Background

[0001] Streptomyces is a relatively well-studied genus of Actinomycete. Streptomycetes are known, among other things, for the production of antibiotics. Partly due to the importance of some of the compounds produced by Streptomyces, many methods of cloning and expressing genes in Streptomyces have been established. This genus is particularly useful for expressing heterologous genes with high G + C content.

[0002] A variety of cloning vectors are used to clone and express genes in Streptomyces, including integrative vectors which contain attachment sites to facilitate site-specific integration of the vector into a Streptomyces host. When these cloning vectors are transferred into Streptomycetes through conjugation, transformation, or electroporation, the vectors integrate into the Streptomyces genome in a stable fashion. This produces Streptomyces clones with stable gene inserts, which are especially useful when expression of the gene insert, and production of the gene product, is desired.

[0003] However, Streptomycetes are complex organisms that are not simple to work with. During its life cycle, a Streptomycete can take many different forms, including the form of a spore, mycelium, and aerial hypha. Streptomycetes also have complex media requirements, which can be necessary to induce development of different morphologies. Due to the complex life cycle of Streptomycetes, it can be difficult to scale up the expression and production of compounds in Streptomycetes.

[0004] Rhodococcus is also a genus of Actinomycete. This genus has slightly lower G+C content compared to Streptomyces. However, Rhodococci are easier to manipulate than Streptomycetes because they have less complex life cycles. Rhodococci are also capable of growing in diverse types of media which makes them more versatile.

[0005] There is a need for improved methods of cloning and expressing heterologous genes in Rhodococcus.

Summary of Invention

[0006] The invention pertains to a method of biosynthetically producing heterologous compounds in Rhodococci, the method comprising the steps of identifying genes which are involved in the biosynthesis of the desired compound, cloning the genes into an integrative vector, transferring the vector into Rhodococci, growing the Rhodococci to express the genes so that products of the genes synthesize, or are synthesized into, assemble, or are assembled into, the desired compound, and isolating the desired compound from the Rhodococci. The desired product may also comprise the gene products themselves.

[0007] In one aspect of the invention, the desired compound is selected from the group consisting of an antibiotic, an anti-viral, an anti¬ fungal, an anti-tumour agent, a chemotherapeutic agent, a peptide, and a protein. In one embodiment of the invention, the compound is an antibiotic, which includes polyketides and non-ribosomal peptides. In another embodiment of the invention, the antibiotic is calcium-dependent and is effective against gram-positive bacteria. In a specific embodiment, the compound is daptomycin.

[0008] In another aspect of the invention, the method uses a Rhodococci comprising an attachment site for an integrative vector. In some embodiments of the invention, the Rhodococci comprise a ΦC31 attachment site. In other embodiments, the Rhodococci comprise a pSAM2 attachment site. In one embodiment of the invention, the Rhodococci is Rhodococcus sp. RHAl. In other embodiments of the invention, the Rhodococci is selected from Rhodococcus equi, Rhodococcus eiythropolis, Rhodococcus rhodochrous, and Rhodococcus sp. 124.

[0009] In another aspect of the invention, the method uses an integrative vector that can attach at a ΦC31 attachment site, a pSAM2 attachment site, a tRNA sequence site, a transposon site, an IS site, or any other suitable site. In one embodiment of the invention, the vector is a ΦC31 -derived vector. In another embodiment of the invention, the vector is a pSAM2-derived vector. In another embodiment of the invention, the vector is derived from pStreptoBAC V. In a specific embodiment of the invention, the vector carrying the gene of interest is pCVl .

[0010] Another aspect of the invention is the use of Rhodococci to biosynthetically produce a heterologous compound. Brief Description of Drawings

[0011 ] In drawings which illustrate various embodiments of the invention and which are not intended to limit the scope of the invention:

[0012] Figure 1 illustrates the transfer of the daptomycin biosynthetic gene cluster carried on pCVl from E. coli (donor) to Rhodococcus sp. RHAl (recipient) via conjugation.

