WO2006111561A1

WO2006111561A1 - Improved microbial production of anthracyclins

Info

Publication number: WO2006111561A1
Application number: PCT/EP2006/061708
Authority: WO
Inventors: Van Den Marco Alexander Berg
Original assignee: Dsm Ip Assets B.V.
Priority date: 2005-04-21
Filing date: 2006-04-20
Publication date: 2006-10-26

Abstract

The present invention provides a microbial strain that produces at least 0.1 g.l-1 culture broth of epidaunorubicin and/or epirubicin. The strain is preferably obtained from the genus Streptomyces. Furthermore, the present invention provides a method for the production of epidaunorubicin and/or epirubicin comprising fermentation of the abovementioned microbial strain under conditions conducive to the production of epidaunorubicin and/or epirubicin.

Description

IMPROVED MICROBIAL PRODUCTION OF ANTHRACYCLINS

Field of the invention

The present invention relates to the fermentative production of epidaunorubicin and epirubicin in a host cell.

Background of the invention

Anthracyclins are clinically important cancer chemotherapeutic agents. Compounds belonging to this group are, amongst others, daunorubicin, doxorubicin and rhodomycin. Although these products are widely applied, the annual production is only several hundred kilograms due to the complex and expensive manufacturing routes. Besides this, there are several serious drawbacks such as undesirable acute and long- term toxic effects during clinical application, stressing the need for less toxic derivatives, with equal clinical effect and at lower cost.

A stereo isomer of doxorubicin, the anticancer drug epirubicin (also referred to as 4'-epidoxorubicin), has less cardio toxic effects than both daunorubicin and doxorubicin, and therefore is a more suitable drug to use in clinical applications. Unfortunately, epirubicin is very difficult to obtain due to low yields of the overall synthetic pathway.

The primary starting point of all anthracyclins produced today is the fermentative production of daunorubicin by actinomycetes (J. Ind. Microbiol. & Biotechnol. (1999) 23, 647-652). Several species have been described that can produce daunorubicin, with

Streptomyces peuceticus (also referred to as Streptomyces peucetius) ATCC29050 as the preferred type strain, but only one can produce doxorubicin, namely Streptomyces peuceticus subspecies caesius ATCC27952. The latter is a mutant strain derived from the ATCC29050 strain and the relative low amount of doxorubicin is formed by C- 14 hydroxylation of its immediate precursor, daunorubicin. Many efforts have been put in increasing the titers of final products and conversion ratio of daunorubicin into doxorubicin. Current production strains have a daunorubicin titer over 1 g.l^"1 but the complicated recovery process makes it a costly product.

R₁ R₂ R₃ R₄ R₅

Daunorubicin C(O)CH₃ H OH OCH₃ H

Doxorubicin C(O)CH₂OH H OH OCH₃ H

Rhodomycin D CH₂CH₃ H OH OH C(O)OCH₃

Epidaunorubicin C(O)CH₃ OH H OCH₃ H

Epirubicin C(O)CH₂OH OH H OCH₃ H

Epirhodomycin D CH₂CH₃ OH H OH C(O)OCH₃

Doxorubicin can also be synthesized chemically starting from daunorubicin. This requires three steps with a yield of approximately 50%. The replacement of these three Chemical steps by a single enzymatic oxidation (doxA hydroxylase) is attractive, but this enzyme has very low in vivo activity. There is quite some engineering needed to increase the fluxes in Streptomyces peuceticus towards increased doxorubicin production (J. Bacteriol. (1999) 181, 305-318; J. Bacteriol. (1996) 178, 7316-7321 ). In US 5,695,966, the use of the DNA fragment encoding such an enzyme in other species is described, but also here the yields remain low. This biotransformation of daunorubicin into doxorubicin is improved by equipping other species like Streptomyces lividans with several genes of the Streptomyces peuceticus instead of doxA hydroxylase alone (WO 00/55829). Also for doxorubicin the complicated recovery process increases the costs on top of these low conversion ratios.

