WO2001073096A1 - Production amelioree de metabolites secondaires en presence d'additifs organiques - Google Patents

Production amelioree de metabolites secondaires en presence d'additifs organiques Download PDF

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WO2001073096A1
WO2001073096A1 PCT/US2001/040398 US0140398W WO0173096A1 WO 2001073096 A1 WO2001073096 A1 WO 2001073096A1 US 0140398 W US0140398 W US 0140398W WO 0173096 A1 WO0173096 A1 WO 0173096A1
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streptomyces
organism
organic additive
secondary metabolites
metabolite
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PCT/US2001/040398
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English (en)
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Julian E. Davies
Genhui Chen
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Cubist Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

Definitions

  • the present invention relates to a method for increasing the amount of a secondary metabolite of interest produced by cultured organisms.
  • the present invention is also directed to a method for enhancing the molecular diversity of secondary metabolites produced by an organism. More specifically, the invention relates to a method for culturing organisms in the presence of organic additives such as dimethyl sulfoxide (DMSO), ethanol and dimethyl sulfone to increase the production of useful secondary metabolites.
  • organic additives such as dimethyl sulfoxide (DMSO), ethanol and dimethyl sulfone
  • Primary metabolites include the common, essential molecules synthesized by most, if not all, living cells. Secondary metabolites, in contrast, do not appear to be required for cellular growth and specific secondary metabolites are frequently synthesized by only relatively few organisms. Synthesis of secondary metabolites has been demonstrated in bacteria, fungi and plants, and it appears that the generation of these compounds is a response to, for example, nutrient deprivation or other environmental stress. Elaboration of secondary metabolites frequently commences after the cessation of the exponential growth phase of the organism and generally requires the expression of a set of differentially-regulated genes involved only in secondary metabolism. The extraordinary number of secondary metabolites that are produced and the striking molecular diversity represented within this group of natural products have provided a valuable resource in the continuing search for novel chemical structures that may be of clinical and commercial importance.
  • Secondary metabolites useful in the prevention, amelioration and cure of human disease. Secondary metabolites also have provided "lead structures" that can be chemically modified to diminish their toxicity and to improve their specificity and pharmacokinetic traits. These natural products and their semi-synthetic derivatives have found application as antibacterial, antifungal, antiviral, antihelminthic and anticancer agents. Other secondary metabolites have been discovered to have immunosuppressive traits and anticholesterolemia properties.
  • Discovery programs designed to detect and identify novel chemical structures elaborated by newly-isolated or newly-constructed organisms, frequently employ a relatively large number of different growth media to increase the probability that at least one will support synthesis of a secondary metabolite having a desired activity. Nevertheless, such an approach will fail to detect those secondary metabolites whose production level does not reach the threshold of detection.
  • the object of the present invention is to manipulate the composition of the cell culture media to obtain an increase in the production of desirable and useful secondary metabolites.
  • the invention provides in one embodiment, a method for increasing the production of a secondary metabolite by an organism, comprising culturing the organism in a medium comprising an amount of at least one organic additive, which amount is sufficient to increase the production of the secondary metabolite.
  • the method of the invention comprises increasing the production of useful metabolites by culturing microbial and plant cells in a culture medium suitable for growth containing at least one of the organic additives dimethyl sulfoxide (DMSO), ethanol or dimethyl sulfone, the additive being present at a concentration between about 0.1% and about 10% v/v, preferably between about 0.5 and 7.5% v/v, and more preferably between about 1 % and about 5% v/v.
  • DMSO dimethyl sulfoxide
  • ethanol or dimethyl sulfone the additive being present at a concentration between about 0.1% and about 10% v/v, preferably between about 0.5 and 7.5% v/v, and more preferably between about 1 % and about
  • the invention provides a culture medium for production of secondary metabolites by organisms, comprising a nutrient medium capable of supporting the growth of the organisms and at least one organic additive at a concentration between about 0.1% to about 10% v/v.
  • the preferred organic additives are dimethyl sulfoxide (DMSO), ethanol and dimethyl sulfone.
  • Figure 1 is a graph showing the variation of the secondary metabolite tetracenomycin (TCMC) produced by Streptomyces glaucescens grown in TSB medium in the presence of various concentrations of DMSO.
  • TCMC secondary metabolite tetracenomycin
  • Figure 2 is a graph showing the variation of the secondary metabolite chloramphenicol produced by Streptomyces venezuelae grown in soy flour medium in the presence of various concentrations of DMSO.
  • Figure 3 is a graph showing the variation of the secondary metabolite chloramphenicol produced by Streptomyces venezuelae grown in GNY medium in the presence of various concentrations of DMSO.
  • Figure 4 is a graph showing the variation of the secondary metabolite thiostrepton produced by Streptomyces azureus grown in R5 medium in the presence of various concentrations of DMSO.
  • Figures 5a and 5b are graphs showing the variation of low molecular weight secondary metabolites produced by transformant SI OH 10 o ⁇ Streptomyces lividans grown in R5 medium containing 25 ⁇ g/ml apramycin, in the presence and absence of DMSO, respectively.
  • Figures 6a and 6b are graphs showing the variation of low molecular weight secondary metabolites produced by transformant 446-S3-102H4 of Streptomyces lividans grown in R5 medium containing 25 ⁇ g/ml apramycin, in the absence and presence of DMSO, respectively.
