EP2658855A2 - Novel polyketide compounds and methods of making same - Google Patents

Novel polyketide compounds and methods of making same

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Publication number
EP2658855A2
EP2658855A2 EP11813782.7A EP11813782A EP2658855A2 EP 2658855 A2 EP2658855 A2 EP 2658855A2 EP 11813782 A EP11813782 A EP 11813782A EP 2658855 A2 EP2658855 A2 EP 2658855A2
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Prior art keywords
seq
polyketide
compound
nascent
coa
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EP11813782.7A
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German (de)
French (fr)
Inventor
Gregor Kosec
Dusan GORANOVIC
Jaka HORVAT
Stefan Fujs
Branko Jenko
Hrvoje PETKOVIC
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Acies Bio doo
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Acies Bio doo
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • Polyketides are a large and diverse class of natural products that includes many compounds with antibacterial, antifungal, antihelminthic, immunosuppressant, anticancer or other pharmacological activities, such as tetracyclines, erythromycins, avermectins, rapamycins as well as FK506. They are synthesized by ordered condensation of acylthioesters similarly to the synthesis of fatty acids. Great structural diversity of polyketides or related compounds (e.g. mixed polyketide synthase (PKS)/non-ribosomal peptide synthases (NRPS)) as compared to fatty acids arises from greater choices that these enzymatic systems can make during carbon chain assembly.
  • PPS mixed polyketide synthase
  • NRPS non-ribosomal peptide synthases
  • starter units can be selected from acetate, propionate, butyrate and often other unusual CoA- activated carboxylic acids such as shikimate-derived cyclohexane-carboxylic acid.
  • extender units are selected from CoA-activated carboxylic acids such as malonic, methylmalonic, ethylmalonic acid and only rarely from other derivatives.
  • Nascent ⁇ -keto unit, resulting from the condensation reaction can be further reduced to different degrees ( ⁇ -keto, ⁇ -hydroxyacyl, 2-enoyl or saturated thioester) and the stereochemistry of the substituents is also specified in each cycle of chain extension.
  • products of the P S or PKS-related enzyme systems are often further modified by site-specific oxidative enzymes, methyltransferases or glycosyl transferases, thus introducing expanded structural diversity.
  • polyketide synthases Several classes of polyketide synthases have been described. Among them, modular type I polyketide synthases, encoding for example erythromycin, rapamycin and avermectin biosynthesis have been extensively studied. These large enzymes are organized in modules in a manner that each module catalyzes one step of polyketide chain extension. Each module consists of at least three protein domains, namely ⁇ -ketoacyl ACP synthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) where the AT domain determines the choice of the extender (or starter) unit for the relevant chain elongation step. In addition, some modules may also contain additional domains involved in the reduction of the ⁇ -keto group, i.e.
  • KS ⁇ -ketoacyl ACP synthase
  • AT acyl transferase
  • ACP acyl carrier protein
  • some modules may also contain additional domains involved in the reduction of the ⁇ -ket
  • ⁇ -ketoreductase KR
  • dehydratase DH
  • enoylreductase ER domains.
  • the module involved in the final elongation step often contains a thioesterase (TE) domain which releases the nascent polyketide chain from the PKS enzyme and it is believed, that TE domain can also influence cyclisation pattern of the macrolide or macrolactone structure.
  • TE thioesterase
  • the acyltransferase domain of the module 1 of DEBS1 TE which specifically incorporates methylmalonyl-CoA extender unit into the polyketide chain was replaced by the AT domain of the module 2 of rapamycin producing PKS, specifically incorporating malonyl-CoA. Indeed, the resulting products lacked a methyl group at the carbon 4 of the lactone ring.
  • Another example of efficient replacement of acyltransferase domain replacement (AT swap) of erythromycin PKS is the incorporation of ethylmalonyl-CoA specific AT domain from niddamycin PKS instead of native AT domain encoding methylmalonyl-CoA.
  • extender units incorporated into the polyketide chain can be effectively used to vary the moiety that extends away from the backbone of the molecule, which can have effect on its interaction with its biological target.
  • efficient supply of ethylmalonyl-CoA extender unit had to be assured during cultivation in order to enable efficient production of 6-desmethyl-6-ethylerythromycin A (Stassi, Kakavas et al. 1998).
  • the same group was able to obtain a series of novel erythromycin analogues by swapping AT-domains of DEBS genes with heterologous AT-domains from different organisms.(Ruan, Pereda et al. 1997; Katz, Stassi et al. 2000).
  • Adequate supply of the extender unit is necessary in addition to the genetic exchange of the AT domain in the PKS, in order to generate the target product.
  • Substrate supply can be provided by metabolic processes taking place in the host organism or alternatively, extender unit precursors can be added exogenously to cultivation broths.
  • Ethyl-substituted erythromycin derivatives were only produced when cultures of Saccharopolyspora erythraea with engineered erythromycin PKS were transfected with a heterologous gene encoding crotonyl-CoA reductase/carboxylase activity (CCR) or when a precursor of ethylmalonyl-CoA, diethylethylmalonate was added to fermentation broths (Stassi, Kakavas et al. 1998).
  • CCR crotonyl-CoA reductase/carboxylase activity
  • N-acetylcysteamine (SNAC) thioester derivatives of malonic acid or other extender units were used for feeding experiments as SNAC derivatives resemble closely the naturally occurring CoA thioesters thus enable incorporation of exogenously added compounds into polyketide chains.
  • AT domains of extender modules In contrast to the AT domain of the loading module of avermectin PKS which, which is able to incorporate a variety of exogenously added synthetic starter units, AT domains of extender modules generally have very limited spectrum of the extender units that can be incorporated.
  • AT domains can select for malonyl-CoA and methylmalonyl-CoA, and less often ethylmalonyl-CoA and methoxymalonyl-CoA extender units are selected only by limited number of AT domains.
  • AT domains show specificity for unusual extender units, whose supply is often encoded in gene clusters governing the biosynthesis of secondary metabolites.
  • One example is the incorporation of an unusual five carbon extender unit by the acyltransferase domain of the module 4 (AT4) of PKS governing the biosynthesis of FK506.
  • this extender unit results in the presence of allyl group at the carbon 21 of the FK506 structure (see Formula I, below), a feature which distinguishes FK506 from a structurally and biosynthetically related compound FK520, possessing an ethyl group at equivalent position.
  • Biosynthetic origin of the five carbon extender unit in FK506 producing organism Streptomyces tsukubaensis has been recently elucidated (Goranovic, Kosec et al. 2010).
  • these authors demonstrated that when synthetically-derived allylmalonyl-SNAC thioester was supplemented into cultivation broth of the mutant strain of S.
  • AT domains are generally believed to be specific for a certain extender unit, e.g. malonyl- CoA, methylmalonyl-CoA or ethylmalonyl-CoA, however, non-specific or promiscuous AT domains have also been described, which can incorporate more than one naturally occurring extender units.
  • Polyketide synthases comprising such domains usually catalyze the biosynthesis of a mixture of closely related polyketides.
  • One of the best known examples is a polyketide synthase of Streptomyces cinamonnensis, which simultaneously produces a mixture of monensin A and monensin B, depending on whether ethylmalonyl- CoA or methylmalonyl-CoA, both produced by metabolic pathways of this organism, is incorporated into the chain by one of the AT domains (Liu and Reynolds 1999).
  • this promiscuity (relaxed specificity) is very limited, and can only accommodate very closely related extender units such as malonyl-CoA and methylmalonyl-CoA or methylmalonyl-CoA and ethylmalonyl-CoA.
  • only structurally very similar compounds that differ in one methyl group at the a-carbon atom of the nascent polyketide chain can be generated using existing methodology, hence little change in pharmacological properties can be expected, as confirmed in the past.
  • all these "natural" extender units i.e. extender units synthesized by the producing microorganism
  • all these "natural" extender units lack simple chemical reactivity of the groups, which would enable further semi-synthetic chemical derivatization at a-carbon positions of the polyketide backbone.
  • these extender units are bound to ACP they cannot be added to fermentation broth and genes encoding the biosynthesis of these extender units must be effectively expressed in the engineered strain (Emmert, Klimowicz et al. 2004; US2008254508).
  • the PKS enzyme from B. cereus utilize a reaction mechanism that differs importantly from type I PKS from Actinomyces ⁇ Streptomyces), which encodes majority of medically important polyketide- derived structures. Most notably, it lacks an AT domain in enzymatic modules that would select the extender unit for incorporation in each elongation step of polyketide chain biosynthesis. Therefore, although it seems straightforward to produce these two extender units inside cells no AT domain of type I PKS from Actinomyces (Streptomyces) has been demonstrated system to be able to incorporate hydroxymalonyl-ACP and aminomalonyl- ACP into polyketide chains of clinically relevant polyketide molecules.
  • a system for incorporation of extender units with chemically more reactive side chains would be composed of an AT domain of type I PKS that would retain its specificity when replacing such an AT domain with diverse AT domains from heterologous PKS genes and a set of extender units or extender unit analogues that could be supplemented to cultivation broths which would be further incorporated into polyketide chains at the specific (desired) positions.
  • the extender unit analogues would be esters or thioesters of malonic acid substituted at position of the carbon 2. This substituent would thus represent a chemically reactive group moiety sticking out of the polyketide chain.
  • malonic acid can be synthesized by methods known in the art, for example (but not limited to): A) alkylation of malonic esters on position 2 by substituted alkyl halides (Eglinton and Whiting 1953) and B) ring opening of cyclopropyl dicarboxylates with HBr or HC1. (Demjanov 1939).
  • Polyketides are a large group of biogenetically related compounds, bio-synthesized by the large group of closely related PKS enzymes.
  • the compounds belonging to this group ob biosynthetically related structure display huge structural diversity which reflects also large spectrum of pharmacological activities, such as antibacterial, antifungal, anti- cancer, anti-helminthic, coccidiostatic, insecticide, immunosuppressive, neuroregenerative and other biological activities.
  • pharmacological activities such as antibacterial, antifungal, anti- cancer, anti-helminthic, coccidiostatic, insecticide, immunosuppressive, neuroregenerative and other biological activities.
  • further improvements of their pharmacologically-related features have been achieved by additional modifications of these compounds using semi-synthetic chemistry and biosynthetic engineering approaches.
  • FK506 (tacrolimus), has been used for many years as an immunosuppressant after organ transplantation.
  • FKBPs cytosolic receptors
  • Many immunophilins are encoded in the human genome and are known to associate with other cellular proteins of diverse functions. Immunophilin ligands generate various down-stream biological activities by disruption of the natural FKBP-containing complexes or by formation of novel ternary complexes.
  • the binary complex of FK506 (or FK520) with FKBP12 binds to calcineurin, a calmodulin dependent protein phosphatase. Calcineurin dephosphorilates and thereby activates a transcription factor termed "nuclear factor of activated T cell" (NFATC), which is then translocated from the cytoplasm to the nucleus where it upregulates the expression of interleukin 2. Interleukin 2 then stimulates growth and differentiation of T lymphocytes. When calcineurin is inhibited by immunosuppressive drugs T-cell mediated immune response is largely inhibited. As calcineurin is widespread among eukaryotic organisms FK506 and FK520 also have antifungal and antiprotozoan activities.
  • F 506 has been used mainly as immunosuppressant used to decrease the possibility of organ rejection after transplantation. Intravenous or oral administration after allogeneic liver, kidney or bone marrow transplantation represent the most examples of use.
  • FK506 has shown potential for use in treatment of several autoimmune, inflammatory or respiratory diseases such as: Bechet syndrome, Crohn's disease, atopic dermatitis, uveitis, psoriasis, nephritic syndrome, rheumatoid arthritis, asthma, aplastic anemia, biliary cirrhosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease.
  • autoimmune, inflammatory or respiratory diseases such as: Bechet syndrome, Crohn's disease, atopic dermatitis, uveitis, psoriasis, nephritic syndrome, rheumatoid arthritis, asthma, aplastic anemia, bili
  • FK.506 as well as pimecrolimus (a derivative of FK520) are also used in topical formulations such as creams and ointments for localized treatment of skin disorders, particularly atopic dermatitis.
  • FK506 is a widely used immunosuppressant to prevent the graft-versus-host disease after organ transplantation, its use is complicated due to the narrow therapeutic window, toxicity in the kidney, central nervous system and pancreas and most importantly large inter- and intra-individual variability in bioavailability and pharmacokinetics of the drug. Frequent therapeutic drug monitoring using enzyme immunoassay is therefore required to control the concentration of tacrolimus in the blood.
  • enzyme immunoassay is therefore required to control the concentration of tacrolimus in the blood.
  • L-732,531 a C32 derivative of FK520, found to posses significantly superior pharmacologic properties to tacrolimus as it shows lower affinity for erythrocytes and therefore a lower and concentration-independent blood-to-plasma ratio.
  • Elucidation of biosynthetic mechanisms of FK506 and FK520 based on the sequences of their gene clusters has also enabled the production of several analogues by biosynthetic engineering.
  • acyl transferase domains of PKS extender modules 13- and 15- desmethoxy derivatives of FK520 have been produced, which lack immunosuppressive activity and show high potential as neuroregenerative drugs.
  • WO 2004/096822 discloses polyketide compounds including a residue having a -CCH triple bond residue adjacent an ether bond. The triple bond is introduced by chemical modification of a precursor polyketide.
  • US 65558942 discloses a polyketide compound having an ethinyl residue covalently bound to a terminal carbon atom of a polyketide carbon backbone. A structure according to Structure I hereinbelow is not shown.
  • WO 2000/01838 discloses methods of making non-natural polyketides using PKS modified to accept non-natural starter units or non- natural extender units. The documents speculates on the use of N-acetyl cysteamine thioesters having an alkinyl (1-8C) residue (page 15).
  • WO 2000/01838 does not contain an enabling disclosure of how to add extender units including ethinyl residues to a nascent polyketide in a PKS reaction.
  • the present invention serves this need by providing novel polyketide compounds, in particular chemically reactive precursor polyketides that can easily be chemically modified to yield therapeutically active polyketide compounds derived from said precursor polyketide compounds.
  • the present invention is based on the unexpected finding that the AT4 domain of FK506 producing PKS, more specifically, the FkbB protein of the FK506 producing PKS, is promiscuous to an extent that it not only accepts the known substrates methylmalonyl- CoA, ethylmalonyl-CoA, propylmalonyl-CoA and allylmalonyl-CoA, but is sufficiently promiscuous to also accept extender units including very reactive triple bonds, in particular extender units including a structure comprising -G ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • polyketide compounds including such triple bonds are produced by PKS enzymes comprising an AT domain similar or homologous to an AT domain of the fourth module of an FK506 producing PKS (an "FK506 AT4 domain", e.g. SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17).
  • PKS enzymes comprising an AT domain similar or homologous to an AT domain of the fourth module of an FK506 producing PKS (an "FK506 AT4 domain", e.g. SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17).
  • Triple bond containing polyketides are produced by incubating the suitable PKS with suitable triple-bond containing extender units.
  • the cloning new PKS enzymes using the known AT swap methodology yields new PKS enzymes carrying an FK506 AT4 domain at one or multiple "non-natural" positions.
  • modified PKS enzymes allow for the biosynthetic production of new chemically reactive polyketide compounds by incubating the modified enzymes with the triple-bond containing extender units.
  • the resulting polyketide compounds will carry the triple-bond containing side chains at various positions of the polyketide carbon backbone.
  • the present invention thus also relates to the triple-bond containing polyketide compounds.
  • These triple-bond containing polyketide compounds can chemically be modified to obtain new biologically active polyketide compounds.
  • the triple-bond containing polyketide compounds, according to the invention serve as precursors for new pharmaceutically active polyketide compounds.
  • Compounds of the present invention can be used in drug screening, or can be used as drugs.
  • the present invention relates to:
  • polyketide synthase comprising a sequence set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or comprising a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17 for adding an extender unit having the structure
  • the invention also relates to the use of the above polyketide synthase for incorporating an extender unit of the above structure into a polyketide compound.
  • # 1 or 2 wherein said polyketide compound is a macrolide antibiotic, a macrolactone antibiotic, a polyene antibiotic, a polyether antibiotic, or a acetogenins.
  • said nascent polyketide compound is a nascent macrolide antibiotic, a nascent macrolactone antibiotic, a nascent polyene antibiotic, a nascent polyether antibiotic, or a nascent acetogenin.
  • polyketide compound is selected from the group consisting of: erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, emphotericin, nystatin, candicidin, monensin and salinomycin.
  • nascent polyketide compound is selected from the group consisting of: nascent erythromycin A, nascent pikromycin, nascent oleandromycin, nascent tylosin, nascent medicamycin, nascent rifamycin, nascent avermectin, nascent spinosyn, nascent rapamycin, nascent FK506, nascent meridamycin, nascent geldanamycin, nascent epothilone, nascent emphotericin, nascent nystatin, nascent candicidin, nascent monensin and nascent salinomycin
  • a method of making a polyketide compound comprising: a) providing a microorganism functionally expressing a polyketide synthase, said polyketide synthase comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; b) providing a substrate compound having the structure
  • X4 is any organic residue, preferably X4 is selected from NAc, CoA
  • R s is any organic or inorganic residue, preferably a residue selected from the list consisting of CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CHCH 2 , CH 2 CCH, CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 CH 2 CH 2 C1, CH 2 CH 2 F, CH 2 OCH 3 , C 6 H 6 (phenyl),
  • R k is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates, monosaccharides, disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups;
  • R a and R b are each independently at each occurrence a residue selected from the list consisting of H, CH 3 , CH 2 CH 3 , and CH 2 CH 2 CH 3 ; wherein R a and R b can also be connected so as to form a 5-, 6- or 7-membered ring, preferably a pyrrolidine or piperidine ring;
  • -Y is -H, -OH, -CH 3 , preferably -H or -OH, most preferred -H;
  • Method of # 9 wherein said at least one enzyme involved in the biosynthesis of allylmalonyl-CoA or ethylmalonyl-CoA is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, or from the group consisting of sequences at least 70%, 80%, 90% or 95% identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
  • a method of making a chemically modified polyketide compound comprising:
  • Method of # 11 wherein said chemical modification is selected from the group consisting of: oxidation by KMn0 4 , addition of water, alkylation, formation of a halomagnesium compound, halogenation, addition of borane including subsequent oxidation with hydrogen peroxide, C-C coupling with substituted alkenes, and oxytallation with Tl 3+ salts, and combinations thereof.
  • the chemical modification is at the
  • a polyketide compound comprising the structure:
  • Y is -H or -OH; n is 1 or 0; p is 0, 1 or 2; and
  • C* is a carbon atom; and Chainl -C*-Chain2 is the carbon backbone of said polyketide compound.
  • the covalent bond between Chainl and C*, and/or between Chain2 and C* in Structure I above, and elsewhere in the description can be a single bond, a double bond, or an epoxide group; preferably a single bond or a double bond.
  • R s is any organic or inorganic residue, preferably a residue selected from the list consisting of CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CHCH 2 , CH 2 CCH, CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 (phenyl), aaminophenyl), and ; preferably R s is selected from CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH2CHCH2, CH 2 CCH, CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 CH 2 C1, and CH 2 CH 2 F.
  • R k is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates such as monosaccharides and disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups;
  • -X 3 is -H, -OH, or double bonded O, wherein, if -X 3 is double bonded O then RJO is non-existent;
  • -Ri to -R 10 are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -CH 2 -C ⁇ CH, wherein Rio can also be non-existent; and at least one of -Ri to -R 10 is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -CH 2 -C ⁇ CH.
  • a chemically modified polyketide compound having the structure:
  • -X 3 is -H, -OH, or double bonded O, wherein, if -X 3 is double bonded O then R 20 is non-existent;
  • Hal is selected from F, CI, Br and I,
  • R21, R22 are any organic moiety
  • -X is -CH 3 or -C 2 H 5 ;
  • -Ri to -Re are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -CH 2 -C ⁇ CH; and at least one of -R ⁇ to -R6 is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -CH 2 -C ⁇ CH.
  • a chemically modified polyketide compound having the structure:
  • -X is -CH 3 or -0 2 3 ⁇ 4;
  • the invention relates to recombinant polyketide synthases which carry a promiscuous AT4 domain of a FK506 PKS at a position where such a promiscuous AT4 domain is not normally occurring.
  • PKS enzymes are useful for producing non-natural polyketide compounds when fed with non-natural extender units of the invention, such as ethynylmalony-SNAC, or propargylmalonyl-SNAC, which polyketide compounds are amenable to chemical modification by virtue of carrying a non- natural triple bond.
  • a recombinant polyketide synthase said recombinant polyketide synthase having multiple functional modules, each functional module comprising at least one keto- synthase (KS) domain, one acyltransferase (AT) domain, and one acyl carrier protein (ACP) domain, wherein at least one of said modules comprises the sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90%, or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17, wherein said at least one of said modules is not the fourth module of a FK506 polyketide synthase.
  • KS keto- synthase
  • AT acyltransferase
  • ACP acyl carrier protein
  • FK506 polyketide synthase is an enzyme having the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or a sequence at least 70%, 80%, 90%, or 95% identical to any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
  • the present invention also relates to a compound having the structure:
  • X4 is any organic residue, preferably X4 is selected from NAc, CoA
  • a second aspect of the invention relates to a polyketide compound comprising the structure:
  • Chain is a part of a polyketide carbon backbone, respectively; and p is 0, 1 or 2, preferably 0 or 1, most preferred 1.
  • the polyketide compound comprises only a single Structure I.
  • the polyketide compound is selected from the group consisting of macrolides, macrolactones, polyene antibiotics, polyether antibiotics, tetracyclines, acetogenins.
  • the polyketide compound is obtainable from a natural polyketide compound, preferably selected from the group consisting of macrolides erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, macrolactones rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, polyene antibiotics, emphotericin, nystatin, candicidin; polyether antibiotics monensin and salinomycin; by substitution in said natural polyketide compound of a structure: wherein X I and X 2 are naturally occurring substituents of the natural polyketide compound or H, and
  • Chain are independently a part of the carbon backbone of the natural polyketide compound, respectively; by the structure: wherein Z is CH or N, preferably CH; Y is H or OH, preferably H; p is 0, 1 or 2, preferably 0 or 1 , more preferred 1 ; and Chain is as defined above.
  • Ri to Rio are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -C ⁇ N, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N, wherein R 10 can also be nonexistent; and at least one of Ri to Rio is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -C ⁇ N, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • Rj to Ri 0 are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -C ⁇ N, wherein Rio can also be non-existent; and at least one of Ri to Rio is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -C ⁇ N.
  • Ri to Rio is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • Ri to Rio is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • Ri, R 2 , R 5 , Re ,R9 are -CH 3 ;
  • R 3 is H;
  • R4 is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -C ⁇ N, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N;
  • R 7 , Rg are -OCH 3;
  • X 3 is double bonded O and R 10 is non-existent.
  • the chemically active triple bond is introduces at position 21 of the structure according to Formula I (above).
  • X is -CH 3 or -C2H5;
  • Ri to R6 are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -C ⁇ N, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N; and at least one of Ri to Re is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -C ⁇ N, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • Ri to Re are individually selected from -H, -CH 3 , -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -C ⁇ N, and at least one of Ri to Re is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, and -CH 2 -C ⁇ N.
  • Such polyketides are thus erythromycin-derived polyketides. These polyketides likely show antibiotic properties.
  • the invention thus also relates to the use of such polyketide compounds as an antibiotic.
  • Rj to R 6 only a single one of Rj to R 6 is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N. Hence, only a single chemically active triple-bond is introduced.
  • Rj to R 6 is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -C ⁇ N and -CH 2 -CH 2 -C ⁇ N.
  • R 2 to Re are -CH 3
  • R ⁇ is selected from -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -ON and -CH 2 -CH 2 -ON, preferably -OCH, -CH 2 -OCH, and -CH 2 -C ⁇ N.
  • These polyketides correspond to the structure shown in Formula II (below).
  • R 2 , R 3 , R 5 , Re are -CH 3
  • R4 is selected from -OCH, -CH 2 - CH, -CH 2 -CH 2 -C ⁇ CH, -ON, -CH 2 -ON and -CH 2 -CH 2 -C ⁇ N, preferably -C ⁇ CH, -CH 2 -OCH, and -CH 2 -ON.
  • the present invention also relates to a chemically modified polyketide compound obtainable from the FK506-derived polyketides, above, said chemically modified polyketide compound having the structure:
  • Hal is selected from F, CI, Br and I;
  • R21 and R 22 are preferably linear or branched alkyl.
  • R 21 and R 22 can be hetero alkyl.
  • R 21 and R22 are may independently be hydrogen, methyl, ethyl, propyl, n-butyl or tert- butyl.
  • Rn to R20 is a chemically modified (formerly triple-bonded) side chain.
  • Rn to R20 is a chemically modified - formerly triple-bonded - side chain.
