# Patent

## Title
Methods for producing secondary metabolites

## Abstract

The invention relates to methods for producing secondary metabolites using transformed Synechocystis sp. bacteria, and secondary metabolites produced by transformed Synechocystis sp. bacteria. The invention further relates to phosphopantetheinyl transferase enzymes.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application of International Application No. PCT/AU2008/000474, filed Apr. 2, 2008; which claims the benefit of Australian Patent Application No. 2007901738 filed Apr. 2, 2007. These applications are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The invention relates to methods for producing secondary metabolites using transformed Synechocystis sp. bacteria, and secondary metabolites produced by transformed Synechocystis sp. bacteria. The invention further relates to phosphopantetheinyl transferase enzymes.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 690159—401_USPC_SEQUENCE_LISTING.txt. The text file is approximately 13 KB, was created on Nov. 4, 2012, and is being submitted electronically via EFS-Web.
BACKGROUND
Non-ribosomal synthesis allows microorganisms to produce a diverse range of novel compounds including carboxy acids, heterocyclic rings, fatty acids and non-proteinogenic modified amino acids. Small polypeptides are assembled by peptide synthetases just as other compounds, like fatty acids, are linked by other synthetases during synthesis on the ribosome.
Non-ribosomal synthesis provides a pathway of synthesizing compounds which would be expensive or unobtainable using synthetic chemical methods. Nonribosomally synthesised peptides share certain characteristics. These small bioactive peptides are usually between 2 and 50 amino acid residues long and possess potent biological activities. Most examples of these compounds are also highly resistant to physical and chemical degradation making them ideal for use as oral therapeutics. The valuable products of microbial nonribosomal peptide synthesis include the immunosuppressant cyclosporin A and antibiotics such as penicillin, gramicidin S, vancomycin, cephalosporin, and surfactins.
Such compounds are synthesized by complex secondary metabolism pathways involving polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and fatty acid synthases (FAS). These enzymes are activated by phosphopantetheinyl transferases (PPTs). PPTs activate carrier proteins that are essential for PKS, NRPS and FAS activity. PPTs convert inactive carrier proteins to their active, cofactor-bearing holo-forms via transfer of the essential prosthetic 4′ phosphopantetheine moiety from co-enzyme A (CoA) (FIG. 1).
Cyanobacteria constitute a rich source of secondary metabolites with the majority being derived from PKS, NRPS and FAS. Due to their broad intergeneric distribution of integrated enzyme systems, cyanobacteria provide a source of many uncharacterised amino acid activating and modifying peptide synthetase modules. Consequently freshwater and marine cyanobacteria have been screened for bioactivity revealing natural products with novel biological applications and clearly implicating cyanobacteria as a rich source of potentially useful compounds.
Analysis of cyanobacterial non-ribosomal peptide, polyketide and fatty acid synthesis pathways has revealed unforeseen biochemical structures displaying the potential for novel products from cyanobacteria. However the strains of cyanobacteria which synthesize natural compounds are usually strains associated with slower growth or complicated culture requirements. Thus species which produce natural products may be excluded in culture-based screening. Furthermore current natural product screening relies on organism propagation. As less than 1% of microorganisms are estimated to be culturable, however, the bulk of potential bioactivities present in nature is not detectable. Alternatively, time-consuming organic syntheses have been used to create a modified natural product. Although molecular methods may yield the genetic information, recombinant expression in a suitable host organism is required to allow the isolation and production of novel compounds.
The ability to heterologously express and biochemically characterize recombinant proteins and biosynthesis pathways remains a significant problem with respect to cyanobacteria.
SUMMARY
The cyanobacterium Synechocystis sp. PCC6803 does not produce any secondary metabolites even though it possesses a PPT that could potentially allow the production of active carrier proteins. The Synechocystis PPT has broad substrate specificity and is capable of activating PKS, NRPS and FAS despite the fact that this species does not naturally encode either nonribosomal peptide or polyketide genes. This null background allows for the rapid identification of products potentially produced by heterologous expression in this species. This indicates the potential of Synechocystis as a suitable and efficient microorganism for the production of novel nonribosomal peptides and polyketides.
According to a first aspect there is provided a method for the production of secondary metabolites, the method comprising the steps of transforming Synechocystis sp. bacteria with one or more of a peptide synthetase gene, a polyketide synthase gene, or a fatty acid synthase gene required for production of secondary metabolites, culturing the Synechocystis sp. bacteria under conditions suitable for the expression of the one or more genes required for production of the secondary metabolites, and purifying the secondary metabolites from the bacteria.
In one embodiment, the method further comprises prior to step (c), the step of screening the Synechocystis sp. bacteria for the production of secondary metabolites. In one embodiment, the screening step comprises high performance liquid chromatography or mass spectrometry.
In one embodiment, the Synechocystis sp. bacteria is Synechocystis sp. PCC6803. Preferably the secondary metabolite is a peptide, a polyketide, a fatty acid, or a derivative thereof. In one embodiment the peptide, polyketide, fatty acid or a derivative thereof is a bioactive compound selected from the group consisting of an anti-microbial agent, an anti-viral agent, an anti-fungal agent, an anti-cancer agent, an immunosuppressive agent, an anaesthetic, an analgesic, an antitumour product, an antibiotic, an anti-cholesterolemic, an anti-parasitic agent, a veterinary therapeutic agent, an agrochemical and a cosmetic.
In one embodiment, the one or more synthetase genes is operably linked to a promoter active in Synechocystis sp. Preferably the promoter is an inducible promoter, such as a promoter inducible by varying the light intensity to which the bacteria are exposed during culture.
In one embodiment, the one or more synthetase genes is in a plasmid, phosmid or cosmid. In one embodiment, transformation of the Synechocystis sp bacteria is transposon-mediated. In one embodiment, the bacteria are transformed with multiple peptide synthetase, polyketide synthase, or fatty acid synthase genes forming a biosynthetic gene cluster.
In one embodiment, the Synechocystis sp. bacteria express an exogenous phosphopantetheinyl transferase (PPT). Preferably, the exogenous PPT is stably integrated into the Synechocystis sp. genome. Preferably, the exogenous PPT is a cyanobacterial PPT, such as a PPT from Nodularia spumigena. In one embodiment, the PPT is from Nodularia spumigena NSOR10. Preferably, the PPT comprises the amino acid sequence as set forth in SEQ ID NO: 1 or a variant of fragment thereof.
In another aspect there is provided a secondary metabolite produced in accordance with the method of the first aspect.
In another aspect, there is provided a transformed Synechocystis sp. bacteria comprising one or more of a peptide synthetase gene, a polyketide synthase gene, or a fatty acid synthase gene required for production of secondary metabolites.
In another aspect, there is provided use of Synechocystis sp. bacteria for the production of secondary metabolites, wherein the Synechocystis sp. are transformed with one or more of a peptide synthetase gene, a polyketide synthase gene or a fatty acid synthase gene required for production of secondary metabolites, and the Synechocystis sp. bacteria are cultured under conditions suitable for the expression of the one or more genes required for production of the secondary metabolites.
In one embodiment, the Synechocystis sp. bacteria is Synechocystis sp. PCC6803. In one embodiment the Synechocystis sp. bacteria express an exogenous PPT. In one embodiment the exogenous PPT is stably integrated into the Synechocystis sp. genome. In one embodiment, the exogenous PPT is a cyanobacterial PPT, such as a PPT from Nodularia spumigena. In one embodiment, the PPT is from Nodularia spumigena NSOR10. More preferably, the PPT comprises the amino acid sequence as set forth in SEQ ID NO: 1, or a variant of fragment thereof.
In another aspect, there is provided a phosphopantetheinyl transferase comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.
In another aspect, there is provided a phosphopantetheinyl transferase comprising the amino acid sequence set forth in SEQ ID NO: 2, or a variant or fragment thereof.
In one embodiment, the phosphopantetheinyl transferase encoded by the amino acid set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a functional variant of fragment thereof is provided in a vector. The vector may be provided in a host cell. The host cell may be a Synechocystis sp. host cell, such as Synechocystis sp. PCC6803.
Also provided are vectors comprising sequences of the invention, and host cells comprising the sequences and vectors of the invention. The invention also provides pharmaceutical compositions comprising at least one secondary metabolite prepared in accordance with the invention, together with a pharmaceutically acceptable carrier, excipient, adjuvant or vehicle.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing PPT activation of apo-carrier proteins. A PPT catalyzes the nucleophilic attack of the hydroxyl side chain of the conserved carrier protein serine residue on the 5′-β pyrophosphate linkage CoA. This causes the transfer of the phosphopantetheinyl moiety of CoA to the side chain of the conversed serine residue, converting the carrier protein from an inactive apo-form to an active holo-form (shown on the right of FIG. 1).
FIG. 2 is a table listing the source of cyanobacterial PPT sequences utilised by the inventors. Cyanobacterial PPTs were obtained from published database sequences (genome), or amplified from degenerate primers. N/a (not applicable)—refers to sequences obtained from online genome databases.
FIG. 3 shows a box shade amino acid sequence alignment of representatives of the “Sfp-like” PPT family. Black shading indicates identical residues while the grey shading indicates similar residues. Two subfamilies are shown. F/KES (top alignment) and W/KEA (lower alignment) sequences are separated and a consensus (cons) line is shown beneath. Sequences include; Pse, Pseudomonas aeruginosa, AAG04554; Xan, Xanthomonas albicans, AAG28384; Vib, Vibrio cholerae, AAD48884; Pho, Photorhabdus luminescens, AAK16071; Bac, Bacillus subtilis, P39135; Syn, Synechocystis PCC6803, BAA10326; Cae, Caenorhabdus, elegans, A89451; Dro, Drosphilia melanogaster, AAM12253. PPT motifs are boxed and numbered, including 1* as described. Numbering for Sfp from Bacillus subtilis is shown in brackets.
FIG. 4 is a phylogenetic tree diagram showing analysis of a selection of Sfp-like PPTs. The tree shows AcpS and Sfp-like PPT clades with accession numbers given in parenthesis. Significant bootstrap data (over 500 out of 1000 repeats) are displayed. E. coli AcpS was chosen as an outgroup for the Sfp-like PPT Glade. Two subgroups were observed and distinguished as the W/KEA and FACES clades respectively. Letters/symbols in superscript refer to PKS biosynthesis (P); NRPS biosynthesis (N); hybrid PKS/NRPS biosynthesis (H); siderophore biosynthesis (S); sequences obtained through the translation of contiguous sequences from unfinished genome projects (*); Sfp-like PPTs found in genomes without an AcpS (A−); cyanobacterial PPTs associated with heterocyst glycolipid synthesis (HET); and PPTs associated with lysine biosynthesis (L).
FIG. 5 is a photograph showing Synechocystis sp. PCC6803 Sppt knockout transformants on BGTS plates with chloramphenicol. FIG. 5A shows colonies of Synechocystis sp. PCC6803 wildtype Sppt knockouts; FIG. 5B shows colonies of Synechocystis sp. PCC6803 wildtype chloramphenicol resistance plasmid positive control; FIG. 5C shows colonies of N. punctiform phosphopantetheinyl transferase complemented (ΔNsPPT+) Sppt knockouts; FIG. 5D shows a ΔNsPPT+ negative control which did not produce any colonies.
FIG. 6 is a photograph of a gel used for electrophoresis of PCR screening products of Sppt disrupted and NsPPT-complemented Synechocystis sp. PCC6803 strains. Lane M, molecular weight marker; Lane 1, Synechocystis sp. PCC6803 WT Sppt gene; Lanes 2-4, Synechocystis sp. PCC6803 wildtype (WT) Sppt knockout heterozygous mutant with repeated subculturing on selective media; Lane 5, ΔNsPPT+ complemented Sppt gene; Lanes 6-7, ΔNsPPT+ complemented Sppt heterozygous mutant with repeated subculturing on selective media; Lane 8, ΔNsPPT+ fully segregated Sppt knockout mutant; Lane 9, negative PCR control; Lane 10, knockout plasmid, pGCSlr, with chloramphenicol cassette inserted into Sppt gene. Arrows indicate segregation of Sppt into chromosomal copies of the genome with repeated subculturing.
FIG. 7 is a photograph of an SDS-PAGE gel of purified Synechocystis sp. PCC8603 phosphopantetheinyl (sPPT) (FIG. 7A) and Synechocystis sp. PCC8603 fatty acid synthesis carrier protein (SACP) (FIG. 7B). M: Broadrange and Prestained Markers (New England Biolabs).
FIG. 8 shows mass spectra of SACP (FIG. 8A) and SACP after a phosphopantetheinylation assay with Sppt (FIG. 8B).
FIG. 9 provides graphs showing the enzymatic characterisation of Sppt with respect to SACP3. FIG. 9A shows the effect of pH on phosphopantetheinyl transferase activity as measured by HPLC assay. FIG. 9B shows the effect of CoA, the 4′-phosphopantetheinyl group donor, on Sppt activity.
FIG. 10 shows mass spectra of N. punctiforme ATCC29133 aryl carrier protein (NpArCP) control (FIG. 10A), NpArCP after incubation with Sppt (FIG. 10B), M. aeruginosa PCC7806, peptidyl carrier protein (MPCP) control (FIG. 10C); and MPCP after incubation with Sppt (FIG. 10D).
FIG. 11 shows mass spectra of N. punctiforme ATCC29133 acyl carrier protein (NpACP) control (FIG. 11A), NpACP with Sppt (FIG. 11B), Bacillus brevis ATCC8185 nonribosomal peptide synthetase peptidyl-carrier protein (TycPCP) control (FIG. 11C) and TycPCP after incubation with Sppt (FIG. 11D).
FIG. 12 is a table showing phosphopantetheinylation of non-cognate carrier proteins by the Synechocystis sp. PCC6803 PPT, Sppt. * Holo-CP was detected by the mass addition of a phosphopantetheinyl moiety (340 Da) via mass spectrometry. † The % holo-CP was estimated by comparison of holo- and apo-CP abundance in mass spectra. ND: not detected.
FIG. 13 is a phylogenetic tree diagram showing analysis of cyanobacterial PPTs. Accession numbers given in parenthesis, and underlined sequences indicate those isolated during this study. Significant bootstrap data (greater than 500 out of 1000 repeats) is shown. The PPT from the green sulphur bacterium Chlorobium tepidium was chosen as an outgroup. Distinct phylotypes are observed and depicted as subgroup A (associated with heterocyst forming cyanobacteria) and B respectively.
FIG. 14 shows a boxshade amino acid alignment of PPT representative sequences. The NhcS PPT from N. spumigena NSOR10 was aligned with the all5359 from Nostoc sp. PCC7120 (P37695), slr0495 from Synechocystis sp. PCC 6803 (BAA10326) and Sfp from B. subtilis (P39135). NhcS numbering is shown and percentage similarity to NchS is displayed in brackets. PPT motifs are boxed and numbered.
FIG. 15 is a diagram showing a comparison of the hetMNI loci from Nostoc punctiforme ATCC29133 and Nodularia spumigena NSOR10. Arrows indicate gene direction. A partial segment of the 1520 base pair (bp) hetM sequence is depicted by the broken arrow.
FIG. 16 shows N. spumigena NSOR10 southern hybridisation data. FIG. 16A is a photograph of a gel showing of southern hybridisations of PPT probes to N. spumigena NSOR10. A “+” indicates a band was detected by chemiluminescence, the “−” indicates no bands were visible. Positive controls utilised linearised plasmid DNA. FIG. 16B is a table summarising the results of the southern hybrisation experiments. Bracketed numbering in the table shown in FIG. 16B corresponds to the lanes shown in the gel of FIG. 16A. Slr0495 refers to the PPT (BAA10326) from Synechocystis sp. PCC 6803, Nppt (ZP—00110892) refers to a PPT from N. punctiforme ATCC29133 and nchS is the PPT found in N. spumigena NSOR10.
FIG. 17 shows spectra derived from nanospray mass spectrometry of aryl carrier protein (ArCP), peptidyl carrier protein (PCP) and acyl carrier protein (ACP) pantetheinylation. FIG. 17A: mass spectrum of Arcp1. FIG. 17B: Mass spectrum of pantetheinylated Arcp after incubation with NhcS. FIG. 17C: mass spectrum of Mpcp2. FIG. 17D: mass spectrum of pantetheinylated Mpcp after incubation with NhcS. FIG. 17E: mass spectrum of Nppcp3. FIG. 17F: mass spectrum of pantetheinylated Nppcp after incubation with NhcS.