  • Figures 7a and 7b are graphs showing the variation of low molecular weight secondary metabolites produced by transformant 446-S3-81 D3 o ⁇ Streptomyces lividans grown in R5 medium containing 25 ⁇ g/ml apramycin, in the absence and presence of DMSO, respectively.
  • Figure 8 is a graph showing the variation of the secondary metabolite tetracenomycin (TCMC) produced by Streptomyces glaucescens grown in TSB medium in the presence of ethanol (EtOH).
  • TCMC secondary metabolite tetracenomycin
  • Figure 9 is a graph showing the variation of the secondary metabolite tetracenomycin produced by Streptomyces glaucescens grown in R5 medium in the presence of ethanol.
  • Figure 10 is a graph showing the variation of the metabolite chloramphenicol produced by Streptomyces venezuelae grown in GNY medium in the presence of dimethyl sulfone.
  • the present invention relates to an improved process for the production of secondary metabolites by cultured organisms.
  • the present invention is directed both to methods through which the level of production of a secondary metabolite is increased and to methods through which the component profile of secondary metabolites produced by a cultured organism is enhanced and diversified.
  • the method of the invention involves culturing microbial and plant cells in a nutrient medium capable of supporting their growth and viability, and adding to the medium a sufficient quantity of at least one organic additive to increase the production of secondary metabolites of interest.
  • the organisms are fungi, bacteria, or plants.
  • the culture medium for the organisms is medium which is known to support the growth and viability of the particular species of organisms.
  • organic additive includes organic molecules having a molecular weight of less than about 250, wherein the organic molecule comprises at least one heteroatom selected from the group consisting of sulfur, oxygen and combinations thereof.
  • Organic additives of the present invention include, but are not limited to dimethylsulfoxide (DMSO), dimethly sulfone and lower alcohols including methanol, ethanol, propanol, isopropanol, butanol, n-butanol, iso-butanol, t-butanol, amyl alcohol and iso-amyl alcohol, and combinations thereof.
  • DMSO which is a natural product and exhibits low toxicity, is the preferred additive.
  • organic additives include organic molecules having a molecular weight of less than about 250, wherein the organic molecule consists essentially of carbon, hydrogen and at least one heteroatom selected from the group consisting of sulfur, oxygen and combinations thereof.
  • the organic additive is preferably added to the culture of organisms at the time of innoculation, but it can be added at any time during the culture period.
  • the concentration of the organic additive added does not take into account, for example, the production of ethanol by an organism synthesizing one or more secondary metabolites.
  • a nutrient medium comprises "on a nutrient medium” as well; that is, it includes growing one or more organisms on media solidified by the inclusion of a solidifying agent, for example, agar or agarose, to produce a "semi-solid" nutrient medium.
  • a solidifying agent for example, agar or agarose
  • the invention is applicable to any type of culturing or fermentation system or process, including batch, fed-batch, and continuous fermentation processes.
  • the invention may also be applied to processes for the production of one or more secondary metabolites using immobilized cells.
  • the invention may be used to increase the production of one or more secondary metabolites, or to enhance the component profile and chemical diversity of secondary metabolites elaborated by an organism grown on semi-solid media, including media comprising, for example, agar or agarose, which may be used as solidifying agents.
  • the invention may also be used for the production of secondary metabolites by marine organisms grown in media comprising a relatively high salt content.
  • the optimal concentration of the organic additive in the culture medium will depend on several factors, including the nature of the organic additive, the strain of organism being cultured, as well as the particular culture medium used. The optimal concentration for a particular organism can easily be determined using assays similar to the ones outlined herein. However, the optimal concentration will generally be found within the range of 0.1 to 10% v/v, and preferably within the range of 0.5 to 7.5% v/v, and most preferably within the range of 1.0 to 5% v/v.
  • the invention also contemplates a complete culture medium for producing secondary metabolites from organisms, wherein the culture medium comprises an organic additive.
  • the organic additive is present in the culture medium at concentrations ranging from about 0.1% to about 10% v/v. Examples of such culture media are provided in Section 6.
  • compositions of the invention can be used in conjunction with other culture medium additives which promote growth of organisms and production of secondary metabolites.
  • organisms includes, bacteria, fungi and plant cells.
  • Bacteria include, but are not limited to the Actinomycetes family and, particularly, species o ⁇ Streptomyces, including but not limited to Streptomyces hygroscopicus, Streptomyces halstedii, Streptomyces erythrae (now known as Saccharopolyspora erythrae), Streptomyces mediterranei, Streptomyces aureofaciens, Streptomyces venezuelae, Streptomyces spheroides, Streptomyces cinnamonensis, Streptomyces albus, Streptomyces caespitosus, Streptomyces avermitilis, Streptomyces parvulus, Streptomyces antibioticus, Streptomyces azureus, Streptomyces griseus, Streptomyces clavuligeris, Strepto
  • Non-Streptomyces Actinomycetes include, but are not limited to species of Actinomadura, Actinoplanes, Actinosporangium, Actinosynnema, Ampullariella, Chainia, Dactylosporangium, Kibdelosporangium (including Kibdelosporangium sp.
  • Representative fungi include, but are not limited to species of Penicillium, Cephalosporium, Taxomyces, Monascus, Heterobasidion, Genisculosporium, Byssochlamys and Aspergillus.
  • Other representative fungi include Mycoleptodiscus atromaculans (ATCC accession number 74336), Zalerion arboricola (ATCC accession number 20868), Nodulisporium sp.