  • the present invention also relates to a chemically modified polyketide compound obtainable from the erythromycin-derived polyketide compounds above, said chemically modified polyketide compound having the structure:
  • Hal is selected from F, CI, Br and I; and R 21 , R 22 are any organic moiety; and
  • Rn to R 16 is a chemically modified (formerly triple-bonded) side chain.
  • Rn to R 16 is a chemically modified (formerly triple-bonded) side chain.
  • the present invention also relates to a method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a PKS enzyme, said
  • PKS enzyme comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70%, 80%, 90% or 99% identical to SEQ ID NO:l or SEQ ID NO:2 and accepting a compound of the structure: as a substrate, wherein Z is CH or N; p is 0, 1 or 2, preferably 0 or 1, most preferred 1; and X4 is selected from CoA and NAC, preferably NAC; providing a substrate compound of said structure c) incubating said microorganism in a medium comprising said substrate compound; and d) obtaining said polyketide compound from said medium.
  • esters or thioesters of the extender units can be used, such as thioglycolate thioesters and diethyl ester.
  • esters or thioesters of the extender units can be used, such as thioglycolate thioesters and diethyl ester.
  • single and double esters or single and double thioesters, or mixtures thereof can also be used.
  • SNAC extender units are preferred, because they readily pass the cell membrane, thus can be fed externally, and they are available in greater amounts and for a more attractive price than are the CoA extender units.
  • this invention also relates to biotechnological production methods for compounds of the present invention.
  • the incubation is preferably under permissive conditions. Incubation is also preferably for a time sufficient to produce sufficient amounts of said polyketide compound.
  • the methods can include submerse culture of the microorganism, such as fermentation in a bioreactor.
  • the microorganism is preferably provided with a nutrient medium such as to allow growth of the microorganism.
  • a bi-functional thioester e.g., bis SNAC
  • SNAC a bi-functional thioester
  • the present invention also relates to methods of making modified FK506 compounds by using microorganisms that normally produce FK506.
  • the triple-bond containing extender unit is added in excess, e.g. at a concentration of above 0.0001 M, 0.001 M, 0.01 M, or above 0.1 M in the medium.
  • the concentration is preferably below 0.1 M or below 1 M.
  • said microorganism is deficient in at least one enzyme selected from the group consisting of AHA, A11K, A11R and A11D, preferably A11R
  • the natural pathway for producing the allylmalonyl-CoA extender unit is inoperative (Goranovic, Kosec et al. 2010)), thus the triple-bond containing extender unit will more efficiently be built into the polyketide compound.
  • said microorganism is selected from the group consisting of Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces tsukubaensis, Streptomyces sp.
  • the invention further relates to methods of making a polyketide compound, said method comprising: a method as described above; and chemical modification of the product of said method, so as to obtain a chemically modified polyketide compound.
  • said chemical modification is selected from the group consisting of: oxidation by KMnC»4, addition of water, alkylation, formation of halomagnesium compounds, halogenation, addition of borane and subsequent oxidation with hydrogen peroxide, C-C coupling with substituted alkenes, Ruthenium catalyzed C-C coupling and oxytallation with Tl 3+ salts.
  • said amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70%, 80%, 90% or 99% identical to SEQ ID NO:l or to SEQ ID NO:2 is not within the 4th module of a FK506 producing PKS. Hence, the corresponding PKS is not the (natural) FK506 producing enzyme.
  • Figure 1 shows examples of chemical derivatization of polyketides containing a terminal triple bond (propargyl or ethynyl group).
  • Figure 2 shows possible C-C coupling reactions with substituted alkenes using ruthenium catalysts yielding 2,4-diene moieties on position 21 of FK506.
  • FIG. 3 shows chemical modification of halogenated side chains according to the invention. Detailed description of invention
  • a precursor means one precursor or more than one precursor.
  • analogue as used herein means a chemical compound that is structurally similar to another but whose composition is slightly different. For example, one atom may be replaced by another or a particular functional group can be present or absent.
  • polyketide refers to any compounds whose biosynthesis comprises at least one step which may be catalyzed by a polyketide synthase enzyme or any compound containing part of the structure generated by PKS enzyme.
  • a polyketide is a compound synthesized (or producible) by a PKS enzyme.
  • cent polyketide is to be understood as relating to any precursor polyketide compound in the biosynthesis of a polyketide compound (i.e., of the mature polyketide compound).
  • the precursors in the biosynthetic pathways of polyketide compounds are well known in the art and can be deduced from the knowledge of the biosynthetic pathway.
  • the term “nascent polyketide” can be understood as relating to an unfinished polyketide chain which is being synthetized by PKS and has not yet been completed or cyclised.
  • the term “nascent polyketide” can also be understood as relating to an unfinished polyketide chain, which is being synthesized by PKS and has not yet been completed or folded (or cyclised) to reach its final polyketide structure.
  • the term “extender unit” as used herein means any compound that can be recognized by AT domains of PKS module and incorporated into polyketide chains by catalytic action of PKS.
  • the term “hybrid polyketide” as used herein means is a genetically modified PKS in which one or more than one domain(s) in the target module of natural PKS has been replaced (swapped) using analogues domain(s) from heterologous PKS enzyme using.
  • a PKS enzyme i.e., a polyketide synthase
  • PKSs can be classified into (1) Type I polyketide synthases, which are large, highly modular proteins, (2) Type II polyketide synthases, which are aggregates of monofunctional proteins, and (3) Type III polyketide synthases do not use ACP domains.
  • Type I PKSs are further subdivided into (la) Iterative PKSs, which reuse domains in a cyclic fashion, and (lb) Modular PKSs, which contain a sequence of separate modules and do not repeat domains.
  • Preferred PKS enzymes are bacterial PKS enzymes. Particularly preferred are PKS enzymes of Actinomyces spp. or Streptomyces sp., preferably of Type I.
  • the term "polyketide synthase”, as used herein, shall be understood as relating only to the FkbB protein (or unit), which forms complexes with the FkbA protein, the FkbC protein, and optionally other proteins, to form a functional PKS.
  • the terms "polyketide synthase” and "FkbB protein of the polyketide synthase” or “FkbB unit of the polyketide synthase” are used synonymously.
  • type I PKS refers to the family of enzymes that produce polyketides, and which are organized as single protein molecules having multiple modules.
  • Each module typically consist of minimal obligatory set of domains, namely: keto-synthase (KS) acyltransferase (AT) and acyl carrier protein (ACP) and further alternative (non-obligatory) set of domains involved in the reduction of ⁇ -keto group of nascent polyketide chain namely ketoreductase (KR), dehydratase (DH) and enoylreductase (ER).
  • KR ketoreductase
  • DH dehydratase
  • ER enoylreductase
  • FK506 polyketide synthase in accordance with the present invention, shall be understood as defining a polyketide synthase enzyme capable of synthesizing FK506.
  • FK506 polyketide synthase shall be understood as defining a polyketide synthase enzyme capable of synthesizing the FK506 compound of Formula I.
  • Preferred polyketide synthases of the invention are FK506 polyketide synthases of Streptomyces sp..
  • the polyketide synthases of the invention are PKS enzymes of Streptomyces sp. KCTC11604BP, Streptomyces sp.
  • MA6858 ATTC 55098 or PKS of Streptomyces tsukubaensis, preferably Streptomyces tsukubaensis NRRL 18488.
  • PKS enzymes of Streptomyces clavuligerus preferably Streptomyces clavuligerus KTC 1056 IBP or CKD 1119, or Streptomyces glaucensens, e.g., MTCC 5115.
  • Preferred FK506 polyketide synthases of the invention are the ones of: Streptomyces sp. KCTC 11604BP (GenBank: ADU56322.1 , SEQ ID NO:22);
  • Streptomyces sp. MJM7001 (GenBank: ADX99524.1, SEQ ID NO:23);
  • Streptomyces kanamyceticus (GenBank: ADU56247.1, SEQ ID NO:24),
  • Streptomyces sp. MA6548 (GenBank: AAC68815.1, SEQ ID NO:25, and
  • Streptomyces tsukubaensis preferably Streptomyces tsukubaensis NRRL 18488.
  • precursor of extender unit means any compound, usually ester or thioester of 2-monosubstituted derivatives of malonic acid, which can be converted, by enzymatic activities inside the cells to respective extender unit and get incorporated into polyketide chains by activity of PKS.
  • non-natural extender unit refers to any compounds which can be incorporated as an extender unit in polyketide synthesis wherein the extender unit is not a compound that is produced in naturally occurring metabolic pathways, such as malonyl- CoA, methylmalonyl-CoA, ethylmalonyl-CoA or methoxymalonyl-ACP, which are recognized by the AT4 domain of FK506 PKS which are non-natural 2-monosubstituted derivatives, either esters or thioesters, of malonic acid, that are derived by synthetic chemistry approaches and/or biotransformation using synthetic substrates. These are extender units generally not selected by ordinary AT domains of PKS.
  • the "carbon backbone" of a polyketide compound produced by a PKS enzyme in the context of the present invention, shall be understood as being the linear chain of covalently connected carbon atoms in said polyketide compound, which linear chain, upon synthesis of said polyketide compound by said PKS enzyme, is being formed by sequential addition of two carbon atoms at a time to said linear chain, through the catalytic activity of said PKS enzyme.
  • the linear carbon backbone forms part of a cyclic structure by cyclization via a hetero atom, such as oxygen, thereby forming, e.g., a lactone ring.
  • the term "AT domain” as used herein means the acyltransferase domain involved in the selection of an extender unit(s) located in the typical module of any type I PKS enzyme.
  • AT4 domain means the acyltransferase domain of the module 4 of any FK506 generating PKS either in the form of a polynucleotide chain (DNA, RNA) or polypeptide chain (protein).
  • Preferred AT4 domain is the AT4 domain of the FK506 PKS of Streptomyces tsukubaensis:
  • AT4 domain of the FK506 PKS of Streptomyces sp. MA6548 is also preferred.
  • AHA is a protein having the following amino acid sequence (SEQ ID NO: 18), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
  • A11K is a protein having the following amino acid sequence (SEQ ID NO: 19), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
  • A11R is a protein having the following amino acid sequence (SEQ ID NO:20), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
  • A11D is a protein having the following amino acid sequence (SEQ ID NO:21), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
  • % identity between to amino acid sequences, as used herein, defines the % identity calculated from two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default BLOSUM62 matrix (see below) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference polypeptide.
  • any organic moiety as used hereinbelow relates to any organic moiety, preferably to represents an unbranched or branched, saturated or unsaturated, optionally at least mono-substituted Q aliphatic group; more preferably to a saturated or unsaturated, optionally at least mono-substituted, optionally at least one heteroatom as ring member containing cycloalkyl group, which may be condensed with an optionally at least mono-substituted mono- or polycyclic ring system; or to a branched or unbranched, optionally at least one heteroatom as ring member containing alkyl-cycloalkyl group in which the cycloalkyl group is optionally at least mono-substituted; or to an optionally at least mono-substituted aryl group; or to an optionally at least mono
  • the present invention is directed to a process for the preparation of novel polyketides wherein the process comprises the step of cultivation of a microorganism, preferably a genetically modified strain of a microorganism, preferably belonging to the order of Actinomycetales, more preferably to the genus Streptomyces, wherein the genetic material of the microorganism comprises of at least one PKS module containing at least one AT domain of the module 4 of FK506 PKS (AT4) and wherein the cultivation of the microorganism is carried out in growth medium supplemented with at least one extender unit or analogue or precursor thereof.
  • a microorganism preferably a genetically modified strain of a microorganism, preferably belonging to the order of Actinomycetales, more preferably to the genus Streptomyces
  • the genetic material of the microorganism comprises of at least one PKS module containing at least one AT domain of the module 4 of FK506 PKS (AT4) and wherein the cultivation of the microorganis
  • the process for the preparation of polyketides according to the present application comprises at least one of the following steps:
  • the present invention relates to a process comprising of steps a) to c) as described above.
  • the invention relates to a process for preparation of polyketides as described above, wherein the process comprises of cultivation of a microorganism, preferably a bacterium, preferably classified to the order Actinomycetales, preferably classified to the genus Streptomyces.
  • a microorganism preferably a bacterium, preferably classified to the order Actinomycetales, preferably classified to the genus Streptomyces.
  • microorganism strains are Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces tsukubaensis, Streptomyces sp.
  • the invention relates to the process for preparation of novel polyketides wherein the process comprises a step of cultivating a microorganism, wherein the cultivation is carried out with external addition of an analogue or precursor of an extender unit, preferably a non-natural extender unit, most preferably this extender unit being selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
  • an extender unit preferably a non-natural extender unit, most preferably this extender unit being selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
  • the invention relates to the process for preparation of novel polyketides wherein the process comprises a step of cultivating a genetically modified microorganism, wherein the cultivation is carried out with external addition of an analogue or precursor of an extender unit, preferably a non-natural extender unit, most preferably this extender unit being selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl- CoA, cyanomethylmalonyl-CoA.
  • the AT domain of the first extender module of the deoxyerythronolide B synthase (DEBS1), part of the entire gene cluster encoding biosynthetic pathway for macrolide erythromycin biosynthesis can be replaced with the AT4 domain of the FK506 PKS.
  • DEBS1 deoxyerythronolide B synthase
  • the process comprises a step of cultivating a genetically modified microorganism, wherein at least one AT domain of a PKS gene encoded in the genome of said microorganism has been replaced by the AT domain encoded in the module 4 (AT4) of the PKS which catalyzes the biosynthesis of FK506, wherein the cultivation is carried out in the production medium supplemented with an analogue or precursor of an extender unit preferably a non-natural extender unit, this extender unit being: allylmalonyl-CoA, prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA. Any analogue or precursor of these extender units may be added, preferably a thioester derivative may be used, preferably N- acetylcysteamine thioester derivatives may be used
  • the process comprises a step of cultivating a genetically modified microorganism, wherein at least one AT domain of a PKS gene encoded in the genome of said microorganism has been replaced by the AT domain encoded in the module 4 (AT4) of the PKS which catalyzes the biosynthesis of FK506, wherein the cultivation is carried in the production medium supplemented with an analogue or precursor of an extender unit, preferably a non-natural extender unit, this extender unit being: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
  • any analogue or precursor of these extender units may be added, preferably a thioester derivative may be used, preferably N- acetylcysteamine thioester derivatives may be used.
  • the PKS is modified to produce a compound of the structure:
  • the origin of the AT domain used in this aspect for replacement of a natural AT domain may be in any module of any PKS type I selectively incorporating allylmalonyl-CoA or propylmalonyl-CoA and ethylmalonyl-CoA but preferably not incorporating methylmalonyl-CoA and malonyl-CoA.
  • said AT domain may be an AT domain of the module 4 of any FK506 generating PKS encoded in the genome of any FK506 producing microorganism.
  • this invention thus provides novel polyketides from the group of macrolide but not limited to the macrolides such as erythromycin, tylosin, medicamycin; from the group of macrolactone but not limited to such as rapamycin, FK506, FK520, rifamycin; from the group of polyene antibiotics but not limited to such as nistatin, amphotericin, candicidin and other polyketide-derived or polyketide containing structure of medical and/or commercial importance produced by methods and processes described above.
  • these polyketides will be characterized by a substituent present at least one of the a-carbon atom which is derived from the precursor of extender unit supplemented in the cultivation medium.
  • this substituent may be selected from but is not limited to the group comprising: prop-2-ynyl, ethynyl, cyanomethyl.
  • this invention also provides semi-synthetic derivatives of these novel polyketides produced by producers and methods described below.
  • AT4 from the fourth module of FK506 PKS does not select the most common extender unit such as malonyl- CoA in the absence of substrate supply providing the allyl group and/or absence of substrate supply providing ethyl group at carbon 21 of FK506.
  • non-natural extender units are readily recognized by the AT4 domain of FK506 PKS and efficiently incorporated into the FK506 structure at the carbon 21 position of FK506, thus generating novel FK506 analogues.
  • FK506 is not produced or is produced in very small amounts in strains lacking efficient supply of the natural extender unit, therefore the exclusive production of novel FK506 analogues with incorporated non-natural extender unit is achieved using this approach, and thus novel FK506 analogues can be easily purified.
  • analogues or precursors of non- natural extender units are fed to the wild type strain, mixtures of compounds are produced.
  • this invention is concerned with the process for preparation of novel polyketides, wherein the process comprises a step of cultivating a microorganism, wherein these novel polyketides are structure analogous of FK506, wherein the allyl substituent at the carbon 21 of FK506 is replaced with a substituent, derived from a non-natural extender unit, particularly with a substituent selected from the group consisting of: prop-2-ynyl, ethynyl, cyanomethyl, wherein these substituents are incorporated into the polyketide structure as a result of adding analogues or precursors of extender units selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
  • Any analogue or precursor of these extender units may be fed to the fermentation broth during S. tsukubaensis fermentation process, preferably a thioester derivative may be used, preferably N-acetylcysteamine thioester derivatives may be used.
  • the cultivated microorganism may be selected from the order Actinomycetales, more preferably the microorganism may be selected from the genus Streptomyces, more preferably the microorganism may be selected from the group consisting of: Streptomyces tsukubaensis NRRL18488, Streptomyces tsukubaensis No. 9993 (Ferm BP 927), Streptomyces sp. MA6548 and Streptomyces clavuligerus CKD1119 (Kim and Park 2008), Streptomyces hygroscopicus subsp. hygroscopicus (DSM40822), Streptomyces sp.
  • the cultivated microorganism may be a recombinant microorganism, particularly the strain may be mutated to have targeted inactivation or deletion of one or more genes that contribute to the biosynthesis or regulation of precursor supply, more particularly, the deleted or inactivated genes may contribute to efficient supply of 5 carbon extender unit allylmalonyl-CoA or propylmalonyl-CoA, most particularly the deleted or inactivated genes are selected from the group consisting of allA, allK, allR, allD (Goranovic, Kosec et al. 2010), also referred to as tcsA, tcsB, tcsC and tcsD, respectively (Mo et al., 2010). Therefore, the deletion or inactivation of precursor supply gene provides a system which enables the incorporation of non-natural extender units ensuring exclusive production of the target compound(s) containing non-natural extender unit.
  • this invention provides a method for producing analogues of FK506 or FK520, said method comprising at least 1 of the following steps:
  • the present invention relates to a process comprising of steps b) to c) as described above, most preferably the present invention relates to a process comprising of steps a) to c) as described above.
  • the present invention provides a method for preparation of a novel polyketides and polyketide structure-containing compounds, wherein the process comprises a step of cultivating a microorganism, wherein these novel polyketides have a structure analogous to the structure of FK506, wherein the allyl substituent at the carbon 21 is replaced with a substituent selected from the group consisting of: prop-2-ynyl, ethynyl, cyanomethyl, wherein these substituents are incorporated into the polyketide structure as a result of adding analogues or precursors of extender units selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
  • This method comprises: a) Generation of a genetically modified microbial strain of a non-FK506 or -FK520 producing microorganism, which contains a heterologous gene cluster encoding proteins involved in the biosynthesis of FK506, FK520 or their analogues.
  • this strain may lack efficient supply of allylmalonyl-CoA and/or propylmalonyl-CoA or ethylmalonyl-CoA.
  • this invention provides novel polyketides of Formula I produced by methods and processes described above.
  • these polyketides may be FK506 analogues characterized by the lack of allyl substituent at the carbon 21, wherein another substituent is present at said carbon atom.
  • This substituent may be selected from but is not limited to the group comprising: prop-2-ynyl, ethynyl, cyanomethyl. Therefore, in this aspect the invention provides novel polyketides: 21 -desallyl-2 l-prop-2-ynyl- FK506, 21 -desallyl-21 -ethynyl-FK506, 21 -desallyl-21 -cyanomethyl-FK506.
  • novel compounds provided by this invention may be used directly or as templates for further chemical derivatization or bioconversion in order to obtain compounds useful as immunosuppressants, antifungal agents, neuroregenerative agents, anticancer agents, antiinflammatory agents or agents useful for treatment of fibrosis, rheumatoid arthritis, psoriasis and other hypreproliferative diseases.
  • Methods for chemical derivatization of FK506 and FK520, at position different than carbon 21 are well known in the art and include but are not limited to those described in (Cooper and Donald 1989).
  • useful derivatives of compounds of Formula I may be obtained by combining the methods described herein with biosynthetic engineering and chemobiosynthetic methods, known in the art, which were used for obtaining analogues of FK506 or FK520 and biogenetically similar compounds such as rapamycin and meridamycin, wherein modification of polyketide structure is achieved at position other than carbon 21.
  • biosynthetic engineering and chemobiosynthetic methods known in the art, which were used for obtaining analogues of FK506 or FK520 and biogenetically similar compounds such as rapamycin and meridamycin, wherein modification of polyketide structure is achieved at position other than carbon 21.
  • biosynthetic engineering and chemobiosynthetic methods known in the art, which were used for obtaining analogues of FK506 or FK520 and biogenetically similar compounds such as rapamycin and meridamycin, wherein modification of polyketide structure is achieved at position other than carbon 21.
  • these compounds can be further derivatized using semi-synthetic approaches described above. It will be recognised by those skilled in the art that introducing into a position of the carbon atom not containing keto group (or reduced keto group), a polyketide chain a substituent selected from the group comprising: prop-2-ynyl, ethynyl, cyanomethyl introduces entirely novel opportunities for chemical derivatization previously unavailable in the FK506 or FK506 analogues produced by numerous Actinomycetes (Streptomycete) wild type strains.
  • this invention provides methods for producing chemical derivatives of compound selected from the group comprising: 21-desallyl-21-prop-2-ynyl- FK506, 21-desallyl-21-ethynyl-FK506, 21-desallyl-21-cyanomethyl-FK506.
  • This invention also provides compounds resulting from said methods.
  • a polyketide compound contains a terminal triple bond (C21 propargyl or C21 ethynyl group) it can be modified by methods well known in the art, as follows: a) Oxidation by KMn0 4 yields an appropriate carboxylic acid derivative b) Addition of water by means of HgSC /tbSO-i yields a keto derivative, which can be further reduced to hydroxyl group by a mild reducing agent such as NaB3 ⁇ 4.
  • a side chain (Ri) of the polyketide compound can be replaced by an alternative side chain (R 2 ), according to the following scheme:
  • physiologically functional derivatives of Formula I (and all its semi-synthetic and biosynthetically engineered derivatives mentioned above) including physiologically acceptable esters, solvates and salts.
  • physiologically acceptable esters include esters which are cleaved in the body for example hydroxyl groups esterified with carboxylic acids.
  • Suitable solvates include hydrates.
  • pharmaceutically acceptable salts are non-toxic acid addition salt forms of the compounds of Formula I. Acceptable acid addition salts can be readily obtained by adding the suitable acid to the base form of the compound.
  • Appropriate acids can be chosen from a group comprising of organic acids, for example acetic, propanoic, hydroxyacetic, lactic, pyruvic oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, benzenesulfonic, ethanesulfonic p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids.
  • Appropriate acids can also comprise inorganic acids, for example, nitric, sulphuric, phosphoric, hydrochloric, hydrobromic and similar acids. If desired, said acid addition salts can also be converted to free base forms by addition of appropriate bases.
  • Compounds of FK506 analogues of Formula I or their semi-synthetic derivatives or their esters or addition salts or solvates are useful as pharmaceuticals for example, but without limitation having potential effectiveness as immunosuppressants, antifungal agents, neuroregenerative agents, anticancer agents, antiinflammatory agents or agents useful for treatment of fibrosis, rheumatoid arthritis, psoriasis and other hypreproliferative diseases.
  • the use of compounds of Formula I is provided in the preparation of a medicament for treatment and/or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, inflammatory disorders, cancer, fibrosis, rheumatoid arthritis, psoriasis and/or other hyperproliferative diseases, and neurodegenerative diseases.
  • this invention provides a method for treatment of said medical conditions using said medicaments containing compounds of Formula I by administering said compounds of Formula I to a subject affected by said medical conditions.
  • this invention provides compounds of Formula I which can be used in preparation of a medicament for prevention of organ allograft rejection.
  • compounds of Formula I can be used for preparation of a medicament for treatment of autoimmune and inflammatory disorders.
  • routine experimentation one skilled in the art would be able to determine the ability of compounds of Formula I to inhibit fungal or protozoan cell growth.
  • routine methods one skilled in the art would be able to determine whether said compounds can inhibit tumour cell growth.
  • compounds of Formula I are able to induce suppression of the subject's immune system. Routine assays to determine the ability of compounds to induce suppression of immune system are known to those skilled in the art. Suitable method can be selected depending on the particular medical condition, for example treatment or prevention of allograft organ rejection after transplantation, autoimmune, inflammatory, proliferative and hyperproliferative diseases.
  • this invention provides and, one skilled in the art would be able to determine, the ability of novel polyketides-derived compounds possessing diverse useful biological activities such as antibacterial activity from the group of macrolide or macrolactone compounds but not limited to the macrolides such as erythromycin, tylosin, midecamycin, or macrolactone such as rifamycin; anticancer and neuroregenerative activity from the group of macrolactone but not limited to such as rapamycin and meridamycin; from the group of polyene antibiotics/antifungals but not limited to such as nystatin, amphotericin, candicidin and other polyketide-derived or polyketide containing structure of medical and/or commercial importance produced by methods and processes described in this patent application.