[0013] Figure 2 is a schematic representation of the plasmid pCVl with the daptomycin insert showing the positions of primer sets used to confirm the insertion of the gene cluster.

[0014] Figure 3 is an agarose gel showing PCR products amplified from Rhodococcus sp. RHAl transconjugants using primers P61 and P62.

[0015] Figure 4 is an agarose gel showing PCR products amplified from Rhodococcus sp. RHAl transconjugants using primers P74 and P75.

[0016] Figure 5 is an agarose gel showing PCR products amplified from Rhodococcus sp. RHAl transconjugants using primers P 117 and P122.

[0017] Figures 6A and 6B depict the results of antibiotic activity assays of supernatants from Rhodococcus sp. RHAl transconjugants against M. luteus.

[0018] Figure 7 depicts an overlay of Staphylococcus aureus grown over a thin-layer chromatogram (TLC) loaded with samples of daptomycin and extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid. [0019] Figure 8 is an HPLC chromatogram of daptomycin.

[0020] Figure 9 is an HPLC chromatogram of extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid.

[0021] Figure 10 is an HPLC chromatograph of daptomycin and extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid.

[0022] Figure 11 is a schematic diagram of a bioassay-guided fractionation strategy for obtaining extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid.

Detailed Description of the Invention

[0023] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0024] This invention pertains to methods of biosynthetically producing heterologous compounds in Rhodococci. In one aspect, the invention takes advantage of the versatility of Rhodococci, their ability to express heterologous genes, particularly genes of high G + C content, and the abundance of cloning and expression vectors readily available for other actinomycetes which are also useful in Rhodococci. [0025] The method includes identifying genes which are involved in the biosynthesis of the desired compound, cloning the genes of interest into a vector, transferring the vector into Rhodococci, growing the Rhodococci to express the genes of interest, which allows the products of the genes of interest to synthesize, or be synthesized into, assemble, or be assembled into, a desired compound, and isolating the desired compound from the Rhodococci. The desired compound can also comprise the products of the genes of interest themselves.

[0026] The genes of interest can comprise genes which code for proteins involved in biosynthesis of a desired compound. Such proteins can be involved in the biosynthesis of heterologous compounds such as antibiotics, anti-fungals, anti-virals, anti-tumour agents, chemotherapeutics, and other medically or industrially useful compounds. The proteins can be enzymes which catalyse various reactions in the biosynthetic pathway of the desired compound. The genes of interest may also code for peptides or proteins of interest, including enzymes. Such enzymes may include degradative enzymes, industrially useful enzymes, or any other enzymes of interest. Thus, the desired compounds may be the products of the genes of interest.

[0027] The genes of interest can be isolated from any organism, including organisms with high G+C content. In some embodiments, the genes are isolated from other Actinomycetes, including Streptomycetes, Nocardiae, Mycobacteria, Corynebacteria, and others. In one specific embodiment, the genes are isolated from Streptomyces roseosporus. [0028] The method of the invention can be used with any species of Rhodococcus. The high G+C content, promoter structures, and growth characteristics of Rhodococcus make it a good expression host, particularly for expression of genes from organisms with high G+C content. When the method is used with integrative vectors, the Rhodococci can comprise any species having an appropriate integration site for integrative vectors. In one embodiment, the species comprises Rhodococcus sp. RHAl which comprises an attachment site for ΦC31- derived vectors and pSAM2 vectors that are normally used with Streptomycetes. In other embodiments, the species comprise Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodochrous, and Rhodococcus sp. 124.

[0029] The vector can be any suitable vector. In one embodiment, the vectors are integrative vectors, which are capable of site-specifically integrating into the Rhodococcus genome through an attachment site. Integrative vectors can be useful for stably inserting a cloned gene sequence into the Rhodococcus genome, as stably inserted sequences can be expressed more efficiently, particularly in fermentation processes. The attachment sites for the integrative vectors can include a ΦC31 attachment site, a pSAM2 attachment site, a tRNA sequence site, a transposon site, an IS site, or any other suitable attachment site. In some embodiments, the vectors comprise integrative vectors used for cloning and expressing genes in Streptomycetes. In one embodiment, the vector comprises a ΦC31 -derived vector. In another embodiment, the vector comprises a pStreptoBAC V derived vector. In yet another embodiment, the vector comprises a pSAM2 derived vector. In a specific embodiment, the vector carrying the gene of interest is pCVl.