  • AtCC accession number 74245 Aspergillus versicolor (ATCC accession number 74035), Aspergillus nidulans, Acremonium chrysogenum, Penicillium chrysogenum, Aspergillus niger, Cephalosporium acremonium, Hypoxylon fragiforme (ATCC accession numbers 20994 and 20995), Fusarium subglutinans (ATCC accession number 74358), and Fusarium pallidoroseum (ATCC accession number 74289).
  • Representative plants include, but are not limited to, species of
  • Catharanthus Perilla, Taxus, Curcuma, Hevea, Pinus, Eschscholzia, Hypericum, and Brassica.
  • organisms are found within genera including Pseudomonas, Bacillus, Myxococcales, Rhizobia, and Cyanobacteria.
  • the term "organisms,” as used herein, also includes those organisms isolated from marine and terrestrial environments that have traits that characterize them as similar to previously-identified organisms although they have not yet been taxonomically classified. (Fenical, W. (1997) Trends in Biotechnology 15: 339-41 ; A. D. Kinghorn, The Discovery of Drugs from Higher Plants, in THE DISCOVERY OF NATURAL PRODUCTS WITH THERAPEUTIC POTENTIAL, 81-108 (V.P. Gullo, ed., Butterworth-Heinemann, Stoneham, MA 1994))
  • McConnell et al. The Discovery of Marine Natural Products with Therapeutic Potential, in THE DISCOVERY OF NATURAL PRODUCTS WITH THERAPEUTIC POTENTIAL, 109-174 (V.P. Gullo, ed., Butterworth-Heinemann, Stoneham, MA 1994)).
  • organisms also encompasses mutant strains of known species of microbes and plants that have arisen spontaneously, have been induced by treatment with mutagenic agents, including for example radiation or mutagenic chemicals, or have been created by genetic engineering.
  • the phrase "component profile” includes all of the secondary metabolites produced by a cultured organism as detected by the means chosen therefor. As known to those skilled in the relevant art, the component profile may be assessed in many different ways. Non-limiting examples of component profiles are depicted in Figures 5, 6 and 7, which correspond to the HPLC analyses of the fermentations carried out in Examples V, VI and VII. A "peak” or detectable signal within a component profile is referred to herein as “a component” and includes peaks derived from the properties of one, or from more than one, secondary metabolite. Therefore, as used herein the phrase “component profile” and the term “complex” are used interchangeably to refer to the mixture of secondary metabolites that are co-produced by an organism.
  • an enhanced component profile refers to a component profile, obtained from an organism cultured in the presence of an organic additive, which has at least one component or peak that was not detected when the organism was cultured in the absence of the organic additive.
  • the new peaks or components disclosed, for example, in Figures 5, 6 and 7 may represent an increase in the level of production of one or more secondary metabolites, in the presence of the organic additive, which now surpasses the threshold of the detection method employed.
  • an enhanced component profile may comprise an increase in the level of one or more peaks or the presence of a previously-undetected peak.
  • an enhanced component profile may also be characterized by the absence or diminution of a previously-detected peak, especially in those instances in which the molecules represented by that peak interfere with the detection or purification of a secondary metabolite of interest.
  • An enhanced component profile also comprises one exhibiting a combination of more than one of these phenomena.
  • growth of an organism in a culture medium for the production of a secondary metabolite includes those processes, known in the art, referred to as cosynthesis, mutasynthesis, and biotransformation. It also includes, as demonstrated in Examples V, VI and VII, the synthesis of secondary metabolites by recombinant organisms.
  • Cosynthesis comprises the growth of a plurality of organisms in a culture medium whereby at least one secondary metabolite is synthesized.
  • Mutasynthesis comprises growth of at least one organism, wherein the organism carries a mutation preventing the synthesis of a first secondary metabolite that its non-mutant parent can synthesize, in a culture medium comprising a second metabolite, whereby that second metabolite is modified by the mutant strain.
  • Biotransformation comprises the growth of at least one organism in a culture medium comprising a metabolite, whereby the metabolite is modified.
  • a secondary metabolite produced by biotransformation is disclosed in U.S. Patent No. 5,468,771 , which discloses the formation of a cholesterol-lowering compound by cultivating Streptomyces cyanus (ATCC 5214) in a culture medium comprising the precursor molecule to be modified.
  • a similar procedure is disclosed in U.S. Patent No. 5,194,377 to Schwartz et al., where the growth medium comprised amino acid analogues that were incorporated into the antifungal secondary metabolites produced by Mycoleptodiscus atromaculans.
  • Recombinant processes include the growth of at least one recombinant organism in a culture medium for the production of at least one secondary metabolite.
  • Recombinant processes comprise those performed with an organism carrying at least one gene, which has been modified by or incorporated within the organism using recombinant DNA tools and methods which are well known to those of ordinary skill in this art. Examples of such processes are disclosed in U.S. Patent Nos. 5,843,718; 5,830,750; 5,712,146 and 5,672,491 to Khosla et al, U.S. Patent No. 5,824,485 to Thompson et al, U.S. Patent No. 5,783,431 to Peterson et al, U.S. Patent No.
  • the term "daptomycin” refers to the n-decanoyl derivative of A-21978C 0 type antibiotic. "Daptomycin” is synonymous with LY 146032.
  • An exemplary process for production of daptomycin is as follows. Streptomyces roseosporus is fermented with a feed of n-decanoic acid, as disclosed in United States Patent No. 4,885,243, with the modification that the decanoic acid feed is kept at the lowest levels possible without diminishing the overall yield of the fermentation.