  • Halogenated side chains It has been found that the above methods, which were described above with reference to triple-bond containing extender units and triple-bond containing side chains can equally be used with extender units comprising halogenated residues, hence producing halogenated side chains in the polyketide compounds of the invention. These halogenated side chains are also suitable precursors for producing a wide variety of novel polyketide compounds. The invention hence also relates to the any of the above compounds and methods, in which halogenated extender units are used instead of the above described triple-bond compounds.
  • the FK506 AT4 domain not only accepts prop-2- ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA as a substrate (see above), but the FK506 AT4 domain also accepts e.g. 2-chloroethylmalonyl-CoA, 2- fluoroethylmalonyl-CoA, 3-fluoropropylmalonyl-CoA, 2-fluoropropylmalonyl-CoA, 3- fluoroprop-2-enylmalonyl-CoA, 2-fluoroprop-2-enylmalonyl-CoA as a substrate.
  • 2-chloroethylmalonyl-CoA 2- fluoroethylmalonyl-CoA
  • 3-fluoropropylmalonyl-CoA 2-fluoropropylmalonyl-CoA
  • Halogenated extender units and the resulting moieties in the FK506 polyketide, according to this aspect of the invention, are summarized in Table 3. It is to be understood that corresponding side chains can similarly be introduced in the erythromycin polyketide structures of the invention (e.g. Formula II, III, IV).
  • the invention thus further relates to the process for preparation of novel polyketides as described above, where the extender unit is selected from the group consisting of: 2-chloroethylmalonyl-Co A, 2-fluoroethylmalonyl-Co A, 3 -fluoropropylmalonyl-Co A, 2-fluoropropylmalonyl-Co A, 3 -fluoroprop-2-enylmalonyl-Co A, 2-fluoroprop-2- enylmalonyl-CoA.
  • the extender unit is selected from the group consisting of: 2-chloroethylmalonyl-Co A, 2-fluoroethylmalonyl-Co A, 3 -fluoropropylmalonyl-Co A, 2-fluoropropylmalonyl-Co A, 2-fluoroprop-2- enylmalonyl-CoA.
  • the single or double esters or thioesters of precursors thereof are preferably used, such as but not limited to SNAC, diethyl , thioglycolate version of the respective extender units is the preferred added substance, because they readily pass the cell membrane and are available for a more attractive price than the Co A esters.
  • the present invention also relates to polyketide compounds as described above, wherein the modified (non-natural) side chains are: 2-chloroethyl , 2-fluoroethyl, 3- fluoropropyl, 2-fluoropropyl, 3-fluoroprop-2-eny or 2-fluoroprop-2-enyl.
  • the invention also relates to 21-desallyl- 21 -prop-2-ynyl-FK506, 21 -desallyl-21 -ethynyl-FK506, 21 -desallyl-21 -cyanomethyl- FK506, 21 -desallyl-21 -(2-chloroethyl)-F 506, 21 -desallyl-21 -(2-fluoroethyl)-FK506, 21 - desallyl-21 -(3-fluoropropyl)-FK506, 21 -desallyl-21 -(2-fluoropropyl)-FK506, 21 -desallyl- 21 -(3-fluoroprop-2-enyl)-FK506, 21 -desallyl-21 -(2-fluoroprop-2-enyl)-FK506, 21 -desallyl-21 -(2-fluoroprop-2-enyl)-FK506.
  • Halogenated side chains in polyketide compounds of the invention can further be chemically modified in a reaction
  • one aspect of the invention relates to a polyketide compound comprising the structure:
  • R x is a halogenated C1-C6, preferably C1-C3, alkyl moiety, saturated or non- saturated.
  • Rx is one of the moieties shown as possible "side chains" in the second column of Table 3 and/or in the first column of Table 4.
  • the polyketide compound is preferably selected from the group consisting of macrolides, macrolactones, polyene antibiotics, polyether antibiotics synthesised by the type I polyketide synthase enzymes.
  • the polyketide compound is obtainable from a natural polyketide compound, preferably selected from the group consisting of macrolides erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin; macrolactones rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone; polyene antibiotics such as emphotericin, nystatin, candicidin; polyether antibiotics monensin and salinomycin, by substitution in said natural polyketide compound of a structure: wherein X 1 and X 2 are naturally occurring substituents of the natural polyketide compound or H, and Chain are independently a part of the carbon backbone of the natural polyket
  • X 3 is -H, -OH, or double bonded O, wherein, if X 3 is double bonded O then Rio is nonexistent; Ri to R 10 are individually selected from -H, -CH 3 , and Rx as defined above; wherein Rio can also be non-existent; and at least one of Ri to RJO is Rx as defined above.
  • polyketide compound has the structure: ; wherein
  • X is -CH3 or -C2H5; Ri to R6 are individually selected from -H, -CH 3 and Rx as defined above; and at least one of R ⁇ to R $ is Rx as defined above.
  • Another embodiment relates to a chemically modified polyketide compound obtainable from the polyketide compound above, said chemically modified polyketide compound having the structure:
  • X is -H, -OH, or double bonded O, wherein, if X is double bonded O then R 20 is nonexistent;
  • Rn to R 2 o are independently selected from -H, -CH3 and R 2 , wherein R 2 is defined as in column 3 of Table 4 (R and R ⁇ being any organic moiety); wherein R 2 o can also be nonexistent; and at least one of R 1 to R 20 is R 2 as defined in column 3 of Table 4 (R and Ri being any organic moiety).
  • Another embodiment relates to a chemically modified polyketide compound obtainable from the polyketide compound defined above, said chemically modified polyketide compound having the structure:
  • X is -CH 3 or -C2H5;
  • Rn to R 16 are independently selected from -H, -C3 ⁇ 4 and R 2 as defined in column 3 of Table 4 (R and R ⁇ being any organic moiety); wherein at least one of Rn to Ri 6 is R 2 as defined in column 3 of Table 4 (R and Ri being any organic moiety).
  • One aspect of the invention relates to a method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a P S enzyme, said PKS enzyme comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70% identical to SEQ ID NO:l or SEQ ID NO:2 and accepting a compound of the structure: wherein Rx is a halogenated C1-C3 alkyl moiety, preferably any on the moieties shown as possible "side chains" in the second column of Table 3; X4 is selected from CoA and NAc; as a substrate; b) providing a substrate compound of said structure c) incubating said microorganism in a medium comprising said substrate compound; and d) obtaining said polyketide compound from said medium.
  • bi-functional thioesters e.g., bis-SNAC
  • said microorganism is deficient in at least one enzyme selected from the group consisting of AHA, A11K, A11R and A11D, preferably in A11R, or in A11R only.
  • One aspect of the present invention also relates to a method of making a polyketide compound, said method comprising:
  • said chemical modification is selected from nucleophilic substitution with NaCN, nucleophilic substitution with amines, substitution with potassium ethylxantogenate and subsequent hydrolysis, reaction with dimethylcianimido- dicarbonate, reaction with dimethyltio-2-nitroethen and alkylation.
  • Example 1 Cultivation of Streptomyces tsukubaensis strains for production of analogues ofFK506
  • the ISP4 sporulation agar medium soluble starch, 1%, K2HPO4, 0.1%, MgSC x 7H2O, 0.1%, NaCl, 0.1%, (NH 4 ) 2 S0 4 , 0.2%, CaCC- 3 , 0.2%, FeS0 4 * 7H 2 0, 0.000001%, MnCl 2 * 4 H 2 0, 0.000001%, ZnS0 4 * 7 H 2 0, 0.000001%, bacteriological agar 2%) was used for spore stock preparation.
  • Streptomyces tsukubaensis strains were cultivated as a confluent lawn on the ISP4 agar medium for 8-14 days at 28 °C.
  • spores of Saccharopolyspora erythraea strains were inoculated in seed medium TSB (Tryptone Soya Broth 3%, Oxoid CM1065) and incubated at 30°C and 220 rpm for 24-48 h.
  • Cultivation was carried out at 30 °C, 220 rpm for 6-7 days.
  • 10 mM concentration of a precursor was used In the feeding experiments with unnatural extender units 10 mM concentration was used.
  • Example 3 Isolation of genomic DNA Spores of Streptomyces tsukubaensis NRRL 18488 strains were used to inoculate 50 ml of TSB medium (Kieser et al., 2000, Practical Streptomyces genetics, A laboratory Manual. ISBN-0-7084-0623-8) in a 250-ml Erlenmeyer flask, which was maintained with shaking (210 rpm) at 28 °C for 24 hours. Cultures were grown for 24 hours at 28 °C. Mycelium was recovered by centrifugation and genomic DNA was prepared using PureLink Genomic DNA Mini Kit (Invitrogen) according to the instructions of the kit manufacturer. DNA was resuspended in 100 ⁇ TE buffer (Sambrook, and Russell, 2000, Molecular Cloning: A Laboratory Manual, ISBN-978-087969577-4).
  • Example 4 Transformation of Streptomyces tsukubaensis and Saccharopolyspora erythraea strains Plasmid constructs based on either pSET152 (Bierman, Logan et al. 1992) or pKC1139 (Bierman, Logan et al. 1992) were introduced by transformation into electrocompetent E. coli strain ET 12567 containing the conjugative plasmid pUZ8002 (Paget, Chamberlin et al. 1999). The plasmid pUZ8002, contains all the necessary genes for construction of conjugative pili, however it lacks the origin of transfer and, thus, remains in the host cell (Jones, Paget et al. 1997).
  • Example 5 Chemical synthesis of non-natural extender units
  • Example 6 Feeding of diverse non-natural extender units to the S. tsukubaensis strain with inactivated allR gene and analysis of the produced FK506 analogues
  • the gradient program was: 60 % A, 0 min; 60-20 % A, 0-17 min; 20-60 % A, 17-18 min; 60 % A, 18-30 min and the injection volume 10 ⁇ at temperature of the column 45 °C was used.
  • the mass selective detector (Waters, Quattro micro API) was equipped with an electrospray ionisation using a cone voltage of 20 V and capillary voltage of 3.5 kV for positive ionization of the analytes. Dry nitrogen was heated to 350 °C, the drying gas flow was 400 1/h and collision energy was 20 eV.
  • Table 5 LC-MS/MS data confirming the presence of novel FK506 analogues in fed fermentation broths.
  • Example 7 Replacement of the AT domain in the first extender module of DEBS 1 with the AT4 domain of the FK506 PKS
  • the starting point for construction of chimeric gene was the DEBS 1 gene of erythromycin PKS encoding 3 polyketide synthase modules involved in incorporation of the starter unit and two extender units into the erythromycin polyketide chain.
  • the target ATI domain of DEBS 1 is flanked by BstBI and BpulOI restriction sites. Therefore, our strategy was to exchange the BpulOI-BstBI fragment with a new fragment in which the native AT domain had been replaced by the AT domain of the fourth module of FK506 PKS (AT4).
  • the new hybrid BpulOI-BstBI fragment was constructed from 3 PCR products.
  • the first product was amplified using DEBSSwpFl and DEBSSwpRl oligonucleotide primers and Saccharopolyspora erythraea NRRL2338 genomic DNA as template.
  • Another product was amplified using the same template and oligonucleotide primers DEBSSwpF2 and DEBSSwpR3.
  • the two products were cloned together into pUC19 plasmid and joined with their Pstl sites.
  • the plasmid containing both fragments was then opened using Xhol and Avrll restriction enzymes and the third fragment containing the AT domain of the module 4 of the FK506 PKS was inserted. This fragment was obtained by PCR amplification.
  • DEBS-AT4 a hybrid gene termed "DEBS-AT4" was obtained.
  • Introduction of the Xhol site in the region just upstream of the conserved GQG motif of AT domains caused a replacement of a threonine residue to a serine residue.
  • the Avrll restriction site at the carboxy terminus of the AT domain did not require any change in the predicted amino acid sequence.
  • Mscl restriction site was used instead of the Xhol site at the amino terminus of the AT domain.
  • DEBSSwpR2 oligonucleotide primer was used instead of DEBSSwpRl and AT4SwpF2 was used instead of AT4SwpFl.
  • the primers used in this procedure are given in Table 6.
  • AT4SwpRl ACCTAGGACGACGGTCGGCCAGTCGACGGCC Avrll (SEQ ID NO: 10)
  • PCR amplification of "DEBSSwpFl-DEBSSwpRl" DNA fragment Isolated plasmid DNA of pCJR65 obtained by standard procedures was PCR amplified using a Promega Thermal Cycler. The PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 ⁇ dNTP, 3% DMSO, 0.5 ⁇ of each primer (i.e. DEBSSwpFland DEBSSwpRl), approximately 50 ng of template plasmid DNA and 2.5 units of enzyme in a final volume of 50 ⁇ for 30 cycles.
  • the thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 64°C for 20 sec (annealing step), and 72°C for 35 s (extension step).
  • the PCR-amplified product was cloned into a pUC19 cloning vector.
  • the sequence analysis of the cloned PCR product confirmed its respective partial DEBS1-TE sequence.
  • PCR amplification of the template "AT4TemplF 1 -AT4TemplRl" DNA fragment: S. tsukubaensis genomic DNA obtained by the procedure described above was PCR amplified using a Biometra Thermal Cycler.
  • the PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 ⁇ dNTP, 3% DMSO, 0.5 ⁇ of each primer (i.e. AT4TemplFl and AT4TemplRl), approximately 50 ng of template plasmid DNA and 2.5 units of enzyme in a final volume of 50 ⁇ for 30 cycles.
  • the thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 67°C for 20 sec (annealing step), and 72°C for 37 s (extension step).
  • PCR amplification of the template "AT4SwpFl-AT4SwpRl" DNA fragment PCR- product "AT4TemplFl-AT4TemplRl” obtained by the procedure described above was PCR amplified using a Promega Thermal Cycler.
  • the PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 ⁇ dNTP, 3% DMSO, 0.5 ⁇ of each primer (i.e.
  • AT4SwpFl and AT4SwpRl 1.5 ⁇ of said PCR reaction mixture and 2.5 units of enzyme in a final volume of 50 ⁇ for 30 cycles.
  • the thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 68°C for 20 sec (annealing step), and 72°C for 37 s (extension step).
  • the PCR-amplified product was cloned into a pUC19 cloning vector.
  • the sequence analysis of the cloned PCR product confirmed its respective sequence of the AT4 domain.
  • Example 8 Replacement of the AT domain in the extender module 4 of erythromycin PKS (DEBS2) with the AT4 domain of the FK506 PKS
  • the replacement of the AT domain was carried out similarly to the procedure analogous to the one described in Example 4
  • the Mscl and Avrll domain splice sites were chosen in the equivalent positions in erythromycin-AT4 and FK506-AT4 as described by Oliynyk et al. (1996).
  • To construct the replacement cassette for AT4 of DEBS2 the fragments corresponding to 1.2 kb flanking region upstream of the engineered Mscl site and 0.5 kb flanking region downstream of the Avrll site were obtained by PCR amplification from S. erythraea genomic DNA and joined together in the pUC19 based vector.
  • the oligonucleotide primers used to amplify the Mscl-upstream region were DEBSUpRev 5'- TTTTTCTGCAGCGCCCTGGCCAGGGAAGACCAGGACCG-3 , (SEQ ID NO: 13) and DEBSUpFwd S'-TTTTTAAGCTTCCTGCGAGGCACCGACACCGGCG-S' (SEQ ID NO: 14), the former introducing the Mscl site plus a Pstl site located just before the Mscl site, and the latter introducing a Hindlll site and priming across a Sfil site.
  • the amplified product was digested with Pstl and Hindlll and ligated into pUC19 that had been digested with Pstl and Hindlll.
  • the oligonucleotide primers used to amplify the Avrll-downstream region were DEBSDnRev 5'-TTTTTGAATTCCGTCCTCCGGCGGCCACTGCTCGG-3' (SEQ ID NO: 15) and DEBSDnFwd 5'- TTTTTCTGCAGCCTAGGGGGACGGCCGGCCGAGCTGCCCACC-3' (SEQ ID NO: 16), the former introducing an EcoRI site and the latter introducing an Avrll site plus a Pstl site located just after the Avrll site.
  • Both fragments were combined in pUC19 vector using the Pstl restriction site.
  • the fragment corresponding to the FK506-AT4 domain was also amplified by PCR as described in detail in Example 4.
  • specific primers AT4TemplFl and AT4TemplRl were used to specifically amplify the region encoding the AT domain of the module 4 of FK506 PKS from genomic DNA of Streptomyces tsuk baensis NRRL 18488 as template.
  • specific primers AT4SwpF2 and AT4SwpRl were used where AT4SwpF2 introduced the engineered Mscl restriction site and AT4SwpR2 included the Avrll site.
  • the PCR product was cloned into pUC19 and subsequently excised using Mscl and Avrll. This fragment was then inserted into the pUC19 based vector, containing the flanking regions of erythromycin- AT4, also previously digested by Mscl and Avrll, whereby "DEBS2-AT4" fragment was obtained.
  • the primers used in this procedure are given in Table 6.
  • the chimeric fragment described above was then excised and inserted into the p C1139 plasmid which was used for transformation of Saccharopolyspora erythraea NRRL2338 by conjugation from E. coli as described.
  • Example 9 Production of erythromycin analogues with incorporated non-natural extender units in Saccharopolyspora erythraea
  • Vector pKCl 139 contains a normal pUC19-based Ori for replication in E. coli, but a temperature- sensitive Ori for replication in Streptomyces, which is unable to function at elevated temperatures above 34°C (Bierman, Logan et al. 1992). This plasmid was inserted into S. erythraea genome using the procedure described above.
  • the identity of the produced erythromycin analogues in the fed cultivation broths was determined by LC-MS MS analysis.
  • the gradient program was: 80 % A, 0 min; 60-20 % A, 0-17 min; 30-70 % A, 17-18 min; 80 % A, 18-30 min and the injection volume 10 ⁇ at temperature of the column 45 °C was used.
  • Table 7 LC-MS/MS data confirming the presence of novel erythromycin analogues in fed fermentation broths.
  • brine solution is added.
  • An extraction step may be performed, preferably with dichlorometane.
  • the organic phase may be washed with water, and dried (e.g., with Na 2 S04).
  • a purification step may be performed, e.g. by distillation.
  • ethynylpropanedioic acid is prepared from dipropyl ethynylpropanedioate by dissolving the dipropyl ethynylpropanedioate in EtOH; adding hydrochloric acid (preferably concentrated HC1), and optionally water; performing an evaporation step to remove EtOH, and extracting with ethyl acetate.
  • the resulting product is washed, e.g., with saturated NaCl solution, and dried.
  • ethynylpropanedioic acid is suspended in dichloromethane and DMF.
  • Oxallyl chloride is added at low temperature ( ⁇ 5°C) and the mixture is allowed to react at room temperature (i.e., 15-30°C).
  • an evaporation step is performed, e.g., under reduced pressure (e.g., P ⁇ 0.8 bar).
  • the ethynylpropanedioic acid is obtained.
  • ethynylpropanedioyl dichloride (1,15 mM) is dissolved in 7 ml of THF, cooled on ice and 274 mg of N- Acetyl cysteamine is added within 15 min. 0,5 ml of TEA is slowly added and suspension is stirred for additional 2 h at temp. 0 °C . Solvent is evaporated under reduced pressure, 20 ml of water is added and extracted twice with 30 ml of dichloromethane. Extracts were washed twice with 30 ml of water, dried with Na 2 S0 4 and evaporated to dryness.
  • ethynylpropanedioyl dichloride is dissolved in THF, cooled on ice (i.e., T ⁇ 5°C) and N-Acetyl cysteamine is added.
  • TEA is added and the resulting suspension is preferably allowed to react for at least 30 min at a low temperature (T ⁇ 5°C).
  • an evaporation step is performed, e.g., under reduced pressure (P ⁇ 0.8 bar).
  • water is added.
  • the product is extracted from the aqueous solution in an organic solvent, e.g., dichloromethane.
  • washing steps can be performed and the product can be dired.

Abstract

The present invention relates to novel polyketide compounds and to enzymes and production organisms for obtaining such novel polyketide compounds. Polyketides of the present invention include non-natural, chemically active extender units, which are incorporated into the polyketide molecule by virtue of promiscuous acyl transferase domains of the polyketide synthase enzyme. Such polyketides including chemically active side chains can easily be chemically modified to yield novel polyketide drugs or drug candidates.

Description

Novel polyketide compounds and methods of making same Technical field
The present invention relates to novel polyketide compounds and to enzymes and production organisms for obtaining such novel polyketide compounds. Polyketides of the present invention include non-natural, chemically active extender units, which are incorporated into the polyketide molecule by virtue of promiscuous acyl transferase domains of the polyketide synthase enzyme. Such polyketides including chemically active side chains can easily be chemically modified to yield novel polyketide drugs or drug candidates. Background of the invention
Polyketides are a large and diverse class of natural products that includes many compounds with antibacterial, antifungal, antihelminthic, immunosuppressant, anticancer or other pharmacological activities, such as tetracyclines, erythromycins, avermectins, rapamycins as well as FK506. They are synthesized by ordered condensation of acylthioesters similarly to the synthesis of fatty acids. Great structural diversity of polyketides or related compounds (e.g. mixed polyketide synthase (PKS)/non-ribosomal peptide synthases (NRPS)) as compared to fatty acids arises from greater choices that these enzymatic systems can make during carbon chain assembly. For example, starter units can be selected from acetate, propionate, butyrate and often other unusual CoA- activated carboxylic acids such as shikimate-derived cyclohexane-carboxylic acid. Most often, extender units are selected from CoA-activated carboxylic acids such as malonic, methylmalonic, ethylmalonic acid and only rarely from other derivatives. Nascent β-keto unit, resulting from the condensation reaction can be further reduced to different degrees (β-keto, β-hydroxyacyl, 2-enoyl or saturated thioester) and the stereochemistry of the substituents is also specified in each cycle of chain extension. In addition, products of the P S or PKS-related enzyme systems are often further modified by site-specific oxidative enzymes, methyltransferases or glycosyl transferases, thus introducing expanded structural diversity.
Several classes of polyketide synthases have been described. Among them, modular type I polyketide synthases, encoding for example erythromycin, rapamycin and avermectin biosynthesis have been extensively studied. These large enzymes are organized in modules in a manner that each module catalyzes one step of polyketide chain extension. Each module consists of at least three protein domains, namely β-ketoacyl ACP synthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) where the AT domain determines the choice of the extender (or starter) unit for the relevant chain elongation step. In addition, some modules may also contain additional domains involved in the reduction of the β-keto group, i.e. β-ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains. The module involved in the final elongation step often contains a thioesterase (TE) domain which releases the nascent polyketide chain from the PKS enzyme and it is believed, that TE domain can also influence cyclisation pattern of the macrolide or macrolactone structure. This modular structure of type I PKS inspired many investigators to design diverse approaches for altering the nature of the polyketide products produced by PKS by genetic biosynthetic engineering approaches.
