region - the so- called "scar" (temperature required for induction recombinase repression is 42 ⁰C, E. coli DH5α cells with plasmid BT340 and cosmid LK6 were therefore cultivated at this temperature). The mutated scar-bearing cosmid was then transferred by transformation of protoplasts prepared according to the protocol Jandova Z., Tichy P.: Transformation of Streptomyces Hncolnensis protoplasts with plasmid vectors, in Folia Microbiologica (Praha), 37(3), 181 -7. (1992). Resulting cells of the streptomycete strain had functional gene lmbX replaced with the inactivation cassette.

Preparation of S. Hncolnensis protoplasts

Inoculum was prepared by inoculating 50 ml of medium YEME containing 10 % sucrose and 5 mmol/1 MgCl₂ with S. Hncolnensis spores bearing the inactivation cassette from an agar slant and incubating on a shaker at 30 ⁰C for 24 hours. One-ml of the inoculum was then re- inoculated on a fresh YEME medium containing 10 % sucrose and 5 mmol/1 MgCl₂ 0.5 % glycine and the culture was incubated on a shaker at 28 ⁰C for 16-17 hours. The cells were harvested by centrifugation (10 min, 12 ⁰C, 6000 g) washed twice with 15 ml of buffer P under the same conditions and resuspended in 10 ml of buffer P with lysozyme (2 mg/ml). The cell suspension was incubated at 30 ⁰C with occasional shaking. Protoplast formation was followed under the microscope. Mycelium residues were removed by centrifugation (10 min, 20 ⁰C₅ 1 000 g). Protoplasts were filtered through a sterile cotton filter, centrifuged (10 min, 20 ⁰C, 3 000 g) and washed twice with buffer P under the same conditions. The sedimented protoplasts were finally resuspended in 200 μl of buffer P and immediately used for transformation.

Transformation of S. lincolnensis protoplasts

Five-hundred ng in a volume of 5 μl plasmid DNA of the mutated scar bearing plasmid were mixed with 5 μl of 50 % sucrose and 100 μl of the protoplast suspension were added to the mixture. After mixing 200 μl of buffer T were added, the protoplast suspension was mixed again and immediately spread on R2YE agar. After a 16-h incubation at 30 ⁰C both Petri dishes were overlaid with 2 ml of aqueous kanamycin (200 μg/ml of R2YE agar) and incubated at

30 ⁰C for additional 7-10 days.

The inactivation cassette was exchanged by the scar by homologous recombination. Double recombinants of the obtained Streptomyces lincolnensis AlmbX strain were selected for the loss of apramycin resistance.

E. coli cells were cultivated in liquid or solid media (LB, SOC) at 37 ⁰C. The strain

BW25113/LK6/pIJ790 was cultivated at 30 ⁰C. After transformation with the cassette the strain was cultivated at 37 ⁰C. The strain DH5α containing plasmid pBT340 was cultivated at 30 ⁰C.

Liquid cultures were incubated on a rotary shaker at 200 rpm.

Streptomyces lincolnensis cells were cultivated in liquid or solid media (R2YE, MS, DNA, YT) at 28 - 30 ⁰C. Liquid cultures were incubated on a rotary shaker at 230 rpm and stored in 500- ml Erlenmeyer flasks with inserts preventing formation of mycelium clumps. After selection of cells carrying the vector appropriate antibiotics were added to the cultivation media.

Example 2

Streptomyces lincolnensis AlmbX defective in PPL biosynthesis was at a concentration of 10⁸ spores inoculated in 50 ml of inoculation medium YEME and cultivated at 28 ⁰C for 30 hours. Twenty-ml of production medium AVM containing 4-L-butylproline (100 mg/1) were then inoculated with 5 % of the inoculation culture. The culture was then cultivated for 120 hours at 28 C . The culture liquid was separated from the grown culture by centrifugation and the supernatant was used for the analysis of the produced lincomycin derivative - BuLIN. Example 3

Streptomyces Hncolnensis Δ lmbX defective in PPL biosynthesis was at a concentration of 10⁸ spores inoculated in 50 ml of inoculation medium YEME and cultivated at 28 ⁰C for 30 hours. Twenty ml of production medium AVM containing 4-L-pentylproline (100 mg/1) were then inoculated with 5 % of the inoculation culture. The culture was then cultivated for 120 hours at 28 C . The culture liquid was separated from the grown culture by centrifugation and the supernatant was used for the analysis of the produced lincomycin derivative - PeLIN.

Example 4

Acquity UPLC system (Waters, Milford, Massachusetts) with 2996 PDA detector set at 194 nm was used for the analysis of BuLIN. The results were evaluated by computer program Empower 2 (Waters). Supernatant of cultivation medium was purified on SPE Oasis HLB 3cc extraction columns. The sample (4 ml) was applied to the column preconditioned and equilibrated with methanol (3 ml) and water (3 ml). A part of the sample matrix was washed with water (3 ml) and 10 % methanol (v/v, 1 ml). Fractions containing lincomycin derivatives were eluted with 80 % methanol (v/v, 1 ml), evaporated to dryness and re-diluted in 200 μl methanol. Samples were analyzed on a chromatography column BEH Cl 8 (50 x 2.1 mm, particle diameter 1.7 μm, Waters); mobile phase consisted of components A, 1 mmol/1 amonium formiate (pH 9.0) and B, acetonitrile; isocratic elution (24 % B); flow rate: 0.4 ml rnin^"1, column temperature: 35 ⁰C , data collection rate: 20 pts s^"1 , dose volume: 5 μl, analysis time: 3.5 min. After each analysis the column was washed (100 % acetonitrile, 1 min) and equilibrated (1.5 min). Eluate containing BuLIN (retention time 5.05 min) was collected, evaporated to dryness and used for MS analysis which confirmed the presence of BuLIN in the sample.