DEFINITIONS
In the context of this specification, the term “comprising” means “including principally, but not necessarily solely”. Furthermore, variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings.
Throughout this specification, reference to “a” or “one” element does not exclude the plural, unless context determines otherwise. For instance, reference to “a nucleic acid construct” should not be read as excluding the possibility of multiple copies of such nucleic acid constructs.
The term “at least one” when used in the context of a group of selectable elements includes any and all members of the group individually selected and includes any combination of the members of the group. Similarly, the term “at least two” when used in the context of a group of selectable elements includes any selection of two or more members of the group in any combination.
As used herein, a “bioactive compound” is a compound which influences the biological structure, function, or activity of a cell or tissue of a living organism. A bioactive compound may, in general, elicit a biological response or effect or activity within the targeted end-user. For example, a bioactive compound may have a beneficial effect on the metabolism of a human, plant or animal, and/or provide therapeutic and/or cosmetic effects. Examples of bioactive compounds include, but are not limited to anti-viral agents, anti-fungal agents, anti-cancer agents, immunomodulatory agents, immunosuppressive agents, anaesthetics, analgesics, antitumour products, antibiotics, anti-cholesterolemics, anti-parasitic agents, veterinary therapies, agrochemicals, cosmetic agents, polynucleotides, proteins, peptides, polysaccharides, hormones, drugs, vitamins, steroids, anti oxidants, anti-inflammatory agents, moisturizers, carotenoids, UV absorbing agents, UV protecting agents and the like.
To the extent that it is permitted, all references cited herein are incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to methods for producing secondary metabolites, and in some embodiments to secondary metabolites produced by the methods of the invention. Despite having a null background and not naturally encoding either nonribosomal peptide synthetase or polyketide synthase genes, cyanobacteria Synechocystis sp. can produce secondary metabolites when transformed with a peptide synthetase, polyketide synthase and/or fatty acid synthase gene. Consequently Synechocystis sp. provides excellent potential as a heterologous host in which to produce secondary metabolites.
In one aspect, there is provided a method for the production of secondary metabolites, the method comprising transforming Synechocystis sp. bacteria with one or more of a peptide synthetase gene, a polyketide synthase gene or a fatty acid synthase gene required for production of secondary metabolites, culturing the Synechocystis sp. bacteria under conditions suitable for the expression of the one or more genes required for production of the secondary metabolites and purifying the secondary metabolites from the bacteria. The peptide synthetase gene may be a non-ribosomal peptide synthetase gene.
The Synechocystis sp. bacteria may be selected from any suitable species or strain, or derivative thereof. For example, the Synechocystis sp. bacteria may be strains PCC6301, 6307, 6701, 6906, 7008, or 9632 (see Archives of Microbiology, Christiansen et al. 2001). In a particular embodiment, the Synechocystis sp. is PCC6803 (Pasteur Culture Collection, Paris). It is envisaged that the method may be suitable for production of any secondary metabolites such as a peptide or polyketide, fatty acid or a derivative thereof. The peptide may be a non-ribosomal peptide. The secondary metabolites may further comprise an anti-microbial agent, an anti-viral agent, an anti-fungal agent, an anti-cancer agent, an immunosuppressive agent, an anaesthetic or an analgesic, antitumour products, antibiotics, anti-cholesterolemics, anti-parasitic agents, veterinary therapies and agrochemicals and cosmetics. Specific examples of such agents include, but are not limited to vancomycin, microcystin, nodularin, saxitoxin, cyclosporin, and penicillin. The Synechocystis sp. bacteria may be selected from any suitable species or strain, or derivative thereof. For example, the Synechocystis sp. bacteria may be strains PCC6301, 6307, 6701, 6906, 7008, or 9632 (see Archives of Microbiology, Christiansen et al. 2001). In a particular embodiment, the Synechocystis sp. is PCC6803 (Pasteur Culture Collection, Paris). It is envisaged that the method may be suitable for production of any secondary metabolites such as a peptide, polyketide, fatty acid or derivatives or combinations thereof. The peptide may be a non-ribosomal peptide. The secondary metabolites may further comprise an anti-microbial agent, an anti-viral agent, an anti-fungal agent, an anti-cancer agent, an immunosuppressive agent, an anaesthetic or an analgesic, antitumour products, antibiotics, anti-cholesterolemics, anti-parasitic agents, veterinary therapies and agrochemicals and cosmetics. Specific examples of such agents include, but are not limited to vancomycin, microcystin, nodularin, saxitoxin, cyclosporin, and penicillin.
The peptide synthetase gene, polyketide synthase gene and/or fatty acid synthase gene used to transform Synechocystis sp. bacteria may be derived from any suitable source. For example, peptide synthetase, polyketide synthase and fatty acid synthase genes may be derived from various other bacteria and/or fungi. Examples of potential organisms from which the genes may be derived include, but are not limited to microalgae such as rhodophytes, chlorphytes, chromophytes and cyanobacteria (e.g. Chroocales, Chamaesiphonales, Nostcales, Nostocacaea, Rivulariaceae, Scytonemataceae, Stigonematales), Streptomyces, Bacillus, and various marine bacteria including as well as microbial symbionts of animals and plants.
By way of a non-limiting example only, suitable nucleotide sequences of peptide synthetase, polyketide synthase and/or fatty acid synthase genes and their corresponding protein sequences may be obtained from a cyanobacterial source, such as Sorangium cellulosum (obtainable through GenBank accession number NC—010162), Acaryochloris marina MBIC11017 (obtainable through GenBank accession number CP000843), Synechoccus sp. PCC 7002 (obtainable through GenBank accession number CP000951), Nodularia spumigena CCY 9414 (obtainable through GenBank accession number NZ_AAVW00000000), Fulvimarina pelagi HTCC2506 (obtainable through GenBank accession number AATP01000000) and Synechococcus sp. WH 5701 (obtainable through GenBank accession number NZ_AANO00000000).
By way of an additional non-limiting example, suitable nucleotide sequences of peptide synthetase, polyketide synthase and/or fatty acid synthase genes and their corresponding protein sequences may be obtained from Streptomycetaceae such as Mycobacterium ulcerans Agy99 (obtainable through GenBank accession number NC—008611), Streptomyces avermitilis MA-4680 (obtainable through GenBank accession number NC—003155), Mycobacterium ulcerans Agy99 4680 (obtainable through GenBank accession number CP000325), Mycobacterium tuberculosis H3Rv (obtainable through GenBank accession number NC—000962) and Streptomyces coelicolor A3 (2) (obtainable through GenBank accession number NC—003888).
By way of a further non-limiting example, suitable nucleotide sequences of peptide synthetase, polyketide synthase and/or fatty acid synthase genes and their corresponding protein sequences may be obtained from Bacillus sp. such as Bacillus weihenstephanensis KBAB4 (obtainable through GenBank accession number NC—010184), Bacillus amyloliquefaciens FZB42 (obtainable through GenBank accession number NC—009725) and Corynebacterium glutamicum ATCC 13032(obtainable through GenBank accession number NC—003450).
By way of a further non-limiting example, a suitable polyketide synthase gene or protein sequence may be derived from Aspergillus niger and/or Aspergillus nidulans (wA gene, Mayorga et. al., Genetics 126 (1990) 73-79 (obtainable through GenBank accession number X65866).
The skilled addressee will understand that each of the accession numbers stated herein incorporate by reference all sequences and accession numbers obtainable through the stated number, such as specifically identified nucleotide and protein sequences of peptide synthetase, polyketide synthase and/or fatty acid synthase genes.
The Synechocystis sp. bacteria may be transformed or transfected with one or more of a peptide synthetase gene, a polyketide synthase gene or a fatty acid synthase gene using any suitable method. Such methods are generally known in the art and are described in, for example, Molecular Cloning: A Laboratory Manual (Joseph Sambrook, David W Russell, 3rd. Edition, Cold Spring Harbour Press 2001), Current Protocols in Molecular Biology (Ausubel F. M. et al. (Eds), John Wiley and Sons, Inc 2007), Molecular Cloning (Maniatis et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982) and Current Protocols in Microbiology (Coico et al (Eds), John Wiley and Sons, Inc, 2007), the entire contents of which are incorporated herein by cross-reference.
In particular embodiments, the peptide synthetase, polyketide synthase or fatty acid synthase gene may be cloned into a vector. The vector may be a plasmid vector, a viral vector, a phosmid, a cosmid or any other suitable vehicle construct for the insertion of foreign sequences, their introduction into cells and the expression of the introduced sequences. The vector may be an expression vector comprising expression control and processing sequences such as a promoter, an enhancer, polyadenylation signals and transcription termination sequences.
The construct may also include a selectable marker, for example, an antibiotic-resistance gene such as chloramphenicol or tetracycline. Genetic material for insertion into the construct may be generated, for example, by performing polymerase chain reaction (PCR) assays on target DNA. The resulting nucleic acids may then be inserted into the construct, for example, by restriction-ligation reactions or by the TA cloning method.
Suitable methods for the introduction of vector constructs and other foreign nucleic to acid material into Synechocystis sp. bacteria are generally known in the art, and described, for example, in Current Protocols in Molecular Biology, Ausubel et al. (Eds), New York: John Wiley & Sons, 2007) and Molecular Cloning: A Laboratory Manual, (Sambrook et al. 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). By way of example, Synechocystis sp. may be transformed by the “heat shock” method. Under this method the cells are chilled in the presence of divalent cations such as Ca2+, which causes cell wall permeability. Cells are incubated on ice with the construct and briefly heat shocked (e.g. at 42° C. for 0.5-2 minutes) causing the vector construct to enter the cell. Alternatively, Synechocystis sp. may be transformed with the vector construct by electroporation, a method involving briefly shocking the cells with an electric field causing the cells to briefly develop holes through which the construct may enter the cell. Natural membrane-repair mechanisms rapidly close these holes after the shock.
The peptide synthetase, polyketide synthase or fatty acid synthase gene/s may be inserted into the Synechocystis sp. genome by means of one or more transposons, or other mobile elements. These have been shown to mobilise large DNA fragments up to 59 kb in size. Mobilisation of transposons is mediated by transposases, usually resulting in the insertion of the DNA into target sequences in the genome. Putative transposases have been found to be associated with several biosynthetic gene clusters such as the microcystin and nodularin biosynthesis gene clusters. The ability of transposons to mobilise large gene clusters provides a DNA transfer system suitable for the transfer engineered biosynthetic gene clusters into null hosts for the expression of secondary metabolites.
Following entry of the construct into the cell, the Synechocystis sp. bacteria may be cultured under conditions suitable to facilitate reproduction. Methods for the culture of bacteria are well known in the art and described in, for example, Current Protocols in Microbiology, (Coico, et al. (Eds), John Wiley & Sons, Inc., 2007). The culture may be performed in medium containing a substrate which facilitates the identification of transformed strains, for example, an antibiotic such as chloramphenicol, kanamycin or tetracycline.
