# Patent

## Title
Biosynthetic gene cluster for ambruticins and the encoded proteins

## Abstract

Domains of ambruticin polyketide synthase and modification enzymes and polynucleotides encoding them are provided. Methods to prepare ambruticin in pharmaceutically useful quantities are described, as are methods to prepare ambruticin analogs and other polyketides using the polynucleotides encoding ambruticin synthase domains or modifying enzymes.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. § 119 to U.S. provisional application Ser. Nos. 60/551,103, filed Mar. 8, 2004, and 60/568,290, filed May 4, 2004, the entire contents of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to nucleic acid encoding polypeptides capable of synthesizing compounds having anti-fungal activity, methods for their preparation, and methods for their use.
2. Description of Related Art
Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146 and 6,410,301; Fu et al. (1994), Biochemistry 33:9321-26; McDaniel et al. (1993) Science 262: 1546-1550; Kao et al. (1994) Science, 265:509-12, and Rohr (1995) Angew. Chem. Int. Ed. Engl. 34: 881-88, each of which is incorporated herein by reference.
Biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two-carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide-modifying enzymatic activities, or domains. In classical Type I PKSs, the number and order of modules, and the types of ketide-modifying domains within each module, determine the structure of the resulting product. Recently, variants of Type I PKSs have been found in which single modules may be used in an iterative fashion to add more than one two-carbon unit to the growing polyketide chain (see, for example, Müller (2004) Chem. Biol. 11(1):4-6). Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundaries of the various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced. It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson (1998) Curr. Opin. Microbiol. 1:319-29; Carreras and Santi (1998) Curr. Opin. Biotech. 9:403-11; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference).
One valuable class of polyketides includes the ambruticins and their analogs (FIG. 1), produced by the myxobacterium Polyangium cellulosum (also known as Sorangium cellulosum) var. fulvum, deposited as ATCC 25532. Ambruticin S (also known as “Acid S” or “W7783”) was described in U.S. Pat. Nos. 3,651,216 and 3,804,948. Ambruticins VS1, VS3, and VS5 and a fermentative process for producing them using Sorangium cellulosum So ce10, deposited as DSM 5386, are disclosed in PCT publication WO91/00860. The ambruticins are anti-fungal agents. It is believed that the ambruticins kill fungi through interference with osmoregulation (Knauth and Reichenbach (2000) J. Antibiotics 53:1182-1190).
Given the promise of ambruticins in the treatment of fungal infections, there exists an unmet need for a production system that can provide large quantities of ambruticins. The present invention meets this need by providing the biosynthetic genes responsible for the production of ambruticins and providing for their expression in heterologous hosts.
BRIEF SUMMARY OF THE INVENTION
The present invention provides recombinant nucleic acids encoding polyketide synthases and polyketide modification enzymes. The recombinant nucleic acids of the invention are useful in the production of polyketides, including but not limited to ambruticins and ambruticin analogs and derivatives in recombinant host cells. The biosynthesis of ambruticin is performed by a modular PKS together with polyketide modification enzymes. The ambruticin synthase is made up of several proteins, each having one or more modules. Each module has one or more domains with specific synthetic functions.
The present invention also provides domains and modules of the ambruticin PKS and corresponding nucleic acid sequences encoding them and/or parts thereof. Such compounds are useful in the production of hybrid PKS enzymes and the recombinant genes that encode them.
The present invention also provides modifying genes of the ambruticin biosynthetic gene cluster, including but not limited to isolated and recombinant forms and forms incorporated into a vector or the chromosomal DNA of a host cell.
The present invention also provides recombinant host cells that contain the nucleic acids of the invention. In one embodiment, the host cell provided by the invention is a Streptomyces host cell that produces an ambruticin modification enzyme and/or a domain, module, or protein of the ambruticin PKS. Methods for the genetic manipulation of Streptomyces are described in Kieser et al, “Practical Streptomyces Genetics,” The John Innes Foundation, Norwich (2000), which is incorporated herein by reference in its entirety. In other embodiments, the host cells provided by the invention are eubacterial cells such as E. coli, yeast cells such as Saccharomyces cerevisiae, or myxobacterial cells such as Myxococcus xanthus. 
Accordingly, there is provided a recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of the ambruticin polyketide synthase. Preferably at least an entire domain of a module of the ambruticin synthase is included. Representative ambruticin PKS domains useful in this aspect of the invention include, for example, β-ketoreductase (KR), dehydratase (DH), enoylreductase (ER), acyltransferase (AT), acyl carrier protein (ACP) and β-ketoacylsynthase (KS) domains. In one embodiment of the invention, the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for PKS domains, wherein at least one encoded domain corresponds to a domain of ambruticin PKS. In such DNA molecules, the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective. In this manner, ambruticin PKS coding sequences or modules and/or domains can be made to encode PKS to biosynthesize compounds having antibiotic or other useful bioactivity other than ambruticin.
These and other aspects of the present invention are described in more detail in the Detailed Description of the Invention, below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows various members of the ambruticin family of natural polyketides.
FIG. 2A shows the organization of the portion of the ambruticin biosynthetic cluster as deduced from SEQ ID NO:1. FIG. 2B shows the organization of the portion of the upstream region of the ambruticin biosynthetic cluster as deduced from SEQ ID NO:2. The open bars indicate the cosmid inserts, the hatched arrows indicate ORFs, and the open circles indicate transposon integration sites that result in disruption of ambruticin biosynthesis or export.
FIG. 3 shows the nucleotide sequence of the ambruticin biosynthetic gene cluster (SEQ ID NO:1), and the sequence of the deduced translated proteins of the ambA, ambB, ambC, ambD, ambE, ambF, ambG, ambH, ambI, ambJ, ambM, ambN, ambO, ambP, ambQ, ambR, and ambS genes (SEQ ID NOS:3-19, respectively). Also shown is the sequence of the deduced translated proteins of ORFs 9-11 (SEQ ID NOS:28-30, respectively).