  • the residual decanoic acid is maintained at less than 50 parts per million (ppm) during aerobic fermentation. In a more preferred embodiment, the residual decanoic acid is maintained between one and 20 ppm during the aerobic fermentation.
  • the residual decanoic acid is maintained at approximately ten ppm during the aerobic fermentation.
  • the concentration of residual decanoic acid is measured throughout the fermentation and the feed level of decanoic acid is adjusted to continuously keep the residual decanoic acid levels within the preferred parameters.
  • the prior art does not describe the in situ specific and low residual constant decanoic acid concentration required to achieve optimal expression of daptomycin containing lower levels of impurities.
  • the process disclosed herein will also facilitate the detection and isolation, for example, of new secondary metabolites that are structurally related to known chemical entities.
  • secondary metabolites may be detected using the methods of the present invention, which were not known to be produced by prior art processes.
  • the process of the present invention will also facilitate the detection, isolation and characterization of novel chemical structures by providing increased production levels and enhanced component profiles for secondary metabolites.
  • Mixtures of secondary metabolites comprising new activities or new peaks may be fractionated chromatographically, for example by high-performance liquid chromatography (HPLC). Appropriate fractions of the profile may be collected, concentrated as necessary and further purified using methods well known to those of ordinary skill in the art.
  • the methods of the present invention can be applied to those discovery approaches in which newly-discovered or newly-constructed organisms are grown in or on a number of different media, assembled empirically over time, such that at least one set of conditions will be suitable for the synthesis of secondary metabolites at a detectable level.
  • testing of each organism will result in the generation of a correspondingly large set of samples to be examined for compounds exhibiting useful properties.
  • Each of these samples is then tested in a wide array of activity screens employing a number of assays of varying degrees of sensitivity and specificity, generating a signal where the material examined has a desired trait.
  • High-throughput screens can be designed within which a population of organisms, which may include newly-isolated or newly-constructed strains, are distributed onto semi-solid media or into individual wells containing growth media. Subsequently, secondary metabolites produced under these conditions are screened for biological activity by sampling the growth media surrounding the colony.
  • an indicator organism may be overlaid onto the semi-solid medium upon or within which the secondary-metabolite-producing organisms are growing.
  • the indicator may simply be an organism sensitive to an antibiotic produced as a secondary metabolite or the indicator can be an organism that will generate, or has been genetically engineered to generate, a detectable signal in the presence of a compound having a desired activity.
  • Examples of the latter include indicator strains, isolated or constructed, which will generate a detectable signal in the presence of an agonist or an antagonist of receptor binding.
  • Such systems are only limited by the imagination and creativity of the investigator and many examples are known to those of ordinary skill in the art.
  • Non-limiting examples of screening methods are found in the following representative patents: U.S. Patent Nos. 5,780,294 to Siehl et al, 5,914,323 to Mundy et al, 5,948,612 to Bascomb et al, and publications: Stockwell et al. (1999) Chem Biol 6(2): 71-83; Kunkel et al. (1997) Anticancer Drug Res 12(8): 659-670; Shawar et al (1997) Antimicrob Agents Chemother 41 .
  • Application of the methods of the present invention is expected to increase the probability of detecting interesting secondary metabolites both by increasing the level of production thereof, and by enhancing the component profile of secondary metabolites elaborated by the population of producing organisms examined.
  • the methods of the present invention can be applied to those processes initiated once a potentially-useful molecule has been detected, and a sufficient amount of that compound is to be produced to verify preliminary activity data, to provide a preliminary evaluation of the compound, and to permit a determination of its structure.
  • the amount of material required for these pilot and range-finding experiments is often considerable, when compared to the level of production of the secondary metabolite by the organism, and can create a serious impediment to development of a secondary metabolite. Accordingly, supplementation of the growth media with one or more organic additives is expected to facilitate such work by increasing the level of production of the secondary metabolite of interest or by inducing the production of that metabolite.
  • the level of production of a secondary metabolite of interest can be improved by carrying out a coordinated development program involving both medium optimization and strain improvement.
  • the level of production of a secondary metabolite by an organism can be improved using, for example, empirical methods, directed procedures, molecular biology, genetic engineering, and combinations of these methods in one or more stages of overall process improvement.
  • Empirical procedures for improving the production of secondary metabolites were developed, historically, and generally rely upon random mutagenesis of the producing organism coupled with, essentially, trial-and-error media development.
  • This approach requires little information concerning the organism, the biosynthetic pathway involved or even the structure of the secondary metabolite of interest.
  • considerable labor and time may be required since large numbers of candidate strains must be evaluated to find those isolates that are, in fact, stable derivatives that are significantly improved producers of the secondary metabolite.
  • Multiple rounds of mutagenesis and screening which may be automated, are frequently necessary to develop a useful strain. Even with a demonstrably improved strain, media development must still be carried out to optimize production of the, secondary metabolite.
  • One of the liabilities of this approach is that it is very inflexible.
  • the ultimate process developed comprising the multiply-mutated strain and the corresponding medium optimized for production of the secondary metabolite of interest by that strain, is not generally applicable to a process for the production of any other secondary metabolite.