Transformed Synechocystis sp. strains may be selected and propagated. For example, if the target vector contains one or more selectable markers, the transformed Synechocystis sp. bacterial cells may be identified by expression of the marker or markers. Using the example of a drug resistance gene such as a chloramphenicol resistance gene, Synechocystis sp. transformants that grow in the selection media containing chloramphenicol can be identified as transformants. In the case of Synechocystis sp. transformants expressing more than one selectable marker, double transformants may be identified by the ability to grow in the selection media containing multiple selection determinants.
For the purpose of exemplification only, a specific method that may be utilised to transform Synechocystis sp. bacteria is as follows. Synechocystis sp. bacteria may be transformed with a gene deletion construct comprising sequences that are homologous to Synechocystis DNA sequences flanking the peptide synthetase, polyketide synthase or fatty acid synthase gene/s of interest. DNA is added to a 0.1-0.5 ml volume of concentrated Synechocystis culture, followed by incubation for 1-6 hours and plating out. After 20-24 hours selective conditions can be applied. Colonies of transformants come up in about one week, and may then be restreaked on plates with increasingly higher concentration of the antibiotic for which a resistance marker has been introduced. If a pure mutant phenotype is required, all wild-type genome copies need to be replaced. Two factors are important to readily obtain segregation: (1) a gradual increase in antibiotic selection pressure, and (2) selection of growth conditions under which the mutant phenotype has a competitive advantage or is not very much impaired when compared with the wild type. A convenient and rapid method to screen for segregation of wild-type and mutant genotypes is to prepare DNA from propagated Synechocystis transformants and to amplify the region of the mutation by PCR.
It will be appreciated that in accordance with the methods disclosed herein, the Synechocystis sp. bacteria may be transformed with multiple peptide synthetase, polyketide synthase and/or fatty acid synthase genes, allowing the generation of a biosynthetic gene cluster in the transformed Synechocystis sp. bacterial host. The biosynthetic cluster may comprise a hybrid of one or more peptide synthetase, polyketide synthase and/or fatty acid synthase genes. The polyketide synthase gene of the cluster or hybrid may be a non-ribosomal polyketide synthase gene.
Regulation of the expression of the peptide synthetase, polyketide synthase and/or fatty acid synthase gene sequences may be achieved by the use of regulatory sequences. For example, an expression vector may be used comprising expression control and processing sequences such as a promoter, an enhancer, polyadenylation signals and transcription termination sequences. Promoters may include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The regulatory sequence may be regulated by an external stimulus. For example, light-inducible promoters are particularly useful due to the photosynthetic nature of Synechocystis sp. bacteria. The present invention is not limited in this respect and various inducible promoters may be used as known to the person skilled in the art. Such promoters include promoters induced by low temperature and by the action of a chemical compound such as tryptophan-inducible promoters, salt-inducible promoters, nitrite-inducible promoters, tyrosine-inducible promoters, and arabinose-inducible promoters.
Synechocystis sp. bacteria transformed with one or more of a peptide synthetase gene, a polyketide synthase gene or fatty acid synthase gene may be screened for production of the secondary metabolites. The secondary metabolites may then be purified. Any suitable methods of screening or purification may be used, taking into account various factors such as structural, enzymatic, functional, size features of the desired secondary metabolite. Methods and assays suitable for screening of transformed Synechocystis sp. bacteria and the purification of secondary metabolites are known in the art, and are described, for example, in Current Protocols in Protein Science, Coligan et al., (Eds) John Wiley and Sons, Inc. 2007). The screening and purification step may comprise, for example, chromatography methods, accelerated solvent extraction, or mass spectrometry or a combination thereof. Chromatography methods may include, for example, reverse phase chromatography, normal phase chromatography, affinity chromatography, thin layer chromatography, counter current chromatography, ion exchange chromatography and reverse phase chromatography. Examples of other methods include precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation, isoelectric focusing, gel electrophoresis, selective precipitation techniques, and combinations of those and other techniques.
Secondary metabolites produced in accordance with the methods described herein may also be genetically engineered to contain various affinity tags or carrier proteins that aid purification. For example, the use of histidine and protein tags engineered into an expression vector comprising a peptide synthetase gene, a polyketide synthase gene and/or a fatty acid synthase gene may facilitate purification by, for example metal-chelate chromatography (MCAC) under either native and denaturing conditions. The purification of secondary metabolites may also be “scaled-up” for large-scale production purposes.
By way of exemplification only, a sample of Synechocystis sp. bacteria transformed with one or more peptide synthetase, a polyketide synthase and/or fatty acid synthase genes may be chemically screened by analysis using LC/MS coupled to UV photodiode array detection. Separation is performed and UV spectra are recorded and molecular weight information is obtained by MS with thermospray, continuous-flow fast atom bombardment, atmospheric pressure chemical ionization or electrospray ionization. Fragment information is obtained by tandem MS/MS or multiple stage MSn experiments while LC/NMR is used for confirmation of compound identity. Purification may also be performed by other methods known in the art such as solid phase extraction.
The skilled addressee will appreciate that the methods described herein are not limited by the methods of screening or purification described above and that any other method may be used to identify and/or isolate secondary metabolites derived from the transformed Synechocystis sp. bacteria.
In accordance with the methods disclosed herein, the secondary metabolite may be any compound derived from expression of the one or more peptide synthetase, polyketide synthase or fatty acid synthase genes used for transformation of the Synechocystis sp. bacterial host. The genes may be expressed alone or in combination. The expression of one or more of the peptide synthetase, polyketide synthase or fatty acid synthase genes may be combined to produce a hybrid product. Non-limiting examples of the secondary metabolites that may be produced in accordance with the methods disclosed herein are described in the Dictionary of Natural Products (Chapman & Hall, John Buckingham, Chapman & Hall/CRC, England CRC Press 1994), the entire contents of which are incorporated herein by cross-reference.
In certain embodiments, the secondary metabolite is a bioactive compound. Accordingly, the methods disclosed herein encompass the production of secondary metabolites including peptides, polyketides, fatty acids and hybrids or derivatives thereof that are bioactive compounds. In general, a bioactive compound is a compound which influences the biological structure, function, or activity of a cell or tissue of a living organism. The inventors have identified that in some species of Synechocystis sp., for example PCC6803, the production of secondary metabolites may be improved further through the use of exogenous/heterologous PPT. Preferably, the exogenous PPT is stably integrated into the Synechocystis sp. bacterial genome. For example, as shown herein, the Nodularia spumigena PPT is capable of being engineered to act in each of fatty acid synthesis, heterocyst glycolipid synthesis and biosynthesis of secondary metabolites. The inventors have demonstrated that the N. spumigena PPT phosphopantetheinylates carrier proteins from the microcystin synthetase gene cluster in M. aeruginosa PCC7806 and the nostopeptolide biosynthesis gene cluster in N. punctiforme. Other PPTs may be suitable for transformation into Synechocystis sp., bacterial hosts, examples of which are shown in but not limited to those PPTs provided in the figures herein.
Examples of secondary metabolites that may be produced by the methods disclosed herein include, but are not limited to amino acids, fatty acids, macrolides, amides and lipopeptides or hybrids or derivatives thereof.
Fatty acids that may be produced include, but are not limited to omega 3-polyunsaturated fatty acids such as eicosapentanoic acid (EPA).
Lipopeptides produced by the methods disclosed herein generally comprise one or more amino acid derived fragments linked to one or more fatty acid derived portions. The lipopeptide may be cyclic or linear. An example of a lipopeptide that may be produced is hapalosin, a compound which is known to reverse multi-drug resistance derived from P-glycoprotein. The lipopetides may be cytotoxic, antitumour, antiviral, antibiotic, antimalarial, antimycotic, multi-drug resitance reversing, antifeedants, herbicides, immunosuppressive agents, improved metabolism, cholesterol lowering effects, antioxidant properties.
Other non-limiting examples of smetabolites that may be obtained in accordance with the methods disclosed herein include the antibiotic erythromycin, the immunosuppressant FK506, and the antitumor compound epothilone. See also PCT patent publication No. 93/13663 (erythromycin); U.S. Pat. No. 6,303,342 B1 (epothilone); U.S. Pat. No. 6,251,636 B1 (oleandolide); PCT publication WO 01/27284 A2 (megalomicin); U.S. Pat. No. 5,098,837 (tylosin); U.S. Pat. No. 5,272,474 (avermectin); U.S. Pat. No. 5,744,350 (triol polyketide); and European patent publication No. 791,656 (platenolide), each of which is entirely incorporated herein by cross-reference.
Other non-limiting examples of metabolites that may be produced in accordance with the methods disclosed herein include cyanovirin-N,borophycin, cryptophycin, docosahexaenoic acid, β-Carotene, phycobiliprotein, phycocyanin, polysaccharides and protease inhibitors (e.g. micropeptins, aerugenosins, microginins, anabaenopeptins and microverdins)
Secondary metabolites produced in accordance with the methods disclosed herein may possess synergistic activity.
The PPTs disclosed herein include, but are not limited to those comprising the polypeptides set forth SEQ ID NO: 1 or SEQ ID NO: 2, or a variant or fragment thereof. The skilled addressee will appreciate that sequence variations, including both natural variations and engineered variations, may be made to the PPT-encoding polypeptides. Accordingly it will be appreciated that the sequences disclosed herein, including for example the PPT sequences provided in SEQ ID NOS: 1 and 2, encompass variants and/or fragments thereof.
The term “variant” as used herein refers to a substantially similar sequence. Polynucleotide and polypeptide sequence variants may share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference sequence. In general, polypeptide sequence variants possess qualitative biological activity in common. Polynucleotide sequence variants generally encode polypeptides which generally possess qualitative biological activity in common. Also included within the meaning of the term “variant” are homologues of polynucleotides and polypeptides of the invention. A polynucleotide homologue is typically from a different bacterial species but sharing substantially the same function or biological activity as the corresponding polynucleotide disclosed herein. A polypeptide homologue is typically derived from a different species but sharing substantially the same function or biological activity as the corresponding polypeptide disclosed herein. For example, homologues of the polynucleotides and polypeptides disclosed herein include, but are not limited to those from different species of cyanobacteria.
Further, the term “variant” also includes analogues of the polypeptides disclosed herein. A polypeptide “analogue” is a polypeptide which is a derivative of a polypeptide disclosed herein, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function. The term “conservative amino acid substitution” refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.
In general, the degree of sequence homology between the sequences of the invention and a “variant” can be determined conventionally using known computer programs include, including, but not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters.
The BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman to find the best segment of homology between two sequences (Advances in Applied Mathematics 2:482-489 (1981)). When using BESTFIT or any other sequence alignment program to determine the degree of homology between sequences, the parameters may be set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