Although the above-described developments resulted in some improvements in daunorubicin and doxorubicin synthesis, this did not solve the problem of an economically attractive synthesis of the less toxic, and therefore most wanted, analogues like epidaunorubicin (also referred to as 4'-epidaunorubicin) and, specifically, epirubicin. Currently, there are two options to synthesize epidaunorubicin and epirubicin:

(i) Semi-synthetic, using the daunorubicin or doxorubicin produced via fermentation as building blocks followed by many complex chemical steps, or

(ii) Fermentative, in very low yields by complex metabolic engineering of the Streptomyces peuceticus type strain ATCC29050 (Nat. Biotechnol. (1998) 16, 69-74) The drawbacks of the first method are the already stipulated complex, expensive and low-yield chemical conversions but also the requirement for highly purified starting materials in order to avoid unwanted side reactions. Unfortunately this is difficult to realize due to the complicated separation of daunorubicin and doxorubicin.

The drawbacks of the second method are that the yields of epirubicin and its precursor epidaunorubicin are so low compared to the other anthracyclins produced that industrial application is not feasible. Low productivities for epidaunorubicin up to 0.065 g.r¹ were reported (Nat. Biotechnol. (1998) 16, 69-74), and although the production of epirubicin was indeed detected, the overall productivity of this compound was still lower than that of epidaunorubicin. In the 7 years that have passed since this disclosure, a method by which these unfavorable yields could be improved, or strains displaying economically feasible levels of epidaunorubicin and/or epirubicin productivity, have not been reported. Therefore, an economically feasible way of producing epirubicin and epidaunorubicin as pure compounds is not available and is extremely desirable.

Summary of the invention

It is an object of the present invention to provide a microbial strain with improved productivity of epidaunorubicin or epirubicin or a mixture of epidaunorubicin and epirubicin. Surprisingly, we have found that microbial strains with the desired improved productivity can be obtained by incubating a population of microbial cells, selecting a subpopulation of viable cells, and isolating cells out of the subpopulation of cells that produce at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin. Furthermore, it is an object of the present invention to provide an improved method for the production of epidaunorubicin and/or epirubicin.

The present invention provides a microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin and a method to obtain said microbial strain comprising plating out a population of microbial cells, selecting a subpopulation of viable cells, and isolating cells out of the subpopulation of cells that produce at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin.

Furthermore, the present invention provides a method for the production of epidaunorubicin and/or epirubicin comprising fermentation of a microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin under conditions conducive to the production of epidaunorubicin and/or epirubicin.

Detailed description of the invention

In the first aspect of the present invention there is provided a microbial strain, in particular an actinomycete strain, that produces at least 0.1 g.l^"1 culture broth of an α-L-arabino-hexopyranoside-derived anthracyclin like epidaunorubicin or epirubicin. Preferably, the actinomycete strain of the invention produces at least 1 g.l^"1 culture broth of an α-L-arabino-hexopyranoside-derived anthracyclin, more preferably at least 10 g.l^"1 culture broth. The skilled person will understand that an upper limit in anthracyclin titers may be around 50-70 g.l^"1 , although this may be further increased by in situ product removal. Alternatively and more preferably, the production levels are given in yields: at least 5 mg.g^"1 biomass dry-weight, more preferably at least 50 mg.g^"1 biomass dry- weight and most preferably at least 500 mg.g^"1 biomass dry-weight of an α-L-arabino-hexopyranoside derived anthracyclin. Preferably, the microbial strain is from the genus Streptomyces, such as Streptomyces peuceticus.

In one embodiment, the ratio of the sum of both epidaunorubicin and epirubicin over the sum of all other anthracyclins present, like daunorubicin, doxorubicin and rhodomycin D is higher than 0.1 , more preferably higher than 1 , even more preferably higher than 10 and most preferably higher than 100. In a preferred situation the ratio of epirubicin over epidaunorubicin is higher than 0.01 , preferably higher than 0.1 , more preferably higher than 1 , even more preferably higher than 10 and most preferably higher than 100.