[0030] The biosynthetic genes may code for proteins involved in the production of all or part of a compound of interest. The biosynthetic genes may also code for proteins or peptides of interest. In some embodiments, the genes encode proteins involved in the biosynthesis of antibiotics, including polyketides and non-ribosomal peptides. In a specific embodiment, the gene of interest is the daptomycin biosynthetic gene cluster from S. roseosporus which codes for proteins involved in the biosynthesis of daptomycin, a calcium dependent antibiotic effective against gram-positive organisms. In other embodiments, the cloned genes code for proteins involved in the biosynthesis of tetracycline, mithramycin, gentamicin C, and astaxanthin. As will be appreciated by persons skilled in the art, the method of the invention can be used to produce any heterologous compound of interest.

[0031] Once the desired genes are cloned into a vector, the vector can be transferred into Rhodococcus through a variety of means. The vector can be transferred by conjugation, transformation, electroporation, transduction, or any other suitable means of transfer which are known in the art. In one embodiment, the vector is transferred to Rhodococcus through conjugation.

[0032] The method of the invention also comprises the step of screening for successful Rhodococcus transferants. A transferant refers to any Rhodococcus organism which has received a vector through any of the transfer mechanisms described above. Many methods of screening for transferants are known in the art, such as screening for selection markers present in the vectors but which are not normally present in Rhodococcus. Commonly used selection markers include antibiotic resistance genes or genes that code for proteins that catalyze reactions with chromogenic end products. In some embodiments, transferants can be selected by growing the organisms in the presence of the antibiotic for which the vector encodes resistance and to which the organism is normally sensitive. If the vector has been successfully transferred to the organism, the antibiotic resistance marker in the vector will be expressed, and the organism will grow in the presence of the antibiotic. In embodiments of the invention where the vector is an integrative vector, the vector can integrate into the Rhodococcus genome and the genes in the vector are expressed as part of the genome. In a specific embodiment of the invention, the vector pStreptoBAC V, which carries an apramycin marker, is transferred into Rhodococcus by conjugation, and successful transconjugants are selected using apramycin.

[0033] Once successful transferants are selected, the method of the invention comprises growing the transferant Rhodococci, detecting the products of the genes of interest, or the desired compounds produced by reactions catalyzed by the products of the genes of interest, and collecting the products or desired compounds. In one embodiment, the compound comprises daptomycin, a calcium-dependent antibiotic, which is effective against gram-positive organisms.

[0034] Another aspect of the invention involves the use of Rhodococci as expression hosts for the expression of heterologous compounds. Because Rhodococci are metabolically versatile organisms which are capable of growing on a variety of media, Rhodococci can be used to produce heterologous compounds in the presence of various other compounds or chemicals. Rhodococci are also amenable to fermentation culture.

Examples

[0035] The following examples illustrate various embodiments of the invention and are not intended to limit the scope of the invention.

Example I: Transfer of Daptomvcin biosvnthetic gene cluster in Rhodococcus sp. RHA I

[0036] The plasmid pStreptoBAC V is a Streptomyces integrative shuttle vector having the φC31 attachment site and an apramycin resistance marker. The plasmid pCVl comprises pStreptoBAC V with the ~ 130 KB insert of the daptomycin biosynthetic gene cluster from S. roseosporus. Construction of this plasmid is discussed in PCT Publication Nos. WO 02/059322 and WO 03/014297, and in Miao et al., 2005, which are incorporated herein by reference.