FIG. 4 shows the nucleotide sequence of the region upstream of the ambruticin biosynthetic gene cluster (SEQ ID NO:2), and the sequence of the deduced translated proteins of ORFs 1-8 (SEQ ID NOS:20-27, respectively).
FIG. 5 shows the nucleotide sequence of plasmid pKOS375-151.1 (SEQ ID NO:61). Example 3 describes the construction of plasmid pKOS375-151.1.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides recombinant materials for the production of polyketides. In one aspect, the invention provides recombinant nucleic acids encoding at least one domain of a polyketide synthase required for ambruticin biosynthesis. Methods and host cells for using these genes to produce a polyketide in recombinant host cells are also provided.
The nucleotide sequences encoding ambruticin PKS domains, modules and polypeptides of the present invention were isolated from Sorangium cellulosum So ce10(hereafter “So ce10”) as described in Example 1. Given the valuable properties of ambruticin and its derivatives and analogs, means to produce useful quantities of these molecules in a highly pure form are of great potential value. The compounds produced may be used as antitumor agents or for other therapeutic uses, and/or intermediates for further enzymatic or chemical modification. The nucleotide sequences of the ambruticin biosynthetic gene cluster encoding domains, modules and polypeptides of ambruticin synthase, and modifying enzymes, and other polypeptides can be used, for example, to make both known and novel polyketides.
The present invention provides isolated or recombinant nucleic acid comprising a nucleotide sequence that encodes at least one polypeptide involved in or required for the biosynthesis of ambruticin. Preferably, the complement of the nucleotide sequence hybridizes, under highly stringent conditions, to a nucleic acid encoding at least one domain or module as disclosed in Table 1. In one aspect, the nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:3-19, or comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:33-49. Preferably, the nucleic acid comprises the nucleotide sequence of SEQ ID NOS:33-49 or SEQ ID NO:1.
The invention also provides for a vector comprising the purified or recombinant nucleic acid, wherein said polypeptide is operatively linked to a promoter. The invention further provides for a host cell comprising the vector, wherein said polypeptide is capable of expression in said host cell. Preferably, the host cell is a Streptomyces spp., E. coli, yeast, or myxobacteria.
The invention also provides for a method of producing an ambruticin or ambruticin analog, said method comprising culturing a cell comprising the vector under conditions under which said cell produces said ambruticin or ambruticin analog, wherein said polypeptide is capable of expression in said cell.
The invention further provides for a purified or recombinant polypeptide involved in or required for the biosynthesis of ambruticin comprising a domain as listed in Table 1. Preferably, the purified or recombinant polypeptide comprises a module as listed in Table 1. In one aspect, the purified or recombinant polypeptide comprises the amino acid sequence of one selected from SEQ ID NOs:3-19.
In one aspect of the invention, purified and isolated DNA molecules are provided that comprise one or more coding sequences for one or more domains or modules of ambruticin synthase. Examples of such encoded domains include ambruticin synthase KR, DH, ER, AT, ACP, and KS domains. Domains will herein be referred to according to the module in which they are found as “domain(module)”; for example, the module 1 AT domain will be referred to as “AT(1).” In one aspect, the invention provides DNA molecules in which sequences encoding one or more polypeptides of ambruticin synthase are operably linked to expression control sequences that are effective in suitable host cells to express said polypeptides and produce ambruticin, its analogs or derivatives, or novel polyketides.
The sequence of the ambruticin gene cluster were assembled from sequences deduced from cosmids 10K10B3 (pKOS344-112E) and 10T5F7 (pKOS344-135F), and plasmid pKOS375-77Bg, and is shown as SEQ ID NO:1 in FIG. 3. The organization of the ambruticin gene cluster is shown in FIG. 2A. This gene cluster is found to comprise at least seventeen open reading frames (ORFs), named ambA, ambB, ambC, ambD, ambE, ambF, ambG, ambH, ambI, ambJ, ambM, ambN, ambO, ambP, ambQ, ambR, and ambS. The ambA gene encodes the loading module of the ambruticin PKS, also referred to herein as “module 0,” and comprises KS and AT domains. The KS(0) domain is apparently inactive as a ketosynthase, having the active site cysteine residue replaced with a serine, and is thought to act as a decarboxylase to prime the PKS with a propionate group derived from methylmalonate. The AT(0) domain comprises the signature amino acid sequences (GHSQ and YASH (SEQ ID NOs:31 and 32, respectively)) of a methylmalonyl-specific AT domain. The ambB gene encodes modules 1 and 2 of the ambruticin PKS, the ambC gene encodes modules 3 and 4, the ambD gene encodes module 5, and the ambE gene encodes modules 6 and 7, the ambF gene encodes an ACP domain and module 8, together with a thioesterase domain and an aminotransferase domain. Unlike in canonical modular PKS, the ACP domain of the ambF gene is located on a different polypeptide from the remainder of the domains of module 7. Further, KS(8) is followed by a pair of ACP domains, labeled ACP(8a) and ACP(8b), and there is no clear AT domain in module 8. The ambH gene encodes module 9 and a terminal thioesterase (TE) domain. The ambG gene encodes a didomain protein having a CoA-ligase and an ACP domain, while the ambJ gene encodes a sequence with strong homology to a family including known and putative FAD-dependent monooxygenases, and is likely to be involved in oxidation of the polyketide. The ambJ gene encodes a sequence with strong homology to those of MonCI and NanO, predicted to be responsible for the formation of epoxide functionalities during the biosynthesis of monensin and nanchangmycin respectively, and may play a similar role in the biosynthesis of ambruticin. The ambP gene encodes a dioxygenase. The ambO gene encodes a flavin monooxygenase. The ambN gene encodes an aldehyde dehydrogenase. The ambQ gene encodes a desaturase. The ambR gene encodes a glutamate semialdehyde aminotransferase. The ambS gene encodes a N-methyltransferase. The ambM gene encodes a C-methyltransferase. The function of each of the amb genes in ambruticin biosynthesis is shown in FIG. 6. The downstream region of the ambruticin gene cluster resides ORF9-11. ORF9 (amb7) encodes a histidine kinase response regulator containing multiple domains. ORF10 (amb8) encodes a transcription regulator. ORF11 (amb9) encodes a NADH:flavin oxidoreductase.
Table 1 provides a description of the genes, modules, and domains/activities of the ambruticin proteins.