The microbial strain of the present invention can be obtained by incubating a population of microbial cells, selecting a subpopulation of viable cells, and isolating cells out of the subpopulation of cells that produce at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin. Preferably, the population of microbial cells already produces epidaunorubicin and/or epirubicin, albeit at low concentrations, i.e. ranging from 0.005 g.l^"1 to 0.075 g.l^"1 culture broth. An example of such microbial cells are the genetically engineered strains of Streptomyces peuceticus described by Madduri etal. (Nat. Biotechnol. (1998) 16, 69-74). Alternatively, such epidaunorubicin and/or epirubicin producing microbial cells can be obtained by isolating the anthracyclin metabolic pathway structural genes in the form of synthetic DNA, which is in vitro adapted for producing epidaunorubicin and/or epirubicin. Notwithstanding the above, the method described above is also suitable to convert a microbial strain that produces at least 0.075 g.l^"1 culture broth of epidaunorubicin and/or epirubicin into a strain that produces more epidaunorubicin and/or epirubicin and/or in a different ratio than the starting strain. Preferably, the said population of microbial cells may be incubated in the presence of a suitable toxin and the said subpopulation of cells is isolated (selected) as being resistant against said toxin. The toxin that is applied should be toxic to the starting population of microbial cells, i.e. the toxin should have the effect that the cells are unable to grow or to survive when incubated in the presence of a suitable concentration of the toxin. Upon incubation in the presence of a suitable concentration of the toxin, a subpopulation of the cells is isolated that displays resistance to the toxin, i.e. is able to grow in the presence of the toxin. A subset of these toxin-resistant cells may have obtained one or more mutations providing, for instance, an improved production level of the anthracyclins epidaunorubicin and/or epirubicin.

A list of typical toxins one could apply for such a selection consist of, but is not limited to:

- Anthracyclins like aclacinomycin, cinerubin A, daunorubicin, 3'-deamino-3'-hy- droxy-4'-amino-adriamycin, doxorubicin, epidaunorubicin, epirhodomycin D, epirubicin, iodorubicin, marcellomycin, rhodomycin D or rubidazone, which are toxic as inhibitors of RNA and/or DNA synthesis, topoisomerase I and/or II, gyrase and/or inductor of DNA single-strand breaks (Cancer Res. (1993) 53, 1072-1078; Biochem. Pharmacol. (1994) 47, 2269-2278; Ann. Hematol.

(1993) 66, 27-31 ; FEMS Microbiol. Lett. (1996) 145, 281 -286), resistant cells might be non-sensitive anthracyclin overproducing cells;

- Compounds like cerulenin or apoenzyme AcylCarrierProtein (Biochem. J. (1998) 330, 933-937; J. Biol. Chem. (1999) 274, 25108-251 12), which are inhibitors of polyketide synthases (PKS), resistant cells might be cells with a increased flux through the PKS enzyme at the start of the anthracyclin pathway;

- Compounds like novobiocin, Novel Ribosome Inhibitors, Simocyclinone D8 or salvicine (EMBO J. (1986) 5, 3305-331 1 ; Antimicrob. Agents Chemother.

(2003) 47, 3831 -3839; Antimicrob. Agents Chemother. (2005) 49, 1093-1 100; Cancer Chemother. Pharmacol. (2005) 55, 286-294), which are compounds with an analogous mode of action as anthracyclins, resistant cells might be cells that produce an altered level and/or composition of anthracyclins; - Precursors like fluoropropionate, which becomes toxic to the cells upon consumption as carbon source for growth, resistant cells might be cells that have an increased flux through the anthracyclin biosynthetic pathway due to a decreased co-consumption of the common substrate, propionate;

- Sugar analogues like 2-deoxyglucose, which cannot be consumed by microorganisms but induces carbon repression, resistant cells might be cells that have an increased flux through the anthracyclin pathway irrespective of the main carbon source used.