  • incorporation of the methods of the present invention is expected to provide an increase in the level of production of at least one secondary metabolite in the production process developed in this manner. More directed methods have been developed and applied in those instances where the structure of the secondary metabolite has been established and the biosynthetic pathway for its synthesis has been experimentally determined or is reasonably apparent.
  • a second directed approach can be employed if the secondary metabolite has antimicrobial activity or if an indicator organism has been isolated or constructed, which will provide a detectable signal in the presence of a desired secondary metabolite.
  • Improved producers of that secondary metabolite may readily be identified from within a larger population grown on an agar medium by overlaying the producing colonies with a sensitive indicator organism.
  • This method for improved production of a secondary metabolite is limited to those having antibiotic activity or those for which a signal-generating indicator organism is available; this approach also relies upon the availability of a suitable growth medium.
  • application of the methods of the present invention is expected to increase the level of expression of the secondary metabolite produced as well as to enhance the component profile of the secondary metabolites synthesized under these conditions.
  • A-factor which is a gamma-butyrolactone derivative, binds to a specific receptor protein in Streptomyces griseus and triggers the expression of streptomycin biosynthetic pathway.
  • Second-site mutations have, in some instances, been shown to affect the level of synthesis of antibiotics. Shima et al. have demonstrated that specific
  • the present invention provides improved methods for the production of secondary metabolites by Streptomyces roseosporus, especially the A-21978C complex of antibiotics, and even more particularly the n-decanoyl derivative of the A-21978C "peptide core" that has been named daptomycin.
  • the invention also provides a method for improving the production level of a secondary metabolite o ⁇ Streptomyces roseosporus, comprising culturing Streptomyces roseosporus in a nutrient medium, in which Streptomyces roseosporus synthesizes an amount of at least one secondary metabolite, and wherein the nutrient medium comprises an amount of at least one organic additive sufficient to increase the production level of the secondary metabolite relative to that in the absence of at least one organic additive.
  • the invention provides a method for improving the production level of a secondary metabolite o ⁇ Streptomyces roseosporus, comprising culturing Streptomyces roseosporus in a nutrient medium, in which Streptomyces roseosporus synthesizes an amount of the secondary metabolite daptomycin, and wherein the nutrient medium comprises an amount of at least one organic additive sufficient to increase the production level of daptomycin relative to that in the absence of the at least one organic additive.
  • the present invention provides a method for enhancing the component profile of secondary metabolites produced by Streptomyces roseosporus, comprising culturing Streptomyces roseosporus in a nutrient medium, in which Streptomyces roseosporus elaborates a component profile of secondary metabolites, wherein the nutrient medium comprises an amount of at least one organic additive sufficient to enhance the component profile of secondary metabolites produced by Streptomyces roseosporus relative to that in the absence of the at least one organic additive.
  • the present invention provides a method for enhancing the component profile of secondary metabolites produced by Streptomyces roseosporus, comprising culturing Streptomyces roseosporus in a nutrient medium, in which Streptomyces roseosporus elaborates components of the A-21978C complex of secondary metabolites, wherein the nutrient medium comprises an amount of at least one organic additive sufficient to enhance the component profile of A-21978C complex of secondary metabolites produced by Streptomyces roseosporus relative to that in the absence of the at least one organic additive.
  • the invention provides a method for identifying a novel secondary metabolite, comprising culturing Streptomyces roseosporus in a nutrient medium, wherein the nutrient medium comprises an amount of at least one organic additive sufficient to enhance the component profile of secondary metabolites produced by Streptomyces roseosporus, and in which, at least on novel secondary metabolite is identified in the component profile of secondary metabolites produced by Streptomyces roseosporus, relative to that in the absence of the at least one organic additive.
  • Spore suspensions of the various species o ⁇ Streptomyces ( ⁇ 1-2 x 10 8 cfu/ml) used in the present invention were obtained as follows.
  • the bacteria were initially grown on ISP (Difco) agar plates until sporulation. The plate surface was then washed with sterile water to harvest the spores.
  • the spore solution was mixed with an equal volume of 50% glycerol and stored as frozen stocks.
  • the frozen spore stocks which were titered to establish the viable count, were used as inoculum in the following examples.
  • the general culturing conditions for the Streptomyces species were as follows. Culture medium was first inoculated with the spore suspension and mixed thoroughly. Ten ml of the inoculated culture medium were added to 50 ml test tubes and the cells were grown aerobically at 30° C in the absence or presence of either DMSO, 5 ethanol or dimethyl sulfone.
  • Cell biomass for each culture of the various Streptomyces species was 15 determined as follows. The remaining spent medium with cells was centrifuged at 4,000 rpm for 10 minutes. The supernatant was discarded and the remaining pellet was resuspended in 40 ml of distilled water. The cell suspension was centrifuged again. The cells were resuspended in distilled water and harvested by filtering the cell suspension through Whatman #1 filter paper. The filter paper with cells was then baked in an oven at 20 80°C overnight and the dry biomass weighed.
  • TLB Tryptic Soy Broth
  • Figure 1 shows that tetracenomycin production was highest when 3 % v/v DMSO was added. Tetracenomycin production was increased 4.6-fold as compared to control cultures, where no DMSO was added.
  • DMSO was added and production started to decline at 5 % v/v DMSO.
  • Example III Streptomyces venezuelae Cells of the wild type strain o ⁇ Streptomyces venezuelae were grown in GNY medium of the following composition:
  • Figure 4 shows that at 1, 3 and 5 % v/v DMSO the production of thiostrepton almost doubled.