Domain swaps were shown several times to enable relatively efficient production of hybrid polyketides. When a chain terminating cyclase domain (TE) was fused to the carboxyl terminus of DEBS1, a PKS multi enzyme catalyzing the first two chain elongation steps of erythromycin biosynthesis, triketide lactone was efficiently synthesized (Cortes, Wiesmann et al. 1995). This hybrid PKS gene was termed DEBS1- TE and often used as a model system to test novel approaches of biosynthetic engineering. In one example, the acyltransferase domain of the module 1 of DEBS1 TE which specifically incorporates methylmalonyl-CoA extender unit into the polyketide chain, was replaced by the AT domain of the module 2 of rapamycin producing PKS, specifically incorporating malonyl-CoA. Indeed, the resulting products lacked a methyl group at the carbon 4 of the lactone ring. Another example of efficient replacement of acyltransferase domain replacement (AT swap) of erythromycin PKS is the incorporation of ethylmalonyl-CoA specific AT domain from niddamycin PKS instead of native AT domain encoding methylmalonyl-CoA. Changing extender units incorporated into the polyketide chain can be effectively used to vary the moiety that extends away from the backbone of the molecule, which can have effect on its interaction with its biological target. However, in addition to domain exchange, efficient supply of ethylmalonyl-CoA extender unit had to be assured during cultivation in order to enable efficient production of 6-desmethyl-6-ethylerythromycin A (Stassi, Kakavas et al. 1998). The same group was able to obtain a series of novel erythromycin analogues by swapping AT-domains of DEBS genes with heterologous AT-domains from different organisms.(Ruan, Pereda et al. 1997; Katz, Stassi et al. 2000). Similarly to "extender modules", "loading" or "starter" modules were also successfully engineered. The loading module of platenolide producing PKS, incorporating acetyl-CoA was exchanged by the loading module of tylactone producing PKS, specific for propionyl-CoA. Indeed, the engineered PKS was able to efficiently produce a novel compound, 16-methyl platenolide resulting from incorporation of propionate into platenolide (Kuhstoss, Huber et al. 1996). In a study that clearly reveals the potential of PKS engineering technologies the entire loading module of DEBS 1, able to incorporate acetate or propionate, was replaced by the loading module of avermectin PKS (Marsden, Wilkinson et al. 1998). Namely, the avermectin loading module had been shown to incorporate a wide variety of unnatural starter units, added exogenously to cultivation mixtures, which has allowed the production of diverse analogues of avermectin (Dutton, Gibson et al. 1991). When this promiscuous loading module was fused with erythromycin PKS (DEBS1) novel biologically active erythromycin analogues were successfully produced (Marsden, Wilkinson et al. 1998; Leadlay, Staunton et al. 2005). Adequate supply of the extender unit is necessary in addition to the genetic exchange of the AT domain in the PKS, in order to generate the target product. Substrate supply can be provided by metabolic processes taking place in the host organism or alternatively, extender unit precursors can be added exogenously to cultivation broths. Ethyl-substituted erythromycin derivatives, for example, were only produced when cultures of Saccharopolyspora erythraea with engineered erythromycin PKS were transfected with a heterologous gene encoding crotonyl-CoA reductase/carboxylase activity (CCR) or when a precursor of ethylmalonyl-CoA, diethylethylmalonate was added to fermentation broths (Stassi, Kakavas et al. 1998). Often, N-acetylcysteamine (SNAC) thioester derivatives of malonic acid or other extender units were used for feeding experiments as SNAC derivatives resemble closely the naturally occurring CoA thioesters thus enable incorporation of exogenously added compounds into polyketide chains. In contrast to the AT domain of the loading module of avermectin PKS which, which is able to incorporate a variety of exogenously added synthetic starter units, AT domains of extender modules generally have very limited spectrum of the extender units that can be incorporated. It is almost a general rule, that the majority of AT domains can select for malonyl-CoA and methylmalonyl-CoA, and less often ethylmalonyl-CoA and methoxymalonyl-CoA extender units are selected only by limited number of AT domains.
In addition to the commonly used extender units, malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA, which are also intermediates of primary metabolic pathways, some AT domains show specificity for unusual extender units, whose supply is often encoded in gene clusters governing the biosynthesis of secondary metabolites. One example is the incorporation of an unusual five carbon extender unit by the acyltransferase domain of the module 4 (AT4) of PKS governing the biosynthesis of FK506. The incorporation of this extender unit results in the presence of allyl group at the carbon 21 of the FK506 structure (see Formula I, below), a feature which distinguishes FK506 from a structurally and biosynthetically related compound FK520, possessing an ethyl group at equivalent position. Biosynthetic origin of the five carbon extender unit in FK506 producing organism Streptomyces tsukubaensis has been recently elucidated (Goranovic, Kosec et al. 2010). In addition, these authors demonstrated that when synthetically-derived allylmalonyl-SNAC thioester was supplemented into cultivation broth of the mutant strain of S. tsukubaensis which lacks supply of natural extender unit, allylmalonyl-SNAC was efficiently incorporated into nascent FK506 polyketide chain by the AT4 domain of FK506 PKS (Goranovic, Kosec et al. 2010) and yields the following structure (Formula I):
Formula I
AT domains are generally believed to be specific for a certain extender unit, e.g. malonyl- CoA, methylmalonyl-CoA or ethylmalonyl-CoA, however, non-specific or promiscuous AT domains have also been described, which can incorporate more than one naturally occurring extender units. Polyketide synthases comprising such domains usually catalyze the biosynthesis of a mixture of closely related polyketides. One of the best known examples is a polyketide synthase of Streptomyces cinamonnensis, which simultaneously produces a mixture of monensin A and monensin B, depending on whether ethylmalonyl- CoA or methylmalonyl-CoA, both produced by metabolic pathways of this organism, is incorporated into the chain by one of the AT domains (Liu and Reynolds 1999). However, this promiscuity (relaxed specificity) is very limited, and can only accommodate very closely related extender units such as malonyl-CoA and methylmalonyl-CoA or methylmalonyl-CoA and ethylmalonyl-CoA. Thus, only structurally very similar compounds that differ in one methyl group at the a-carbon atom of the nascent polyketide chain can be generated using existing methodology, hence little change in pharmacological properties can be expected, as confirmed in the past.
In addition, all these "natural" extender units (i.e. extender units synthesized by the producing microorganism) lack simple chemical reactivity of the groups, which would enable further semi-synthetic chemical derivatization at a-carbon positions of the polyketide backbone. Progress was made recently, when it was discovered that aminomalonyl-ACP and hydroxymalonyl-ACP are incorporated into polyketide chains of the compound zwittermicin A, produced by Bacillus cereus. However, as these extender units are bound to ACP they cannot be added to fermentation broth and genes encoding the biosynthesis of these extender units must be effectively expressed in the engineered strain (Emmert, Klimowicz et al. 2004; US2008254508). In addition, the PKS enzyme from B. cereus utilize a reaction mechanism that differs importantly from type I PKS from Actinomyces {Streptomyces), which encodes majority of medically important polyketide- derived structures. Most notably, it lacks an AT domain in enzymatic modules that would select the extender unit for incorporation in each elongation step of polyketide chain biosynthesis. Therefore, although it seems straightforward to produce these two extender units inside cells no AT domain of type I PKS from Actinomyces (Streptomyces) has been demonstrated system to be able to incorporate hydroxymalonyl-ACP and aminomalonyl- ACP into polyketide chains of clinically relevant polyketide molecules. Therefore no incorporation of chemically synthesised "non-natural" extender unit(s) into polyketide chain(s) has been demonstrated until now, synthesized by the type I PKS, that would enable fast and simple chemical derivatization of the newly introduced reactive moiety in the resulting side chains of polyketide molecules.
Ideally, a system for incorporation of extender units with chemically more reactive side chains would be composed of an AT domain of type I PKS that would retain its specificity when replacing such an AT domain with diverse AT domains from heterologous PKS genes and a set of extender units or extender unit analogues that could be supplemented to cultivation broths which would be further incorporated into polyketide chains at the specific (desired) positions. Structurally the extender unit analogues would be esters or thioesters of malonic acid substituted at position of the carbon 2. This substituent would thus represent a chemically reactive group moiety sticking out of the polyketide chain. An abundance of 2-substituated derivatives of malonic acid have been synthesized by methods of synthetic chemistry and/or biotransfomation and many synthetic derivatives of malonic acid, such as diethyl (3-chloropropyl)malonate, diethyl (ethoxymethylene)malonate, diethyl 2-(2-cyanoethyl)malonate, diethyl 2-(p-tolyl)malonate and many others are even commercially available. Apart from commercial sources, diverse 2-substituated derivatives of malonic acid can be synthesized by methods known in the art, for example (but not limited to): A) alkylation of malonic esters on position 2 by substituted alkyl halides (Eglinton and Whiting 1953) and B) ring opening of cyclopropyl dicarboxylates with HBr or HC1. (Demjanov 1939).
Polyketides are a large group of biogenetically related compounds, bio-synthesized by the large group of closely related PKS enzymes. However, the compounds belonging to this group ob biosynthetically related structure display huge structural diversity which reflects also large spectrum of pharmacological activities, such as antibacterial, antifungal, anti- cancer, anti-helminthic, coccidiostatic, insecticide, immunosuppressive, neuroregenerative and other biological activities. In spite of the great versatility of use of naturally occurring polyketides further improvements of their pharmacologically-related features have been achieved by additional modifications of these compounds using semi-synthetic chemistry and biosynthetic engineering approaches. Most notably, chemical modifications of naturally occurring compounds often improve pharmacokinetic and pharmacodynamic properties of drugs such as improved biological activity, lowering undesired side-effects, improving bio-availability (solubility), improving general pharmacokinetics and other properties. Improved stability in gastric juices and activity against microorganisms with resistance features are additional important factors that motivate chemists to produce novel semi-synthetic polyketide-based derivatives. As an example, the pharmacological properties and use of the immunosuppressive drug FK506 and some of its derivatives are described in continuation.
FK506 (tacrolimus), has been used for many years as an immunosuppressant after organ transplantation. The pharmacologic mode of action of FK506, similar to that of FK520 (ascomycin), is believed to be mediated by the interaction with cytosolic receptors termed immunophilins or FKBPs, having peptidyl-prolyl cis-trans isomerase activity. Many immunophilins are encoded in the human genome and are known to associate with other cellular proteins of diverse functions. Immunophilin ligands generate various down-stream biological activities by disruption of the natural FKBP-containing complexes or by formation of novel ternary complexes. The binary complex of FK506 (or FK520) with FKBP12 binds to calcineurin, a calmodulin dependent protein phosphatase. Calcineurin dephosphorilates and thereby activates a transcription factor termed "nuclear factor of activated T cell" (NFATC), which is then translocated from the cytoplasm to the nucleus where it upregulates the expression of interleukin 2. Interleukin 2 then stimulates growth and differentiation of T lymphocytes. When calcineurin is inhibited by immunosuppressive drugs T-cell mediated immune response is largely inhibited. As calcineurin is widespread among eukaryotic organisms FK506 and FK520 also have antifungal and antiprotozoan activities.
F 506 has been used mainly as immunosuppressant used to decrease the possibility of organ rejection after transplantation. Intravenous or oral administration after allogeneic liver, kidney or bone marrow transplantation represent the most examples of use. In addition, FK506 has shown potential for use in treatment of several autoimmune, inflammatory or respiratory diseases such as: Bechet syndrome, Crohn's disease, atopic dermatitis, uveitis, psoriasis, nephritic syndrome, rheumatoid arthritis, asthma, aplastic anemia, biliary cirrhosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease. However, widespread use for systemic treatment of these conditions is impaired by severe side effects described below. FK.506 as well as pimecrolimus (a derivative of FK520) are also used in topical formulations such as creams and ointments for localized treatment of skin disorders, particularly atopic dermatitis. Although FK506 is a widely used immunosuppressant to prevent the graft-versus-host disease after organ transplantation, its use is complicated due to the narrow therapeutic window, toxicity in the kidney, central nervous system and pancreas and most importantly large inter- and intra-individual variability in bioavailability and pharmacokinetics of the drug. Frequent therapeutic drug monitoring using enzyme immunoassay is therefore required to control the concentration of tacrolimus in the blood. Despite having blood concentrations in the therapeutic range some patients still experience infections or an acute rejection of the transplanted organ.
The great medical and economic potential of these compounds on one hand, and the above mentioned limitations of the current analogues in clinics on the other, clearly demonstrates the need for development of novel FK506-like compounds that may be less toxic, more resistant to metabolic attack and have more uniform bioavailability. Modification using chemically available positions on this molecule has been addressed and several derivatives have been produced, however this approach has limited utility as no appropriate sites available for chemical modification are accessible. Consequently, chemical modification usually requires multiple protective and deprotective steps and produces mixed compounds in variable yields. Nevertheless, one semi-synthetic derivative of FK520, pimecrolimus, is already used for treatment of inflammatory skin diseases due to its lower permeation through the skin and subsequent lower systemic effect than tacrolimus. Another example is L-732,531, a C32 derivative of FK520, found to posses significantly superior pharmacologic properties to tacrolimus as it shows lower affinity for erythrocytes and therefore a lower and concentration-independent blood-to-plasma ratio. Elucidation of biosynthetic mechanisms of FK506 and FK520 based on the sequences of their gene clusters has also enabled the production of several analogues by biosynthetic engineering. By manipulation of acyl transferase domains of PKS extender modules, 13- and 15- desmethoxy derivatives of FK520 have been produced, which lack immunosuppressive activity and show high potential as neuroregenerative drugs. The reason for such a drastic difference in pharmacological activity of these derivatives seems to be in that 13- and 15- methoxy groups are crucial for binding to calcineurin therefore their absence causes the molecule to still strongly inhibit the activity of FKBP but have no effect on T cell proliferation. Similar effect was observed in the case of FK1706, a semi-synthetic derivative of FK506 in which a polar carbonyl group was introduced into the hydrophobic allyl side chain on the C21 carbon atom presumably also preventing binding to calcineurin.
It is known from the literature, that the AT domain of the 4th module of a FK506 producing PKS displays relaxed specificity and that it accepts other extender units apart of allylmalonyl-CoA; namely naturally occurring methylmalonyl-CoA, ethylmalonyl-CoA, and propylmalonyl-CoA (Goranovic, Kosec et al. 2010) and 2-methylallylmalonyl-CoA and fluoroethylmalonyl-CoA as an extender unit (Mo et al., J. Am. Chem. Soc. 2010, "Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds though a dedicated polyketide synthase and facilitates the mutasynthesis of analogues", preprint, published online: http://pubs.acs.org/JACS).
WO 2004/096822 discloses polyketide compounds including a residue having a -CCH triple bond residue adjacent an ether bond. The triple bond is introduced by chemical modification of a precursor polyketide. US 65558942 discloses a polyketide compound having an ethinyl residue covalently bound to a terminal carbon atom of a polyketide carbon backbone. A structure according to Structure I hereinbelow is not shown. WO 2000/01838 discloses methods of making non-natural polyketides using PKS modified to accept non-natural starter units or non- natural extender units. The documents speculates on the use of N-acetyl cysteamine thioesters having an alkinyl (1-8C) residue (page 15). However, no data or other evidence is provided that a polyketide was produced having included such thioester in the polyketide synthesis. The PKS enzyme employed does not have the required promiscuity to accept the speculated-upon alkinyl extender units (as has the AT4 domain of the FK506 PKS of the current invention). Hence, WO 2000/01838 does not contain an enabling disclosure of how to add extender units including ethinyl residues to a nascent polyketide in a PKS reaction.
Against the background of the above outlined state of the art, there remains a substantial need for new polyketide compounds having altered and improved therapeutic properties. The present invention serves this need by providing novel polyketide compounds, in particular chemically reactive precursor polyketides that can easily be chemically modified to yield therapeutically active polyketide compounds derived from said precursor polyketide compounds.
Summary of the invention
The present invention is based on the unexpected finding that the AT4 domain of FK506 producing PKS, more specifically, the FkbB protein of the FK506 producing PKS, is promiscuous to an extent that it not only accepts the known substrates methylmalonyl- CoA, ethylmalonyl-CoA, propylmalonyl-CoA and allylmalonyl-CoA, but is sufficiently promiscuous to also accept extender units including very reactive triple bonds, in particular extender units including a structure comprising -G≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -ON, -CH2-C≡N and -CH2-CH2-C≡N.
According to the present invention, polyketide compounds including such triple bonds are produced by PKS enzymes comprising an AT domain similar or homologous to an AT domain of the fourth module of an FK506 producing PKS (an "FK506 AT4 domain", e.g. SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17). Triple bond containing polyketides are produced by incubating the suitable PKS with suitable triple-bond containing extender units. The resulting polyketide compound with carry the triple bond containing side-chain instead of the naturally occurring side chain, e.g. a methyl side chain.
The cloning new PKS enzymes using the known AT swap methodology (see above), according to the invention, yields new PKS enzymes carrying an FK506 AT4 domain at one or multiple "non-natural" positions. Such modified PKS enzymes allow for the biosynthetic production of new chemically reactive polyketide compounds by incubating the modified enzymes with the triple-bond containing extender units. The resulting polyketide compounds will carry the triple-bond containing side chains at various positions of the polyketide carbon backbone. The present invention thus also relates to the triple-bond containing polyketide compounds. These triple-bond containing polyketide compounds can chemically be modified to obtain new biologically active polyketide compounds. The triple-bond containing polyketide compounds, according to the invention, serve as precursors for new pharmaceutically active polyketide compounds. Compounds of the present invention can be used in drug screening, or can be used as drugs.
The present invention relates to:
1. Use of a polyketide synthase comprising a sequence set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or comprising a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17 for adding an extender unit having the structure
COOH ; wherein p is 0, 1 or 2, preferably p = 1 ; and X4 is any organic residue, preferably X4 is selected from NAc, CoA
to a polyketide compound, preferably to a nascent polyketide.
The invention also relates to the use of the above polyketide synthase for incorporating an extender unit of the above structure into a polyketide compound.
It is to be understood that the structures of X4 above (and at other places in this application) are connected to the sulphur atom via the left-most carbon atoms shown in the respective structure.
2. Use of # 1, wherein the polypeptide encoded by said sequence has acyl transferase (AT) activity.
3. Use of # 1 or 2, wherein said polyketide compound is a macrolide antibiotic, a macrolactone antibiotic, a polyene antibiotic, a polyether antibiotic, or a acetogenins. Alternatively, the use of # 1 or 2, wherein said nascent polyketide compound is a nascent macrolide antibiotic, a nascent macrolactone antibiotic, a nascent polyene antibiotic, a nascent polyether antibiotic, or a nascent acetogenin.
4. Use of # 1 or 2, wherein said polyketide compound is selected from the group consisting of: erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, emphotericin, nystatin, candicidin, monensin and salinomycin. Alternatively, the use of # 1 or 2, wherein said nascent polyketide compound is selected from the group consisting of: nascent erythromycin A, nascent pikromycin, nascent oleandromycin, nascent tylosin, nascent medicamycin, nascent rifamycin, nascent avermectin, nascent spinosyn, nascent rapamycin, nascent FK506, nascent meridamycin, nascent geldanamycin, nascent epothilone, nascent emphotericin, nascent nystatin, nascent candicidin, nascent monensin and nascent salinomycin
5. A method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a polyketide synthase, said polyketide synthase comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; b) providing a substrate compound having the structure
C00H , wherein p is 0, 1 or 2, preferably p = 1 ; and
X4 is any organic residue, preferably X4 is selected from NAc, CoA
c) incubating said microorganism in a medium comprising said substrate compound, whereby a polyketide compound is produced; and d) obtaining said polyketide compound from said medium.
6. Method of # 5, wherein said polyketide compound comprises at least one structure
Chainl Chain2
[Structure I]; wherein Y is -H or -OH; n is 1 or 0; p is 0, 1 or 2, preferably p is 1; C* is a carbon atom; and Chainl -C*-Chain2 is the carbon backbone of said polyketide compound.
7. Method of # 6, wherein said Structure I is
, wherein
Rs is any organic or inorganic residue, preferably a residue selected from the list consisting of CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3 CH2CH2C1, CH2CH2F, CH2OCH3, C6H6 (phenyl),
06Η5ΝΗ2 (paraaminophenyl), and
^^^COOH
R1 is independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH2CH2(C=0)CH3, CH2CH2C1, CH2CH2F, OCH3, OH and NH2; preferably from a lists consisting of H, CH3, CH2CH3 and OCH3;
Rk is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates, monosaccharides, disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups; Ra and Rb are each independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, and CH2CH2CH3; wherein Ra and Rb can also be connected so as to form a 5-, 6- or 7-membered ring, preferably a pyrrolidine or piperidine ring; -Y is -H, -OH, -CH3, preferably -H or -OH, most preferred -H;
n is 0 or 1 ; p = 0, 1 or 2, preferably 1 ; q is an integer from 0 to 10, preferably from 0 to 5; r is an integer from 0 to 20; s is an integer from 0 to 20; wherein r + s is from 0 to 20, inclusive; t is an integer from 0 to 5; preferably t is 1 ; denotes a single bond, a double bond, or an epoxide group between adjacent carbon atoms; wherein any two residues selected from R1, Rk, Ra and Rb can optionally be connected so as to form a ring.
8. Method of claim 6 or 7, wherein said polyketide compound is derived from a reference polyketide compound of the structure:
by substitution of a structure \ / in said reference polyketide compound, wherein C* is as defined above, and X1 and X2 are independently the naturally occurring substituents in said reference polyketide compound at position C*, or H, or non-existent;
with the structure wherein C*, Y, n and p are as defined above.
9. Method of any one of # 5 to 8, wherein said microorganism is deficient in at least one enzyme involved in the biosynthesis of allylmalonyl-CoA or ethylmalonyl-CoA.
10. Method of # 9, wherein said at least one enzyme involved in the biosynthesis of allylmalonyl-CoA or ethylmalonyl-CoA is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, or from the group consisting of sequences at least 70%, 80%, 90% or 95% identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
11. A method of making a chemically modified polyketide compound, said method comprising:
(i) producing a first polyketide compound using a method of any one of claims 5 to 10; and
(ii) chemically modifying said first polyketide compound, thereby obtaining said chemically modified polyketide compound.
12. Method of # 11, wherein said chemical modification is selected from the group consisting of: oxidation by KMn04, addition of water, alkylation, formation of a halomagnesium compound, halogenation, addition of borane including subsequent oxidation with hydrogen peroxide, C-C coupling with substituted alkenes, and oxytallation with Tl3+ salts, and combinations thereof. Preferably, the chemical modification is at the
carbon-carbon triple bond of, or at the terminal carbon of, the structure
13. A polyketide compound comprising the structure:
Chai Chain2
[Structure I];
Y is -H or -OH; n is 1 or 0; p is 0, 1 or 2; and
C* is a carbon atom; and Chainl -C*-Chain2 is the carbon backbone of said polyketide compound.
It is to be understood that the covalent bond between Chainl and C*, and/or between Chain2 and C* in Structure I above, and elsewhere in the description, can be a single bond, a double bond, or an epoxide group; preferably a single bond or a double bond.
14. A polyketide of # 13, wherein said Structure I is
, wherein
Rs is any organic or inorganic residue, preferably a residue selected from the list consisting of CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3 (phenyl), aaminophenyl), and ; preferably Rs is selected from CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3 CH2CH2C1, and CH2CH2F.
R1 is independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH2CH2(C=0)CH3, CH2CH2C1, CH2CH2F, OCH3, OH and NH2, preferably selected from a lists consisting of H, CH3, CH2CH3 and OCH3;
Rk is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates such as monosaccharides and disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups;
Ra and Rb are each independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, and CH2CH2CH3; wherein Ra and Rb can also be connected so as to form a 5-, 6- or 7-membered ring, such as a pyrrolidine or piperidine ring; -Y is -H, -OH, -CH3, preferably -H or -OH, most preferred -H; n is 0 or 1 ; p = 0, 1 or 2, preferably 1 ; q is an integer from 0 to 10, preferably from 0 to 5; r is an integer from 0 to 20; s is an integer from 0 to 20; wherein r + s is from 0 to 20, inclusive; t is an integer from 0 to 5; t is an integer from 0 to 5; preferably t is 1 ; denotes a single bond, a double bond, or an epoxide group between adjacent carbon atoms; wherein any two residues selected from R1, Rk, Ra and Rb can also be connected so as to form a ring. 15. The polyketide compound of # 13 or 14, wherein the polyketide compound is a macrolide antibiotic, a macrolactone antibiotic, a polyene antibiotic, a polyether antibiotic, or an acetogenin.
16. The polyketide compound of any one of # 13 to 15, wherein the structure of said polyketide compound is derivable from the structure of a reference polyketide compound, said reference polyketide compound being selected from the group consisting of: macrolides, erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, macrolactones, rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, polyene antibiotics, emphotericin, nystatin, candicidin, polyether antibiotics, monensin and salinomycin;
X1 X2
\ /
by substitution of a structure > in said reference polyketide compound, wherein C* is as defined above, X1 and X2 are independently the naturally occurring substituents in said reference polyketide compound at position C*, or H, or non-existent;
with the structure , wherein C*, Y, n and p are as defined above.
17. The polyketide compound of any one of # 13 to 16, wherein said polyketide compound has the structure:
; wherein
-X3 is -H, -OH, or double bonded O, wherein, if -X3 is double bonded O then RJO is non-existent;
-Ri to -R10 are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH, wherein Rio can also be non-existent; and at least one of -Ri to -R10 is selected from -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH.
18. A chemically modified polyketide compound, said chemically modified polyketide compound having the structure:
, wherein
-X3 is -H, -OH, or double bonded O, wherein, if -X3 is double bonded O then R20 is non-existent;
-R„ to -R20 are independently selected from: -H, -CH3, -CH2-CH2-COOH, - CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, - CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21, -CH2-CHR21-COO-R22, wherein R20 can also be nonexistent,
Hal is selected from F, CI, Br and I,
R21, R22 are any organic moiety; and
at least one of -Ri 1 to -R20 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH,
-CH2-C≡C-R2i, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, - CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21; -CH2-CHR2,-COO-R22.
19. The polyketide compound of any one of # 13 to 16, wherein said polyketide compound has the structure:
; wherein
-X is -CH3 or -C2H5;
-Ri to -Re are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH; and at least one of -R\ to -R6 is selected from -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH.