TABLE 1







Genes, modules, and domains/activities of the ambruticin PKS


determined from the nucleotide sequence given in SEQ ID NO: 1.










Gene
Module
Domain/Activity
boundaries





ambA


 765-4145



module 0

 765-4145




KS(0)
1026-2300




AT(0)
2628-3674




ACP(0)
3744-3998


ambB


 4202-15271



module 1

4301-8695




KS(1)
4301-5575




AT(1)
5900-6952




KR(1)
7553-8404




ACP(1)
8438-8695



module 2

 8756-15013




KS(2)
 8756-10033




AT(2)
10358-11425




DH(2)
11459-12034




ER(2)
13019-13894




KR(2)
13910-14707




ACP(2)
14756-15013


ambC


15268-26235



module 3

15367-20637




KS(3)
15367-16644




AT(3)
16969-18021




DH(3)
18055-18606




KR(3)
19534-20358




ACP(3)
20380-20637



module 4

20704-25989




KS(4)
20704-21981




AT(4)
22306-23373




DH(4)
23407-23967




KR(4)
24886-25710




ACP(4)
25732-25989


ambD


26232-31910



module 5

26331-31652




KS(5)
26331-27608




AT(5)
27960-29027




DH(5)
29061-29657




KR(5)
30528-31361




ACP(5)
31395-31652


ambE


31907-42430



module 6

32009-37255




KS(6)
32009-33286




AT(6)
33599-34651




DH(6)
34685-35236




KR(6)
36143-36955




ACP(6)
36998-37255



module 7a

37325-42379




KS(7)
37325-38593




AT(7)
38921-39985




DH(7)
40055-40531




KR(7)
41660-42379


ambF


42427-49020




ACP
42427-42666



module 8

43054-45972




KS(8)
43054-44322




ACP(8a)
45193-45447




ACP(8b)
45766-45972



TE(a)

46102-46785



AmTr

47053-48753


ambG


50524-52734




CoA ligase
50524-52248




ACP
52354-52599


ambH


52776-58076



module 9

52872-57299




KS(9)
52872-54125




AT(9)
54447-55496




KR(9)
56253-56942




ACP(9)
57033-57299



TE(b)

57300-58073


ambI

FAD oxygenase
49017-50336


ambJ

Epoxidase
58073-59494


ambP

Dioxygenase
60013-61083


ambO

Flavin monooxygenase
61086-62216


ambN

aldehyde dehydrogenase
62288-63916


ambQ

Desaturase
63946-65550


ambR

glutamate semialdehyde
65637-66977




aminotransferase


ambS

N-methyltransferase
67076-68155


ambM

C-methyltransferase
68191-69378


ORF9

multidomain 2-component
70491-76754




sensor/regulator


ORF10

transcriptional regulator
76992-77699


ORF11

NADH: flavin oxidoreductase
77581-78699









The sequence of the upstream region of the ambruticin gene cluster was assembled from sequences deduced from cosmids 10K10B3 (pKOS344-112E) and 1-16 (pKOS344-135B), and is shown as SEQ ID NO:2 in FIG. 4. The organization of the upstream region of the ambruticin gene cluster is shown in FIG. 2B. This gene cluster is found to comprise at least eight ORFs. ORF1 encodes a homolog of E. coli AtoC homolog (43% identity), and is a response regulator subunit of a His/Asp kinase regulatory pathway (Grebe and Stock (1999) Adv. Microb. Physiol. 41:139-227). ORF2 (ambl) encodes a homolog of the LysR family of transcription regulators (Henikoff et al. (1988) Proc. Natl. Acad. Sci. USA 85(18):6602-6606). ORF3 encodes a homolog to ORF3 of the jerangolid gene cluster and is a partial homolog of SAV3316 from Streptomyces avermitilis. ORF4 (amb2) encodes a cytochrome P540 hydroxylase. ORF5 (amb3) encodes a two-component sensor, HisK subunit. ORF 6 (amb4) encodes a response regulator subunit in a His/Asp kinase regulatory pathway. ORF5 is transcriptionally coupled to ORF6. ORF7 (amb5) encodes a signal transduction histidine kinase subunit. ORF8 (amb6) encodes a conserved hypothetical protein of the YehP family.
Table 2 provides a description of the genes of the upstream region of the ambruticin proteins.







TABLE 2







Genes in the region upstream of the ambruticin PKS determined


from the nucleotide sequence given in SEQ ID NO: 2.