A suitable concentration of the toxin typically lies around the minimal inhibitory concentration (MIC value) found for the microbial cells that are subjected to the method of the invention. The MIC value will be dependent on the microbial cells that are used and may be between for instance 0.001 and 10 g. I^"1. From the toxin-resistant subpopulation of cells, microbial strains are isolated that produce at least 0.1 g.l^"1 culture broth of the anthracyclins epidaunorubicin and/or epirubicin. Surprisingly, it has been found that a high percentage of toxin-resistant cells produce at least 0.1 g.l^"1 culture broth of the anthracyclins epidaunorubicin and/or epirubicin.

The population of microbial cells that is subjected to the method of the invention may be a population of identical cells derived from one particular parent strain. In this way, typically spontaneous mutants may be isolated. The population of microbial cells that is subjected to the method of the invention may also be a population of cells that is firstly subjected to a mutagenic treatment to deliberately introduce genetic variation into said population. The mutagenic treatment typically may comprise a so-called classical treatment, but also may include DNA-mediated transformation. A classical treatment includes protoplast fusion and/or a treatment with UV radiation or certain chemicals.

In another embodiment of the first aspect of the present invention, adaptive evolution techniques maybe used to isolate cells that produce at least 0.1 g.l^"1 culture broth of anthracyclins epidaunorubicin and/or epirubicin. In this case the said subpopulation of cells, with or without prior mutagenic treatment, will be cultivated in sub- selective conditions, which are conditions were the level of toxin applied is just below or around the determined MIC value. Subsequent dilutions of the culture broth in fresh media and slowly increased levels of the toxin will stimulate the gradual selection and increase of the said subpopulation of cells for with an improved production level or, alternatively, an altered ratio of the anthracyclins epidaunorubicin and/or epirubicin. The person skilled in the art will understand that such adaptive evolution experiments can also be performed by using selective cultivation conditions, like for example a changed temperature, an increased stirrer speed, an altered pH or a difficult to consume carbon source, under which the parent cells do not grow or at least show very poor growth, while the said subpopulation of cells for with an improved production level of the anthracyclins have an evolutionary advantage and start to accumulate. Specifically, this technique can be used to find the very rare cells with the aimed characteristics.

In yet another embodiment, the mutagenic treatment is done on toxin-resistant cells, preferably on toxin-resistant cells that produce at least 0.1 g.l^"1 culture broth of the anthracyclins epidaunorubicin and/or epirubicin. In the context of the present invention, microbial strains are isolated that display, as compared to a parent strain, an improved productivity of the anthracyclins epidaunorubicin and/or epirubicin and/or a change in the ratio of the different anthracyclins produced. A parent strain is a strain that does not substantially produce the anthracyclins epidaunorubicin and/or epirubicin. For instance, a suitable parent strain may be a strain that produces epidaunorubicin and/or epirubicin at a level that is lower than 0.1 g.l^"1 culture broth. Typical examples of such parent strains are disclosed by Madduri etal. (Nat. Biotechnol. (1998) 16, 69-74). The person skilled in the art will know that further improvement of the anthracyclin titers produced by the strains of the present invention can be obtained by transforming the cells that produce 0.1 g.l^"1 culture broth or more of anthracyclins with polynucleotides affecting the anthracyclin biosynthesis pathway. These can be isolated from known anthracyclin producing species like, but not limited to, Streptomyces and

Saccharopolyspora. Preferred examples are Streptomyces peuceticus, Streptomyces peuceticus subspecies caesius, Streptomyces insignis, Streptomyces avermitilis and Saccharopolyspora erythraea. These can be genes encoding enzymes of (related) pathways, transcription factors regulation expression levels of the biosynthetic pathway, additional copies of the gene cluster encoding the biosynthetic pathway, but also anti- sense polynucleotides can be used to hamper competition metabolic pathways.