20. A chemically modified polyketide compound, said chemically modified polyketide compound having the structure:
wherein -X is -CH3 or -02¾;
-Rn to -R16 are independently selected from -H, -CH3, -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R2i; -CH2-CHR21-COO-R22; wherein Hal is selected from F, CI, Br and I; and R21, R22 are any organic moiety; and at least one of -Rn to -R16 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R2,, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R2i, and -CH^CHR^-COO-Rz,. 21. A recombinant polyketide synthase comprising a sequence set forth in SEQ
ID NO: l, SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90%, or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17, wherein the recombinant polyketide synthase is not a FK506 polyketide synthase, preferably the recombinant polyketide synthase is not a wild type (or naturally occurring) FK506 polyketide synthase. Hence the invention relates to recombinant polyketide synthases which carry a promiscuous AT4 domain of a FK506 PKS at a position where such a promiscuous AT4 domain is not normally occurring. These recombinant PKS enzymes are useful for producing non-natural polyketide compounds when fed with non-natural extender units of the invention, such as ethynylmalony-SNAC, or propargylmalonyl-SNAC, which polyketide compounds are amenable to chemical modification by virtue of carrying a non- natural triple bond.
22. The recombinant polyketide synthase of # 21, wherein said recombinant polyketide synthase is an FkbB protein of a polyketide synthase protein complex. 23. The recombinant polyketide synthase of # 21 or 22, wherein said FK506 polyketide synthase is an enzyme having the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or a sequence at least 70%, 80%, 90%, or 95% identical to any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
24. A recombinant polyketide synthase, said recombinant polyketide synthase having multiple functional modules, each functional module comprising at least one keto- synthase (KS) domain, one acyltransferase (AT) domain, and one acyl carrier protein (ACP) domain, wherein at least one of said modules comprises the sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90%, or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17, wherein said at least one of said modules is not the fourth module of a FK506 polyketide synthase.
25. The recombinant polyketide synthase of # 24, wherein said FK506 polyketide synthase is an enzyme having the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or a sequence at least 70%, 80%, 90%, or 95% identical to any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
The present invention also relates to a compound having the structure:
COOH ; wherein
p is 0, 1 or 2, preferably p = 1 ; and
X4 is any organic residue, preferably X4 is selected from NAc, CoA
is selected from CoA and NAc,
It is to be understood that the above structures of X4 are covalently connected to S via the left-most carbon atom in the respective structure, above.
These compound have surprising properties in that they are accepted by promiscuous AT4 domains of FK506 PKS enzymes of the present invention, and the triple bond being a very reactive group. These compounds are thus very suitable for producing polyketides which can easily be further modified chemically The present invention also relates to the synthesis of these compounds.
A second aspect of the invention relates to a polyketide compound comprising the structure:
[structure I]; wherein Z is CH or N, preferably CH; Y is -H or -OH, preferably -H;
Chain is a part of a polyketide carbon backbone, respectively; and p is 0, 1 or 2, preferably 0 or 1, most preferred 1.
In one embodiment the polyketide compound comprises only a single Structure I. In a further embodiment the polyketide compound is selected from the group consisting of macrolides, macrolactones, polyene antibiotics, polyether antibiotics, tetracyclines, acetogenins.
In a further embodiment the polyketide compound is obtainable from a natural polyketide compound, preferably selected from the group consisting of macrolides erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, macrolactones rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, polyene antibiotics, emphotericin, nystatin, candicidin; polyether antibiotics monensin and salinomycin; by substitution in said natural polyketide compound of a structure: wherein X I and X 2 are naturally occurring substituents of the natural polyketide compound or H, and
Chain are independently a part of the carbon backbone of the natural polyketide compound, respectively; by the structure: wherein Z is CH or N, preferably CH; Y is H or OH, preferably H; p is 0, 1 or 2, preferably 0 or 1 , more preferred 1 ; and Chain is as defined above.
In a further embodiment of the invention said polyketide compound has the structure:
; wherein
X is -H, -OH, or double bonded O, wherein, if X is double bonded O then R10 is nonexistent; Ri to Rio are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -C≡N, -CH2-C≡N and -CH2-CH2-C≡N, wherein R10 can also be nonexistent; and at least one of Ri to Rio is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -C≡N, -CH2-C≡N and -CH2-CH2-C≡N. Preferably Rj to Ri0 are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-C≡N, wherein Rio can also be non-existent; and at least one of Ri to Rio is selected from -C≡CH, -CH2-C≡CH, and -CH2-C≡N.
From the above it follows that X3 and Rio in the above structure may collectively be replaced by a double bonded oxygen. Hence, the position 9 of the above polyketide backbone can be substituted with a double bonded oxygen. These polyketides are variants of the FK506, and thus likely candidates for immunosuppressive drugs. The present invention thus also relates to the use of compounds of the invention as immunosuppressive drugs.
In one embodiment only a single one of Ri to Rio is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -ON, -CH2-C≡N and -CH2-CH2-C≡N. Hence, only a single side chain is replaced by the chemically active triple-bond structure.
In one embodiment
Ri, R2, R5, Re ,R9 are -CH3;
R3 is H; R4 is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -C≡N, -CH2-C≡N and -CH2-CH2-C≡N;
R7, Rg are -OCH3; and
X3 is double bonded O and R10 is non-existent.
Hence, the chemically active triple bond is introduces at position 21 of the structure according to Formula I (above).
In another embodiment said polyketide compound has the structure:
; wherein
X is -CH3 or -C2H5;
Ri to R6 are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -C≡N, -CH2-C≡N and -CH2-CH2-C≡N; and at least one of Ri to Re is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -C≡N, -CH2-C≡N and -CH2-CH2-C≡N. Preferably Ri to Re are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-C≡N, and at least one of Ri to Re is selected from -C≡CH, -CH2-C≡CH, and -CH2-C≡N.
Such polyketides are thus erythromycin-derived polyketides. These polyketides likely show antibiotic properties. The invention thus also relates to the use of such polyketide compounds as an antibiotic.
In one embodiment only a single one of Rj to R6 is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -ON, -CH2-C≡N and -CH2-CH2-C≡N. Hence, only a single chemically active triple-bond is introduced. In another embodiment
R2 to Re are -CH3, and R\ is selected from -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -ON, -CH2-ON and -CH2-CH2-ON, preferably -OCH, -CH2-OCH, and -CH2-C≡N. These polyketides correspond to the structure shown in Formula II (below).
In another embodiment Ri , R2, R3, R5, Re are -CH3, and R4 is selected from -OCH, -CH2- CH, -CH2-CH2-C≡CH, -ON, -CH2-ON and -CH2-CH2-C≡N, preferably -C≡CH, -CH2-OCH, and -CH2-ON.
These polyketides correspond to the structure shown as Formula III (below) with a single triple-bond side chain at position 6.
The present invention also relates to a chemically modified polyketide compound obtainable from the FK506-derived polyketides, above, said chemically modified polyketide compound having the structure:
Rn to R2o are independently selected from -H, -CH3, -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21;
-CH2-CHR21-COO-R22, wherein R20 can also be non-existent,
Hal is selected from F, CI, Br and I;
R2i, R22 are any organic moiety; and at least one of Rn to R20 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21; -CH2-CHR2i-COO-R22. R21 and R22 are preferably linear or branched alkyl.
R21 and R22 can be hetero alkyl.
R21 and R22 are may independently be hydrogen, methyl, ethyl, propyl, n-butyl or tert- butyl.
Hence at least one of Rn to R20 is a chemically modified (formerly triple-bonded) side chain.
In one embodiment only one of Rn to R20 is elected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R2i, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21;
-CH2-CHR21-COO-R22. (Hence only one of Rn to R20 is a chemically modified - formerly triple-bonded - side chain.)
The present invention also relates to a chemically modified polyketide compound obtainable from the erythromycin-derived polyketide compounds above, said chemically modified polyketide compound having the structure:
wherein X is -CH3 or -C2H5; Rii to Ri6 are independently selected from -H, -CH3, -CH2-CH2-CO-OH, -CH2-CH=CH- OH, -CH2-C≡C-R2i, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21; -CH2-CHR21-COO-R22; wherein
Hal is selected from F, CI, Br and I; and R21, R22 are any organic moiety; and
at least one of Rn to R16 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2- C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, - CH2-CH=CH-CH=CH-R21, and -CH2-CHR2i-COO-R22.
Hence at least one of Rn to R16 is a chemically modified (formerly triple-bonded) side chain.
In one preferred embodiment only one of Rn to R16 is -CH2-CH2-CO-OH, -CH2-CH=CH- OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21, and -CH2-CHR2i-COO-R22.
Hence at least one of Rn to R16 is a chemically modified (formerly triple-bonded) side chain.
The present invention also relates to a method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a PKS enzyme, said
PKS enzyme comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70%, 80%, 90% or 99% identical to SEQ ID NO:l or SEQ ID NO:2 and accepting a compound of the structure: as a substrate, wherein Z is CH or N; p is 0, 1 or 2, preferably 0 or 1, most preferred 1; and X4 is selected from CoA and NAC, preferably NAC; providing a substrate compound of said structure c) incubating said microorganism in a medium comprising said substrate compound; and d) obtaining said polyketide compound from said medium.
Apart from the SNAC extender units and the CoA-activated extender units disclosed in this patent application, other esters or thioesters of the extender units can be used, such as thioglycolate thioesters and diethyl ester. In addition both, single and double esters or single and double thioesters, or mixtures thereof, can also be used. SNAC extender units are preferred, because they readily pass the cell membrane, thus can be fed externally, and they are available in greater amounts and for a more attractive price than are the CoA extender units.
Thus this invention also relates to biotechnological production methods for compounds of the present invention. The incubation is preferably under permissive conditions. Incubation is also preferably for a time sufficient to produce sufficient amounts of said polyketide compound. The methods can include submerse culture of the microorganism, such as fermentation in a bioreactor. The microorganism is preferably provided with a nutrient medium such as to allow growth of the microorganism.
In an alternative embodiment, a bi-functional thioester (e.g., bis SNAC) is be used instead of the monoester acid in the methods of the invention.
In one embodiment said microorganism comprises a PKS enzyme capable of producing a compound of the structure:
Hence, the present invention also relates to methods of making modified FK506 compounds by using microorganisms that normally produce FK506. In these embodiments it is advantageous to feed the triple-bond containing extender unit to the culture medium, such that these extender units can be built into the polyketide compound by the FK506 AT4 domain. Preferably, the triple-bond containing extender unit is added in excess, e.g. at a concentration of above 0.0001 M, 0.001 M, 0.01 M, or above 0.1 M in the medium. The concentration is preferably below 0.1 M or below 1 M.
In one embodiment said microorganism is deficient in at least one enzyme selected from the group consisting of AHA, A11K, A11R and A11D, preferably A11R In these microorganisms the natural pathway for producing the allylmalonyl-CoA extender unit is inoperative (Goranovic, Kosec et al. 2010)), thus the triple-bond containing extender unit will more efficiently be built into the polyketide compound.
In a further embodiment said microorganism is selected from the group consisting of Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces tsukubaensis, Streptomyces sp. ATCC 55098, Streptomyces griseojuscus, Streptomyces lividans, Streptomyces rimosus, Streptomyces fridae, Streptomyces cinnamonensis, Streptomyces albus, Streptomyces lasaliensis, Streptomyces antibioticus and Amycolatopsis mediterranei.
The invention further relates to methods of making a polyketide compound, said method comprising: a method as described above; and chemical modification of the product of said method, so as to obtain a chemically modified polyketide compound.
Preferably, said chemical modification is selected from the group consisting of: oxidation by KMnC»4, addition of water, alkylation, formation of halomagnesium compounds, halogenation, addition of borane and subsequent oxidation with hydrogen peroxide, C-C coupling with substituted alkenes, Ruthenium catalyzed C-C coupling and oxytallation with Tl3+ salts.
In a preferred embodiment said amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70%, 80%, 90% or 99% identical to SEQ ID NO:l or to SEQ ID NO:2 is not within the 4th module of a FK506 producing PKS. Hence, the corresponding PKS is not the (natural) FK506 producing enzyme.
Brief description of the Figures
Figure 1 shows examples of chemical derivatization of polyketides containing a terminal triple bond (propargyl or ethynyl group). Figure 2 shows possible C-C coupling reactions with substituted alkenes using ruthenium catalysts yielding 2,4-diene moieties on position 21 of FK506.
Figure 3 shows chemical modification of halogenated side chains according to the invention. Detailed description of invention
The articles "a" and "an" as used herein refer to one or to more than one, i.e. at least one, of the grammatical objects of the article. For example "a precursor" means one precursor or more than one precursor. The term "analogue" as used herein means a chemical compound that is structurally similar to another but whose composition is slightly different. For example, one atom may be replaced by another or a particular functional group can be present or absent.
The term "polyketide" as used herein refers to any compounds whose biosynthesis comprises at least one step which may be catalyzed by a polyketide synthase enzyme or any compound containing part of the structure generated by PKS enzyme. In one preferred embodiment of the invention, a polyketide is a compound synthesized (or producible) by a PKS enzyme.
The term "nascent polyketide" is to be understood as relating to any precursor polyketide compound in the biosynthesis of a polyketide compound (i.e., of the mature polyketide compound). The precursors in the biosynthetic pathways of polyketide compounds are well known in the art and can be deduced from the knowledge of the biosynthetic pathway. Alternatively, the term "nascent polyketide" can be understood as relating to an unfinished polyketide chain which is being synthetized by PKS and has not yet been completed or cyclised. Hence, the term "nascent polyketide" can also be understood as relating to an unfinished polyketide chain, which is being synthesized by PKS and has not yet been completed or folded (or cyclised) to reach its final polyketide structure. The term "extender unit" as used herein means any compound that can be recognized by AT domains of PKS module and incorporated into polyketide chains by catalytic action of PKS. The term "hybrid polyketide" as used herein means is a genetically modified PKS in which one or more than one domain(s) in the target module of natural PKS has been replaced (swapped) using analogues domain(s) from heterologous PKS enzyme using.
A PKS enzyme (i.e., a polyketide synthase), according to the invention, shall be understood as being a member of the family of multi-domain enzymes or enzyme complexes that produce polyketides. PKSs can be classified into (1) Type I polyketide synthases, which are large, highly modular proteins, (2) Type II polyketide synthases, which are aggregates of monofunctional proteins, and (3) Type III polyketide synthases do not use ACP domains. Type I PKSs are further subdivided into (la) Iterative PKSs, which reuse domains in a cyclic fashion, and (lb) Modular PKSs, which contain a sequence of separate modules and do not repeat domains.
Preferred PKS enzymes are bacterial PKS enzymes. Particularly preferred are PKS enzymes of Actinomyces spp. or Streptomyces sp., preferably of Type I. In preferred embodiments, the term "polyketide synthase", as used herein, shall be understood as relating only to the FkbB protein (or unit), which forms complexes with the FkbA protein, the FkbC protein, and optionally other proteins, to form a functional PKS. In these embodiments, the terms "polyketide synthase" and "FkbB protein of the polyketide synthase" or "FkbB unit of the polyketide synthase" are used synonymously.
The term "type I PKS" as used herein refers to the family of enzymes that produce polyketides, and which are organized as single protein molecules having multiple modules. Each module typically consist of minimal obligatory set of domains, namely: keto-synthase (KS) acyltransferase (AT) and acyl carrier protein (ACP) and further alternative (non-obligatory) set of domains involved in the reduction of β-keto group of nascent polyketide chain namely ketoreductase (KR), dehydratase (DH) and enoylreductase (ER).
The term "FK506 polyketide synthase", in accordance with the present invention, shall be understood as defining a polyketide synthase enzyme capable of synthesizing FK506. In another embodiment of the invention, the term "FK506 polyketide synthase" shall be understood as defining a polyketide synthase enzyme capable of synthesizing the FK506 compound of Formula I. Preferred polyketide synthases of the invention are FK506 polyketide synthases of Streptomyces sp.. Preferably, the polyketide synthases of the invention are PKS enzymes of Streptomyces sp. KCTC11604BP, Streptomyces sp. MA6858 ATTC 55098, or PKS of Streptomyces tsukubaensis, preferably Streptomyces tsukubaensis NRRL 18488. Also preferred are PKS enzymes of Streptomyces clavuligerus, preferably Streptomyces clavuligerus KTC 1056 IBP or CKD 1119, or Streptomyces glaucensens, e.g., MTCC 5115.
Preferred FK506 polyketide synthases of the invention are the ones of: Streptomyces sp. KCTC 11604BP (GenBank: ADU56322.1 , SEQ ID NO:22);
Streptomyces sp. MJM7001 (GenBank: ADX99524.1, SEQ ID NO:23);
Streptomyces kanamyceticus (GenBank: ADU56247.1, SEQ ID NO:24),
Streptomyces sp. MA6548 (GenBank: AAC68815.1, SEQ ID NO:25, and
Streptomyces tsukubaensis, preferably Streptomyces tsukubaensis NRRL 18488. The term "precursor of extender unit" as used herein means any compound, usually ester or thioester of 2-monosubstituted derivatives of malonic acid, which can be converted, by enzymatic activities inside the cells to respective extender unit and get incorporated into polyketide chains by activity of PKS.
The term "non-natural extender unit" as used herein refers to any compounds which can be incorporated as an extender unit in polyketide synthesis wherein the extender unit is not a compound that is produced in naturally occurring metabolic pathways, such as malonyl- CoA, methylmalonyl-CoA, ethylmalonyl-CoA or methoxymalonyl-ACP, which are recognized by the AT4 domain of FK506 PKS which are non-natural 2-monosubstituted derivatives, either esters or thioesters, of malonic acid, that are derived by synthetic chemistry approaches and/or biotransformation using synthetic substrates. These are extender units generally not selected by ordinary AT domains of PKS.
The "carbon backbone" of a polyketide compound produced by a PKS enzyme, in the context of the present invention, shall be understood as being the linear chain of covalently connected carbon atoms in said polyketide compound, which linear chain, upon synthesis of said polyketide compound by said PKS enzyme, is being formed by sequential addition of two carbon atoms at a time to said linear chain, through the catalytic activity of said PKS enzyme. In many polyketide compounds the linear carbon backbone forms part of a cyclic structure by cyclization via a hetero atom, such as oxygen, thereby forming, e.g., a lactone ring. The term "AT domain" as used herein means the acyltransferase domain involved in the selection of an extender unit(s) located in the typical module of any type I PKS enzyme.
The term "AT4 domain" as used herein means the acyltransferase domain of the module 4 of any FK506 generating PKS either in the form of a polynucleotide chain (DNA, RNA) or polypeptide chain (protein). Preferred AT4 domain is the AT4 domain of the FK506 PKS of Streptomyces tsukubaensis:
AVTGTALTRPRTVFVFPGQGSQWLGMGLKLMAES PVFAARMRECADALAEHTGRDLIA L EDPAVKSRVDVVHPVCWAVMMSLAAV EAAGVRPDAVIGHSQGE IAAACVAGAITLEDGA RLVALRSALLQRELAGHGAMGS IAFPAADVEAAAAQVDNVWVAGRNGTGTT IVSGRPDAV ETL IARYEARGV VTRLWDC PTHT PFVDPLYDE FQRIAAATTSRT PRI PWFSTADER I DS PLDDEYWFRNLRN PVGFAAAVAAAREPGDTVFVEVSAHPVLLPAINGTTVGTLRRGGG ADQWDSLAKAYTAGVAVD PTWAAPGTAHDTTRTASGPVPGPAHDLPTYAFHHERYWI E PSSGTDATGLGLDAVDHPLLAASVAL (SEQ ID NO:l)
Also preferred is the AT4 domain of the FK506 PKS of Streptomyces sp. MA6548:
WTGTALTAPRTVFVFPGQGSQ LGMGRELMAES PVFAAR RQCADALAEHTGRDL IAML DDPAVKSRVDWHPVC AV VSLAAVWEAAGVRPDAVVGHSQGE IAAACVAGAI SLEDGA RLVALRSALLVRELAGRGAMGS IAFAAAARI DGV VAGRNGTATT IVSGRPDAVETL IAD YEARGVWVTRLWDC PTHT PFVDPLYDELQRIVAATTSRAPE I P FSTADER I DAPLDD EY FRNMRNPVGFAAAVAAARE PGDTVFI EVSAHPVLLPAINGTTVGTLRRGGGADRVLD SLAKAHTVGVAVD STWAATGAADDAASVTAHDTGTAHDLPTYAFHHERYWI EPATGT D ASGLGLDAVDH PLLAASVALPDS (SEQ ID NO:2) Also preferred is the AT4 domain of the FK506 PKS of Streptomyces kanamyceticus:
>gb|ADU56247.1| :6502-6891 polyketide synthase [Streptomyces
kanamyceticus ]
VVTGTALTAPRTVFVFPGQGSQWLGMGR LMAESPVFAARMRQCADALAEHTGRDLIAMLDDPAV SRVD VVHPVC AVMVSLAAVWEAAGVRPDAVIGHSQGEIAAACVAGAISLEDGARLVALRSALLVRELAGRGAM GSIAFAAADVEAAAARIDGV VAGRNGTATTIVSGRPDAVETLIADYETRGVWVTRLVVDCPTHTPFVDP LYDELQRIVAATTSRAPEIPWFSTADERWIDAPLDDEYWFRNMRNPVGFAAAVAAAREPGDTVFIEVSAH PVLLPAINGTTVGTLRRGGGADRLLDSLAKAHTVGVAVD PTVVAATGAAHDTARTADGAATGTAHDLPT YAFHHERYWIEPSAGTDASGLGLDAVDHPLLAASVALPDS (SEQ ID NO:17)
The above AT4 domains of the FK506 PKS are advantageous, in that they do not accept the naturally occurring methylmalonyl-CoA and malonyl-CoA extender units as substrates. Hence, the AT4 domain of the FK506 PKS according to the invention favours the production of FK506-related compounds having non-natural extender units, such as the ones comprising a carbon-carbon triple bond, according to the invention. AHA, according to the present invention, is a protein having the following amino acid sequence (SEQ ID NO: 18), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
>gi|295831518|gb|ADG39431.1| AHA [Streptomyces tsukubaensis]
MTSGVAFLFPGQGSYVPGVFAGLGADAGRVATLVAEIDAAVEEFRLKPVRPLLFSPDAPALAELLESDHE RLDVAILATSIALAELLESRHGMSPDHVAGHSLGEFGALAVAGVFTPGDAARAVCERHATLRKAPPPTGG MLAVKADAARAGELIAAARAGTSAVSALNSPSQTVISGAEADLV VQQLAREEGIRTSRLHVPGPFHVPQ LADASALYATTMRTIRISAPRERFFYSHGLGRFLTAQDDWDLMVNDMTRPVRFHDSVRALNAEGVTTYV ECGALDVLTRIVSGSLPRAVTLAPLREATTTPDLSARLRPAGTPAVNGVAAPAGPAPAAEVDPEVLAGVR AVCAEVLEYPLEVITDDADFQADLGVDSLAMTELQAHALQRFGLKETLQDADTGTYGTVSGLAAYITGLL SEGTGSVSGRR
"A11K", according to the present invention, is a protein having the following amino acid sequence (SEQ ID NO: 19), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
>gi I 295831519 I gb| ADG39432.1 I A11K [Streptomyces tsukubaensis]
MISRAPDGEGPHDDRVAWGMGVAVPGACDPEELWKLLCGDRPVFDEPSDRFRLDSFWSADPAAEDRGYV
RTSGFLHDFRPHPALAAEIAAGTLSAAAQNPVWLRHCLLQARDTVTARSTDRYAYHVGTSALVGQRTDEA VLAECVPRAVAERLHRDEPARMAEAEARLRALLRSHHGYGAEEPRDTLPDRVVRAAAAGLLPDDCEFSVV DAACSSSLYAIGLGVASLLAGACDIAYCGGVSGVTPRYNVTFSKLHGLSPSGDVRAFDDDADGTLFSDGA GWALKRLDRAVEDGDPVFGVLVGFGGSSDGRGTAIYAPNPVGQRRCLDRARQASGLTADDVDWVIAHGT GTAVGDAVELRTLAAATDPGSVWCGSNKSLLGHTGWSSGVVSVVQALTALRQGTIPAQRRFTGPGLTAQT GDRVRIPSADVPWHAGGRRSRTAGVSAFGFGGTNAHLLITDREPVRTGPRPARTGPDPWVLAWTAHLPG DPGPEATERLLREGRIPGPRTFGPRYPAPPFPDVRLPPPTVRSTDAGQLMALRVAGLFAAEHGEL APVR ATTGVFAAATGPPPSSMDHLVRCHAADVHRILDEPDRTAFTEWLADLRATTPATTKDTLPGLLPNIIPAR IANRYDLGGPTMLVDTGTTSGLTAVHTAVRQLAAGAVDMALVLGVSATGRPEFARFMGVAAERIAEGAFL LALSRESVALAHGLTPLVRLRTDWTGSPQASADAVPGGPGAAEDTFLGADGVLAVIRALHSTASGVTVGP ADGEPGPVITLSPADGSPLRQTRTSR
"A11R", according to the present invention, is a protein having the following amino acid sequence (SEQ ID NO:20), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
>gi|295831520|gb|ADG39433.1| A11R [Streptomyces tsukubaensis]
MTHVRDAAATDDPQAIAACEVPAGYRAAVVLAADHQALAGSPVEDRDPRKTVQVQEVPTPEPDHGEVLIA TMASSINYNTVWSALFEPVPTFRFLRTLGRTSPEAARHDQPYHVLGSDLSGVVLRTGPGVREWKPGDEVV AHCLQPDLQTPGGHDDTLLDPGQRVWGYETNFGGLAELSLVKANQLMP PAHLTWEEAASLGVALSTAYR QLVSHHGAAMKQGERVLVWGAAGGVGAYATQLALNGGAVPICVVSSQAKADLCRQMGAELVIDRAAEGFS FWEGRDRPRLSEWSRFRGAVRSLAGDDPDIVIEHPGRDTFGVSVMIAARGGKWTCASTTGYQHTYDNRH LWMRV RIIGSHMANYREAWAANELVARGSIHPVLSRVYPLDATGDATHAVANNSHHGKVGVLCLADRPG MGVRDPELRARKLDSINLFRKGQPR
"A11D", according to the present invention, is a protein having the following amino acid sequence (SEQ ID NO:21), or a sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to the following sequence:
>gi I 2958315211 gb I ADG39434.11 A11D [Streptomyces tsukubaensis ]
MSESERLGIVRDFVAREILGREGILDSLADAPLALYERFAETGLMNWWVPKEHGGLGLGLEESVRIVSEL AYGDAGVAFTLFLPVLTTSMIGWYGSEEL ERFLGPLVARRGFCATLGSEHEAGSELARISTTVRRDGDT LVLDGTKAFSTSTDFARFLVVIARSADDPARYTAVTVPRDAPGLRVDKRWDVIGMRASATYQVSFSDCRV PGDNALNGNGLRLLEIGLNASRILIAASALGVARRIRDVCMEYGKTKSLKGAPLVKDGVFAGRLGQFEMQ IDV ANQCLAAARAYDATAARPDAARVLLRQGAQKSALTAKMFCGQTAWQIASTASEMFGGIGYTHDMVI GKLLRDVRHAS11EGGDDVLRDLVYQRFWPTAKRT
Within the context of the present invention, term "% identity" between to amino acid sequences, as used herein, defines the % identity calculated from two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default BLOSUM62 matrix (see below) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference polypeptide.