Gene
Domain/Activity
boundaries





ORF1
response regulator subunit in His/Asp
 1-765



kinase


ORF2 (amb1)
transcriptional regulator
2607-3557


ORF3
homolog of ORF3 of jerangolid gene
3595-4041



cluster


ORF4 (amb2)
cytochrome P540 hydroxylase
5141-6361


ORF5 (amb3)
two-component sensor, HisK subunit
6503-7282


ORF6 (amb4)
response regulator subunit in His/Asp
7279-8664



kinase


ORF7 (amb5)
signal transduction histidine kinase
 9474-11000


ORF8 (amb6)
YehP family
11030-12463









The invention provides for nucleic acids comprising the ORFs and the polypeptides encoded by the ORFs downstream and upstream of the ambruticin gene cluster (ORF1-11).
The involvement of the genes and proteins of the invention in the biosynthesis of ambruticins is clear based upon the results of transposon mutagenesis. Transposon inserted in each of the transposon integration sites indicated in FIG. 2A results in disruption of ambruticin biosynthesis or export.
In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one domain, alternatively at least one module, alternatively at least one polypeptide, involved in the biosynthesis of an ambruticin.
In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to SEQ ID NOS:33-49 or its complement. Hereinafter, each reference to a nucleic acid sequence is also intended to refer to and include the complementary sequence, unless otherwise stated or apparent from context. In an embodiment the subsequence comprises a sequence encoding a complete ambruticin PKS domain, module or polypeptide.
In one aspect, the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes an ORF, module or domain having an amino acid sequence identical or substantially similar to an ORF, module or domain encoded by SEQ ID NOS:33-49. Generally, a polypeptide, module or domain having a sequence substantially similar to a reference sequence has substantially the same activity as the reference protein, module or domain (e.g., when integrated into an appropriate PKS framework using methods known in the art). In certain embodiments, one or more activities of a substantially similar polypeptide, module or domain are modified or inactivated as described below.
In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NOS:33-49, e.g., a polypeptide, module or domain involved in the biosynthesis of an ambruticin, wherein said nucleotide sequence comprises at least 10, 20, 25, 30, 35, 40, 45, or 50 contiguous base pairs identical to a sequence of SEQ ID NOS:33-49. In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NOS:33-49, e.g., a polypeptide, module or domain involved in the biosynthesis of a ambruticin, wherein said polypeptide, module or domain comprises at least 10, 15, 20, 30, or 40 contiguous residues of a corresponding polypeptide, module or domain comprising a sequence of SEQ ID NOS:33-49.
It will be understood that SEQ ID NO:1 was determined using the inserts of cosmids 10K10B3 (pKOS344-112E), 10T5F7 (pKOS344-135F), and plasmid pKOS375-77Bg. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to an ORF encoding sequence of the inserts of cosmids 10K10B3 (pKOS344-112E), 10T5F7 (pKOS344-135F), and plasmid pKOS375-77Bg.
It will be understood that SEQ ID NO:2 was determined using the insert of plasmid pKOS344-135B. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to an ORF encoding sequence of the insert of plasmid pKOS344-135B.
In another aspect, the invention provides the proteins encoded by the genes of SEQ ID NOS:1 and 2 in both purified and recombinant form, as well as proteins having amino acid sequences substantial identity to the proteins encoded by the genes of SEQ ID NOS:1 and 2. The amino acid sequences of these genes are provided in FIGS. 3 and 4, respectively.
Those of skill will recognize that, due to the degeneracy of the genetic code, a large number of DNA sequences encode the amino acid sequences of the domains, modules, and proteins of the ambruticin PKS, the enzymes involved in ambruticin modification and other polypeptides encoded by the genes of the ambruticin biosynthetic gene cluster. The present invention contemplates all such DNAs. For example, it may be advantageous to optimize sequence to account for the codon preference of a host organism. The invention also contemplates naturally occurring genes encoding the ambruticin PKS that are polymorphic or other variants.
As used herein, the terms “substantial identity,” “substantial sequence identity,” or “substantial similarity” in the context of nucleic acids, refers to a measure of sequence similarity between two polynucleotides. Substantial sequence identity can be determined by hybridization under stringent conditions, by direct comparison, or other means. For example, two polynucleotides can be identified as having substantial sequence identity if they are capable of specifically hybridizing to each other under stringent hybridization conditions. Other degrees of sequence identity (e.g., less than “substantial”) can be characterized by hybridization under different conditions of stringency. “Stringent hybridization conditions” refers to conditions in a range from about 5° C. to about 20° C. or 25° C. below the melting temperature (Tm) of the target sequence and a probe with exact or nearly exact complimentarily to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel, 1987, Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory). Typically, stringent hybridization conditions for probes greater than 50 nucleotides are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 50° C., preferably at least about 60° C. As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed. Exemplary conditions include hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minuets at 65° C., or at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 65° C.; wash with 2×SSC, 1% SDS, at 50° C.
Alternatively, substantial sequence identity can be described as a percentage identity between two nucleotide or amino acid sequences. Two nucleic acid sequences are considered substantially identical when they are at least about 70% identical, or at least about 80% identical, or at least about 90% identical, or at least about 95% or 98% identical. Two amino acid sequences are considered substantially identical when they are at least about 60%, more often at least about 70%, at least about 80%, or at least about 90% sequence identity to the reference sequence. Percentage sequence (nucleotide or amino acid) identity is typically calculated using art known means to determine the optimal alignment between two sequences and comparing the two sequences. Optimal alignment of sequences may be conducted using the local homology algorithm of Smith and Waterman (1981) Adv. Appi. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nati. Acad. Sci. U.S.A. 85: 2444, by the BLASTTM (Basic Local Alignment Search Tool, National Library of Medicine) algorithm of Altschul (1990) J. Mol. Biol. 215: 403-410; and Shpaer (1996) Genomics 38: 179-191, or by the algorithm of Needleham et al. (1970) J. Mol. Biol. 48: 443-453; and Sankoff et al.m 1983, Time Warps, String Edits, and Macromolecules, The Theory and Practice of Sequence Comparison, Chapter One, Addison-Wesley, Reading, MA; generally by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.; BLASTTM from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). In each case default parameters are used (for example the BLASTTM program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Nati. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands).
The invention methods may be directed to the preparation of an individual polyketide. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. The resulting polyketides may be further modified to convert them to other useful compounds. Examples of chemical structures of that can be made using the materials and methods of the present invention include known analogs, such as those described in Kalesse and Christmann, 2002, “The Chemistry and Biology of the Ambruticin Family” Synthesis (8):981-1003 and the references cited therein, and novel molecules produced by modified or chimeric PKSs comprising a portion of the ambruticin PKS sequence, molecules produced by the action of polyketide modifying enzymes from the ambruticin PKS cluster on products of other PKSs, molecules produced by the action on products of the ambruticin PKS of polyketide modifying enzymes from other PKSs, and the like.
In one aspect, the cell is native to the ambruticin biosynthetic gene cluster. Alternatively, the cell is a host cell that is either native or heterologous to the ambruticin gene cluster, wherein the ambruticin biosynthetic genes are present, either on a vector or integrated into the chromosome of the cell. A cell native to the ambruticin biosynthetic gene cluster is a cell of the genus Sorangium. Preferably, the cell is a Sorangium cellulosum. More preferably, the cell is the So ce10, NCIMB12601 or So ce307 strain of Sorangium cellulosum. A host cell heterologous to the ambruticin gene cluster includes, but is not limited to, eubacterial cells such as E. coli, yeast cells such as Saccharomyces cerevisiae, or myxobacterial cells such as Myxococcus xanthus. 