[0037] The plasmid pCVl was transferred to Rhodococcus sp. RHAl via conjugation (see Figure 1). The donor used in the mating experiment was a plasmid mobilizing strain of E. coli DHlOB carrying the plasmid pCVl . Rhodococcus sp. RHAl was used as the recipient. Conjugation was performed as described in Kieser et al. (2000). The transconjugants were selected on media containing 50μg/ml apramycin.

[0038] Transconjugant colonies of Rhodococcus sp. RHAl thus obtained were selected for apramycin and daptomycin resistance by growth in liquid as well as solid medium. All transconjugants were resistant to apramycin and daptomycin. In contrast, wild type Rhodococcus sp. RHAl is sensitive to both of these antibiotics. The insertion of the daptomycin biosynthetic gene cluster into the genome of the Rhodococcus sp. RHAl transconjugants was confirmed by performing colony PCR on the transconjugants using different sets of primers to amplify various regions of the daptomycin gene cluster. Primers used in the amplification of the gene cluster are described in Table 1. Primer sequences are also disclosed in PCT Publication Nos. WO 02/059322 and WO 03/014297, and Miao et al. Figure 2 is a schematic representation of the plasmid pCVl, which shows the positions of primer sets used to confirm insertion of the gene cluster.

Table 1: Primer sets used in PCR amplification of transconjugants.

[0040] Amplification of the desired regions of the gene cluster in the transconjugants with different primer sets and the absence of the corresponding bands in the wild type host (unconjugated Rhodococcus sp. RHAl) verified the presence of the daptomycin biosynthetic gene cluster in the Rhodococcus sp. RHAl transconjugants. Figure 3 is an agarose gel of PCR products amplified from Rhodococcus sp. RHAl transconjugants using Primer Set I (P61/P62). If the daptomycin gene cluster is inserted into the genome of the transconjugant, Primer Set I should amplify a 710 b.p. product from the central region of the daptomycin biosynthetic gene cluster. In the gel, lane M = 1 Kb plus DNA ladder, lanes 1-20 = PCR products from transconjugants 1-20, W= wild type RHAl, + = positive control (PCR product amplified from E. coli DHlOB + pUZ8002 + pCVl), and - = negative control (no template). Products that appear to be approximately 710 b.p. in size were amplified from each of the transconjugants as indicated by the band in lanes marked 1-20. No product was amplified from wild type Rhodococcus sp. RHAl.

[0042] Figure 4 is an agarose gel of PCR products amplified from Rhodococcus sp. RHAl transconjugants using Primer Set II (P74/P75). If the daptomycin gene cluster is inserted into the genome of the transconjugant, Primer Set II should amplify a 729 b.p. product from the beginning of the daptomycin biosynthetic gene cluster. In the gel, lane M = 1Kb plus ladder, lanes 1-4 = PCR products from transconjugants 1-4, W = wild type Rhodococcus sp. RHAl, -C = negative control (no template), +C = positive control (pCVl in E. coli DHlOB + pUZ8002). Products that appear to be approximately 729 b.p. in size were amplified from each of the transconjugants as indicated by the band in lanes marked 1-4. No product was amplified from wild type Rhodococcus sp. RHAl.

[0043] Figure 5 is an agarose gel of PCR products amplified from the transconjugants using Primer Set III (Pl 17/122). If the daptomycin gene cluster is inserted into the genome of the transconjugant, Primer Set III should amplify a 592 b.p. product from the end of the daptomycin biosynthetic gene cluster. In the gel, lane M = 1 Kb plus ladder, lanes 1-4 = PCR products from transconjugants 1-4, W=wild type Rhodococcus sp. RHAl, -C = negative control (no template), +C = positive control (pCVl in E. coli DHlOB + pUZ8002). Products which appear to be approximately 592 b. p. in size were amplified from each of the transconjugants as indicated by the band in lanes marked 1-4. No product was amplified from wild type Rhodococcus sp. RHAl.