GAP uses the algorithm described in Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP presents one member of the family of best alignments.
Another method for determining the best overall match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.
Also within the scope of the invention are fragments of the polypeptides disclosed herein. A polypeptide “fragment” is a molecule that comprises a constituent or is a constituent of a polypeptide disclosed herein or a variant thereof. Typically a polypeptide fragment possesses qualitative biological activity in common with the polypeptide of which it is a constituent. The polypeptide fragment may be between about 5 to about 1000 amino acids in length, between about 5 to about 750 amino acids in length, between about 5 to about 500 amino acids in length, between about 5 to about 270 amino acids in length, between about 5 to about 250, between about 5 to about 200 amino acids in length, between about 5 to about 150 amino acids in length, between about 5 to about 100 amino acids in length, between about 5 to about 50 amino acids in length, or between about 5 to about 10 amino acids in length. The fragment may be derived from the full-length PPT or alternatively may be synthesised by some other means, for example chemical synthesis.
Fragments of the polynucleotide sequences set forth herein are also included within the scope of the invention. A polynucleotide “fragment” is a polynucleotide molecule that encodes a constituent or is a constituent of a polynucleotide of the invention or variant thereof. Fragments of a polynucleotide do not necessarily need to encode polypeptides which retain biological activity. The fragment may, for example, be useful as a hybridization probe or PCR primer. The term “primer” as used herein means a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis. An “oligonucleotide” is a single-stranded nucleic acid typically ranging in length from 2 to about 100 bases. The precise length of a primer will vary according to the particular application, but typically ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template. Polynucleotide fragments may be derived from a polynucleotide of the invention. Alternatively, polynucleotide fragments, primers and oligonucleotide probes may be synthesized by some other means, for example chemical synthesis methods (see for example Narang S. A. et al. (1979) Meth. Enzymol. 68:90; Brown, E. L. et al. (1979) Meth. Enzymol. 68:109; and U.S. Pat. No. 4,356,270), Beaucage S. L. et al. (1981) Tetrahedron Letters, 22:1859-1862).
The skilled addressee will understand that the sequences disclosed herein and variants and fragments thereof may be used to screen other organisms or nucleic acid samples for the presence of useful PPTs.
Despite the absolute requirement for PPTs in a wide range of important and fundamental biosynthetic pathways, these enzymes have remained elusive due to their low sequence identity and lack of proximity to their respective biosynthetic clusters. This has hampered many efforts to express polyketide and non-ribosomal peptide products in heterologous host systems, largely due to the inability of the intrinsic E. coli PPTs to activate foreign substrates. This difficulty is further complicated by the observation that cyanobacterial biosynthetic gene clusters do not predominantly encode co-localised PPTs. For example, the Lyngbya majuscula gene clusters encoding barbamide, lyngbyatoxin and curacin A do not encode the essential PPT required for their synthesis.
The prototype PPT for the activation of secondary metabolite gene clusters is from Bacillus subtilis, Sfp. This PPT is required for the activation of carrier proteins incorporated within the biosynthetic pathway responsible for the production of surfactin. Members of this family are approximately 230 amino acids in length and are often found associated with the biosynthetic pathway in which they act. They display a broad range of specificities and the relaxed specificity of this family has been harnessed and manipulated in diverse applications, such as Sfp catalysed phagemid display and Sfp-labelling of carrier proteins.
Sequence alignment and phylogenetic analyses reveals that the PPT from Synechocystis sp. PCC6803 is closely related to the broad range PPT Sfp, and despite the lack of complex secondary metabolite production in Synechocystis sp PCC6803, the intrinsic PPT was predicted to be able to activate the carrier proteins of such metabolic pathways. The activity of the heterologously expressed Synechocystis sp. PPT Slr0495 has now been shown via nanospray mass spectrometry to comprise an additional mass of 355 Da corresponding to the phosphopantetheinyl arm of CoA when transferred to an acyl carrier protein. Preliminary experiments have shown Slr0495 to activate Bacillus subtilis peptidyl carrier proteins from a nonribosomal peptide synthetase, and Nostoc punctiforme PCC73102 acyl carrier proteins from a polyketide synthase.
Modules in peptide synthetases are several hundred amino acids long with the corresponding genes comprising the largest open reading frames known. Each module within a peptide synthetase contains core sequences responsible for amino acid recognition, modification and chain elongation. These core sequences result in a significant diversity of molecules of peptide synthetase origin. Possible modifications include activation, acyladenylation, thioesterification, methylation and racemisation of individual amino acid precursors. In addition, the final peptide may be linear, branched, cyclised or may be integrated with a polyketide side chain. To date more than 300 modified amino acids, including L- and D-forms, have been characterised arising from thiotemplate peptide formation. It has been shown that the genes involved in producing these large enzyme complexes are also modular and contain domains responsible for substrate recognition and modification. These operons encoding peptide synthetases may be manipulated to produce new novel peptides as has been shown for the genetic engineering of polyketides.
Peptide synthetase genes are coupled to strong promoters such as the PpsbA promoter, in specially tailored vectors for high expression. The inventors have developed and used such vectors for the heterologous expression of pili genes. Constructs are designed such that expression may also be regulated by a specific external stimulus, such as light intensity. Using the natural DNA uptake trait (transformability) of Synechocystis sp. PCC6803, these genetic constructs are introduced into the genome via homologous recombination. As the efficiency of this process is dependent on DNA insert size, optimization experiments are carried out to develop a protocol to enable the integration of various sized gene clusters encoding different compounds, which can range from about 10 kb to about 40 kb. Insertion sites target pili genes, which are responsible for both transformability and motility in this cyanobacterium. Insertion of genetic constructs into these sites render the strain non-motile and/or non-transformable, which serve as an intrinsic ‘reporter phenotype’ in addition to antibiotic selection. PCR and northern hybridizations are used in order to confirm stable integrants. Downstream experiments optimize expression of the newly integrated genes. Alternative strategies include the utilisation of phosmid based expression vectors and transposon-based gene transfer.
Transposons and other mobile elements have been shown to mobilise large DNA fragments up to 59 kb in size. Mobilisation of transposons is mediated by transposases, usually resulting in the insertion of the DNA into target sequences in the genome. Putative transposases have been found to be associated with several biosynthetic gene clusters such as the microcystin and nodularin biosynthesis gene clusters. Utilising the ability of transposons to mobilise large gene clusters, a DNA transfer system is suitable for the transfer engineered biosynthetic gene clusters into null hosts for the expression of novel compounds. The ability of the transposases to mobilise gene clusters is determined via activity assays, such as mating-out conjugations. Cyanobacterial transposases found to be active may be used to optimise a DNA transfer system for the introduction of heterologous DNA into the Synechocystis host.
Also provided herein is the use of Synechocystis sp. bacteria for the production of secondary metabolites, wherein the Synechocystis sp. are transformed with one or more of a peptide synthetase, a polyketide synthase or a fatty acid synthase gene required for production of secondary metabolites, and the Synechocystis sp. bacteria are cultured under conditions suitable for the expression of the one or more genes required for production of the secondary metabolites.
The Synechocystis sp. bacteria may be transformed with multiple peptide synthetase, polyketide synthase and/or fatty acid synthase genes, allowing the generation of a biosynthetic gene cluster in the transformed Synechocystis sp. bacterial host. The biosynthetic cluster may comprise a hybrid of one or more peptide synthetase, polyketide synthase and/or fatty acid synthase genes. The polyketide synthase gene of the cluster or hybrid may be a non-ribosomal polyketide synthase gene.
The Synechocystis sp. bacteria may be Synechocystis sp. PCC6803. The Synechocystis sp. bacteria may also expresses an exogenous PPT, which may be stably integrated into the Synechocystis sp. genome. The exogenous PPT may be a cyanobacterial PPT, and may be derived from Nodularia spumigena. In one embodiment, the PPT is from Nodularia spumigena NSOR10.
It will be understood that the description relating to the methods of producing secondary metabolites with transformed Synechocystis sp. bacteria provided herein are similarly applicable to the use of Synechocystis sp. bacteria for the production of secondary metabolites.
The present invention also includes pharmaceutical compositions comprising at least one secondary metabolite produced according to the invention, together with a pharmaceutically acceptable carrier, excipient, adjuvant of vehicle.
In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.
These compositions can be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. More preferably administration is to by the parenteral route.
The carriers, diluents and adjuvants must be “acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.
The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
In order that the invention may be more clearly understood preferred forms will be described with reference to the following examples. It will be understood that these examples are intended to be illustrative of and not limiting to the invention.
EXAMPLES
Example 1
Phylogenetic Analyses
(1) Creation of the sfp-Like Database
Sequences were collected by word-based ENTREZ searches, using any combination of words relating to phosphopantetheinyl transferases, e.g., Acyl carrier protein synthetase, Sfp, pantetheine. The output data of BLAST (Basic Local Alignment Search Tool) PSI-protein searches were utilised to extend the sequence set, and identify sequences that were not recognised through word-based searches. Available (completed and partial) genomes from the National Centre for Biotechnology Information (NCBI), Joint Genome Institute (JGI) and Cyanobase (www.kazusa.or.jp/cyano/cyano.html) were subjected to multiple BLAST screens with a variety of known PPT sequences from different bacterial genera.
(2) Multiple Alignments and Phylogenetics
The sequences were aligned using a program pileup from GCG and the multiple-sequence alignment tool from CLUSTAL X. The Neighbour Joining (N-J) method of Saitou and Nei was used to generate trees in CLUSTAL X. The data sets were bootstrapped (1000 resampling events), and the resulting trees were visualised using NJ plot and Treeview X. Alignments were created for publication via boxshade.
(3) Extraction, Amplification and Sequencing
Cyanobacterial strains (FIG. 2) were obtained from the UNSW Cyanobacterial Culture Collection. Genomic DNA was extracted from cyanobacterial cultures as previously described (Neilan et al., Applied and Environmental Microbiology (1995) “Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus” 61: 3875-83). Amplification of cyanobacterial PPT fragments was performed using the primers PPTF [5′-CAGGAGTAYGGNAARCC-3′] (SEQ ID NO: 3) and PPTR [5′-TTCTCGATRTCDATNCC-3′ (SEQ ID NO: 4) that were specifically designed to correspond to motifs 3 and 4 respectively. Heterocyst PPT sequences were amplified utilising PPT2F [5′-GCCCGTGGTAAACAAATATTAG-3′] (SEQ ID NO: 5) and PPT2R [5-′GCCTCTTTACAAGTCCA-3′] (SEQ ID NO: 6). Thermal cycling was performed in a GeneAmp PCR 2400 thermocycler (Perkin Elmer, Norwalk, Conn.) as previously published (Neilan et al., Applied and Environmental Microbiology (1995), Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. 61:3875-83) with an annealing temperature of 45-55° C. depending on the primer pair utilized. Automated sequencing was performed using the PRISM Big Dye cycle sequencing system and a model 373 sequencer (Applied Biosystems Inc., USA). Sequence analysis was performed using Applied Biosystems Autoassembler software.