In one embodiment, one or more, or all the necessary, ambruticin biosynthetic genes are integrated into the chromosome of a myxobacteria, such as Myxococcus xanthus, and the myxobacteria, when cultured, expresses the ambruticin PKS and produces ambruticin. The ambruticins produced include, but are not limited to, ambruticin VS3, ambruticin VS4, and ambruticin VS5. In another aspect, the ambruticin biosynthetic genes are integrated into the chromosome of a myxobacteria, such as Myxococcus xanthus, using the necessary helper plasmids.
As noted, in one aspect the invention provides recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of the ambruticin polyketide synthase. A polyketide synthase “derived from” a naturally occurring PKS contains the scaffolding encoded by all the portion employed of the naturally occurring synthase gene, contains at least two modules that are functional, and contains mutations, deletions, or replacements of one or more of the activities of these functional modules so that the nature of the resulting polyketide is altered. This definition applies both at the protein and genetic levels. Particular embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS, and derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or wherein any of these activities has been added or mutated so as to change the ultimate polyketide synthesized. There are at least five degrees of freedom for constructing a polyketide synthase in terms of the polyketide that will be produced. See, U.S. Pat. No. 6,509,455 for a discussion.
As can be appreciated by those skilled in the art, polyketide biosynthesis can be manipulated to make a product other than the product of a naturally occurring PKS biosynthetic cluster. For example, AT domains can be altered or replaced to change specificity. The variable domains within a module can be deleted and or inactivated or replaced with other variable domains found in other modules of the same PKS or from another PKS. See e.g., Katz and McDaniel, Med. Res. Rev. 19: 543-558 (1999) and WO 98/49315. Similarly, entire modules can be deleted and/or replaced with other modules from the same PKS or another PKS. See e.g., Gokhale et al., Science 284: 482 (1999) and WO 00/47724 each of which are incorporated herein by reference. Protein subunits of different PKSs also can be mixed and matched to make compounds having the desired backbone and modifications. For example, subunits of 1 and 2 (encoding modules 1-4) of the pikromycin PKS were combined with the DEBS3 subunit to make a hybrid PKS product (see Tang et al., Science. 287: 640 (2001), WO 00/26349 and WO 99/6159).
Mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded. Polypeptides with “altered activity” include those in which one or more domains are inactivated or deleted, or in which a mutation changes the substrate specificity of a domain, as well as other alterations in activity. Mutations include deletions to the entire or substantially the entire gene. Example 4 teaches the construction of a recombinant vector and a recombinant cell deleted for the ambS gene. It is well within the art for one to design similar deletions of one or mote other ambruticin biosynthetic genes. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. (See Zoller and Smith, Methods in Enzymology (1983) 100:468). Primer extension is effected using DNA polymerase. The product of the extension reaction is cloned, and those clones containing the mutated DNA are selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. (See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409). PCR mutagenesis can also be used for effecting the desired mutations. Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by chemical mutagenesis, or by irradiation. In addition to providing mutated forms of regions encoding enzymatic activity, regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS synthase can be recovered, for example, using PCR techniques with appropriate primers. By “corresponding” activity encoding regions is meant those regions encoding the same general type of activity—e.g., a ketoreductase activity in one location of a gene cluster would “correspond” to a ketoreductase-encoding activity in another location in the gene cluster or in a different gene cluster; similarly, a complete reductase cycle could be considered corresponding—e.g., KR/DH/ER could correspond to KR alone.
If replacement of a particular target region in a host polyketide synthase is to be made, this replacement can be conducted in vitro using suitable restriction enzymes or can be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene. One such system involving plasmids of differing temperature sensitivities is described in PCT application WO 96/40968. Another useful method for modifying a PKS gene (e.g., making domain substitutions or “swaps”) is a RED/ET cloning procedure developed for constructing domain swaps or modifications in an expression plasmid without first introducing restriction sites. The method is related to ET cloning methods (see, Datansko and Wanner, 2000, Proc. Natl. Acad. Sci. USA 97, 6640-45; Muyrers et al., 2000, Genetic Engineering 22:77-98). The RED/ET cloning procedure is used to introduce a unique restriction site in the recipient plasmid at the location of the targeted domain. This restriction site is used to subsequently linearize the recipient plasmid in a subsequent ET cloning step to introduce the modification. This linearization step is necessary in the absence of a selectable marker, which cannot be used for domain substitutions. An advantage of using this method for PKS engineering is that restriction sites do not have to be introduced in the recipient plasmid in order to construct the swap, which makes it faster and more powerful because boundary junctions can be altered more easily.
In a further aspect, the invention provides methods for expressing chimeric or hybrid PKSs and products of such PKSs. For example, the invention provides (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-ambruticin producing enzyme, but which includes one or more functional domains, modules or polypeptides of ambruticin PKS; and (2) encoding DNA for a chimeric PKS that is substantially patterned on the ambruticin PKS, but which includes one or more functional domains, modules, or polypeptides of another PKS or NRPS.
With respect to item (1) above, in one embodiment, the invention provides chimeric PKS enzymes in which the genes for a non-ambruticin PKS function as accepting genes, and one or more of the above-identified coding sequences for ambruticin domains or modules are inserted as replacements for one or more domains or modules of comparable function. Construction of chimeric molecules is most effectively achieved by construction of appropriate encoding polynucleotides. In making a chimeric molecule, it is not necessary to replace an entire domain or module accepting of the PKS with an entire domain or module of ambruticin PKS: subsequences of a PKS domain or module that correspond to a peptide subsequence in an accepting domain or module, or which otherwise provide useful function, may be used as replacements. Accordingly, appropriate encoding DNAs for construction of such chimeric PKS include those that encode at least 10, 15, 20 or more amino acids of a selected ambruticin domain or module.
Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and 97/02358. A number of genetic engineering strategies have been used with DEBS to demonstrate that the structures of polyketides can be manipulated to produce novel natural products, primarily analogs of the erythromycins (see the patent publications referenced supra and Hutchinson, 1998, Curr. Opin. Microbiol. 1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83). In one embodiment, the components of the chimeric PKS are arranged onto polypeptides having interpolypeptide linkers that direct the assembly of the polypeptides into the functional PKS protein, such that it is not required that the PKS have the same arrangement of modules in the polypeptides as observed in natural PKSs. Suitable interpolypeptide linkers to join polypeptides and intrapolypeptide linkers to join modules within a polypeptide are described in PCT publication WO 00/47724.
A partial list of sources of PKS sequences for use in making chimeric molecules, for illustration and not limitation, includes Avermectin (U.S. Pat. No. 5,252,474; MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256; MacNeil et al., 1992, Gene 115: 119-25); Candicidin (FRO008) (Hu et al., Mol. Microbiol. 14: 163-72); Epothilone (U.S. Pat. No. 6,303,342); Erythromycin (WO 93/13663; U.S. Pat. No. 5,824,513; Donadio et al., 1991, Science 252:675-79; Cortes et al.,1990, Nature 348:176-8); FK-506 (Motamedi et al., 1998, Eur. J. Biochem. 256:528-34; Motamedi et al., 1997, Eur. J. Biochem. 244:74-80); FK-520 (U.S. Pat. No. 6,503,737; see also Nielsen et al., 1991, Biochem. 30:5789-96); Lovastatin (U.S. Pat. No. 5,744,350); Nemadectin (MacNeil et al., 1993, supra); Niddamycin (Kakavas et al., 1997, J. Bacteriol. 179:7515-22); Oleandomycin (Swan et al., 1994, Mol. Gen. Genet. 242:358-62; U.S. Pat. No. 6,388,099; Olano et al., 1998, Mol. Gen. Genet. 259:299-308); Platenolide (EP Pat. App. 791,656); Rapamycin (Schwecke et al., 1995, Proc. Natl. Acad. Sci. USA 92:7839-43); Aparicio et al., 1996, Gene 169:9-16); Rifamycin (August et al., 1998, Chemistry & Biology, 5: 69-79); Soraphen (U.S. Pat. No. 5,716,849; Schupp et al., 1995, J. Bacteriology 177: 3673-79); Spiramycin (U.S. Pat. No. 5,098,837); Tylosin (EP 0 791,655; Kuhstoss et al., 1996, Gene 183:231-36; U.S. Pat. No. 5,876,991). Additional suitable PKS coding sequences remain to be discovered and characterized, but will be available to those of skill (e.g., by reference to GenBankTM (Genetic Sequence Data Bank, U.S. Department of Health and Human Services).