The following BLOSUM62 matrix is used:
Ala 4
Arg - 1 5
Asn -2 0 5
Asp -2 2 1 6
Cys 0 -3 -3 •3 9
Gin - 1 1 0 0 -3 5
Glu • 1 0 0 2 -4 2 5
Gly 0 -2 0 - 1 -3 -2 -2 6
His •2 0 1 - 1 •3 0 0 •2 8
He - 1 -3 •3 •3 - 1 -3 -3 •4 -3 4
Leu - 1 -2 -3 -4 - 1 -2 -3 -4 -3 2 4
Lys • 1 2 0 ■ 1 -3 1 1 -2 - 1 -3 -2 5
/Met - 1 - 1 -2 -3 - 1 0 -2 -3 -2 1 2 - 1 5
Phe -2 -3 -3 -3 -2 -3 -3 -3 - 1 0 0 -3 0 6
Pro - 1 -2 -2 - 1 -3 • 1 - 1 -2 -2 •3 -3 - 1 -2 -4 7
Ser 1 - 1 1 0 -1 0 0 0 - 1 -2 -2 0 - 1 2 - 1 4
Thr 0 - 1 0 • 1 - 1 • 1 • 1 •2 -2 - 1 - 1 • 1 - 1 •2 - 1 1 5
Trp -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 - 1 1 -4 -3 -2 11
Tyr -2 -2 -2 -3 -2 - 1 -2 •3 2 - 1 - 1 -2 - 1 3 -3 -2 -2 2
Val 0 -3 -3 -3 - 1 -2 -2 -3 -3 3 1 -2 1 - 1 -2 -2 0 -3
Ala Arg Asn Asp Cys In Glu Gly His lie Leu Lys Phe Pro Ser Thr Trp Tyr Val It is to be understood that the expression "any organic moiety", as used hereinbelow relates to any organic moiety, preferably to represents an unbranched or branched, saturated or unsaturated, optionally at least mono-substituted Q aliphatic group; more preferably to a saturated or unsaturated, optionally at least mono-substituted, optionally at least one heteroatom as ring member containing cycloalkyl group, which may be condensed with an optionally at least mono-substituted mono- or polycyclic ring system; or to a branched or unbranched, optionally at least one heteroatom as ring member containing alkyl-cycloalkyl group in which the cycloalkyl group is optionally at least mono-substituted; or to an optionally at least mono-substituted aryl group; or to an optionally at least mono-substituted heteroaryl group which may be condensed with an optionally at least mono-substituted mono- or polycyclic ring system; or to a branched or unbranched alkyl-aryl group in which the aryl group is optionally at least mono- substituted and/or condensed with a mono- or polycyclic ring system.
The present invention is directed to a process for the preparation of novel polyketides wherein the process comprises the step of cultivation of a microorganism, preferably a genetically modified strain of a microorganism, preferably belonging to the order of Actinomycetales, more preferably to the genus Streptomyces, wherein the genetic material of the microorganism comprises of at least one PKS module containing at least one AT domain of the module 4 of FK506 PKS (AT4) and wherein the cultivation of the microorganism is carried out in growth medium supplemented with at least one extender unit or analogue or precursor thereof.
In one embodiment, the process for the preparation of polyketides according to the present application comprises at least one of the following steps:
a) Obtaining a genetically modified strain of a microorganism
b) Cultivation the strain of a microorganism in the suitable cultivation medium supplemented or fed with at least one precursor of a non-natural extender unit in shake flask or bioreactor and production of a target polyketide.
c) Isolation of the produced polyketide from the cultivation broth.
Preferably, the present invention relates to a process comprising of steps a) to c) as described above.
In a preferred embodiment the invention relates to a process for preparation of polyketides as described above, wherein the process comprises of cultivation of a microorganism, preferably a bacterium, preferably classified to the order Actinomycetales, preferably classified to the genus Streptomyces. Examples of microorganism strains are Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces tsukubaensis, Streptomyces sp. ATCC 55098, Streptomyces griseofiiscus, Streptomyces lividans, Streptomyces rimosus, Streptomyces fridae, Streptomyces cinnamonensis, Streptomyces albus, Streptomyces lasaliensis, Streptomyces antibioticus and Amycolatopsis mediterranei.
In another aspect the invention relates to the process for preparation of novel polyketides wherein the process comprises a step of cultivating a microorganism, wherein the cultivation is carried out with external addition of an analogue or precursor of an extender unit, preferably a non-natural extender unit, most preferably this extender unit being selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA.
In another aspect the invention relates to the process for preparation of novel polyketides wherein the process comprises a step of cultivating a genetically modified microorganism, wherein the cultivation is carried out with external addition of an analogue or precursor of an extender unit, preferably a non-natural extender unit, most preferably this extender unit being selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl- CoA, cyanomethylmalonyl-CoA.
The AT domain of the first extender module of the deoxyerythronolide B synthase (DEBS1), part of the entire gene cluster encoding biosynthetic pathway for macrolide erythromycin biosynthesis can be replaced with the AT4 domain of the FK506 PKS. When the strain of Saccharopolyspora erythraea, containing this way engineered erythromycin PKS was cultivated in the medium supplemented with an esterified analogue or precursor of allylmalonyl-CoA the resulting product contained an allyl group at the carbon 12 of the lactone ring. Surprisingly, when the cultivation medium was supplemented with several precursors or analogues of non-natural extender units, the resulting products lacked a methyl group at the carbon 12 of the lactone erythromycin ring. Instead, corresponding moieties were present at the carbon 12 atom of the lactone ring, said chemical moieties originating from the compounds which were fed in the production medium. Thus, when this organism is cultivated in the presence of analogues or precursors of allylmalonyl-CoA or non-natural extender units substituted erythromycin analogues are produced, according to the following formula:
Formula II,
in which X= CH3, or C2H5 and R is = allyl, prop-2-ynyl, ethynyl, cyanomethyl,. Analogously, when the AT domain from the fourth module of the erythromycin PKS (6- DEBS) was replaced by the AT4 domain of the FK506 PKS, and the resulting "DEBS2- AT4" engineered strain of Saccharopolyspora erythraea was cultivated in the production medium supplemented with precursors or analogues of allylmalonyl-CoA or non-natural extender units, erythromycin analogues substituted at the carbon 6 were produced:
Formula III
in which X= CH3, or C2H5 and R= allyl, prop-2-ynyl, ethynyl, cyanomethyl.
Hence, particular aspects of the invention relate to the process for preparation of novel polyketides or polyketide structure- containing compounds wherein the process comprises a step of cultivating a genetically modified microorganism, wherein at least one AT domain of a PKS gene encoded in the genome of said microorganism has been replaced by the AT domain encoded in the module 4 (AT4) of the PKS which catalyzes the biosynthesis of FK506, wherein the cultivation is carried out in the production medium supplemented with an analogue or precursor of an extender unit preferably a non-natural extender unit, this extender unit being: allylmalonyl-CoA, prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA. Any analogue or precursor of these extender units may be added, preferably a thioester derivative may be used, preferably N- acetylcysteamine thioester derivatives may be used.
In another embodiment the process comprises a step of cultivating a genetically modified microorganism, wherein at least one AT domain of a PKS gene encoded in the genome of said microorganism has been replaced by the AT domain encoded in the module 4 (AT4) of the PKS which catalyzes the biosynthesis of FK506, wherein the cultivation is carried in the production medium supplemented with an analogue or precursor of an extender unit, preferably a non-natural extender unit, this extender unit being: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA. Any analogue or precursor of these extender units may be added, preferably a thioester derivative may be used, preferably N- acetylcysteamine thioester derivatives may be used. Using methods of the invention, the PKS is modified to produce a compound of the structure:
Formula IV,
in which X= CH3, or C2H5, and R\, R2, R3, R4, R5, R6 are methyl or allyl, prop-2-ynyl, ethynyl or cyanomethyl. Particularly, the origin of the AT domain used in this aspect for replacement of a natural AT domain may be in any module of any PKS type I selectively incorporating allylmalonyl-CoA or propylmalonyl-CoA and ethylmalonyl-CoA but preferably not incorporating methylmalonyl-CoA and malonyl-CoA. Preferably, said AT domain may be an AT domain of the module 4 of any FK506 generating PKS encoded in the genome of any FK506 producing microorganism.
In yet another aspect this invention thus provides novel polyketides from the group of macrolide but not limited to the macrolides such as erythromycin, tylosin, medicamycin; from the group of macrolactone but not limited to such as rapamycin, FK506, FK520, rifamycin; from the group of polyene antibiotics but not limited to such as nistatin, amphotericin, candicidin and other polyketide-derived or polyketide containing structure of medical and/or commercial importance produced by methods and processes described above. In this particular aspect, these polyketides will be characterized by a substituent present at least one of the a-carbon atom which is derived from the precursor of extender unit supplemented in the cultivation medium. In a more particular aspect this substituent may be selected from but is not limited to the group comprising: prop-2-ynyl, ethynyl, cyanomethyl. In another aspect this invention also provides semi-synthetic derivatives of these novel polyketides produced by producers and methods described below.
Those skilled in the art will be able to recognize the limits of the AT4 domain in the FK506 generating PKS gene as well as the limits of an AT domain in a desired module of any PKS gene, to be replaced by the AT4 domain of FK506 PKS. Based on the published literature those skilled in the art will also recognize likely optimal sites for AT domain exchange, they will also be able to carry out the exchange of AT domains in any PKS gene using diverse standard techniques of molecular cloning.
Using this way engineered polyketide strains, and subsequently carry out cultivation in the presence of the externally added non-natural extender units or precursors thereof, those skilled in the art will be able to introduce substituents, selected from the group consisting of: allyl, prop-2-ynyl, ethynyl, cyanomethyl to diverse positions of polyketide structures or compounds containing polyketide-derived structure, particularly structures of polyketides selected from the group consisting, but not limited to: macrolides, macrolactones, polyethers and polyenes.
Surprisingly, we have also observed new FK506 analogues when precursors of non- natural extender units were fed to the fermentation broths of Streptomyces tsukubaensis lacking supply of the five carbon extender unit providing the allyl group and/or substrate supply providing ethylmalonyl extender unit at carbon 21 of FK506. Surprisingly, we have also observed new FK506 analogues when precursors of non-natural extender units were fed to the fermentation broths of Streptomyces tsukubaensis lacking supply of the five carbon extender unit providing the allyl group and/or substrate supply providing ethylmalonyl extender unit at carbon 21 of FK506. Surprisingly, AT4 from the fourth module of FK506 PKS does not select the most common extender unit such as malonyl- CoA in the absence of substrate supply providing the allyl group and/or absence of substrate supply providing ethyl group at carbon 21 of FK506.
Unexpectedly, non-natural extender units are readily recognized by the AT4 domain of FK506 PKS and efficiently incorporated into the FK506 structure at the carbon 21 position of FK506, thus generating novel FK506 analogues.
FK506 is not produced or is produced in very small amounts in strains lacking efficient supply of the natural extender unit, therefore the exclusive production of novel FK506 analogues with incorporated non-natural extender unit is achieved using this approach, and thus novel FK506 analogues can be easily purified. When analogues or precursors of non- natural extender units are fed to the wild type strain, mixtures of compounds are produced.
Therefore, in a particular embodiment this invention is concerned with the process for preparation of novel polyketides, wherein the process comprises a step of cultivating a microorganism, wherein these novel polyketides are structure analogous of FK506, wherein the allyl substituent at the carbon 21 of FK506 is replaced with a substituent, derived from a non-natural extender unit, particularly with a substituent selected from the group consisting of: prop-2-ynyl, ethynyl, cyanomethyl, wherein these substituents are incorporated into the polyketide structure as a result of adding analogues or precursors of extender units selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA. Any analogue or precursor of these extender units may be fed to the fermentation broth during S. tsukubaensis fermentation process, preferably a thioester derivative may be used, preferably N-acetylcysteamine thioester derivatives may be used.
In one more particular embodiment the cultivated microorganism may be selected from the order Actinomycetales, more preferably the microorganism may be selected from the genus Streptomyces, more preferably the microorganism may be selected from the group consisting of: Streptomyces tsukubaensis NRRL18488, Streptomyces tsukubaensis No. 9993 (Ferm BP 927), Streptomyces sp. MA6548 and Streptomyces clavuligerus CKD1119 (Kim and Park 2008), Streptomyces hygroscopicus subsp. hygroscopicus (DSM40822), Streptomyces sp. AA6554, Streptomyces hygroscopicus var. ascomyceticus MA6475 ATCC14891, Streptomyces hygroscopicus var. ascomyceticus MA6678 ATCC55087, Streptomyces hygroscopicus var. ascomyceticus MA6674, Streptomyces hygroscopicus var. ascomyceticus ATCC55276, Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891, Streptomyces hygroscopicus subsp. yakushimaensis, Streptomyces sp. DSM7348, Micromonospora n. sp. A92-306401 DSM8429.
In one more particular embodiment the cultivated microorganism may be a recombinant microorganism, particularly the strain may be mutated to have targeted inactivation or deletion of one or more genes that contribute to the biosynthesis or regulation of precursor supply, more particularly, the deleted or inactivated genes may contribute to efficient supply of 5 carbon extender unit allylmalonyl-CoA or propylmalonyl-CoA, most particularly the deleted or inactivated genes are selected from the group consisting of allA, allK, allR, allD (Goranovic, Kosec et al. 2010), also referred to as tcsA, tcsB, tcsC and tcsD, respectively (Mo et al., 2010). Therefore, the deletion or inactivation of precursor supply gene provides a system which enables the incorporation of non-natural extender units ensuring exclusive production of the target compound(s) containing non-natural extender unit.
Therefore in this aspect this invention provides a method for producing analogues of FK506 or FK520, said method comprising at least 1 of the following steps:
a) Generating a mutant strain of a FK506 or 520 or FK523 and/or structurally similar compound producing organism which in its genome contains a biosynthetic cluster which encodes polypeptides contributing to synthesis of FK506 analogue in which at least one gene related to the supply of precursors for biosynthesis of polyketides has been deleted or inactivated.
b) Cultivating the strain of a microorganism which in its genome contains a biosynthetic cluster which encodes polypeptides contributing to synthesis of FK506 or FK506 analogue in the suitable cultivation medium supplemented or fed with at least one precursor of a non-natural extender unit in the shake flask or bioreactor and producing the target FK506 analogue.
c) Isolation of the produced analogue of FK506 from the cultivation broth.
Preferably, the present invention relates to a process comprising of steps b) to c) as described above, most preferably the present invention relates to a process comprising of steps a) to c) as described above.
Those skilled in the art are also aware that gene clusters encoding biosynthetic pathways such as PKS and NRPS may be transferred and expressed in heterologous hosts. Therefore this invention includes the expression of FK506 or FK520 or closely related biosynthetic clusters) either intact or modified in any way in a heterologous host. Methods and vectors for heterologous expression as well as microbial strains engineered for efficient heterologous expression are well known in the art (Fuji 2009). Therefore in another aspect the present invention provides a method for preparation of a novel polyketides and polyketide structure-containing compounds, wherein the process comprises a step of cultivating a microorganism, wherein these novel polyketides have a structure analogous to the structure of FK506, wherein the allyl substituent at the carbon 21 is replaced with a substituent selected from the group consisting of: prop-2-ynyl, ethynyl, cyanomethyl, wherein these substituents are incorporated into the polyketide structure as a result of adding analogues or precursors of extender units selected from the group consisting of: prop-2-ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA. This method comprises: a) Generation of a genetically modified microbial strain of a non-FK506 or -FK520 producing microorganism, which contains a heterologous gene cluster encoding proteins involved in the biosynthesis of FK506, FK520 or their analogues. Particularly, this strain may lack efficient supply of allylmalonyl-CoA and/or propylmalonyl-CoA or ethylmalonyl-CoA. b) Cultivation the strain of a microorganism which in its genome contains heterologous biosynthetic cluster which encodes polypeptides contributing to the biosynthesis of FK506 or FK506 analogue in the suitable cultivation medium supplemented with at least one precursor of a non-natural extender unit in shake flask or bioreactor and production of a FK506 analogue. c) Isolation of the produced FK506 analogue from the cultivation broth.
In yet another aspect this invention provides novel polyketides of Formula I produced by methods and processes described above. In one particular aspect, these polyketides may be FK506 analogues characterized by the lack of allyl substituent at the carbon 21, wherein another substituent is present at said carbon atom. This substituent may be selected from but is not limited to the group comprising: prop-2-ynyl, ethynyl, cyanomethyl. Therefore, in this aspect the invention provides novel polyketides: 21 -desallyl-2 l-prop-2-ynyl- FK506, 21 -desallyl-21 -ethynyl-FK506, 21 -desallyl-21 -cyanomethyl-FK506.
Table 1. Examples of used extender unit precursors and novel FK506 analogues produced.
Using methods well known to those skilled in the art compounds of Formula I isolated from fermentation broths.
In another particular aspect, novel compounds provided by this invention may be used directly or as templates for further chemical derivatization or bioconversion in order to obtain compounds useful as immunosuppressants, antifungal agents, neuroregenerative agents, anticancer agents, antiinflammatory agents or agents useful for treatment of fibrosis, rheumatoid arthritis, psoriasis and other hypreproliferative diseases. Methods for chemical derivatization of FK506 and FK520, at position different than carbon 21 , are well known in the art and include but are not limited to those described in (Cooper and Donald 1989). There are also possibilities for modifications of the terminal alkene group at position C21, for example oxidation to keto group as in FK1706 (Cooper and Donald 1989) or coupling of C-C bonds using Grubb's ruthenium catalyst (Marinec, Evans et al. 2009, see also Figure 2).
In another particular aspect, and to those skilled in the art will recognise, that useful derivatives of compounds of Formula I may be obtained by combining the methods described herein with biosynthetic engineering and chemobiosynthetic methods, known in the art, which were used for obtaining analogues of FK506 or FK520 and biogenetically similar compounds such as rapamycin and meridamycin, wherein modification of polyketide structure is achieved at position other than carbon 21. These include incorporating novel starter units and incorporating novel pepicolate analogues. Engineering of the genes involved into the so-called post-PKS modifications can also further extend structural diversity of the generated analogues based on the engineered microorganisms and procedures described in this patent application. Finally, these compounds can be further derivatized using semi-synthetic approaches described above. It will be recognised by those skilled in the art that introducing into a position of the carbon atom not containing keto group (or reduced keto group), a polyketide chain a substituent selected from the group comprising: prop-2-ynyl, ethynyl, cyanomethyl introduces entirely novel opportunities for chemical derivatization previously unavailable in the FK506 or FK506 analogues produced by numerous Actinomycetes (Streptomycete) wild type strains. Therefore in another aspect this invention provides methods for producing chemical derivatives of compound selected from the group comprising: 21-desallyl-21-prop-2-ynyl- FK506, 21-desallyl-21-ethynyl-FK506, 21-desallyl-21-cyanomethyl-FK506. This invention also provides compounds resulting from said methods.
It will be recognized by those skilled in the art that introduction of the said groups into different positions of FK506 analogues as well as into different positions of other polyketides or polyketide containing structures, whose biosynthesis is mediated by type I PKS opens entirely new possibilities for semi-synthetic approaches. These functional groups enable new semi-synthetic modifications due to their selective reactivity compared to functional groups usually present in polyketide compounds at the corresponding positions. It will be recognized by those skilled in the art that individual positions on the polyketide derived scaffolds can be modified selectively without affecting numerous natural moieties usually present in polyketides. Below examples are presented of specific chemical reactions of some of the introduced functional groups.
As non-limiting examples, when a polyketide compound contains a terminal triple bond (C21 propargyl or C21 ethynyl group) it can be modified by methods well known in the art, as follows: a) Oxidation by KMn04 yields an appropriate carboxylic acid derivative b) Addition of water by means of HgSC /tbSO-i yields a keto derivative, which can be further reduced to hydroxyl group by a mild reducing agent such as NaB¾. c) Alkylation of acetylenic group with various alkylating agents by means of sodium amide as base and optionally subsequent addition of hydrogen halogenides d) Formation of halomagnesium compounds with Grignard reagents and further derivatization according to the chemistry of Gringnard compounds e) Addition of halogens and hydrohalogenids yields different mono- di- or poly halo compounds f) Addition of borane and subsequent oxidation with hydrogen peroxide g) C-C coupling with substituted alkenes using ruthenium catalysts yielding 2,4-diene moieties on C21 h) Oxytallation with Tl3+ salts in trialkyl ortoformiate or alcohol can transform acetylenic group into an ester of corresponding acid i) Other commonly used methods for modification of triple bonds
Various reactions are shown in Figure 1. According to the invention, a side chain (Ri) of the polyketide compound can be replaced by an alternative side chain (R2), according to the following scheme:
in connection with Table 2 below.
Table 2. Semi-synthetic possibilities arising from incorporation of propargyl group into polyketide compounds.
In one additional aspect this invention provides physiologically functional derivatives of Formula I (and all its semi-synthetic and biosynthetically engineered derivatives mentioned above) including physiologically acceptable esters, solvates and salts. Physiologically suitable esters include esters which are cleaved in the body for example hydroxyl groups esterified with carboxylic acids. Suitable solvates include hydrates. Examples of pharmaceutically acceptable salts are non-toxic acid addition salt forms of the compounds of Formula I. Acceptable acid addition salts can be readily obtained by adding the suitable acid to the base form of the compound. Appropriate acids can be chosen from a group comprising of organic acids, for example acetic, propanoic, hydroxyacetic, lactic, pyruvic oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, benzenesulfonic, ethanesulfonic p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids. Appropriate acids can also comprise inorganic acids, for example, nitric, sulphuric, phosphoric, hydrochloric, hydrobromic and similar acids. If desired, said acid addition salts can also be converted to free base forms by addition of appropriate bases.
Compounds of FK506 analogues of Formula I or their semi-synthetic derivatives or their esters or addition salts or solvates are useful as pharmaceuticals for example, but without limitation having potential effectiveness as immunosuppressants, antifungal agents, neuroregenerative agents, anticancer agents, antiinflammatory agents or agents useful for treatment of fibrosis, rheumatoid arthritis, psoriasis and other hypreproliferative diseases. In another aspect, the use of compounds of Formula I is provided in the preparation of a medicament for treatment and/or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, inflammatory disorders, cancer, fibrosis, rheumatoid arthritis, psoriasis and/or other hyperproliferative diseases, and neurodegenerative diseases. In a further aspect this invention provides a method for treatment of said medical conditions using said medicaments containing compounds of Formula I by administering said compounds of Formula I to a subject affected by said medical conditions. In one specific embodiment, this invention provides compounds of Formula I which can be used in preparation of a medicament for prevention of organ allograft rejection. In another embodiment compounds of Formula I can be used for preparation of a medicament for treatment of autoimmune and inflammatory disorders. Using routine experimentation, one skilled in the art would be able to determine the ability of compounds of Formula I to inhibit fungal or protozoan cell growth. Similarly using routine methods one skilled in the art would be able to determine whether said compounds can inhibit tumour cell growth. Additionally, compounds of Formula I are able to induce suppression of the subject's immune system. Routine assays to determine the ability of compounds to induce suppression of immune system are known to those skilled in the art. Suitable method can be selected depending on the particular medical condition, for example treatment or prevention of allograft organ rejection after transplantation, autoimmune, inflammatory, proliferative and hyperproliferative diseases. Examples of such conditions are, without limitation, rejection of transplanted organ, autoimmune diseases, asthma, tumours, diabetes type I, rheumatoid arthritis, eczema, psoriasis, fibrosis and allergies. Similarly, related to another aspect of this invention, one skilled in the art would be able by routine experimentation to determine the potential of compounds of Formula I as neuroregenerative agents, antiangiogenic agents and components of stents.