(4) Sequence Conservation within the Sfp-Like Family
The resulting alignments reveal additional examples of sequences diverging from the amino acids considered critical for PPT function. For example, it is noted that H90 from Motif 2, associated with binding the 3′-phosphate of CoA, is absent in several sequences including all Methanosarcina, some Staphylococcus sp., and a Streptomyces sp. An alignment of Sfp-like PPT representatives is shown in FIG. 3. Two distinct subfamilies were observed. Motif 4 of the first subfamily is depicted as F(S/C)KES (hereafter referred to as “the F/KES subfamily”). The second subfamily included the Sfp sequence from B. subtilis. This group displays the peptide sequence W(T/C)KEA as motif 4 (hereafter referred to as “the W/KEA subfamily”).
(5) Phylogenetic Analysis
The phylogenetic trees presented show distinct, novel clades and support the subfamilies observed in alignment analyses. The Sfp-like PPT subfamilies were separated and supported by bootstrap data (FIG. 4). The AcpS PPT was utilized to serve as an outlier.
Representatives that harbour multiple Sfp-like PPTs were shown to have PPTs falling within both F/KES and W/KEA branches of the phylogeny, including Streptomyces, Escherichia, Microbacterium, Pseudomonas, Xanthomonas, and Salmonella (FIG. 4). Sfp-like PPTs from organisms without an AcpS-like PPT are also present in both clades. For example, Pseudomonas aeruginosa and Haemophilus influenzae (AAC21831) were observed in the F/KES and W/KEA subfamilies respectively. PPTs found in hybrid (PKS-NRPS) biosynthetic clusters are also represented in both subfamilies.
The F/KES subfamily encompassed the majority of PPTs associated with peptide synthetases and siderophore synthesis, including all enterobactin EntD enzymes and the subset of Streptomyces PPTs described in Weissman et al., (2004) Identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea”, ChemBioChem 5:116-25.
The second subfamily of W/KEA subgroup included the B. subtilis PPT Sfp. This phylotype included the diverse heterocyst glycolipid biosynthetic PPTs the lysine biosynthesis PPTs and invertebrate and eukaryotic PPT sequences. PPT enzymes involved in polyketide biosynthesis are predominant in the W/KEA group, such as MupN (AAM12928) associated with mupirocin production in Pseudomonas fluorescens. 
Example 2
Gene Disruption of Synechocystis PPT (Sppt)
Sppt was insertionally inactivated to determine whether it was the only enzyme capable of phosphopantetheinylation in Synechocystis sp. PCC6803. Genomic DNA for PCR amplification was extracted as previously described (Moffitt, M. C. and B. A. Neilan. 2004. Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Appl Environ Microbiol 70:6353-62). All restriction enzymes were supplied by New England Biolabs (Ipswich, Mass.) or Promega (Madison, Wis.).
A 2.5 kb fragment including Sppt (slr0495, Swissprot accession number Q55185) was amplified with the primers sirup (5′-GTAAACTCCATTAACGCTGGC-3′) (SEQ ID NO: 7) and slrdn (5′-GGTGCAAATCCGTTACATGGA-3′) (SEQ ID NO: 8). This fragment was cloned into pGEM-T-Easy (Promega) and digested with the restriction enzyme AvaI. A chloramphenicol (ChlR) resistance cassette was ligated into this site and the resulting plasmid, pGCSlr, was naturally transformed into Synechocystis sp. PCC6803 to insertionally inactivate Sppt using methods described in Eaton-Rye, J., 2004 The construction of gene knockouts in the cyanobacterium Synechocystis sp. PCC 6803, Methods Mol. Biol.; 274:309-24. The Synechocystis sp. PCC6803 wildtype (WT) was also transformed with a chloramphenicol resistance knockout plasmid as a positive control for strain viability. Briefly, approximately 2 ml Synechocystis sp. PCC6803 cells were centrifuged and washed in BG-11 media. After resuspension in 300 μA BG-11, 3 μg ml−1 plasmid was added and the cells were incubated at 25° C. in 30 μmol m−2 sec−1 constant light for 6 hours. The cells were spread onto sterile nitrocellulose filters (0.45 μm, Millipore, Billerica Mass.) on non-selective BGTS plates (BG-11, 10 mM TES, 0.3% sodium thiosulfate, 1% agar). After 36 hours, the filters were transferred to selective BGTS plates with 10 μg ml−1 chloramphenicol.
Initial transformants were visible on selective chloramphenicol plates and were subcultured repeatedly under increasing selection (FIG. 5A). However, PCR screening of these transformants revealed that the insertionally inactivated Sppt gene could not completely segregate throughout the multiple genome copies (FIG. 6, lane 4). In contrast, Sppt was effectively disrupted in the NsPPT-complemented strain (ΔNsPPT+), (FIG. 6, lane 8). Transformation of the ΔNsPPT+ strain with ddH2O showed that this strain was not resistant to chloramphenicol before gene disruption with pGCSlr (FIG. 5D).
The lack of complete segregation of Sppt throughout the multiple chromosomes suggests that Sppt is essential for phosphopantetheinylation in fatty acid synthesis. Complementation with the broad range activity NsPPT from Nodularia spumigena NSOR10 allowed for the successful disruption of Sppt. This confirms Sppt is required for primary metabolism in Synechocystis sp. PCC6803. It also shows, for the first time, the ability of NsPPT to activate non-cognate carrier proteins in vivo.
Example 3
Expression and Purification of Sppt and SACP
Sppt and SACP were expressed and purified to determine the activity of Sppt via phosphopantetheinylation assays. Sppt was amplified by the primer pair SynPpF (5′-TGTTTAAACTCACCTG-3′) (SEQ ID NO: 9) and SynPpR (5′-CCCAAGGTTACGAAAC-3′) (SEQ ID NO: 10). The resulting PCR product was cloned into pGEM-T (Promega), digested with the restriction enzymes SacI and EcoRV and ligated into pET30 (Novagen, San Diego, Calif.). The Synechocystis sp. PCC6803 fatty acid synthesis (FAS) ACP, designated SACP (AcpP BA000022), was amplified by the primer pair ssl2084Efw (5′-GGAATTCTGAATCAGGAAATTT-3′) (SEQ ID NO: 11) and ssl12084 Hry (5′-CTCGGCTCCAAAAAGCTTTGGG-3′) (SEQ ID NO: 12), digested with the restriction enzymes EcoRI and HindIII, and cloned into pET30. A truncated SACP, SACP3, was cloned and expressed for HPLC kinetic analysis using the primers 2084NdeF (5′-GCATATGAATCAGGAAATTT-3′) (SEQ ID NO: 13) and 2083XhoR (5′-CCTCGAGTAATTTACTTTCGATATGCTCAAC-3′) (SEQ ID NO: 14).
The constructs were transformed into BL21(DE3) Rosetta (Novagen) for expression. Sppt was expressed at 37° C., with an induction concentration of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), for 3 hours. SACP and SACP3 were expressed at 37° C. with 1 mM IPTG for 20-40 min. Cultures were harvested at 5,000×g and cell pellets were stored at −80° C. Cell pellets were resuspended in Hepes buffer (Hepes 50 mM, NaCl 150 mM pH 7.4) and passaged three times through a cooled French pressure cell at 200 psi (Paton Scientific, Victor Harbor, South Australia).
After centrifugation at 20,000×g for 30 min, proteins were purified using HiTrap affinity columns (Amersham Biosciences, Piscataway, N.J.) using a 300 mM imidazole gradient as previously described in Copp J. N., Roberts A. A., Marahiel M. A., Neilan B. A., 2007, Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10. J. Bacteriol.:189(8):3133-9. Fractions containing the purified proteins were desalted with Amicon centrifuge columns (Millipore) and snap frozen with 8% glycerol for storage at −80° C.
The protein concentrations were determined based on the calculated extinction coefficients: SACP 6,970 cm−1M−1, Sppt 53,110 cm−1M−1. Due to the absence of aromatic amino acids in SACP3, the concentration of this protein was established using molecular imaging (FUJIFILM LAS-3000) compared to a standard curve of known BSA concentration.
SACP was expressed and purified as an approximately 17 kDa protein, as determined by SDS-PAGE. However, mass spectrometry analysis revealed this protein to be 14.7 kDa, which corresponded to the theoretical mass. Unusual migration of ACPs, as visualised by SDS-PAGE, has been previously described in the expression of a Streptomyces sp. PKS ACP (Crosby et al. (9995) Polyketide synthase acyl carrier proteins from Streptomyces: expression in Escherichia coli, purification and partial characterisation, Biochim Biophys Acta. 16:32-42). Mass spectrometry analysis of purified SACP3 showed species present in the apo-, holo- and dimerised holo-form (data not shown). The appearance of phosphopantetheinylated SACP results from the partial conversion of apo- to holo-SACP by the intrinsic PPTs within the E. coli host. Phosphopantetheinylation activity of Sppt was confirmed with the further conversion of apo- to holo-SACP in a 10 min assay (FIG. 8B).
Example 4
Sppt in vitro Phosphopantetheinylation of SACP
Sppt activity towards SACP was determined in a 10 min phosphopantetheinylation assay at 37° C. as previously described in Copp J. N., Roberts A. A., Marahiel M. A., Neilan B. A., 2007, Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10. J Bacteria 189(8):3133-9. Briefly, the 100-200 μl reactions comprised of 50 mM Tris-HCl pH 7.4, 12.5 mM MgCl2, 0.5 mM CoA, 2 μM DTT, 100 μM carrier protein and 300 nM Sppt. Reactions were incubated for 10 min at 37° C., terminated by the addition of 1 ml trichloroacetic acid and precipitated overnight at −20° C. before centrifugation for 15 min at 18,000×g. Samples were dissolved in water:acetonitrile:formic acid (50:49:1). Phosphopantetheinylation was observed by an increase in mass of 340 Da as visualised by electrospray ionisation mass spectrometry (ESI-MS). Spectra were acquired using an API Qstar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems) as previously described in Copp J. N., Roberts A. A., Marahiel M. A., Neilan B. A., 2007, Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10. J Bacteriol.: 189(8): 3133-9.
Although sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis shows the purified SACP at an observed molecular weight of 17 kDa (FIG. 7), mass spectrometry revealed the purified protein to be 14.594 kDa (FIG. 8A), corresponding to a loss of the N-terminal methionine residue when compared to the theoretic mass of 14.73 kDa. The ratio of holo- to apo-carrier protein after heterologous expression in E. coli was analysed by mass spectrometry. SACP was partially phosphopantetheinylated after heterologous expression in E. coli, with a holo:apo ratio of 70:30. After in vitro incubation with Sppt, SACP showed an increased abundance of phosphopantetheinylated carrier protein, indicating that Sppt can activate the cognate SACP (FIG. 8B).
Example 5
Kinetic Analysis of Sppt Activity
The CoA inhibition and pH activity range for Sppt was determined using in vitro phosphopantetheinylation assays with the truncated SACP3. Assays were carried out as above in 200 μL reactions comprising of 75 mM MES (pH 5.5-6.5), Tris (pH 7-8.5) or CAPSO (pH 9-10) and 1-3 mM CoA. After precipitation, the pellets were resuspended in 30 μl 55% Solvent B (acetonitrile, 0.1% trifluoroacetic acid). An Alltech 5 μm Nucleosil C18 column (250×4.6 mm) was equilibrated with 55% Solvent B at 45° C. before sample injection. Samples were eluted with a linear gradient from 55% to 68% Solvent B over 15 min with 0.9 ml min−1 flow rate. The absorbance at 200 nm was constantly monitored. The amount of holo-SACP3 formed was determined by comparative analysis of the holo-ACP peak area of control assays without PPT. Holo-SACP3 present after heterologous expression in E. coli, was subtracted from the total amount phosphopantetheinylated.
Phosphopantetheinylation assays, and subsequent HPLC analysis, were carried out to determine the pH and CoA activity range of Sppt towards its cognate SACP3 carrier protein The optimum activity for Sppt was approximately pH 7.7, with a relatively broad pH activity range where approximately 25% of the activity was retained at pH 5.5 and 9.8 (FIG. 9). Sppt activity increased with increasing CoA concentrations and reached a maximum activity with concentrations greater than 1500 μM. The Michaelis-Menten fit of the experimental data yielded a Km value of 169.17±37.48 μM for Sppt with respect to CoA and a kcat value of 28.41±2.12 mind. The catalytic efficiency of Sppt for CoA was 0.168 μM−1 min−1.