In the second aspect of the present invention, there is provided a method for the production of epidaunorubicin and/or epirubicin comprising fermentation of the microbial strain of the first aspect of the invention under conditions conducive to the production of epidaunorubicin and/or epirubicin.

The cells according to the invention may be cultured using procedures known in the art. Of course many procedures are available to the skilled person and many variations within these procedures are possible. As a non-limiting example, reference is made to a suitable procedure as reported by Lomovskaya etal. (J. Bacteriol. (1999) 181, 305-318). After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. The anthracyclins epidaunorubicin and/or epirubicin may then be recovered and, if desired, purified and isolated by conventional means. Said means include crystallization, chromatographic procedures, extraction techniques and the like. The persons skilled in the art understands that due of the toxic nature of these compounds advanced techniques like in situ product recovery may increase the final yield of the anthracyclins produced per amount of carbon consumed, as they are taken away continuously without disturbing the fermentation. In one embodiment of the second aspect of the present invention, the anthracyclins may be produced in a two-step process. First, daunorubicin and/or, more preferably, doxorubicin may be produced fermentative by an optimized cell, which may be converted in a second step to epidaunorubicin and/or epirubicin by a second cell, which is equipped to do so. Examples

General methods Culturing of cells (J. Bacteriol. (1999) 181, 305-318) Five ml of seed medium containing glucose (25 g.l^"1), yeast extract (4 g.l^"1), malt extract (10 g.l^"1), NaCI (2 g.r¹), 3-(morpholino)propanesulfonic acid (MOPS sodium salt) (15 g.l ¹), and MgSO₄ (0.1 g.l^"1) and 10 ml of trace elements, consisting Of ZnCI₂ (40 mg.l^"1), FeCI₃* 6I₂ ¹O (200 mg.l^"1), CuCI₂* 2I₂ ¹O (10 mg.l^"1), MnCI₂* 4HO (10 mg.l^"1), Na₂B₄O₇* 10HO (10 mg.l^"1), and (NhU)₆Mo₇O₂₄* 4HO (10 mg.l^"1) is inoculated with spores or mycelium of a typical strain and incubated at 3OO and 300 rpm in baffled

Erlenmeyer flasks. After 24 h of incubation, the seed culture is transferred to 25 ml of the APM production medium containing glucose (60 g.l^"1), yeast extract (8 g.l^"1), malt extract (20 g.l^"1), NaCI (2 g.l^"1), MOPS sodium salt (15 g.l^"1), MgSO₄ (0.1 g.l^"1), FeSO₄* 7HO (0.01 g.l^"1), ZnSO₄* 7HO (0.01 g.l^"1), and antifoam B emulsion (4 ml; Sigma) and incubated in a 250-ml baffled flask as described above for 72 h. Cultures are extracted with chloroform and analyzed by high-performance liquid chromatography (HPLC). The persons skilled in the art will understand that several iterative rounds of adapting the exact media composition and cultivation conditions to the isolated cells capable of producing at least 0.1 g.l^"1 of the anthracyclins epidaunorubicin and/or epirubicin, may be needed to optimize the yield by using said cells.

Example 1 Isolation of anthracyclin resistant Streptomyces peuceticus mutants High anthracyclin producing Streptomyces peuceticus strains were isolated using the following procedure. First, the natural sensitivity of Streptomyces peuceticus strains for the toxic compounds daunorubicin, doxorubicin and epirubicin were determined by adding different concentrations of these anthracyclins to agar plates. The minimal inhibitory concentration (=MIC-value) was determined by using 2xYT plates (Molecular cloning: a laboratory manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) containing various concentrations between 0.001 and 10 g.l^"1 of each compound. On each plate with the different concentrations 100 μl of a freshly grown culture (OD_{600 nm} =0.4) was plated and incubated at 37⁰C for 1 -3 days. Low epirubicin producing cells (Nat. Biotechnol. (1998) 16, 69-74) were grown under selective conditions as described and UV-irradiated. To isolate anthracyclin resistant variants the irradiated cells were spread on plates with the anthracyclin in the amount that was shown to be lethal to the parent cells. A hundred isolates were colony purified on standard agar plates and stored for later applications.