In yet another wider aspect this invention provides and, one skilled in the art would be able to determine, the ability of novel polyketides-derived compounds possessing diverse useful biological activities such as antibacterial activity from the group of macrolide or macrolactone compounds but not limited to the macrolides such as erythromycin, tylosin, midecamycin, or macrolactone such as rifamycin; anticancer and neuroregenerative activity from the group of macrolactone but not limited to such as rapamycin and meridamycin; from the group of polyene antibiotics/antifungals but not limited to such as nystatin, amphotericin, candicidin and other polyketide-derived or polyketide containing structure of medical and/or commercial importance produced by methods and processes described in this patent application.
Halogenated side chains It has been found that the above methods, which were described above with reference to triple-bond containing extender units and triple-bond containing side chains can equally be used with extender units comprising halogenated residues, hence producing halogenated side chains in the polyketide compounds of the invention. These halogenated side chains are also suitable precursors for producing a wide variety of novel polyketide compounds. The invention hence also relates to the any of the above compounds and methods, in which halogenated extender units are used instead of the above described triple-bond compounds.
In particular, it has been found that the FK506 AT4 domain not only accepts prop-2- ynylmalonyl-CoA, ethynylmalonyl-CoA, cyanomethylmalonyl-CoA as a substrate (see above), but the FK506 AT4 domain also accepts e.g. 2-chloroethylmalonyl-CoA, 2- fluoroethylmalonyl-CoA, 3-fluoropropylmalonyl-CoA, 2-fluoropropylmalonyl-CoA, 3- fluoroprop-2-enylmalonyl-CoA, 2-fluoroprop-2-enylmalonyl-CoA as a substrate.
Halogenated extender units and the resulting moieties in the FK506 polyketide, according to this aspect of the invention, are summarized in Table 3. It is to be understood that corresponding side chains can similarly be introduced in the erythromycin polyketide structures of the invention (e.g. Formula II, III, IV).
Table 3:
The invention thus further relates to the process for preparation of novel polyketides as described above, where the extender unit is selected from the group consisting of: 2-chloroethylmalonyl-Co A, 2-fluoroethylmalonyl-Co A, 3 -fluoropropylmalonyl-Co A, 2-fluoropropylmalonyl-Co A, 3 -fluoroprop-2-enylmalonyl-Co A, 2-fluoroprop-2- enylmalonyl-CoA.
When the extender unit is to be fed to the medium, then the single or double esters or thioesters of precursors thereof are preferably used, such as but not limited to SNAC, diethyl , thioglycolate version of the respective extender units is the preferred added substance, because they readily pass the cell membrane and are available for a more attractive price than the Co A esters.
Accordingly, the present invention also relates to polyketide compounds as described above, wherein the modified (non-natural) side chains are: 2-chloroethyl , 2-fluoroethyl, 3- fluoropropyl, 2-fluoropropyl, 3-fluoroprop-2-eny or 2-fluoroprop-2-enyl. The invention further relates to polyketide compounds of Formula I above, wherein R = 2-chloroethyl , 2-fluoroethyl, 3-fluoropropyl, 2-fluoropropyl, 3-fluoroprop-2-enyl or 2-fluoroprop-2-enyl. In another embodiment the invention relates to polyketide compounds of Formula II or III above, wherein R = 2-chloroethyl , 2-fluoroethyl, 3-fluoropropyl, 2-fluoropropyl, 3- fluoroprop-2-enyl or 2-fluoroprop-2-enyl. Hence, the invention also relates to 21-desallyl- 21 -prop-2-ynyl-FK506, 21 -desallyl-21 -ethynyl-FK506, 21 -desallyl-21 -cyanomethyl- FK506, 21 -desallyl-21 -(2-chloroethyl)-F 506, 21 -desallyl-21 -(2-fluoroethyl)-FK506, 21 - desallyl-21 -(3-fluoropropyl)-FK506, 21 -desallyl-21 -(2-fluoropropyl)-FK506, 21 -desallyl- 21 -(3-fluoroprop-2-enyl)-FK506, 21 -desallyl-21 -(2-fluoroprop-2-enyl)-FK506.
Halogenated side chains in polyketide compounds of the invention can further be chemically modified in a reaction
Chain with Ri and R2 being as defined in Table 4.
Table 4
(Those compounds and methods comprising/using side chains of Table 4 which are not halogenated shall also be regarded as an aspect of the present invention.)
Accordingly, one aspect of the invention relates to a polyketide compound comprising the structure:
wherein Rx is a halogenated C1-C6, preferably C1-C3, alkyl moiety, saturated or non- saturated. In a preferred embodiment Rx is one of the moieties shown as possible "side chains" in the second column of Table 3 and/or in the first column of Table 4.
The polyketide compound is preferably selected from the group consisting of macrolides, macrolactones, polyene antibiotics, polyether antibiotics synthesised by the type I polyketide synthase enzymes. In another embodiment the polyketide compound is obtainable from a natural polyketide compound, preferably selected from the group consisting of macrolides erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin; macrolactones rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone; polyene antibiotics such as emphotericin, nystatin, candicidin; polyether antibiotics monensin and salinomycin, by substitution in said natural polyketide compound of a structure: wherein X1 and X2 are naturally occurring substituents of the natural polyketide compound or H, and Chain are independently a part of the carbon backbone of the natural polyketide compound, respectively; by the structure:
Rx as defined above.
In one embodiment the polyketide compound has the structure:
; wherein
X3 is -H, -OH, or double bonded O, wherein, if X3 is double bonded O then Rio is nonexistent; Ri to R10 are individually selected from -H, -CH3, and Rx as defined above; wherein Rio can also be non-existent; and at least one of Ri to RJO is Rx as defined above.
In another embodiment the polyketide compound has the structure: ; wherein
X is -CH3 or -C2H5; Ri to R6 are individually selected from -H, -CH3 and Rx as defined above; and at least one of R\ to R$ is Rx as defined above.
Another embodiment relates to a chemically modified polyketide compound obtainable from the polyketide compound above, said chemically modified polyketide compound having the structure:
, wherein
X is -H, -OH, or double bonded O, wherein, if X is double bonded O then R20 is nonexistent;
Rn to R2o are independently selected from -H, -CH3 and R2, wherein R2 is defined as in column 3 of Table 4 (R and R\ being any organic moiety); wherein R2o can also be nonexistent; and at least one of R 1 to R20 is R2 as defined in column 3 of Table 4 (R and Ri being any organic moiety).
Another embodiment relates to a chemically modified polyketide compound obtainable from the polyketide compound defined above, said chemically modified polyketide compound having the structure:
wherein X is -CH3 or -C2H5;
Rn to R16 are independently selected from -H, -C¾ and R2 as defined in column 3 of Table 4 (R and R\ being any organic moiety); wherein at least one of Rn to Ri6 is R2 as defined in column 3 of Table 4 (R and Ri being any organic moiety).
One aspect of the invention relates to a method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a P S enzyme, said PKS enzyme comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2, or a sequence at least 70% identical to SEQ ID NO:l or SEQ ID NO:2 and accepting a compound of the structure: wherein Rx is a halogenated C1-C3 alkyl moiety, preferably any on the moieties shown as possible "side chains" in the second column of Table 3; X4 is selected from CoA and NAc; as a substrate; b) providing a substrate compound of said structure c) incubating said microorganism in a medium comprising said substrate compound; and d) obtaining said polyketide compound from said medium. In other embodiments, also bi-functional thioesters (e.g., bis-SNAC) is used can be used instead of the monoester - acid.
In one embodiment said microorganism comprises a PKS enzyme capable of producing a compound of the structure:
In one embodiment said microorganism is deficient in at least one enzyme selected from the group consisting of AHA, A11K, A11R and A11D, preferably in A11R, or in A11R only.
One aspect of the present invention also relates to a method of making a polyketide compound, said method comprising:
a) the method defined above; and
b) chemical modification of the product of said method defined above, so as to obtain a chemically modified polyketide compound.
In one embodiment said chemical modification is selected from nucleophilic substitution with NaCN, nucleophilic substitution with amines, substitution with potassium ethylxantogenate and subsequent hydrolysis, reaction with dimethylcianimido- dicarbonate, reaction with dimethyltio-2-nitroethen and alkylation.
Examples
Example 1 : Cultivation of Streptomyces tsukubaensis strains for production of analogues ofFK506 The ISP4 sporulation agar medium (soluble starch, 1%, K2HPO4, 0.1%, MgSC x 7H2O, 0.1%, NaCl, 0.1%, (NH4)2S04, 0.2%, CaCC-3, 0.2%, FeS04 * 7H20, 0.000001%, MnCl2 * 4 H20, 0.000001%, ZnS04 * 7 H20, 0.000001%, bacteriological agar 2%) was used for spore stock preparation. Streptomyces tsukubaensis strains were cultivated as a confluent lawn on the ISP4 agar medium for 8-14 days at 28 °C. For liquid cultures, spores of Streptomyces tsukubaensis strains (1% v/v) were inoculated in seed medium VG3 (soy meal, 0.25% (w/v), dextrin, 1%, glucose, 0.1%, yeast extract, 0.5%, casein hydrolysate, 0.7%, K2HPO4, 0.02%, NaCl, 0.05%, MnCh x 4H2O, 0.0005%, FeS04 x 7H2O, 0.0025%, ZnSC-4 x 7H2O, 0.0001%, MgS04 7H2O, 0.0005%, CaCh, 0.002%, pH=7.0) and incubated at 28°C and 250 rpm for 24-48 h. 10 % (v/v) of the above seed culture was used for the inoculation of a 250 ml Erlenmeyer flask containing 50 ml of production medium PG3 (dextrin 9%, glucose 0.5%, soy meal 1%, soy peptone 1%, glycerol 1%, L-lysine 0.25%, K2HPO40.1%, CaCOs 0.15%, PEG (1000) 0.1%, pH=6.5). Cultivation was carried out at 28 °C, 250 rpm for 6-7 days. Example 2: Cultivation of Saccharopolyspora erythraea strains for production of erythromycin analogues
The TWM sporulation agar medium (glucose 0.5%, sucrose 1%, tryptone 0.5%, yeast extract 0.25%, EDTA 0.0036% and bacteriological agar 2%, pH = 7.1) was used for spore stock preparation. Saccharopolyspora erythraea NRRL 2338 strains were cultivated as a confluent lawn on the TWM agar medium for 8-14 days at 30 °C. For liquid cultures, spores of Saccharopolyspora erythraea strains (1% v/v) were inoculated in seed medium TSB (Tryptone Soya Broth 3%, Oxoid CM1065) and incubated at 30°C and 220 rpm for 24-48 h. 10 % (v/v) of the above seed culture was used for the inoculation of a 250 ml Erlenmeyer flask containing 25 ml of production medium SM3 (glucose 0.5%, maltodextrin 5%, soya meal 2.5%, beet molasses 0.3%, K2HP04 0.025%, CaC03 0.25% and pH=7.0). Cultivation was carried out at 30 °C, 220 rpm for 6-7 days. 10 mM concentration of a precursor was used In the feeding experiments with unnatural extender units 10 mM concentration was used.
Example 3: Isolation of genomic DNA Spores of Streptomyces tsukubaensis NRRL 18488 strains were used to inoculate 50 ml of TSB medium (Kieser et al., 2000, Practical Streptomyces genetics, A laboratory Manual. ISBN-0-7084-0623-8) in a 250-ml Erlenmeyer flask, which was maintained with shaking (210 rpm) at 28 °C for 24 hours. Cultures were grown for 24 hours at 28 °C. Mycelium was recovered by centrifugation and genomic DNA was prepared using PureLink Genomic DNA Mini Kit (Invitrogen) according to the instructions of the kit manufacturer. DNA was resuspended in 100 μΐ TE buffer (Sambrook, and Russell, 2000, Molecular Cloning: A Laboratory Manual, ISBN-978-087969577-4).
Example 4: Transformation of Streptomyces tsukubaensis and Saccharopolyspora erythraea strains Plasmid constructs based on either pSET152 (Bierman, Logan et al. 1992) or pKC1139 (Bierman, Logan et al. 1992) were introduced by transformation into electrocompetent E. coli strain ET 12567 containing the conjugative plasmid pUZ8002 (Paget, Chamberlin et al. 1999). The plasmid pUZ8002, contains all the necessary genes for construction of conjugative pili, however it lacks the origin of transfer and, thus, remains in the host cell (Jones, Paget et al. 1997). Conjugation procedure was carried out as described in ieser et al., 2000 (Practical Streptomyces genetics, A laboratory Manual. ISBN0-7084-0623-8). Exconjugants were grown at 28°C on ISP4 sporulation medium which is described earlier, with addition of 50 μg/ml apramycin.
Example 5: Chemical synthesis of non-natural extender units
Synthesis of 2-chloroethyl dimethyl malonate
2.2 g of HC1 (g) is introduced into 23.4 g of 1 ,2-dimethoxy ethane. 6.8 g of cyclopropyl 1,1-dimetylcarboxylate and 23 mg of AICI3 is added and stirred 5 h at 50 °C. Rkc mixture is diluted with 100 ml of water and extracted with 70 ml of diethyl ether. Extract is washed with water (150 ml), dried with Na22S04 and evaporated under vacuum. Thus we obtained 7.47 g of crude product ( 93.4 %) which is pure enough for further steps ( GC : 97%).
Preparation of diethyl prop-2-yn-l-ylpropanedioate Product is prepared according to modified procedure from literature (by repeating procedures from literature we have obtained only mixture of products and starting material) (Eglinton and Whiting 1953). To 200 ml of abs. EtOH 11.8 g of sodium was slowly added under cooling (T< 15 °C). 80 g of diethyl malonate is slowly added under cooling and stirred for 1 h. In 1 h 53.2 g of 70% of propargyl chloride in toluene is added under cooling ( T< 15 °C ). Suspension is stirred for additional 17 h at room temp. About half of EtOH is evaporated under reduced pressure and 400 ml of water is added. Solution is extracted twice with 300 ml of MTBE, pooled extracts are washed twice with 300 ml of water, dried with Na2S04 and evaporated under reduced pressure. Resulted raw product (76.65 g) is purified by repeated fractional vacuum distillation. Fractions with purity >97% are pooled and used for next step. Yield : 34.75 g
Preparation of Prop-2-yn-l-ylpropanedioic acid (2)
6.15 g of Diethyl prop-2-yn-l-ylpropandioate is mixed with solution of 4.62 g of KOH in 5.2 ml of water under ice cooling. After stirring overnight a suspension is acidified with 20% HCL to pH=l-2, 20 ml of water is added and extracted twice with 60 ml of diethyl ether. Combined extracts are washed twice with 30 ml of water, dried with Na22S04 and evaporated under vacuum. Thus we obtained 2.37 g of (2) in the form of amorphous solid. Product was not purified and was used directly in next step.
Preparation of Prop-2-yn-l-ylpropanedioyl dichloride (3)
2.0 g of (2) was suspended in 23.5 ml of dichloromethane and 150 μΐ of DMF. Within 1 h 5.22 g of oxallyl chloride was added under ice cooling. Solution is stirred over night under room temperature. Volatiles were evaporated under reduced pressure and remaining acid chloride (2.3 g) was used without further purification.
Preparation of Sl,S3-bis(acetylamino)ethyl) diprop-2-yn-l-ylpropanebis (thioate) (4)
2.3 g of (3) is dissolved in 55 ml of THF, cooled on ice and 3,04 g of N- Acetyl cysteamine is added within 15 min. 5 ml of TEA is slowly added and suspension is stirred for additional 2 h at temp. 0 °C . Solvent is evaporated under reduced pressure, 80 ml of water is added and extracted twice with 120 ml of dichloromethane. Extracts were washed twice with 80 ml of water, dried with Na2SC<4 and evaporated to dryness. Raw product (1.6 g) was purified by column chromatography (silica, MF : chloroform/MeOH = 88:12). Pure fractions were pooled and evaporated to dryness. Thus we obtained 0.53 g of (4) as a yellow-brown oil. LCMS : 344 ( M+H)
Preparation of methyl [(2-[[(2-methoxy-2-oxoethyl)sulfanyl]carbonyl]pent-4- enoyl)sulfanyl]acetate
0.722 g of prop-2-en-l-ylpropanedioic acid is suspended in 10 ml of dichloromethane, 52 μΐ of DMF is added and then 1 ,982 g of oxallyl chloride under ice cooling. Mixture is stirring at room temp, overnight, evaporated under reduced pressure and without purifying used in next step.
To acid chloride from previous step 22 ml of THF is added and under ice cooling 1.06 g of methyl thioglykolate is added followed by 1.5 ml of TEA. After 2 h of at room temp, it is evaporated under reduced pressure, 60 ml of water is added and extracted twice with 50 ml of dichloromethane. Pooled extracts were washed with water (2x 50 ml), dried with sodium sulphate and evaporated under reduced pressure. Yellow oily residue (1.01 g) is pure enough to be used as non-natural extender unit. LCMS : 321 (M+H).
Example 6. Feeding of diverse non-natural extender units to the S. tsukubaensis strain with inactivated allR gene and analysis of the produced FK506 analogues
In our experiments, spores of a Streptomyces tsukubaensis strain NRRL18488in which the allR gene encoding crotonyl-CoA reductase/carboxylase (Goranovic, Kosec et al. 2010), were inoculated in the seed medium VG3 (1% (v/v)) and cultivated for 24 to 48 hours as described earlier. The production medium PG3 was inoculated with the seed medium (10% (v/v)). After 72 hours 10 ml of production phase culture broth was transferred to 250 ml Erlenmeyer flasks containing 20 ml of fresh production medium and 0.15 ml of 10% (w/v) DMSO solution of a non-natural extender unit analogue were added. The culture was incubated for further 6 days as described in the Materials and methods section.
After cultivation was completed, the broth was extracted with the equal volume of methanol (1 :1). The identity of the produced F 506 analogues in the fed cultivation broths was determined by LC-MS/MS analysis. We used the Agilent 1100 series LC-MS system coupled with Waters Micromass Quattro micro detector using reversed phase column (Gemini CI 8 column, 5 μηι, 150 mm χ 2 mm i.d.) from Phenomenex. The separation was performed at a flow rate of 0.250 ml/min by gradient elution with 0.5 % TFA as solvent A and acetonitrile as solvent B. The gradient program was: 60 % A, 0 min; 60-20 % A, 0-17 min; 20-60 % A, 17-18 min; 60 % A, 18-30 min and the injection volume 10 μΐ at temperature of the column 45 °C was used. The mass selective detector (Waters, Quattro micro API) was equipped with an electrospray ionisation using a cone voltage of 20 V and capillary voltage of 3.5 kV for positive ionization of the analytes. Dry nitrogen was heated to 350 °C, the drying gas flow was 400 1/h and collision energy was 20 eV. When the presence of the original FK506 compound is detected using an ESI+ positive mode, an ion of m/z = 826.5 that corresponds to a capture of a sodium ion ([M+Na]+ is most intensive in accordance with the results of other investigators. For FK506 identity confirmation, multiple reaction-monitoring mode can be used and the transition FK506 m/z 826.5 [M+Na]+→m/z 616.4 can be recorded. In order to determine the presence of FK506 analogues we used a similar approach, however, the molecular masses of both ions in multiple reaction-monitoring transition were adapted according to the predicted molecular mass of each analogue (Table 5).
Table 5: LC-MS/MS data confirming the presence of novel FK506 analogues in fed fermentation broths.
Example 7: Replacement of the AT domain in the first extender module of DEBS 1 with the AT4 domain of the FK506 PKS
The starting point for construction of chimeric gene (DEBS1 + AT4) was the DEBS 1 gene of erythromycin PKS encoding 3 polyketide synthase modules involved in incorporation of the starter unit and two extender units into the erythromycin polyketide chain. We realized that the target ATI domain of DEBS 1 is flanked by BstBI and BpulOI restriction sites. Therefore, our strategy was to exchange the BpulOI-BstBI fragment with a new fragment in which the native AT domain had been replaced by the AT domain of the fourth module of FK506 PKS (AT4). The new hybrid BpulOI-BstBI fragment was constructed from 3 PCR products. The first product was amplified using DEBSSwpFl and DEBSSwpRl oligonucleotide primers and Saccharopolyspora erythraea NRRL2338 genomic DNA as template. Another product was amplified using the same template and oligonucleotide primers DEBSSwpF2 and DEBSSwpR3. The two products were cloned together into pUC19 plasmid and joined with their Pstl sites. The plasmid containing both fragments was then opened using Xhol and Avrll restriction enzymes and the third fragment containing the AT domain of the module 4 of the FK506 PKS was inserted. This fragment was obtained by PCR amplification. In the first step, specific primers AT4TemplFl and AT4TemplRl were used to specifically amplify the region encoding the AT domain of the module 4 of FK506 PKS from genomic DNA of Streptomyces tsukubaensis NRRL 18488 as template. In the second step, the PCR product of the first reaction was used as template and oligonucleotide primers AT4SwpFl and AT4SwpRl were used to amplify the fragment which was cloned in-frame into the plasmid containing "DEBSSwpFl-DEBSSwpRl" and "DEBSSwpF2- DEBSSwpR3" fragments using Xhol and Avrll restriction sites. In this way, a hybrid gene termed "DEBS-AT4" was obtained. Introduction of the Xhol site in the region just upstream of the conserved GQG motif of AT domains caused a replacement of a threonine residue to a serine residue. On the other hand, the Avrll restriction site at the carboxy terminus of the AT domain did not require any change in the predicted amino acid sequence. Alternatively, Mscl restriction site was used instead of the Xhol site at the amino terminus of the AT domain. In this case DEBSSwpR2 oligonucleotide primer was used instead of DEBSSwpRl and AT4SwpF2 was used instead of AT4SwpFl. The primers used in this procedure are given in Table 6.
Table 6. Oligonucleotide primers used for construction of the new BpulOI-BstBI fragment which was inserted in the DEBS1 gene.
AT4SwpF2 ATGGCCAAGGCTCCCAGTGGCTCGGCATGG Mscl (SEQ ID NO:9)
AT4SwpRl ACCTAGGACGACGGTCGGCCAGTCGACGGCC Avrll (SEQ ID NO: 10)
AT4TemplFl CGCGTACCGTCGCCGCCGCGCTGTTCTC /
(SEQ ID NO: 11)
AT4TemplRl CGGTGGCGTCCGTACCGGATGACGGTTC /
(SEQ ID NO: 12)
PCR amplification of "DEBSSwpFl-DEBSSwpRl" DNA fragment: Isolated plasmid DNA of pCJR65 obtained by standard procedures was PCR amplified using a Promega Thermal Cycler. The PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 μΜ dNTP, 3% DMSO, 0.5 μΜ of each primer (i.e. DEBSSwpFland DEBSSwpRl), approximately 50 ng of template plasmid DNA and 2.5 units of enzyme in a final volume of 50 μΐ for 30 cycles. The thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 64°C for 20 sec (annealing step), and 72°C for 35 s (extension step). The PCR-amplified product was cloned into a pUC19 cloning vector. The sequence analysis of the cloned PCR product confirmed its respective partial DEBS1-TE sequence. Identical procedure was used for amplification of the "DEBSSwpF2-DEBSSwpR3" fragment where oligonucleotides DEBSSwpF2 and DEBSSwpR3 were used and "DEBSSwpFl-DEBSSwpR2" where oligonucleotides DEBSSwpFl and DEBSSwpR2 were used.