Sppt behaves similar to AcpS-like PPTs with specific carrier protein activation, a high pH optimum and low catalytic efficiencies with respect to CoA. Although most characterised PPTs show significantly higher catalytic efficiencies, Sppt has similar values to AcpS from E. coli which has reported Km values of 50 or 150 μM and a kcat value of approximately 10 mind (21,40). Sfp-like PPTs such as the B. subtilis Sfp and P. aeruginosa PcpS have pH optima between 6-7 while the Sppt optimum is more similar to the B. subtilis and E. coli AcpS PPTs with pH optima between 8-9. This high pH requirement may allow for appropriate electrostatic interactions between Sppt and the acidic SACP; and may explain its poor activation of the neutral MPCP. Optimum Sppt activity at higher pH's may also reflect the environmental conditions (pH 7-9) that favour cyanobacterial bloom formation over phytoplankton growth. Sppt's pH range is broader than other characterised PPTs, which may reflect the dynamic pH of cyanobacterial environments during eutrophication and subsequent bloom cycles.
Example 6
Sppt in vitro Phosphopantetheinylation of Non-Cognate Carrier Proteins
The ability of Sppt to activate carrier proteins from a range of secondary metabolism pathways was tested, including those from unicellular and filamentous, heterocyst-forming cyanobacteria. The carrier proteins tested were the N. punctiforme ATCC29133 PKS aryl carrier protein (ArCP) from HetM of glycolipid synthesis (NpArCP) and PKS ACP of nostopeptolide biosynthesis (NpACP); and the M. aeruginosa PCC7806 NRPS PCP from the loading module (McyG) of microcystin synthesis (MPCP). The Bacillus brevis ATCC8185 NRPS PCP involved in tyrocidine biosynthesis (TycPCP) was also used to test the ability of Sppt to phosphopantetheinylate carrier proteins from alternative bacterial kingdoms.
The M. aeruginosa PCC7806, peptidyl carrier protein, MPCP; and the N. punctiforme ATCC29133 aryl carrier protein, NpArCP, and acyl carrier protein, NpACP, were expressed and purified as previously described in Copp J. N., Roberts A. A., Marahiel M. A., Neilan B. A., 2007, Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10. J. Bacteriol.: 189(8):3133-9. The Bacillus brevis ATCC8185 TycPCP was expressed and purified according to the methods of Reuter et al. (1999), Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4′-phosphopantetheinyl transferase superfamily, EMBO J. 18:6823-6831. Protein concentrations were determined based on the calculated extinction coefficients: NpArCP 8,250 cm−1M−1, NpACP 13,940 cm−1M−1, MPCP 6,970 cm−1M−1 and TycPCP 9,530 cm−1M−1. Phosphopantetheinylation assays were carried out in 30 min reactions and analysed by ESI-mass spectrometry described in Example 4 above.
No phosphopantetheinylation of NpACP, MPCP, NpArCP and TycPCP by the E. coli PPTs was detected after heterologous expression.
Sppt was unable to phosphopantetheinylate the N. punctiforme ATCC29133 NpArCP as shown by mass spectrometry (FIG. 10B). The spectrum of M. aeruginosa PCC7806MPCP, after incubation with Sppt, showed a small mass peak corresponding to the addition of a 340 Da phosphopantetheinyl moiety (22,319 Da) (FIG. 10D). This low intensity peak was consistently present in repeated experiments. Phosphopantetheinylated NpACP was not reproducibly detected after incubation with Sppt (FIG. 11B) and the mass peak of 20,153 Da was only slightly above that of the spectrum background. In contrast, approximately 54% of the P. aeruginosa PAO1 NRPS TycPCP was phosphopantetheinylated by Sppt (FIG. 11D, FIG. 12).
The inability of Sppt to activate non-cognate carrier proteins from other species was shown with the N. punctiforme ATCC29133 glycolipid synthesis NpArCP. This result was not surprising as Synechocystis sp. PCC6803 is unable to form heterocysts or fix nitrogen. The ability of Sppt to phosphopantetheinylate carrier proteins within NRPS and PKS pathways was also analysed using MPCP from microcystin synthesis in M. aeruginosa PCC7806 and NpACP from nostopeptolide synthesis in N. punctiforme ATCC29133. Phosphopantetheinylation of MPCP and NpACP was detected, but only slightly above that of the spectrum background. NpACP phosphopantetheinylation could not be consistently reproduced in replicate assays. The inability of Sppt to activate carrier proteins from secondary metabolite pathways correlates with the lack of natural products synthesised by Synechocystis sp. PCC6803. However, the presence of an Sfp-like PPT in a species that only encodes carrier proteins for FAS raises interesting questions regarding the evolution of PPTs and secondary metabolite synthesis in cyanobacteria.
The ability of Sppt to phosphopantetheinylate the Bacillus brevis ATCC8185 carrier protein TycPCP was incongruous to the lack of activity towards other cyanobacterial carrier proteins. This may be explained by the size of the expressed TycPCP which is less than half the size of NpACP and MPCP. Therefore, the inability to phosphopantetheinylate the larger MPCP and NpACP carrier proteins could be due to electrostatic considerations and poor accessibility of active sites in these larger recombinant proteins.
Example 7
HetMNI Gene Cluster
Cyanobacterial PPTs present an interesting group of PPTs due to the presence of multiple secondary metabolites including polyketide, peptide synthetase and hybrid polyketide/peptide synthetase biosynthesis, PPTs associated with development of specialist cells for nitrogen fixation (heterocysts), Sfp-like PPTs with the notable absence of an AcpS-like PPT, and integrated Sfp-like PPTs within polyketide clusters. The majority of sequenced cyanobacterial genomes contain a singular Sfp-like PPT, However Nostoc punctiforme ATCC 2913 and Gloeobacter violaceus PCC 7421 both display three distinct PPTs within their respective genome sequences.
The divergent range of PPTs within cyanobacteria required more rigorous sequence analysis. A cyanobacterial screen for PPT enzymes was performed utilizing the available sequence data from published genomes. Conserved motif alignments (motif 3 and 4) from Sfp-like PPTs were targeted for the design of degenerate PCR primers. Novel cyanobacterial PPT genes were isolated from toxic, non-toxic, unicellular, and filamentous, heterocyst-forming cyanobacterial species. A phylogenetic tree constructed from published and partial cyanobacterial PPT sequences (FIG. 13) revealed the new phylotypes designated A and B. All cyanobacterial PPT fall in the W/KEA subfamily of Sfp-like PPTs.
PPTs from known heterocyst-forming cyanobacteria formed a distinct Glade supported by bootstrap data and were designated het-type PPTs (A in FIG. 13). The heterocyst-forming Nostoc punctiforme PCC73102 species harbours three PPTs from the W/KEA subfamily within its genome. The N. punctiforme PPT associated with the heterocyst HETMNI gene loci falls in the Het-type Glade, whereas the other two PPTs fall in alternative clades.
A second Glade (B in FIG. 13) includes PPTs from Prochlorococcus, Synechococcus, Gloeobacter and N. punctiforme species. Several of the sequences within this group were located adjacent to an ATPase gene. Alternatively, the G. violaceus and N. punctiforme PPTs in this group were both associated with polyketide biosynthetic clusters.
Sequencing of the N. spumigena HetMNI Locus
The N. spumigena PPT fragment identified by phylogenetic analysis as a het-type PPT potentially associated with heterocyst synthesis was subsequently selected for characterization. A flanking region of 3450 by was sequenced to allow analysis of the surrounding genomic region, revealing a heterocyst related HetMNI gene cluster (Genbank Accession No. AY836561).
Functional classification has not previously been observed in PPT phylogenetics. Analysis of the divergent range of cyanobacterial PPTs has allowed the designation of PPTs associated with heterocyst formation. Using gene alignments to screen PPT sequences has allowed the detection of heterocyst-associated PPTs within cyanobacterial genomes and the characterisation of a heterocyst biosynthesis locus in N. spumigena NSOR10.
Example 8
Isolation of N. Spumigena PPT
(1) Media/Culturing
N. spumigena NSOR10 was cultured at room temperature on a 24 hour light/dark cycle in ASM media (see Provasoli, L., et al. 1957. The development of artificial media for marine algae. Arch Mikrobiol 25:392-428). DNA was extracted as previously described in (Moffitt, M. C. and B. A. Neilan. 2004. Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Appl Environ Microbiol 70:6353-62).
(2) DNA Amplification, Sequencing and Analysis
PCR and sequencing reactions were performed as previously described in Neilan, B. A. et al. 1994, 16S ribosomal RNA gene sequence and phylogeny of toxic Microcystis sp. (cyanobacteria). DNA Seq 4:333-7. Panhandle-based gene walking by adaptor based and specific primers (Moffitt, M. C. and B. A. Neilan. 2004. Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Appl Environ Microbiol 70:6353-62) was utilised at the annealing temperature of 45-55° C. to amplify the unknown genomic regions flanking the N. spumigena PPT fragment (SEQ ID NO: 1). The output from BLAST (Basic Local Alignment Search Tool), Pileup from GCG and the multiple-sequence alignment tool from CLUSTAL X were utilized for the analysis and alignment of sequences. Automated sequencing was performed using the PRISM Big Dye cycle sequencing system and a model 373 sequencer (Applied Biosystems Inc., USA). Sequence analysis was performed using Applied Biosystems Autoassembler software.
(3) Southern Hybridisation Methods
Pure genomic DNA samples from Nodularia spumigena NSOR10 ( ̃10 μg) were digested overnight with Xba I or Xmn I as per manufacturers recommendations (Promega, Australia). Digests and positive controls (0.5-1.0 ng of linearised plasmid pGEM-T-Easy (Promega) were separated on 0.8% agarose gels at 60 mV for approximately 2.5 hours and vacuum-blotted to a nylon membrane (Amersham). The DIG-High Prime DNA labelling kit (Roche, Australia) was utilised for Southern hybridisations. PPT probes were amplified with specific primers as follows. The primer pair slrup (5′-TGTTTAAACTCACCTGTG-3′) (SEQ ID NO: 15) and slrdn (5′-CCCAAGGTAACGAAACGA-3′) (SEQ ID NO: 16) was utilised to amplify Slr0495 from Synechocystis sp. PCC 6803. The primer pair Npunfw (GGATCCGCGATCGCCAGTCTGAGTTC) (SEQ ID NO: 17) and Npunry (GAGCTCTTTGTGTAGTAGCGAATTATC) (SEQ ID NO: 18) was utilised to amplify a PPT from Nostoc punctiforme ATCC 29133. The primer pair NpptF (5′-CATGAAAGATATCACGGCGCTT-3′) (SEQ ID NO: 19) and NpptR (5′-GAAGATAACAAGCTTGTATTGCC-3′) (SEQ ID NO: 20) was used to amplify nchS from Nodularia. spumigena. Probes were labelled by PCR with digoxigenin (DIG) as per manufacturer's instructions (Roche) and tested for efficiency from 100 fg to 10 ng. Hybridisations were performed with overnight at 40° C., stringency washes were performed with 0.5% SSC with 0.1% SDS at 65° C. and signals were analysed by chemiluminescent detection with CPSD by a FUJIFILM Luminescent Image Analyzer LAS-3000.
(4) Creation of Expression Plasmids
The N. sp. heterocyst carrier protein synthase (hereafter nhcS) from N. spumigena NSOR10 (AY836561) was amplified by the primer pair NpptF and NpptR (as described in “(3) Southern Hybridisation Methods”) above). The 720 bp PCR-amplified product was cloned into pGEM-T (Promega) and this plasmid was then digested and the appropriate fragment ligated into pET30 (Novagen) to yield the expression plasmid pNhcS.
The N. punctiforme PCC73102 HetM was amplified from the heterocyst hetMNI locus encoding the ArCP/ketoreductase HetM (ZP—00107100). The primer pair HetMF (5′-GCCATGGCTATAAAACAGTCTTTC-3′) (SEQ ID NO: 21) and HetMR (5′-GGGATCCGAGATTCAAGAAACC-3′) (SEQ ID NO: 22) were utilised to amplify a 1.7 Kb fragment, which was cloned into the pGEM staging vector. Subsequent restriction and cloning into the pET30 expression vector created pHetM. The pArCP expression vector was constructed in a similar manner utilising the primers HetMF (as above) and ArCPR (5′-TAGCTCGAGAACCATCTTGCAC-3′) (SEQ ID NO: 23), to amplify and clone the 260 bp ArCP domain of hetM and create pArCP.
The N. spumigena NSOR10 peptidyl carrier protein (hereafter PCP) and acyl carrier protein (hereafter ACP) (hereby called Npcp and Nacp) were amplified from the hybrid NRPS-PKS ndaC (AA064404) within the ndaS gene cluster responsible for the production of the hepatotoxin Nodularin (see Moffitt, M. C. and B. A. Neilan. 2004. Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Appl Environ Microbiol 70:6353-62). The primer pairs NpcpF (5′-CTCGAGCAGCCTCTACAACTGCA-3′) (SEQ ID NO: 24) and NpcpR (5′-GGATCCGCCAGGAGAACGGCGG-3′) (SEQ ID NO: 25) and NacpF (GGAGCTCTTTTCCAAACATTCT) (SEQ ID NO: 26) and NacpR (5′-GGGATCCTCTAAGCATTCCATCAGTC-3′) (SEQ ID NO: 27) were utilised. The resulting fragments were manipulated as described above to yield pNACP and pNPCP respectively.
The Synechocystis sp. fatty acid synthesis acyl carrier protein (hereafter FAS ACP), hereby called Sacp (BA000022), was constructed from the primer pair 2084NdeF (5′-GCATATGAATCAGGAAATTT-3′) (SEQ ID NO: 28) and 2084XhoR (5′-CCTCGAGTAATTTACTTTCGATATGCTCAAC-3′) (SEQ ID NO: 29) and cloned as above to yield pSACP.