Example 5 .

Acquity UPLC system (Waters, Milford, Massachusetts) with 2996 PDA detector set at 194 nm was used for the analysis of PeLIN. The results were evaluated by computer program Empower 2 (Waters). Supernatant of cultivation medium was purified on SPE Oasis HLB 3cc extraction columns. The sample (4 ml) was applied to the column preconditioned and equilibrated with methanol (3 ml) and water (3 ml). A part of the sample matrix was washed with water (3 ml) and 10 % methanol (v/v, 1 ml). Fractions containing lincomycin derivatives were eluted with 80 % methanol (v/v, 1 ml), evaporated to dryness and re-diluted in 200 μl methanol. Samples were analyzed on a chromatography column BEH Cl 8 (50 x 2.1 mm, particle diameter 1.7 μm, Waters); mobile phase consisted of components A, 1 mmol/1 amonium formiate (pH 9.0) and B, acetonitrile; isocratic elution (24 % B); flow rate: 0.4 ml min^"1, column temperature: 35 ⁰C , data collection rate: 20 pts s^"1 , dose volume: 5 μl, analysis time: 3.5 min. After each analysis the column was washed (100 % acetonitrile, 1 min) and equilibrated (1.5 min). The eluate containing PeLIN (retention time 5.05 min) was collected, evaporated to dryness and used for MS analysis which confirmed the presence of PeLIN in the sample.

Example 6

With the aim of obtaining Streptomyces lincolnensis ATCC 25466 defective in propylproline biosynthesis and final N-demethyllincomycin methylation, genes ImbX and ImbJ were simultaneously inactivated with the aid of the kit for directed mutagenesis of streptomycetes (REDIRECT OCR targeting system, Proc Natl Acad Sci U S A₅ 100(4), 1541-1546, 2003). In cosmid LK6 carrying the whole lmb cluster gene ImbX was replaced with apramycin resistance (50 mg/L) coding inactivation cassette aac(3)IV/oriT. The inactivation cassette was prepared by means of PCR reaction with primer Xf and Xr (table 2) containing homologous flanking regions of gene ImbX. A 1383 pb fragment formed by cleaving of plasmid pIJ773 with restriction enzymes EcoRI and HindIII served as template. In the same cosmid additional gene ImbJ was replaced with a vph cassette coding viomycin (30 mg/L) resistance. The inactivation cassette was prepared by PCR reaction using primers Jf and Jr containing homologous flanking regions of gene ImbJ. A 1622 pb fragment formed by cleaving of plasmid pIJ781 with restriction enzymes EcoRI and HindIII served as template. E. coli BW25113 containing cosmid LK6 and plasmid pIJ790 with genes required for recombination was gradually transformed with inactivation cassettes. By recombination the cassettes were integrated in cosmid LK6 in exchange for genes ImbX and ImbJ. The cosmid prepared in this way was by conjugation transferred to S. lincolnensis ATCC 25466. The cassettes containing S. lincolnensis clones were selected for apramycin and viomycin resistance. To eliminate the effect of the inactivation cassettes on transcription of genes in the same transcription unit the cassettes were cleaved off by FLP recombinase coded by a gene carried on plasmid pBT340. FLP recombinase removed central parts of the inactivation cassettes and left 81 pb long DNA regions - "scars". The mutated cosmid with scars was then introduced to a streptomycete strain in which genes ImbX and ImbJ had already been replaced with the inactivation cassettes. By homologous recombination the inactivation cassettes were exchanged for the scars. Cultivation, selection and transformation conditions were identical with those described in Example 1. Industrial Applicability

The newly prepared derivatives of lincomycin antibiotics can be used for the development of new biologically active compounds against infections caused by pathogenic microorganisms, i.e. in pharmaceutical industry and medicine.

Claims

C L A I M S

1. The method of biotechnological production of lincomycin derivatives characterized in that the strain Streptomyces lincolnensis ATCC 25466, in which at least one gene of the lmb cluster of genes coding for 4-L-propylproline has been inactivated, whereas a substituent of a component whose synthesis is blocked by inactivation must be added to the cultivation medium.

2. The method of biotechnological production of lincomycin derivatives according to claim 1 characterized in that gene lmbX coding for biosynthesis of 4-L-propylproline is inactivated.

3. The method of production of lincomycin derivatives according to claim 2 characterized in that a proline derivative is added to the medium as a substitute for a component.

4. The using of the strain according to claim 1 for biotechnological production of biologically more active lincomycin derivatives.

5. The using of the strain according to claim 4 with inactivated genes lmb J and lmbX for biotechnological production of biologically more active lincomycin derivatives.

6. The using of the strain according to claim 4 with inactivated gene lmbX for biotechnological production of biologically more active lincomycin derivatives.

7. The using of the strain according to claim 4 with inactivated gene lmb J for biotechnological production of biologically more active lincomycin derivatives.