Example II: Antibiotic Activity of Rhodococcus sp. RHAl Transconiugants

[0044] The transconjugants were grown in liquid medium at 300C and the culture supernatant was tested for antibiotic activity against Micrococcus luteus, a gram-positive bacteria. Antibiotic activity assays were performed at 24h intervals using well assays. For the well assays, 150μl of transconjugant culture supernatant was added to wells made in LB agar plates containing calcium (5mM) and without calcium. Soft agar overlays containing M. luteus were poured overtop. The assay plates were incubated at 370C. Antibacterial activity against M. luteus is indicated by the appearance of zones of inhibition around the wells.

[0045] The results of the antibacterial activity assays are shown in Figures 6 A and 6B. After 4-5 days of growth of the Rhodococcus sp. RHAl transconjugants in liquid medium, supernatant from the liquid cultures of transconjugants exhibited antibacterial activity against M. luteus (wells 1 to 6). Supernatant from unconjugated, wild type Rhodococcus sp. RHAl did not have any antibacterial activity (well 7). Moreover, results from the antibacterial activity assays also demonstrate that the antibacterial activity of the supernatants from the Rhodococcus sp. RHAl transconjugant cultures is calcium dependent. The zone of inhibition increased in the presence of Ca (Figure 6B), and in some cases, inhibition was demonstrated only in the presence of calcium. No activity was observed in the assays against E. coli MGl 655, a gram- negative organism (results not shown).

Example III: TLC and HPLC Analysis of Extract of Rhodococcus sp. RHAl Transconjugants

[0046] Extracts of recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid were fractionated using a bioassay-guided strategy (Barsby et al. 2001, 2002) as depicted in Figure 11. Fraction 47D 3-2-1 was analyzed by TLC (silica gel, solvent system: n-BuOH : H2O : CH3COOH = 4 : 2 : 1) and HPLC (ODS, 4.6 X 250 mm, linear gradient from 20% aq. CH3CN containing 0.05% TFA to 100% CH3CN containing 0.05% TFA in 60 minutes and isocratic in 100% CH3CN containing 0.05% TFA for 20 minutes).

[0047] The Rf values of active components on TLC were found to be the same as that of authentic daptomycin by analysis of bioautography. Figure 7 depicts an overlay of Staphylococcus aureus grown over a thin- layer chromatogram (TLC) loaded with samples of daptomycin and extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid. The TLC plate was loaded with 3 different samples (left to right): (L) a mixture of authentic daptomycin and extract from the recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid; (M) authentic daptomycin; and (R) extract of recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid alone. The compounds were spotted at the bottom of the plate (origin) and developed upwards as described above. The circular zones of clearing result from inhibition of the growth of S. aureus. An extract of wild-type RHAl does not inhibit the growth of S. aureus (results not shown).

[0048] Figures 8, 9, and 10 depict HPLC chromatograms of daptomycin and extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid. Figure 8 depicts the HPLC chromatogram of daptomycin alone. Figure 9 depicts the HPLC chromatogram of an extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid. The peak which is believed to correspond to daptomycin is indicated. Figure 10 depicts a chromatogram of daptomycin and extract from recombinant Rhodococcus sp. RHAl containing the integrated pCVl plasmid together. Comparing Figures 9 and 10, the marked peak in Figure 9 corresponds to the marked peak in Figure 10 which is significantly larger than the peak marked in Figure 9, due to the additional presence of pure daptomycin.

[0049] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope. References

Barsby T.5 Kelly M.T., Gagne S.M., Andersen RJ., Bogorol A produced in culture by a marine Bacillus sp. reveals a novel template for cationic peptide antibiotics, Org. Lett. 2001 Feb 8; 3(3):437-40.

Barsby T., Kelly M.T., Andersen RJ., Tupuseleiamides and basiliskamides, new acyldipeptides and antifungal polyketides produced in culture by a Bacillus laterosporus isolate obtained from a tropical marine habitat, J. Nat. Prod. 2002 Oct; 65(10):1447-51.

Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A., Practical Streptomyces Genetics (2000), The John Innes Foundation, Norwich, United Kingdom.

Miao, V. et al., Daptomycin Biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemist}y, Mιcvobio\ogy {2W5), 151, 1507-1523.