  • Clone SI OH 10 contained in a Streptomyces lividans host, is a shuttle vector containing a DNA insert derived from a genomic DNA library, which was made from DNA fragments of approximately 13 Kb from a mixture of previously unknown soil isolates of Streptomyces. This clone was examined to ascertain the effect of organic additives on the component profile of secondary metabolites produced by a host Streptomyces containing exogenous genetic material.
  • Clone SI OH 10 was generated using the shuttle vector plasmid pWHM601. Plasmid pWHM601 was digested with BamHI followed by dephosphorylation with shrimp alkaline phosphotase using standard protocols. Insert DNA with a fragment size of 13 Kb was prepared from partially Sau3AI-digested genomic DNA mixture often unknown soil Streptomyces isolates 73B, 77D, 830, 85D, 871, 88A, 9 IE, 93 A, 115C and 1 18B.
  • the 13 Kb fragments were then ligated to the dephosphoylated plasmid at a ratio of 1 :3 using T4 DNA ligase, and directly transformed into the host Streptomyces lividans TK23 by protoplast transformation.
  • the transformed protoplasts were spread on R5 agar plates and, after a 24 hour outgrowth period, were overlaid with a concentrated solution of apramycin, yielding a final concentration of 50 ⁇ g apramycin/ml.
  • Cells of clone SI OH 10 were cultured in R5 medium containing 25 ⁇ g/ml apramycin in the presence and absence of 3% DMSO. One ml samples were extracted using an equal volume of ethyl acetate and subjected to HPLC analysis.
  • Figure 5b shows the HPLC profile of the low molecular weight metabolites produced by clone SI OH 10 in the absence of DMSO.
  • Figure 5a shows the HPLC profile when 3% DMSO was added to the culture medium.
  • a comparison of the two profiles shows that DMSO is effective in enhancing the component profile of secondary metabolites of clone SI OH 10.
  • Clone 446-S3-81D3 is a recombinant consisting of a cosmid vector containing a DNA fragment derived from an environmental DNA sample.
  • the clone was generated using the shuttle cosmid vector pOJ446.
  • the vector was linearized by digestion with Hpal and dephosphorylated with alkaline phosphatase using standard protocols. The linearized vector was further digested with BamHI.
  • Insert DNA with a fractionation size ranging from 23 to 40 Kb was prepared by partial Sau3AI digestion of DNA directly extracted from forestry soil.
  • a cosmid library was generated by ligating the vector to the insert DNA at a ratio of 1 : 1 using T4 DNA ligase, and packaging in vitro using a Gigapack III Gold Packaging Extract kit.
  • the library was first amplified using Escherichia coli XL 1 Blue MR as a primary host. Cosmids with insert DNA were then extracted and transferred to the expression host Streptomyces lividans TK23 by protoplast transformation. Transformants were selected by growing them on R5 agar plates containing 25 ⁇ g apramycin ml. Cells of clone 446-S3-8 1 D3 were cultured in R5 medium containing 25 ⁇ g apramycin/ml in the presence and absence of various concentrations DMSO. One ml samples were extracted using an equal volume of ethyl acetate and subjected to HPLC analysis.
  • Figure 6a shows the HPLC profile of the low molecular weight metabolites produced by clone 446-S3-8 1 D3 in the absence of DMSO.
  • Figure 6b shows the HPLC profile when 2% DMSO was added to the culture medium.
  • a comparison of the two profiles shows that DMSO effectively increased the production of many of the secondary metabolites that are produced when the 446-S3-8 1 D3 cells are grown in the absence of DMSO.
  • the component profile obtained demonstrates that the concentration of some metabolites has increased by as much as six-fold.
  • Clone 446-53- 102H4 was generated as described in Example VI.
  • Cells of clone 446-53- 102H4 were cultured in R5 medium containing 25 ⁇ g/ml apramycin in the presence and absence of 2% DMSO.
  • One ml samples were extracted using an equal volume of ethyl acetate and subjected to HPLC analysis.
  • Figure 7a shows the HPLC profile of the low molecular weight metabolites produced by clone 446-53- 102H4 in the absence of DMSO.
  • Figure 7b shows the HPLC profile when 2% DMSO was added to the culture medium.
  • TCMC tetracenomycin
  • Figure 9 shows that tetracenomycin production was highest when 3% v/v ethanol was added, resulting in a 1.6-fold increase in the production of tetracenomycin.
  • Streptomyces venezuelae cells were cultured in GNY medium with 1, 3, and 5% dimethyl sulfone (w/v) and analyzed as described in Example III.
  • Figure 10 shows that production of chloramphenicol was almost doubled at 1% dimethyl sulfone.
  • Taxol Microbial Culture Medium of the following composition:
  • At least one organic additive preferably DMSO, ethanol or dimethyl sulfone, is added to the culture medium.
  • concentration of the organic additives in the culture medium is in the range of about 0.1 to about 10% v/v.
  • Spore suspensions of the Streptomyces roseosporus are obtained as follows.
  • the bacteria are initially grown on ISP (Difco) agar plates until sporulation.
  • the plate surface is then washed with sterile water to harvest the spores.
  • the spore solution is mixed with an equal volume of 50% glycerol and stored as frozen stocks.
  • the frozen spore stocks which are titered to establish the viable count, are used as inoculum in the following example.
  • the frozen spore stocks are used to inoculate the fermentation medium directly.