The ambruticin PKS-encoding polynucleotides of the invention may also be used in the production of libraries of PKSs (i.e., modified and chimeric PKSs comprising at least a portion of the ambruticin PKS sequence. The invention provides libraries of polyketides by generating modifications in, or using a portion of, the ambruticin PKS so that the protein complexes produced by the cluster have altered activities in one or more respects, and thus produce polyketides other than the natural ambruticin product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native PKS cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities. Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be transformed into the appropriate host cells to construct a polyketide library. In one approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected for successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. A variety of strategies can be devised to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, more preferably at least 50, reflecting similar numbers of different altered PKS gene-clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.
Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of candidate polyketides. The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can be included.
As noted above, the DNA compounds of the invention can be expressed in host cells for production of proteins and of known and novel compounds. Preferred hosts include fungal systems such as yeast and procaryotic hosts, but single cell cultures of, for example, mammalian cells could also be used. A variety of methods for heterologous expression of PKS genes and host cells suitable for expression of these genes and production of polyketides are described, for example, in U.S. Pat. Nos. 5,843,718; 5,830,750 and 6,262,340; WO 01/31035, WO 01/27306, and WO 02/068613; and U.S. Patent Application Pub. Nos. 20020192767 and 20020045220.
Appropriate host cells for the expression of the hybrid PKS genes include those organisms capable of producing the needed precursors, such as malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-ACP, and having phosphopantotheinylation systems capable of activating the ACP domains of modular PKSs. See, for example, U.S. Pat. No. 6,579,695. However, as disclosed in U.S. Pat. No. 6,033,883, a wide variety of hosts can be used, even though some hosts natively do not contain the appropriate post-translational mechanisms to activate the acyl carrier proteins of the synthases. Also see WO 97/13845 and WO 98/27203. The host cell may natively produce none, some, or all of the required polyketide precursors, and may be genetically engineered so as to produce the required polyketide precursors. Such hosts can be modified with the appropriate recombinant enzymes to effect these modifications. Suitable host cells can be heterologous to the nucleotide sequences encoding ambruticin PKS domans, modules or polypeptides, heterologous to the promoter operatively linked to the nucleotide sequences, or both. Suitable host cells include Streptomyces, E. coli, yeast, and other procaryotic hosts that use control sequences compatible with Streptomyces spp. Examples of suitable hosts that either natively produce modular polyketides or have been engineered so as to produce modular polyketides include but are not limited to actinomyctes such as Streptomyces lividans, Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens, and Saccharopolyspora erythraea, eubacteria such as E. coli, myxobacteria such as Myxococcus xanthus, Stigmatella, Cystobacter, Archangium, and Angiococcus, and yeasts such as Saccharomyces cerevisiae. 
In one embodiment, any native modular PKS genes in the host cell have been deleted to produce a “clean host,” as described in U.S. Pat. No. 5,672,491, incorporated herein by reference.
In some embodiments, the host cell expresses, or is engineered to express, a polyketide “tailoring” or “modifying” enzyme. Once a PKS product is released, it is subject to post-PKS tailoring reactions. These reactions are important for biological activity and for the diversity seen among polyketides. Tailoring enzymes normally associated with polyketide biosynthesis include oxygenases, glycosyl- and methyl-transferases, acyltransferases, halogenases, cyclases, aminotransferases, and hydroxylases. In addition to biosynthetic accessory activities, secondary metabolite clusters often code for activities such as transport.
Tailoring enzymes for modification of a product of the ambruticin PKS, a non-ambruticin PKS, or a chimeric PKS, can be those normally associated with ambruticin biosynthesis or “heterologous” tailoring enzymes. Tailoring enzymes can be expressed in the organism in which they are naturally produced, or as recombinant proteins in heterologous hosts. In some cases, the structure produced by the heterologous or hybrid PKS may be modified with different efficiencies by post-PKS tailoring enzymes from different sources. In such cases, post-PKS tailoring enzymes can be recruited from other pathways to obtain the desired compound. For example, the tailoring enzymes of the ambruticin PKS gene cluster can be expressed heterologously to modify polyketides produced by non-ambruticin synthases or can be inactivated in the ambruticin producer.
Alternatively, the unmodified polyketide compounds can be produced in the recombinant host cell, and the desired modification (e.g., oxidation) steps carried out in vitro (e.g., using purified enzymes, isolated from native sources or recombinantly produced) or in vivo in a converting cell different from the host cell (e.g., by supplying the converting cell with the unmodified polyketide).
It will be apparent to one of skill in the art that a variety of recombinant vectors can be utilized in the practice of aspects of the invention. As used herein, “vector” refers to polynucleotide elements that are used to introduce recombinant nucleic acid into cells for either expression or replication. Selection and use of such vehicles is routine in the art. An “expression vector” includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those that integrate into the host cell genome.
The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes may be chosen to contain control sequences operably linked to the resulting coding sequences in a manner that expression of the coding sequences may be effected in an appropriate host. Suitable control sequences include those that function in eucaryotic and procaryotic host cells. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This can be done individually, or using a pool of isolated encoding nucleotide sequences, which can be inserted into host vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies. Suitable control sequences for single cell cultures of various types of organisms are well known in the art. Control systems for expression in yeast are widely available and are routinely used. Control elements include promoters, optionally containing operator sequences, and other elements depending on the nature of the host, such as ribosome binding sites. Particularly useful promoters for procaryotic hosts include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including those from Type I or aromatic (Type II) PKS gene clusters. Examples are act promoters, tcm promoters, spiramycin promoters, and the like. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, are also useful. Additional examples include promoters derived from biosynthetic enzymes such as for tryptophan (trp), the β-lactamase (bla), bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can be used.
As noted, particularly useful control sequences are those which themselves, or with suitable regulatory systems, activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the plasmid identified as pCK7, i.e., the actI/actIII promoter pair and the actII-ORF4 (an activator gene), is particularly preferred. Particularly preferred hosts are those that lack their own means for producing polyketides so that a cleaner result is obtained. Illustrative control sequences, vectors, and host cells of these types include the modified S. coelicolor CH999 and vectors described in PCT publication WO 96/40968 and similar strains of S. lividans. See U.S. Pat. Nos. 5,672,491; 5,830,750; 5,843,718; and 6,177,262, each of which is incorporated herein by reference.
Other regulatory sequences may also be desirable which allow for regulation of expression of the PKS sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored, and this characteristic provides a built-in marker for screening cells successfully transformed by the present constructs.