Example 2 Epidaunorubicin and epirubicin formation The isolates obtained as described in example 1 were cultivated as follows. Five ml of seed medium containing glucose (25 g.l^"1), yeast extract (4 g.l^"1), malt extract (10 g. I^"1), NaCI (2 g.l^"1 ), 3-(morpholino)propanesulfonic acid (MOPS sodium salt) (15 g.l^"1 ), and MgSO₄ (0.1 g.l^"1) and 10 ml of trace elements, consisting Of ZnCI₂ (40 mg.l^"1), FeCI₃* 6HO (200 mg.l^"1), CuCI₂* 2HO (10 mg.l^"1), MnCI₂* 4HO (10 mg.l^"1), Na₂B₄O₇* 10HO (10 mg.l^"1), and (NhU)₆Mo₇O₂₄* 4HO (10 mg.l^"1) is inoculated with spores or mycelium of a typical strain and incubated at 3OO and 300 rpm in baffled Erlenmeyer flasks. After 24 h of incubation, the seed culture is transferred to 25 ml of the APM production medium containing glucose (60 g.l^"1), yeast extract (8 g.l^"1), malt extract (20 g.l^"1), NaCI (2 g.l^"1), MOPS sodium salt (15 g.l^"1), MgSO₄ (0.1 g.l^"1), FeSO₄* 7HO (0.01 g.l^"1), ZnSO₄* 7HO (0.01 g.l^"1), and antifoam B emulsion (4 ml; Sigma) and incubated in a 250-ml baffled flask as described above for 72 h. Selection markers as thiostrepton or apramycin were applied where needed. Cultures are extracted with chloroform and analyzed by high-performance liquid chromatography (HPLC). Both epidaunorubicin and epirubicin were produced significantly higher as compared to the levels in the parent strains.

Claims

1 . A microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin.

2. A method to obtain a microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin comprising plating out a population of microbial cells, selecting a subpopulation of viable cells, and isolating cells out of the subpopulation of cells that produce at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin.

3. Method according to claim 2, wherein said population of microbial cells is treated with a mutagen.

4. Method according to any one of claims 2 to 3, wherein said population of microbial cells and/or said subpopulation of viable cells is treated with a toxin.

5. Method according to claim 4, wherein the toxin is selected from the group consisting of daunorubicin, doxorubicin, epidaunorubicin, epirhodomycin D, epirubicin, rhodomycin D and sugar analogs.

6. Method according to any one of claims 2 to 5, wherein said population of microbial cells and/or said subpopulation of viable cells is subjected to DNA-mediated transformation with a polynucleotide encoding one or more enzymes effecting the anthracyclin biosynthetic pathway.

7. Method according to claim 6, wherein the polynucleotide is derived from an anthracyclin producing microorganism.

8. A method for the production of epidaunorubicin and/or epirubicin comprising fermentation of a microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin under conditions conducive to the production of epidaunorubicin and/or epirubicin.

9. Method according to claim 8, further comprising recovery of epidaunorubicin and/or epirubicin from the fermentation broth.

10. Method according to any one of claims 8 to 9, further comprising purification of epidaunorubicin and/or epirubicin.

1 1 . Method according to any one of claims 8 to 10, wherein a first cell produces daunorubicin and/or doxorubicin and said microbial strain converts said daunorubicin and/or doxorubicin into epidaunorubicin and/or epirubicin.

12. Use of a microbial strain that produces at least 0.1 g.l^"1 culture broth of epidaunorubicin and/or epirubicin in the production of epidaunorubicin and/or epirubicin.