The Microcystis aeruginosa PCP was amplified from the hybrid NRPS-PKS mcyG (AAX73195) from the mcyS gene cluster responsible for the production of the hepatotoxin microcystin (see Tittett, D., et al. 2000. Structural organization of microcystin biosynthesis in Microcystits aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem. Biol 7:753-64). The PCP primers MpcpF (5′-GGATCCTGAACAGGGA-3′) (SEQ ID NO: 30) and MpcpR (5′-CTCGAGATGGCGACGGCTCC-3′) (SEQ ID NO: 31) were used to construct the expression vectors as outlined above to create pMPCP.
The Nostoc punctiforme NSOR10 NRPS PCP, from this point called Nppcp, (ZP—00110897) was amplified from an uncharacterised gene cluster. This locus is hypothesised to be responsible for nostopeptolide production in N. punctiforme ATCC29133 due to its similarity to the characterised gene cluster in Nostoc sp. GSV224 (see Hoffmann, D., et al. 2003. Sequence analysis and biochemical characterization of the nostopeptolide A biosynthetic gene cluster from Nostoc sp. GSV224. Gene 311:171-80). NosA, a large NRPS gene within this cluster, comprises of four NRPS modules. The C-terminal NosA PCP was amplified from the putative nostopeptolide biosynthetic cluster utilising the primer pairs NppcpBF (5′-GGATCCTAAAATCTAGGCTAG-3′) (SEQ ID NO: 32) NppcpSR (5′-GAGCTCAAATTGTTATT′TCTT-3′) (SEQ ID NO: 33) and cloned as above to yield pNpPCP.
(5) Protein Expression, Purification and Enzyme Activity Analysis
Expression plasmids were separated, transfected into E. coli as host cells and expression analysis performed as follows. Sacp was expressed at 37° C. for 2 hours with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside). NhcS, Arcp and Mpcp were expressed at 30° C. for 4 h with 0.2 mM IPTG. Nppcp was expressed at 22-24° C., 0.1 mM IPTG for 6 hours. HetM was expressed at 18° C., overnight with 0.1 mM IPTG, visualised as a soluble 63.3 kDa protein and verified via western blot as per manufacturer's instructions (Qiagen). However, analysis of HetM via nanospray ion trap mass spectrometry could not be performed due to inadequate yields. The plasmid encoding the ArCP domain of hetM was therefore constructed and the 15.6 kDa Arcp was subsequently expressed in adequate yields. Attempts to resolve the low solubility of the Nacp and Npcp carrier proteins included variations in expression time (2-24 hours), temperature (18-37° C.) and IPTG concentration (0.1-1 mM). After expression cells were pelleted at 4,000 rpm and frozen at −80° C. overnight. Pellets were then thawed on ice, resuspended in 5 ml 50 mM Hepes (Sigma) pH 7.4 and either subjected to 3 passages through a cooled French press at 1000 psi or sonicated at 4° C., 30% amplitude, for 25 seconds with a 0.5 second pulse. The soluble fraction was collected after centrifugation at 20,000 g for 30 min at 4° C. An Amersham HighTrap Ni-chelating column was utilised for purification of the recombinant proteins. Fractions containing the desired protein (as determined by SDS-PAGE) were pooled, desalted and snap frozen (with 8% glycerol) in liquid nitrogen for storage at −80° C. Concentration of the purified proteins was calculated using the absorbance at OD280 and the respective absorbance factors of the recombinant proteins (NhcS 37650, HetM 67430, ArCP 8250, Sacp 6970, Mpcp 6970 and Nppcp 13940).
PPT assays were carried out as previously described in Finking, R., et al., (2002) Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa. J Biol Chem 277:50293-302). In brief, a 100-400 μL reaction comprising of 50 mM Tris-HCl pH 7.4, 12.5 mM MgCl, 0.5 mM CoA, 2 μM DTT, 30 μg of the respective carrier proteins (CPs) and 300 nM of PPT was incubated at 37° C. for 30 minutes. Reactions were terminated by the addition of 1 ml of 10% TCA. Assays were precipitated overnight at −20° C. before at centrifugation 4° C., for 15 mins at 16,000 g. Protein pellets were analysed by electrospray ionisation mass spectrometry (ESI-MS). Spectra were acquired using an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City Calif.). Samples ( ̃200-400 fmol) were dissolved in water:acetonitrile:formic acid (50:49:1), loaded (1 μl) into nanospray needles (Proxeon, Denmark) and the tip positioned  ̃10 mm from the orifice. Nitrogen was used as curtain gas and a potential of 900 V applied to the needle. A T of MS scan was acquired (m/z 550-2000, 1 second) and accumulated for  ̃1 minute into a single file. Spectra were deconvoluted using the Bayesian reconstruction method contained within the Analyst QS software.
(6) Activation of NpACP by Coexpression with NPPT in E. coli. 
The Nodularia spumigena PPT, NPPT, was amplified by PCR using the primers NpptF (5′-TGCATATGACGGCGCTTAATCATT-3′) (SEQ ID NO: 34) and NpptR (5′TCTCGAGTCAGTATTGCCAACAC-3′) (SEQ ID NO: 35) and was subsequently cloned into pGEM-T-Easy (Promega). NPPT was then subcloned into the expression vector pET30a (Novagen) by digestion with NcoI and XhoI (New England Biolabs) to yield pNPPT2. NPPT, complete with the T7 promoter and transcriptional terminator originally from pET30a, was released from pNPPT2 with DraIII and SphI (New England Biolabs) and blunt-ended with Klenow enzyme (Promega). The fragment was subsequently cloned into AvaI-linearised, blunt-ended and phosphorylated pRARE (Novagen) to yield pNPPT3.
The constructs pNPPT3 and pNpACP were sequentially transformed into BL21(DE3) (Novagen) for coexpression of the NPPT and NpACP proteins. In the control experiments, NpACP was expressed with pRARE only. NpACP proteins which were coexpressed with pNPPT3 or pRARE were purified on a Hi-Trap nickel column and precipitated with 10% trichloracetic acid. Molecular masses were determined with Nanospray-MS analysis.
Example 9
Activity of nhcS
(1) Sequencing and Analysis of the HetMNI Locus
Flanking regions of the partial PPT gene fragment were amplified to allow sequencing of 3450 by of the N. spumigena NSOR10 hetMNI locus (AY836561). Sequence analysis revealed the 240aa nhcS PPT, a 27,555 Da protein with an isoelectric point (pI) of 6.1. NhcS displayed 81% similarity to HetI of Nostoc sp. PCC 7120, 58% similarity to the Synechocystis sp. PCC 6803 Slr0495 and 55% similarity to Sfp from B. subtilis (FIG. 14). This PPT is encoded in reverse orientation to the hetM and hetN genes, as observed in the hetMNI locus of the heterocyst forming cyanobacteria Nostoc sp. PCC 7120, A. variabilis ATCC 29413, and N. punctiforme ATCC 29133 (FIG. 14).
The predicted protein products of the genes downstream of nhcS in the hetMNI loci were also analysed and compared to homologous proteins. The partial fragment of the N. spumigena iterative PKS gene hetM was sequenced and showed 90% similarity to the corresponding fragment of N. punctiforme hetM (also called hglB). The N. spumigena HetN is a 126-residue protein that displayed 83% similarity to the C-terminal half of A. variabilis HetN (a 263 residue protein). This domain encodes a unique reductase protein that has a role in heterocyst suppression via inhibition of HetR (Li et al., 2002 “Expression of hetN during heterocyst differentiation and its inhibition of hetR up-regulation in the cyanobacterium Anabaena sp. PCC7120” FEBS Letters 517:87-91). No ORFs were detected in the sequenced region extending 900 by upstream of nhcS.
(2) Southern Hybridisation
Southern probes were utilised to ascertain the number of PPTs encoded within the N. spumigena NSOR10 genome. Probes were constructed from a diverse range of cyanobacterial PPTs including nhcS, slr0495 from Synechocystis sp. PCC 6803 (BAA10326) and a PPT from Nostoc punctiforme ATCC 29133 (ZP—00110892). These PPTs are placed in distinct cyanobacterial phylogenetic clades. Probe efficiency and specificity was detected to 10 pg as verified through controls (not shown). Hybridisations performed with the nhcS probe revealed a singular band from N. spumigena NSOR10 (FIG. 16) and no hybridisation was detected with the slr0495 or nppt probes. Together, these experiments suggest that nhcS is the single PPT encoded by N. spumigena NSOR10.
(3) Expression and Purification of Recombinant Proteins
In order to confirm the pantetheinyl transferase activity of nhcS, this enzyme was expressed as a 27.6 kDa His-tagged, soluble protein. The Arcp from the hetMNI locus in N. punctiforme ATCC29133 was chosen due to the similarity of the hetMNI genes to N. spumigena NSOR10. No ArCP pantetheinylation was seen after heterologous expression in E. coli. Sacp was expressed as a 14.8 kDa protein. After expression of the ACP in E. coli 40% of the protein was in the holo form. Production of soluble Nacp and Npcp from N. spumigena NSOR10, despite multiple variations in expression parameters, did not yield soluble protein for analysis as deduced by western blot (not shown). Mpcp and Nppcp were expressed as 22.0 kDa and 20.8 kDa proteins respectively; no pantetheinylation was seen after expression in E. coli. 
(4) Analysis of nhcS Activity
Activity was detected by ionisation mass spectrometry through the mass addition of 350 da, relating to the incorporation of the pantetheinyl arm of CoA transferred by the PPT to the CP (FIG. 17).
NhcS activity was initially confirmed utilising the cognate HetM ArCP from the associated hetMNI gene cluster (in N. punctiforme ATCC29133). The conversion of the apo-ArCP (15.75 kDa) to the holo-ArCP (16.05 kDa) is seen in FIG. 17B), and shows the role of this enzyme in the synthesis of heterocyst glycolipids.
Enzymatic analysis of NhcS activity toward secondary metabolite PCPs from NRPS was tested with Nppcp and Mpcp. Mass spectroscopy analyses show the 350 Da addition of the pantetheinyl moiety from CoA to each of the respective CPs tested. The successful expression of the PCP from the Nostopeptolide (nosA) and the Microcystin (mcyG) biosynthetic gene clusters allowed the detection and analysis of nhcS phosphopantetheinylation in secondary metabolism. Complete pantetheinylation of Mpcp was achieved in a 30 min assay. Nppcp was pantetheinylated, showing that nhcS can activate carrier proteins from biosynthetic pathways that are not present in N. spumigena NSOR10.
Control reactions were performed without nhcS. There was no detectable pantetheinylation of the PCPs after heterologous expression in E. coli. 
NhcS can therefore act in each of FAS, heterocyst glycolipid synthesis and biosynthesis of secondary metabolites such as the hybrid polyketide/non-ribosomal peptide nodularin. The ability of nhcS to activate non-cognate secondary metabolite CPs has great potential for application in biotechnological fields. Pantetheinylation of non-cognate carrier proteins from alternative hosts demonstrates the application of this PPT for active synthesis of biosynthetic clusters from a diverse range of species.
Example 10
Complementation of Synechocystis sp. PCC6803 with NsPPT
The Nodularia spumigena NSOR10 PPT, NsPPT (Genbank accession number AY646183), was expressed under the control of a nitrate inducible promoter, PnirA, from Synechococcus sp. strain PCC7942, to complement Sppt activity in vivo (34). PnirA was amplified with the primers nirAF (5′-TTCTAGATCCCTCTCAGATCAAAAAG-3′) (SEQ ID NO: 36) and nirAR (5′TGCATATGGGATTCATCTGCCTAC-3′) (SEQ ID NO: 37) and the fragment was cloned into pET30a with XbaI and NdeI to yield pPnirA (Novagen). NsPPT, was amplified by with the primers NpptF (5′-TGCATATGACGGCGCTTAATCATT-3′) (SEQ ID NO: 34) and NpptR (5′-TCTCGAGTCAGTATTGCCAACAC-3′) (SEQ ID NO: 35) and was subsequently cloned into pPnirA with NdeI and XhoI. NsPPT, complete with the PnirA promoter and T7 transcriptional terminator, was released from this construct with DraIII and SphI and blunt-ended with Klenow enzyme (Promega) for ligation into PstI-linearised pKW1188, which harbours the slr0168 photosystem II gene flanking regions and a kanamycin resistance cassette. The resulting plasmid was naturally transformed into Synechocystis sp. PCC6803 for homologous recombination into slr0168.
Transformants were selected on BGTS plates supplemented with 10 μml−1 kanamycin. The NsPPT-complemented strain was designated Synechocystis sp. PCC6803 ΔNsPPT+ (ΔNsPPT+). The Sppt knockout plasmid, pGCSlr, was naturally transformed into this strain for disruption of the Sppt gene. ddH2O was also transformed into ΔNsPPT+ as a negative control. NsPPT-complementated colonies were PCR-screened for complete segregation of Sppt gene disruption using the primers slrup and slrdown. Complementation and knockout experiments were carried out three times in duplicate.
While the invention has been described in the manner and detail as above, it will be appreciated by persons skilled in the art that numerous variations and/or modifications including various omissions, substitutions, and/or changes in form or detail may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art in Australia or elsewhere.