The various PKS nucleotide sequences, or a mixture of such sequences, can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements or under the control of a single promoter. The PKS subunits or components can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunits so that hybrid or chimeric PKSs can be generated. The design of such restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR. Methods for introducing the recombinant vectors of the present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl2 or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.
Thus, the present invention provides recombinant DNA molecules and vectors comprising those recombinant DNA molecules that encode at least a portion of the ambruticin PKS and that, when transformed into a host cell and the host cell is cultured under conditions that lead to the expression of said ambruticin PKS enzymes, results in the production of polyketides including but not limited to ambruticin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors.
Suitable culture conditions for production of polyketides using the cells of the invention will vary according to the host cell and the nature of the polyketide being produced, but will be know to those of skill in the art. See, for example, the examples below and WO 98/27203 and WO 01/83803. The polyketide product produced by host cells of the invention can be recovered (i.e., separated from the producing cells and at least partially purified) using routine techniques (e.g., extraction from broth followed by chromatography). The compositions, cells and methods of the invention may be directed to the preparation of an individual polyketide or a number of polyketides. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it.
EXAMPLES
The following Examples are intended to illustrate, but not limit, the scope of the invention.
Example 1
Isolation of Transposon Insertions in So ce10
Transposons insertions in So ce10 were isolated by methods developed by Julien and Fehd (2003) Appl. Environ. Microbiol. 69:6299-6301; incorporated herein by reference). So ce10was grown in 307 seed medium to an OD600 of 1.0, and 10 ml of cells were centrifuged. 307 seed medium consists of (per liter): 9 gm casitone (Difco), 3 gm fructose, 0.5 gm MgSO4·7H2O, 0.5 gm CaCl2·2H2O, and 25 mL of 1 M HEPES, pH 7.6.
The So ce10pellet was resuspended in  ̃200 μl of 307 seed medium and mixed with 10 ml of E. coli strain harboring plasmids pGZ119EH, pKOS111-47, and pKOS249-52B or pKOS375-57B1 that also had been pelleted. The mixed cells were spotted on an S42 plate and incubated at 30° C. overnight. The next day, the cells were resuspended in 10 ml 307 seed medium and aliquots were plated on S42 plates (Jaoua et al., 1992, Plasmid 28:157-165, incorporated herein in its entirety by reference) containing phleomycin (50 μg/ml) and kanamycin (50 μg/ml). The plates were incubated 7 days at 32° C.
To screen the transposon insertion strains for production of ambruticin, individual colonies were picked into 96 well microtiter plates containing S4s2 medium. After 7 days of incubation, the plates were overlaid with Candida parapsilopsis in YPD top agar. After two days of incubation at 30° C., the plates were examined for growth of the Candida. Those that showed growth indicate the lack of ambruticin production, and thus are candidates for transposon insertion in genes for biosynthesis or export of ambruticin.
Example 2
Isolation of Ambruticin PKS Cosmids
Genomic DNA was isolated from Sorangium cellulosum So ce10 using an established protocol (Jaoua, S., Neff, S., and Schupp, T. “Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome,” 1992 Plasmid 28:157-165). The DNA was partially digested with Sau3AI using a serial dilution method and libraries were constructed in SuperKOS (a smaller derivative of SuperCos-1) using the protocol for SuperCos-1 from Stratagene. Colonies were picked, cosmid DNA was isolated on the Qiagen robot, and the DNA was submitted for end sequencing. The data was analyzed by BLASTTM and all PKS positive cosmids were prepared in larger amounts for further analysis.
From about 1000 end sequences of the So ce10cosmid library, six cosmids were found which encoded multimodular PKS at one end. The other end of each of these six did not encode a gene that was obviously from a PKS cluster. The six sequences fell roughly into three types: two that matched cis-AT type PKS typical of myxobacteria (10K2C2, 10K7F6), two that look like possible trans-AT type PKS (10K10B3, 10K7F10), and two that matched polyether/polyene type PKS (10K1B5, 10K10D10). By restriction digestion and gel analysis, the banding patterns indicated that no pair of these cosmids shared a significant overlap; however, subsequent DNA sequence analysis revealed overlaps that were not apparent from restriction analysis.
The sequence of the insertion site of one of the transposon mutants described in Example 1 above was identical to the PKS sequence at the end of cosmid 10K10B3 so this cosmid was shotgun sequenced. This cosmid was found to contain a PKS loading module and seven PKS extender modules having a domain organization consistent with the structure of ambruticin (except for an apparent malonyl-specific AT in module 4 where incorporation of a methylmalonyl unit was expected, but this is not unprecedented). Additional transposon insertions that knocked out ambruticin production also matched sequence in this cosmid, verifying that it encodes the ambruticin PKS.
Cosmids or plasmids representing regions upstream and downstream of cosmid 10K10B3 were isolated from libraries prepared from genomic DNA of So ce10strains having transposon insertions at the ends of cosmid 10K10B3 (strains with insertions 1A11, 2B8 and 5F7 were used). The libraries were plated on LB medium containing both carbenicillin and phleomycin to select only those cosmids (or plasmids) having the transposon. After end sequencing the clones obtained, those that covered the greatest amount of sequence upstream or downstream of 10K10B3 were chosen for complete shotgun sequencing.
Example 3
Heterologous Expression of the Ambruticin Biosynthetic Genes and Production of Ambruticin in Myxococcus xanthus 
Chromosomal DNA is embedded in agarose using the Chef bacterial genomic DNA kit from Biorad. So ce10is grown in 307 seed medium (see Example 1) to an OD600  ̃4.0 ( ̃4×108 cells/ml). A 20 ml aliquot of cell is centrifuged, resuspended in 2 ml of milli Q water, and added to 2 ml of melted 2% agarose from the kit. The agarose cell suspension is transferred to plug molds. After hardening, the agar plugs are placed into lysozyme buffer and lysozyme is added to 1 mg/ml. The plugs are incubated at 37° C. overnight. The next day the plugs are washed 2 times with 50 ml of milli Q water and placed in proteinase K buffer containing proteinase K. The plugs are incubated at 50° C. overnight. The next day the plugs are washed several times with TE (10 mM Tris pH7.6, 1 mM EDTA). The BAC library is constructed by Amplicon Express (Pullman, Wash.) by partially digesting the DNA with BamHI and ligating into pEC1BAC cleaved with BamHI. The average insert size of the library is  ̃100 kb.
To modify the BAC, plasmid pKOS375-151.1 is constructed. This plasmid harbors several important functions necessary for transfer and integration of a plasmid into the M. xanthus. First, it contains oriT, the region required for conjugative transfer of DNA. Second it contains the Mx9 integrase gene and the Mx9 attP required for site-specific integration (Julien (2003) J. Bacteriol. 185921):6324-6330). Third it contains the bleomycin resistance marker for selection in M. xanthus. Fourth, it contains a loxP site that can be used for Cre mediated site-specific integration with the loxP site on the BAC. Finally, it contains the R6Kγ replication origin. This origin requires the pir protein supplied only in certain E. coli strains and thus pKOS375-151.1 is a conditionally replicating plasmid.
The first step in constructing pKOS375-151.1 is to remove the p15A origin of replication from pACYC177 (New England Biolabs, Beverly, Mass.) and replace it with the R6Kγ conditionally replicating origin. This is done by cleaving pACYC177 with NheI and BstBI, making the DNA ends blunt with the Klenow fragment of DNA polymerase I, and ligating it with the blunt HindIII fragment from pKOS249-96.5 harboring the R6Kγ origin. This plasmid, pKOS375-109 replicates only in E. coli cells expressing the pir+ or the pir116 gene, such as E. coli strain EC100D (Epicentre).
Next the Mx9 int gene and attP site are added. Plasmid pKOS249-28 is cleaved with AseI and HindIII, the DNA ends are made blunt with the Klenow fragment of DNA polymerase I, and ligated to pKOS375-109 cleaved with XhoI and BstEII and the DNA ends blunt with the Klenow fragment of DNA polymerase I to create pKOS375-118.
To this plasmid is added the loxP site, oriT, and the bleomycin resistance gene. pKOS375-118 is cleaved with XbaI and EcoRI, the DNA ends made blunt with the Klenow fragment of DNA polymerase I and ligated to the NotI-EcoRI fragment from pKOS375-131.1 harboring the loxP, oriT, and bleomycin resistance genes. This plasmid is designated pKOS375-132.
In the final step to construct pKOS375-151.1, the ampicillin resistance gene is removed by cleaving pKOS375-132.1 with SspI and AhdI, making the DNA ends blunt with the Klenow fragment of DNA polymerase I, and relegating. The complete nucleotide sequence of pKOS375-151.1 is depicted in FIG. 5 (SEQ ID NO:61) and features described in Table 3.