As explained above, two amplification steps were necessary to obtain the fragment encoding the AT4 domain:
PCR amplification of the template "AT4TemplF 1 -AT4TemplRl " DNA fragment: S. tsukubaensis genomic DNA obtained by the procedure described above was PCR amplified using a Biometra Thermal Cycler. The PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 μΜ dNTP, 3% DMSO, 0.5 μΜ of each primer (i.e. AT4TemplFl and AT4TemplRl), approximately 50 ng of template plasmid DNA and 2.5 units of enzyme in a final volume of 50 μΐ for 30 cycles. The thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 67°C for 20 sec (annealing step), and 72°C for 37 s (extension step). PCR amplification of the template "AT4SwpFl-AT4SwpRl" DNA fragment: PCR- product "AT4TemplFl-AT4TemplRl" obtained by the procedure described above was PCR amplified using a Promega Thermal Cycler. The PCR reaction was carried out with Phusion polymerase (Finnzymes) and the buffer provided by the manufacturer in the presence of 200 μΜ dNTP, 3% DMSO, 0.5 μΜ of each primer (i.e. AT4SwpFl and AT4SwpRl), 1.5 μΐ of said PCR reaction mixture and 2.5 units of enzyme in a final volume of 50 μΐ for 30 cycles. The thermal profile of all 30 cycles was 98°C for 15 sec (denaturation step), 68°C for 20 sec (annealing step), and 72°C for 37 s (extension step). The PCR-amplified product was cloned into a pUC19 cloning vector. The sequence analysis of the cloned PCR product confirmed its respective sequence of the AT4 domain.
Once the chimeric fragment "DEBSl-AT4"of the was assembled it was excised from the pUC19-based plasmid and cloned into the pKCl 139 based plasmid (Bierman, Logan et al. 1992), which enables the incorporation of the "DEBS-AT4" fragment into genomes of diverse Streptomyces species encoding phage OC31 integration sites in their genomes.
Example 8: Replacement of the AT domain in the extender module 4 of erythromycin PKS (DEBS2) with the AT4 domain of the FK506 PKS
The replacement of the AT domain was carried out similarly to the procedure analogous to the one described in Example 4 The Mscl and Avrll domain splice sites were chosen in the equivalent positions in erythromycin-AT4 and FK506-AT4 as described by Oliynyk et al. (1996). To construct the replacement cassette for AT4 of DEBS2 the fragments corresponding to 1.2 kb flanking region upstream of the engineered Mscl site and 0.5 kb flanking region downstream of the Avrll site were obtained by PCR amplification from S. erythraea genomic DNA and joined together in the pUC19 based vector. The oligonucleotide primers used to amplify the Mscl-upstream region were DEBSUpRev 5'- TTTTTCTGCAGCGCCCTGGCCAGGGAAGACCAGGACCG-3, (SEQ ID NO: 13) and DEBSUpFwd S'-TTTTTAAGCTTCCTGCGAGGCACCGACACCGGCG-S' (SEQ ID NO: 14), the former introducing the Mscl site plus a Pstl site located just before the Mscl site, and the latter introducing a Hindlll site and priming across a Sfil site. The amplified product was digested with Pstl and Hindlll and ligated into pUC19 that had been digested with Pstl and Hindlll. The oligonucleotide primers used to amplify the Avrll-downstream region, were DEBSDnRev 5'-TTTTTGAATTCCGTCCTCCGGCGGCCACTGCTCGG-3' (SEQ ID NO: 15) and DEBSDnFwd 5'- TTTTTCTGCAGCCTAGGGGGACGGCCGGCCGAGCTGCCCACC-3' (SEQ ID NO: 16), the former introducing an EcoRI site and the latter introducing an Avrll site plus a Pstl site located just after the Avrll site. Both fragments were combined in pUC19 vector using the Pstl restriction site. The fragment corresponding to the FK506-AT4 domain was also amplified by PCR as described in detail in Example 4. In the first step, specific primers AT4TemplFl and AT4TemplRl were used to specifically amplify the region encoding the AT domain of the module 4 of FK506 PKS from genomic DNA of Streptomyces tsuk baensis NRRL 18488 as template. In the next step, specific primers AT4SwpF2 and AT4SwpRl were used where AT4SwpF2 introduced the engineered Mscl restriction site and AT4SwpR2 included the Avrll site. The PCR product was cloned into pUC19 and subsequently excised using Mscl and Avrll. This fragment was then inserted into the pUC19 based vector, containing the flanking regions of erythromycin- AT4, also previously digested by Mscl and Avrll, whereby "DEBS2-AT4" fragment was obtained. The primers used in this procedure are given in Table 6. The chimeric fragment described above was then excised and inserted into the p C1139 plasmid which was used for transformation of Saccharopolyspora erythraea NRRL2338 by conjugation from E. coli as described.
Example 9: Production of erythromycin analogues with incorporated non-natural extender units in Saccharopolyspora erythraea
The "DEBS1-AT4" "DEBS2-AT4" hybrid gene fragments were cloned into the pKCl 139 plasmid resulting in plasmids pKCl 139-DEBS1-AT4 and pKC1139-DEBS2-AT4. Vector pKCl 139 contains a normal pUC19-based Ori for replication in E. coli, but a temperature- sensitive Ori for replication in Streptomyces, which is unable to function at elevated temperatures above 34°C (Bierman, Logan et al. 1992). This plasmid was inserted into S. erythraea genome using the procedure described above. Later on, secondary recombinant strains were obtained in which the native eryAI gene had the AT domain of its module 1 replaced by the AT4 domain of FK506 PKS or alternatively eryA2 gene had the AT domain of erythromycin PKS module 4 replaced by the AT4 domain of FK506 PKS. Extraction of erythromycin analogues
After cultivation was completed, the pH of broth was adjusted to 10 -10.5 and then mixed with the equal volume of acetonitrile (1:1). Mixture of broth and acetonitrile was agitated for 30-60 min on shaker and than 10% Of NaCl was added. The mixture was agitated slowly till NaCl dissolved, after that mixture separated immediately into acetonitrile- and water-rich phase. The content of erythromycin analogues was determined in acetonitrile- rich phase.
LC-MS/MS analysis of erythromycin analogues
The identity of the produced erythromycin analogues in the fed cultivation broths was determined by LC-MS MS analysis. We used the Agilent 1100 series LC-MS system coupled with Waiters Micromass Quattro micro detector using reversed phase column (Gemini CI 8 column, 5 um, 150 mm x 2 mm i.d.) from Phenomenex. The separation was performed at a flow rate of 0.250 ml/min by gradient elution with 20 mM ammonium acetate buffer as solvent A and acetonitrile as solvent B. The gradient program was: 80 % A, 0 min; 60-20 % A, 0-17 min; 30-70 % A, 17-18 min; 80 % A, 18-30 min and the injection volume 10 μΐ at temperature of the column 45 °C was used. The mass selective detector (Waters, Quattro micro API) was equipped with an electrospray ionisation using a cone voltage of 30 V and capillary voltage of 3 kV for positive ionization of the analytes. Dry nitrogen was heated to 350 °C, the drying gas flow was 400 1/h and collision energy was 30 eV. When the presence of the original erythromycin A compound is detected using an ESI+ positive mode, an ion of m/z = 734.65. For erythromycin A identity confirmation, multiple reaction-monitoring mode can be used and the transition erythromycin A m/z 734.65 [M]+→m/z 158.18 can be recorded. In order to determine the presence of erythromycin analogues we used a similar approach, however, the molecular masses of both ions in multiple reaction-monitoring transition were adapted according to the predicted molecular mass of each analogue (Table 7).
Table 7: LC-MS/MS data confirming the presence of novel erythromycin analogues in fed fermentation broths.
Example 10: Chemical derivatization of the obtained polvketides
PrgFK506 ketone
802 mg ( 1 mM) of Propargyl FK506 is added to 740 mg of HgS04 in 5 ml of 2% H2S04 and stirred 1 h at 50 °C. Slurry is cooled to RT, neutralized and extracted 3 times with EtOAc. Extracts were pooled, washed with water and evaporated. Residue is purified by column chromatography (Silica, EtOAc : n-Hexane). Thus we obtained 180 mg of pure titled compound.
Propinylmethyl or 17-(2-butinyl) FK506 or C21 -(2-butinyl)
To 802 mg (ImM) of propargyl FK506 in 15 ml of THF is added 45 mg of NaNH2 at -10 °C. 170 mg of MeJ in 5 ml of THF is slowly added and stirred 2 h at -10 °C and than 1 h at room temp. H20 ( 50 ml) is slowly added under cooling and extracted with 40 ml of diethyl ether/EtOAc/MeOH ( 4:4:2, 3 times). Extracts are washed with water, dried with Na2S04 and evaporated. Residue is purified by column chromatography (Silica, EtOAc : n-Hexane). Thus we obtained 120 mg of pure titled compound. Example 11 : Chemical synthesis of ethynylmalonate-SNAC
Synthesis of dipropyl 2-ethynyl malonate
To t-butanol (50 mL) 1.15 g of sodium (50 mmol) was added. The mixture was stirred at room temperature for 3 h and then 1.85 mL of dipropyl malonate (10.0 mmol, 1 equiv.) was added. The atmosphere above the mixture was flushed with argon and then 3.16 g of (ethynyl(phenyl)iodonium tetrafluoroborate (10.0 mmol, 1 equiv.) was added. The mixture was heated to gentle reflux for 12 h and then 250 mL of brine was added. The organic products were extracted with 3x200 mL of dichlorometane, the organic phase was additionally washed with water (300 mL), dried with Na2S04, filtered, and evaporated to give a reddish-brown oil. Resulted raw product was purified by repeated fractional vacuum distillation through micro Vigreux column. Fractions with purity >95% are pooled and used for next step. Yield : 680 mg. Briefly, sodium is added to t-butanol and optionally stirred. Thereafter, dipropyl malonate is added to the mixture. (Ethynyl(phenyl)iodonium tetrafluoroborate is added, preferably under argon atmosphere. The mixture is allowed to react under reflux. Optionally brine solution is added. An extraction step may be performed, preferably with dichlorometane. The organic phase may be washed with water, and dried (e.g., with Na2S04). A purification step may be performed, e.g. by distillation.
Preparation of ethynylpropanedioic acid
424 mg (2 mM) of dipropyl ethynylpropanedioate was dissolved in 5 ml of EtOH, followed 25 ml of water and 8 ml of cone. HC1 is added and stirred 3 h under reflux. After evaporation of EtOH 20 ml of water is added and extracted with ethyl acetate (3x35 ml). After washing with saturated NaCl (2x 15 ml) and drying with Na2S04 solution is evaporated under vacuum. Thus we obtained 95 mg (60,9 %) of ethynylpropanedioic acid as white crystals.
Briefly, ethynylpropanedioic acid is prepared from dipropyl ethynylpropanedioate by dissolving the dipropyl ethynylpropanedioate in EtOH; adding hydrochloric acid (preferably concentrated HC1), and optionally water; performing an evaporation step to remove EtOH, and extracting with ethyl acetate. Optionally, the resulting product is washed, e.g., with saturated NaCl solution, and dried.
Preparation of ethynylpropanedioyl dichloride
155 mg of ethynylpropanedioic acid was suspended in 5 ml of dichloromethane and 15 μΐ of DMF. Within 1 h 510 mg of oxallyl chloride was added under ice cooling. Solution is stirred over night at room temperature. Volatiles were evaporated under reduced pressure and remaining acid chloride (210 mg) was used without further purification.
Briefly, ethynylpropanedioic acid is suspended in dichloromethane and DMF. Oxallyl chloride is added at low temperature (<5°C) and the mixture is allowed to react at room temperature (i.e., 15-30°C). Optionally, an evaporation step is performed, e.g., under reduced pressure (e.g., P < 0.8 bar). The ethynylpropanedioic acid is obtained.
Preparation of S1,S3-bis[2-(acetylamino)ethyl] ethynylpropanebis(thioate)
190 mg of ethynylpropanedioyl dichloride (1,15 mM) is dissolved in 7 ml of THF, cooled on ice and 274 mg of N- Acetyl cysteamine is added within 15 min. 0,5 ml of TEA is slowly added and suspension is stirred for additional 2 h at temp. 0 °C . Solvent is evaporated under reduced pressure, 20 ml of water is added and extracted twice with 30 ml of dichloromethane. Extracts were washed twice with 30 ml of water, dried with Na2S04 and evaporated to dryness. Raw product (304 mg) was purified by column chromatography (silica, MF : chloroform/MeOH = 90:10). Pure fractions were pooled and evaporated to dryness. Thus we obtained 180 mg of product as a yellow-brown oil. LCMS : 331 ( M+H).
Briefly, ethynylpropanedioyl dichloride is dissolved in THF, cooled on ice (i.e., T < 5°C) and N-Acetyl cysteamine is added. TEA is added and the resulting suspension is preferably allowed to react for at least 30 min at a low temperature (T < 5°C). Optionally, an evaporation step is performed, e.g., under reduced pressure (P < 0.8 bar). Optionally, water is added. The product is extracted from the aqueous solution in an organic solvent, e.g., dichloromethane. Optionally, washing steps can be performed and the product can be dired.
Example 12: BLAST analysis of various AT4 domains
A BLAST search was run against the AT4 domain of the polyketide synthase of Streptomyces tsukubaensis (SEQ ID NO:l). The results are summarized in Table 8, below.
Table 8
Example 13
A BLAST search was run against the AT4 domain of the polyketide synthase of Streptomyces sp. MA6548 (SEQ ID NO:2). The results are summarized in Table 9, below.
Table 9
kanamyceticus
Streptomyces sp. FK506 87 SEQ ID NO:l KCTC 11604BP
Streptomyces sp. FK506 87
MJM7001
Streptomyces FK520 82 accepts
hygroscopicus subsp. ethylmalonyl-CoA ascomyceticus and methylmalonyl- CoA
Streptomyces rapamycin 56
hygroscopicus
References
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Cortes, J., K. E. Wiesmann, et al. (1995). "Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage." Science 268(5216): 1487-9.
Demjanov, N. (1939). Chem Zentralblatt(II): 2913.
Dutton, C. J., S. P. Gibson, et al. (1991). "Novel avermectins produced by mutational biosynthesis." J Antibiot (Tokyo) 44(3): 357-65.
Eglinton, G. and M. C. Whiting (1953). Researches on Acetylenic Compounds. Part XL. The Synthesis of Some w-Acetylenic acids: 3052 - 3059.
Emmert, E. A., A. K. Klimowicz, et al. (2004). "Genetics of zwittermicin a production by Bacillus cereus." Appl Environ Microbiol 70(1): 104-13. Goranovic, D., G. osec, et al. (2010). "Origin of the Allyl group in FK506 biosynthesis." J Biol Chem 285: 14292 - 14300.
Katz, L., D. L. Stassi, et al. (2000). Polyketide derivatives and recombinant methods for making same. USA. U. S. P. a. T. Office.
Kuhstoss, S., M. Huber, et al. (1996). "Production of a novel polyketide through the construction of a hybrid polyketide synthase." Gene 183(1-2): 231 -6.
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Mo et al. (2010) "Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds though a dedicated polyketide synthase and facilitates the mutasynthesis of analogues", J. Am. Chem. Soc, published online: http://pubs.acs.org/JACS
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Ruan, X., A. Pereda, et al. (1997). "Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives." J Bacteriol 179(20): 6416-25.
Stassi, D. L., S. J. Kakavas, et al. (1998). "Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering." Proc Natl Acad Sci U S A 95(13): 7305-9.

Claims

Claims:
1. Use of a polyketide synthase comprising a sequence set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or comprising a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17 for adding an extender unit having the structure
COOH ; wherein p is 0, 1 or 2, preferably p = 1 ; and
X4 is any organic residue, preferably X4 is selected from NAc, CoA
to a nascent polyketide compound.
2. Use of claim 1, wherein the polypeptide encoded by said sequence has acyl transferase (AT) activity.
3. Use of claim 1 or 2, wherein said nascent polyketide compound is a nascent macrolide antibiotic, a nascent macrolactone antibiotic, a nascent polyene antibiotic, a nascent polyether antibiotic, or a nascent acetogenin.
4. Use of claim 1 or 2, wherein said nascent polyketide compound is selected from the group consisting of: nascent erythromycin A, nascent pikromycin, nascent oleandromycin, nascent tylosin, nascent medicamycin, nascent rifamycin, nascent avermectin, nascent spinosyn, nascent rapamycin, nascent FK506, nascent meridamycin, nascent geldanamycin, nascent epothilone, nascent emphotericin, nascent nystatin, nascent candicidin, nascent monensin and nascent salinomycin.
5. A method of making a polyketide compound, said method comprising: a) providing a microorganism functionally expressing a polyketide synthase, said polyketide synthase comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90% or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; providing a substrate compound having the structure
C00H , wherein p is 0, 1 or 2, preferably p = 1 ; and ganic residue, preferably X4 is selected from NAc, CoA
, and } more preferably X4 is selected from CoA and NAc; c) incubating said microorganism in a medium comprising said substrate compound, whereby a polyketide compound is produced; and d) obtaining said polyketide compound from said medium.
6. Method of claim 5, wherein said polyketide compound comprises at least one structure
[Structure I]; wherein Y is -H or -OH; n is 1 or 0; p is 0, 1 or 2, preferably p is 1; C* is a carbon atom; and Chainl-C*-Chain2 is the carbon backbone of said polyketide compound.
7. Method of claim 6, wherein said Structure I is , wherein
Rs is any organic or inorganic residue, preferably a residue selected from the list consisting of CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3
C6H5NH2 (paraaminophi
^^^COOH
R1 is independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH2CH2(C=0)CH3, CH2CH2C1, CH2CH2F, OCH3, OH and NH2; preferably from a lists consisting of H, CH3, CH2CH3 and OCH3;
Rk is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates, monosaccharides, disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups; Ra and Rb are each independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, and CH2CH2CH3; wherein Ra and Rb can also be connected so as to form a 5-, 6- or 7-membered ring, preferably a pyrrolidine or piperidine ring;
-Y is -H, -OH, -CH3, preferably -H or -OH, most preferred -H; n is 0 or 1 ; p = 0, 1 or 2, preferably 1 ; q is an integer from 0 to 10, preferably from 0 to 5; r is an integer from 0 to 20; s is an integer from 0 to 20; wherein r + s is from 0 to 20, inclusive; t is an integer from 0 to 5; preferably t is 1 ; denotes a single bond, a double bond, or an epoxide group between adjacent carbon atoms; wherein any two residues selected from R1, Rk, Ra and Rb can also be connected so as to form a ring.
8. Method of claim 6 or 7, wherein said polyketide compound is derived from a reference polyketide compound of the structure:
X1 X2
\ /
by substitution of a structure » / in said reference polyketide compound, wherein C* is as defined above, and X and X are independently the naturally occurring substituents in said reference polyketide compound at position C*, or H, or non-existent;
with the structure , wherein C*, Y, n and p are as defined above.
9. Method of any one of claims 5 to 8, wherein said microorganism is deficient in at least one enzyme involved in the biosynthesis of allylmalonyl-CoA or ethylmalonyl-CoA.
10. Method of claim 9, wherein said at least one enzyme involved in the biosynthesis of allylmalonyl-CoA or ethylmalonyl-CoA is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, or from the group consisting of sequences at least 70%, 80%, 90% or 95% identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
11. A method of making a chemically modified polyketide compound, said method comprising:
(i) producing a first polyketide compound using a method of any one of claims 5 to
10; and
(ii) chemically modifying said first polyketide compound, thereby obtaining said chemically modified polyketide compound.
12. Method of claim 11, wherein said chemical modification is selected from the group consisting of: oxidation by KMn04, addition of water, alkylation, formation of a halomagnesium compound, halogenation, addition of borane including subsequent oxidation with hydrogen peroxide, C-C coupling with substituted alkenes, and oxytallation with Tl3+ salts, and combinations thereof.
13. A polyketide compound comprising the structure:
;
Y is -H or -OH; n is 1 or 0; p is 0, 1 or 2; and
C* is a carbon atom; and Chainl -C*-Chain2 is the carbon backbone of said polyketide compound.
14. A polyketide of claim 13, wherein said Structure I is , wherein
Rs is any organic or inorganic residue, preferably a residue selected from the list consisting of CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3
C6H5NH2 (paraaminophenyl),
^^^COOH
R1 is independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, CH2CH2CH3, CH2CHCH2, CH2CCH, CH2CH2(C=0)CH3, CH2CH2C1, CH2CH2F, OCH3, OH and NH2, preferably selected from a lists consisting of H, CH3, CH2CH3 and OCH3;
Rk is independently at each occurrence a residue selected from the list consisting of double bonded oxygen, OH, H, carbohydrates such as monosaccharides and disaccharides, and modified carbohydrates comprising double bonds, primary amine groups, secondary amine groups or methoxy groups; Ra and Rb are each independently at each occurrence a residue selected from the list consisting of H, CH3, CH2CH3, and CH2CH2CH3; wherein Ra and Rb can also be connected so as to form a 5-, 6- or 7-membered ring, such as a pyrrolidine or piperidine ring;
-Y is -H, -OH, -CH3, preferably -H or -OH, most preferred -H; n is 0 or 1 ; p = 0, 1 or 2, preferably 1 ; q is an integer from 0 to 10, preferably from 0 to 5; r is an integer from 0 to 20; s is an integer from 0 to 20; wherein r + s is from 0 to 20, inclusive; t is an integer from 0 to 5; t is an integer from 0 to 5; preferably t is 1 ; denotes a single bond, a double bond, or an epoxide group between adjacent carbon atoms; wherein any two residues selected from R1, Rk, Ra and Rb can also be connected so as to form a ring.
15. The polyketide compound of claim 13 or 14, wherein the polyketide compound is a macrolide antibiotic, a macrolactone antibiotic, a polyene antibiotic, a polyether antibiotic, or an acetogenin.
16. The polyketide compound of any one of claims 13 to 15, wherein the structure of said polyketide compound is derivable from the structure of a reference polyketide compound, said reference polyketide compound being selected from the group consisting of: macrolides, erythromycin A, pikromycin, oleandromycin, tylosin, medicamycin, macrolactones, rifamycin, avermectin, spinosyn, rapamycin, FK506, meridamycin, geldanamycin, epothilone, polyene antibiotics, emphotericin, nystatin, candicidin, polyether antibiotics, monensin and salinomycin;
X1 X2
\ /
by substitution of a structure * / in said reference polyketide compound, wherein C* is as defined above, X1 and X2 are independently the naturally occurring substituents in said reference polyketide compound at position C*, or H, or non-existent;
with the structure , wherein C*, Y, n and p are as defined above.
17. The polyketide compound of any one of claims 13 to 16, wherein said polyketide compound has the structure:
; wherein
-X is -H, -OH, or double bonded O, wherein, if X is double bonded O then R10 is non-existent;
-Ri to -Rio are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH, wherein Rio can also be non-existent; and at least one of -Ri to -Rio is selected from -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH.
18. A chemically modified polyketide compound, said chemically modified polyketide compound having the structure:
, wherein
-X3 is -H, -OH, or double bonded O, wherein, if -X3 is double bonded O then R2o is non-existent;
-Ri i to -R20 are independently selected from: -H, -CH3, -CH2-CH2-COOH, - CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, - CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21, -CH2-CHR21-COO-R22, wherein R20 can also be nonexistent, Hal is selected from F, CI, Br and I,
R21, R22 are any organic moiety; and
at least one of -Ru to -R20 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, - CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R2i; -CH2-CHR2,-COO-R22.
19. The polyketide compound of any one of claims 13 to 16, wherein said polyketide compound has the structure:
; wherein
-X is -CH3 or -C2H5;
-Ri to -Re are individually selected from -H, -CH3, -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH; and at least one of -Ri to -R6 is selected from -C≡CH, -CH2-C≡CH, and -CH2-CH2-C≡CH.
20. A chemically modified polyketide compound, said chemically modified polyketide compound having the structure:
wherein -X is -CH3 or -C2H5; -Rii to -Ri6 are independently selected from -H, -CH3, -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R2i, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R21;
-CH2-CHR2i-COO-R22; wherein Hal is selected from F, CI, Br and I; and R21, R22 are any organic moiety; and at least one of -Rn to -Ri6 is selected from -CH2-CH2-CO-OH, -CH2-CH=CH-OH, -CH2-C≡C-R21, -CH2-C≡C-MgBr, -CH2-CH=CH-Hal, -CH2-CHal=CH-Hal, -CH2-CH2-CHO, -CH2-CH=CH-CH=CH-R2i, and -CH2-CHR2i-COO-R22.
21. A recombinant polyketide synthase comprising a sequence set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90%, or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17, wherein the recombinant polyketide synthase is not a FK506 polyketide synthase.
22. The recombinant polyketide synthase of claim 21, wherein said recombinant polyketide synthase is an FkbB protein of a polyketide synthase protein complex.
23. The recombinant polyketide synthase of claim 21 or 22, wherein said FK506 polyketide synthase is an enzyme having the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or a sequence at least 70%, 80%, 90%, or 95% identical to any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
24. A recombinant polyketide synthase, said recombinant polyketide synthase having multiple functional modules, each functional module comprising at least one keto- synthase (KS) domain, one acyltransferase (AT) domain, and one acyl carrier protein (ACP) domain, wherein at least one of said modules comprises the sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2 or SEQ ID NO: 17; or a sequence at least 85%, 90%, or 95% identical to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO: 17, wherein said at least one of said modules is not the fourth module of a FK506 polyketide synthase.
25. The recombinant polyketide synthase of claim 24, wherein said FK506 polyketide synthase is an enzyme having the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or a sequence at least 70%, 80%, 90%, or 95% identical to any one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
* * * * *
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