## Claims

1. A method for the production of secondary metabolites, the method comprising:
(a) transforming Synechocystis sp. bacteria with one or more bacterial peptide synthetase genes required for production of secondary metabolites;
(b) culturing the Synechocystis sp. bacteria under conditions suitable for expression of the one or more genes required for production of secondary metabolites; and
(c) purifying the secondary metabolites from the bacteria, wherein said Synechocystis sp. bacteria express a Nodularia sp. phosphopantetheinyl transferase (PPT) that is needed for the production of the secondary metabolite, wherein said Synechocystis sp. bacteria comprise an inactivated endogenous phosphopantetheinyl transferase (Sppt) gene.

2. The method of claim 1, further comprising prior to step (c), the step of screening the Synechocystis sp. bacteria for production of secondary metabolites.

3. The method of claim 2, wherein the screening step comprises high performance liquid chromatography, mass spectrometry or a combination thereof.

4. The method of claim 1 wherein the secondary metabolite is a peptide or an analogue thereof which retains substantially the same function as the peptide.

5. The method of claim 4, wherein the peptide or analogue thereof is a bioactive compound.

6. The method of claim 5, wherein the bioactive compound is an anti-microbial agent, an anti-viral agent, an anti-fungal agent, an anti-cancer agent, an immunosuppressive agent, an anaesthetic, an analgesic, an antitumour product, an antibiotic, an anti-cholesterolemic, an anti-parasitic agent, a veterinary therapeutic agent, an agrochemical, or a cosmetic.

7. The method of claim 1 wherein said gene required for production of secondary metabolites is operably linked to a promoter active in Synechocystis sp.

8. The method of claim 7, wherein the promoter is inducible.

9. The method of claim 8, wherein the promoter is inducible by varying intensity of light to which the bacteria are exposed during culture.

10. The method of claim 1 wherein said gene required for production of secondary metabolites is in a plasmid, phosmid or cosmid.

11. The method of claim 1 wherein said transforming is transposon-mediated.

12. The method of claim 1 wherein the bacteria are transformed with multiple bacterial peptide synthetase genes forming a biosynthetic peptide synthetase gene cluster.

13. The method of claim 1 wherein the exogenous PPT is encoded by a gene that is stably integrated into a Synechocystis sp. genome.

14. The method of claim 1, wherein the exogenous PPT is a cyanobacterial PPT.

15. The method of claim 1, wherein the PPT is from Nodularia spumigena.

16. The method of claim 1, wherein the PPT is from Nodularia spumigena NSOR10.

17. The method of claim 16, wherein the PPT comprises the amino acid sequence as set forth in SEQ ID NO: 1, or a variant or fragment thereof having PPT activity.

18. The method of claim 1 wherein the Synechocystis sp. is Synechocystis sp. PCC6803.

19. The method of claim 1, wherein the one or more bacterial peptide synthetase genes are cyanobacterial peptide synthetase genes.