  • the spore stock are used to inoculate 10 ml. of a seed medium, e.g. Tryptic Soy Broth (TSB) that is incubated with shaking for 24 to 48 hrs. at 30° C.
  • TLB Tryptic Soy Broth
  • the culturing conditions for the Streptomyces roseosporus species are as follows. Culture medium, generally 25 ml. of soy flour-molasses medium in a 250 ml. baffled flask is inoculated with the spore suspension or with a seed culture (generally 1 ml) and mixed thoroughly. The cells are grown aerobically at 30 °C in the absence or presence of either DMSO, for seven days.
  • Analysis of the secondary metabolites produced in the cultures is estimated as follows. At various times during the fermentation, generally after five to seven days, aliquots of the fermentation are removed and centrifuged, for example, at 5000 rpm in a bench-top centrifuge, using sufficient force to pellet the cells. Samples of the supernatant are analyzed directly by HPLC or an ethyl acetate extract of the supernatant is prepared and analyzed by HPLC. The cell pellet is extracted with ethyl acetate and the extract analyzed by HPLC. Alternatively, the spent medium, containing the Streptomyces roseosporus cells may be extracted directly as follows. 1 ml of the spent medium with cells is transferred to a 2 ml microfuge tube for extraction.
  • microfuge tube An equal amount of organic extraction solvent, ethyl acetate is added to the microfuge tube and the tube is horizontally shaken for 30 min.
  • the microfuge tube is centrifuged at 14,000 rpm for 5 minutes at which time 0.75 ml of the organic fraction is transferred to a new 2 ml microfuge tube, dried in an evacuated centrifuge with medium heat, approximately 40 °C, and then analyzed using a Beckman System Gold HPLC with a reverse C18 column.
  • Cell biomass for each culture o ⁇ Streptomyces roseosporus is determined as follows. The remaining spent medium with cells is centrifuged at 4,000 rpm for 10 minutes. The supernatant is discarded and the remaining pellet is resuspended in 40 ml of distilled water. The cell suspension is centrifuged again. The cells are resuspended in distilled water and harvested by filtering the cell suspension through Whatman #1 filter paper. The filter paper with cells is then baked in an oven at 80 °C overnight and the dry biomass weighed.
  • Cells are also grown in the absence of DMSO and analyzed as above, for comparison of the effects of DMSO on daptomycin production levels, component profile, and for detection of new components that may be novel secondary metabolites elaborated by Streptomyces roseosporus. Authentic standards of daptomycin are also chromatographed for comparison.

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Abstract

L'invention concerne un procédé relatif à l'amélioration du niveau de production ainsi que du profil de composantes et de la diversité chimique des métabolites secondaires produits par des organismes. Le procédé consiste à ajouter au moins un additif organique au milieu de croissance inoculé avec l'organisme dans lequel sont produits le ou les métabolites secondaires. Parmi les additifs organiques appropriés, on peut citer le sulfoxyde de diméthyle, le sulfone de diméthyle et l'éthanol, la préférence allant au premier cité. Les additifs organiques considérés sont utilisés selon une concentration comprise entre 0,1 % et 10 % (v/v) dans le milieu de croissance, de préférence entre 0,5 % et 7,5 %, et de préférence encore entre 1 % et 5 %.
PCT/US2001/040398 2000-03-28 2001-03-28 Production amelioree de metabolites secondaires en presence d'additifs organiques WO2001073096A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8217085B2 (en) 2009-10-30 2012-07-10 Biogenic Innovations, Llc Methylsulfonylmethane (MSM) for treatment of drug resistant microorganisms
CN101550436B (zh) * 2008-04-01 2012-10-17 上海来益生物药物研究开发中心有限责任公司 一种提高抗菌物质a21978c产量的方法
CN103194502A (zh) * 2013-04-24 2013-07-10 黑龙江大学 树状多节孢内生真菌hdfs4-26生物发酵合成紫杉醇及其前体物的分离与纯化方法
US8546373B2 (en) 2009-10-30 2013-10-01 Biogenic Innovations, Llc Methods of sensitizing drug resistant microorganisms using methylsulfonylmethane (MSM)
US9186297B2 (en) 2005-09-12 2015-11-17 Abela Pharmaceuticals, Inc. Materials for facilitating administration of dimethyl sulfoxide (DMSO) and related compounds
US9186472B2 (en) 2005-09-12 2015-11-17 Abela Pharmaceuticals, Inc. Devices for removal of dimethyl sulfoxide (DMSO) or related compounds or associated odors and methods of using same
US9427419B2 (en) 2005-09-12 2016-08-30 Abela Pharmaceuticals, Inc. Compositions comprising dimethyl sulfoxide (DMSO)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55144897A (en) * 1979-05-01 1980-11-12 Squibb & Sons Inc Improved synthesis of penicillins