TABLE 3







Features on plasmid pKOS375-151.1.












GenBank




Plasmid
accession
GenBank


DNA fragment
nucleotides
number
nucleotides





R6Kγori
367-634
M65025
 434-168


oriT
1040-1405
X54459
4606-4971


Tn5 kanamycin
1435-1759
V00615, V00618
1196-1700, 1-125


resistance


promoter


bleomycin
1785-2165
X01702
 65-446


resistance


gene


loxP
2191-2224
M10494
 14-47


Mx9 int
2438-4099
AY247757
 607-2269









BAC clone, pKOS375-123.5K3, harbors the entire ambruticin biosynthetic gene cluster and is 200 kb, as determined by partial sequencing. To modify this BAC for conjugation and integration into M. xanthus, Cre mediated site-specific recombination with pKOS375-151.1 is performed. The conditions of the in vitro reaction is as follows: 3 μl of Cre buffer, 3 μl pKOS375-123.5K3 (500 ng DNA), 2 μl pKOS375-151.1 (50 ng), 22 μl Milli Q water, 1 μl Cre (New England Biolabs, Beverly, Mass.). The reaction is incubated at 37° C. for 60 minutes and then at 65° C. for 10 minutes to inactivate the Cre protein. A 5 μl aliquot is dialyzed and electroporated into DH10B cells harboring pKOS111-47 (constructed by digesting with NsiI and self-ligating the RP4 helper plasmid to remove the kanamycin resistance gene (Pansegrau et al. (1994) J. Mol. Biol. 239:623-663)) and pRG1 (Griffen and Kolodner (1990) J. Bacteriol. 172:6291-6299), a plasmid expressing the lacI repressor gene, used to repress transcription from the T7A1 promoter that transcribes the Mx9 int gene. This modified BAC is designated pKOS375-162.3.3.
To transfer and integrate the ambruticin genes, M. xanthus strain DZ1 is grown in CYE medium at 30° C. to an OD600 between 0.5 and 1.0. Approximately 1×109 cells are concentrated into 100 μl and mixed with approximately 1×108 DH10B (pKOS375-162.3.3, pRG1, pKOS111-47) cells. The cells are spotted onto the center of a CYE plate and incubated at 30° C. overnight. The next day the cells are resuspended in 5 ml CYE, 5 μl of approximately 1×1012 T3 phage/ml are added, and the cells are incubated at 30° C. for 2 hours to lyse the E. coli cells. Aliquots are plated in CYE top agar containing gentamycin (60 μg/ml) and phleomycin (50 μg/ml). The plates are incubated until colonies appeared, at least 5 days.
To detect the production of ambruticins, individual colonies are fermented in MOM medium with XAD1180 resin. The fermentations are started by growing seed cultures in 3 ml of CYE overnight at 30° C. The next day, 6 μl of methyl oleate is added and the cultures are grown for an additional day. The cultures are subcultured in 50 ml of CYE containing 50 μl of methyl oleate and grown for two days at 30° C. at 150 rpm. The cultures are then diluted into 50 ml fermentation medium (25 ml 2×CTS, 2.5 ml HEPES (1 M pH 7.6), 350 μl methyl oleate, 200 μl trace metals, and 22 ml Milli Q water) containing XAD1180 resin. The cultures are incubated at 30° C. shaking at 125 rpm. After 24 hours, the shaking is increased to 150 rpm and the cultures are incubated an additional 5 days.
The XAD1180 resin is harvested by decanting the medium and cells and washing the XAD twice with Milli Q water. The ambruticins are eluted with 25 ml of 100% methanol. Aliquots are analyzed using LC/MS. Ambruticin VS3 is the major product, and ambruticin VS4 and ambruticin VS5 are produced in detectable amounts.
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and individually indicated to be incorporated herein by reference.
Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description are for purposes of illustration and not limitation of the following claims.

## Claims

1. A purified or recombinant polypeptide comprising the β-ketoacylsynthase, the acyltransferase, or the acyl carrier protein domain of SEQ ID NO:3.

2. The purified or recombinant polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO:3.