US4732680A (en) * 1985-05-08 1988-03-22 Betz Laboratories, Inc. Biochemical conversion processes
US4885243A (en) * 1984-10-09 1989-12-05 Eli Lilly And Company Process for producing A-21978C derivatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55144897A (en) * 1979-05-01 1980-11-12 Squibb & Sons Inc Improved synthesis of penicillins
US4885243A (en) * 1984-10-09 1989-12-05 Eli Lilly And Company Process for producing A-21978C derivatives
US4732680A (en) * 1985-05-08 1988-03-22 Betz Laboratories, Inc. Biochemical conversion processes

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: "Enhanced production of microbial metabolites in the presence of dimethyl sulfoxide", J. OF ANTIBIOTICS., vol. 53, no. 10, October 2000 (2000-10-01), pages 1145 - 1153, XP002944442 *
DATABASE CAPLUS [online] SQUIBB ET AL.: "Modified method or penicillin fermentation", XP002944437, accession no. STN Database accession no. 1981:101311 *
DATABASE CAPLUS [online] YU ET AL.: "The induction effect of methyl-jasmonate on taxol biosynthesis", XP002944433, Database accession no. 2000:132168 *
DOULL ET AL.: "Conditions for the production of jadomycin B by streptomyces venezuelae ISP5230: effects of heat shock, ethanol treatment and phage infection", J. INDUSTRIAL MICROBIOL., vol. 13, 1994, pages 120 - 125, XP002944441 *
OGATA ET AL.: "Screening of compounds stimulating spore formation and mycelial growth of pock-formin plasmid-carrying strains in streptomyces azureus", APPL. MICROBIOL. BIOTECHNOL., vol. 37, 1992, pages 652 - 654, XP002944432 *
SABOURIN ET AL.: "Biodegradation of dimethylsilanediol in soils", APPLIED AND ENVIRONMENTAL MICROBIOL., vol. 62, no. 12, December 1996 (1996-12-01), pages 4352 - 4360, XP002944438 *
TIANRAN CHANWU YANJIU KAIFA, vol. 11, no. 5, 1999, pages 1 - 7, XP002944434 *
TOMBO ET AL.: "Diasteroselective microbial hydroxylation of milbemycin derivatives", AGRIC. BIOL. CHEM., vol. 53, no. 6, 1989, pages 1531 - 1535, XP002944440 *
TOYAMA ET AL.: "Three distinct quinoprotein alcohol dehydrogenases are expressed when pseudomonas putida is grown on different alcohols", J. OF BACTERIOL., vol. 177, no. 9, May 1995 (1995-05-01), pages 2442 - 2450, XP002944439 *
UDVARNOKI ET AL.: "Biosynthetic origin of the oxygen atoms of tetracenomycin C.", ANGEW. CHEM. INT. ED. ENGL., vol. 34, no. 5, 1995, pages 565 - 567, XP002944435 *
YAMAMOTO ET AL.: "Effects of culture conditions on the growth of usneacea lichen tissue cultures", PLANT CELL PHYSIOL., vol. 28, no. 8, 1987, pages 1421 - 1426, XP002944436 *

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US9186297B2 (en) 2005-09-12 2015-11-17 Abela Pharmaceuticals, Inc. Materials for facilitating administration of dimethyl sulfoxide (DMSO) and related compounds
US9427419B2 (en) 2005-09-12 2016-08-30 Abela Pharmaceuticals, Inc. Compositions comprising dimethyl sulfoxide (DMSO)
US9186472B2 (en) 2005-09-12 2015-11-17 Abela Pharmaceuticals, Inc. Devices for removal of dimethyl sulfoxide (DMSO) or related compounds or associated odors and methods of using same
CN101550436B (zh) * 2008-04-01 2012-10-17 上海来益生物药物研究开发中心有限责任公司 一种提高抗菌物质a21978c产量的方法
JP2013509197A (ja) * 2009-10-30 2013-03-14 バイオジェニック イノベーションズ， リミテッド ライアビリティ カンパニー 微生物活性を調節するためのメチルスルホニルメタン（ｍｓｍ）の使用
US8546373B2 (en) 2009-10-30 2013-10-01 Biogenic Innovations, Llc Methods of sensitizing drug resistant microorganisms using methylsulfonylmethane (MSM)
EP2494059A4 (fr) * 2009-10-30 2013-12-04 Biogenic Innovations Llc Utilisation de méthysulfonylméthane (msm) à des fins de modulation de l'activité microbienne
US8841100B2 (en) 2009-10-30 2014-09-23 Biogenic Innovations, Llc Use of methylsulfonylmethane (MSM) to modulate microbial activity
US8217085B2 (en) 2009-10-30 2012-07-10 Biogenic Innovations, Llc Methylsulfonylmethane (MSM) for treatment of drug resistant microorganisms
EP2494059A1 (fr) * 2009-10-30 2012-09-05 Biogenic Innovations, Llc Utilisation de méthysulfonylméthane (msm) à des fins de modulation de l'activité microbienne
US9487749B2 (en) 2009-10-30 2016-11-08 Biogenic Innovations, Llc Use of methylsulfonylmethane (MSM) to modulate microbial activity
US9839609B2 (en) 2009-10-30 2017-12-12 Abela Pharmaceuticals, Inc. Dimethyl sulfoxide (DMSO) and methylsulfonylmethane (MSM) formulations to treat osteoarthritis
US9855212B2 (en) 2009-10-30 2018-01-02 Abela Pharmaceuticals, Inc. Dimethyl sulfoxide (DMSO) or DMSO and methylsulfonylmethane (MSM) formulations to treat infectious diseases
US10596109B2 (en) 2009-10-30 2020-03-24 Abela Pharmaceuticals, Inc. Dimethyl sulfoxide (DMSO) or DMSO and methylsulfonylmethane (MSM) formulations to treat infectious diseases
CN103194502A (zh) * 2013-04-24 2013-07-10 黑龙江大学 树状多节孢内生真菌hdfs4-26生物发酵合成紫杉醇及其前体物的分离与纯化方法

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