# Patent

## Title
Non-ribosomal peptide synthetases and associated biosynthetic genes

## Abstract

The present invention describes the identification of novel non-ribosomal peptide synthetases and associated biosynthetic genes from Streptomyces hygroscopicus. The present invention further provides methods for generating novel compounds, such as antibiotics, from these synthetases and associated genes.

## Background
This application is a Continuation-In-Part of U.S. patent application Ser. No. 10/402,842, filed on Mar. 28, 2003, which claims the benefit of Provisional Patent Application Ser. No. 60/368,713, filed on Mar. 29, 2002, the disclosures of which are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to non-ribosomal peptide synthetases and associated biosynthetic genes. The present invention further relates to methods for generating novel compounds, such as antibiotics, with these synthetases and associated genes.
BACKGROUND OF THE INVENTION
Bioactive molecules that are isolated from plants, bacteria, and fungi are often referred to as natural products. These molecules are synthesized by primary or secondary pathways within the organism or may even be degradation products of another molecule. Many of these molecules have shown a variety of therapeutic uses in humans and other animal species. One of the best known examples is taxol, which was originally isolated from the bark of the Pacific Yew tree. Taxol has been shown to have anti-cancer properties and is currently used in the treatment of breast cancer. Actinomycetes are prolific producers of bioactive small molecules. These molecules may be used chemically as immunosuppressants, antibiotics, and cancer therapeutics. Actinomycetes are Gram-positive bacteria that form long, thread-like branched filaments. The term actinomycetes is used to indicate organisms belonging to Actinomycetales, an Order of the domain Bacteria. The Actinomycetales are divided into 34 Families including Streptomyceteae, to which belongs the Genus Streptomyces (Bergey's Manual of Systematic Bacteriology, Second Edition, 2001; George M. Garrity, Editor-in-Chief, Springer Verlag, New York).
Natural products derived from microbial sources primarily belong to three metabolic families: peptides, polyketides, and terpenes. Peptide natural products can be further classified based on their mode of synthesis: ribosomal and non-ribosomal. Non-ribosomal peptides are synthesized on enzymatic thiotemplates termed non-ribosomal peptide synthetases (NRPS). The non-ribosomal peptides encompass a wide range of compounds having diverse activities including, but not limited to, immunosupressive (such as cyclosporin), surfactant (such as surfactin), siderophores (such as enterobactin), virulence factors (such as yersinabactin), antibacterial (such as penicillin and vancomycin), and anti-cancer (such as actinomycin and bleomycin) activities (Weber et al., Current Genomics 1994; 26:120–25; Ehmann et al., Proc. Nat. Acad. Sci. 2000; 97:2509–14; Gehring et al., Biochemistry 1998; 37:11637; Kallow et al., Biochemistry 1998; 37:5947–52; Trauger et al., Proc. Nat. Acad. Sci. 2000; 97:3112–17; Schauweker et al., J. Bacteriology 1999; 27:2468–74; and Shen et al., Bioorganic Chem 1999; 27:155–71). Non-ribosomal peptides typically range in size from 1–11 amino acids and are produced by a variety of microbes including cyanobacteria, actinomycetes and fungi.
In many cases the non-ribosomal peptides contain non-proteogenic amino acids such as norleucine, β-alanine, ornithine, etc., for which biogenesis pathways, which are secondary to primary metabolism, are required and are post-synthetically modified (e.g., hydroxylated or methylated) by tailoring enzymes. As used herein, the term “proteogenic” indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways. The choice of including a (D)- or (L)-amino acid into a peptide of the present invention depends, in part, on the desired characteristics of the peptide. For example, the incorporation of one or more (D)-amino acids can confer increasing stability on the peptide in vitro or in vivo. As used herein, the term “amino acid equivalent” refers to compounds which depart from the structure of the naturally occurring amino acids, but which have substantially the structure of an amino acid, such that they can be substituted within a peptide that retains biological activity. Thus, for example, amino acid equivalents can include amino acids having side chain modifications and/or substitutions, and also include related organic acids, amides or the like. The term “amino acid” is intended to include amino acid equivalents. The term “residues” refers both to amino acids and amino acid equivalents.
The genes required to make a NRPS and the necessary tailoring enzymes have been shown in all cases to be localized to the chromosome of the producing microbe. NRPSs are modular in nature, where a module may be defined as a segment of the NRPS necessary to catalyze the activation of a specific amino acid and result in the incorporation of that amino acid into a non-ribosomal peptide. A minimal module contains three domains: (1) adenylation domains (about 60 kDa), responsible for selecting and activating an amino acid and transferring the aminoacyl adenylate to a peptidyl carrying center; (2) thiolation domains, also referred to as peptidyl carrier proteins (8–10 kDa), containing a serine residue which is post-translationally modified with a 4-phosphopantetheine group (Ppant) which acts as an acceptor for the aminoacyl adenylate; and (3) condensation domains (50–60 kDa) which catalyze peptide bond-forming chain-translocating steps between an upstream peptidyl-s-Ppant and the downstream aminoacyl-Ppant of the adjacent module (Doekel, S. and Marahiel, M. A. 2000; Chem. Biol. 7:373–384). This minimal module for chain extension is typically repeated within a synthetase and a co-linear relationship exists between the number of modules present and the number of amino acids in the final product with the order of the modules in the synthetase determining the order of the amino acids in the peptide.
There is a continuing need in the art to determine the genes encoding NRPS complexes.
SUMMARY OF THE INVENTION
The present invention provides the nucleic acid and amino acid sequences of a non-ribosomal peptide synthetase (NRPS) complex from Streptomyces hygroscopicus. The NRPS described herein is comprised of two components, designated MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4), and contains the sequences required for the biosynthesis of the peptide core of lipoglycopeptide antibiotic AC98.
The present invention also provides characterization of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4), including the number of modules in each component and the functional domains contained within each module. In particular, MppA (SEQ ID NO:2) is comprised of three modules, each containing an adenylation, thiolation, and condensation domain, and MppB (SEQ ID NO:4) is comprised of two modules, two epimerization domains, and a partial module comprised only of a condensation domain and thiolation domain.
Further provided by the present invention are expression vectors comprising the genes encoding MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4), and host cells transfected with such MppA (SEQ ID NO:2) and/or MppB (SEQ ID NO:4)-encoding vectors.
The present invention also provides nucleic acid and amino acid sequences for several open reading frames (ORFs) encoding associated gene products that modify the amino acids of the core peptide post-biosynthesis, as well as host cells comprising the ORFs.
In yet a further embodiment, the present invention provides a method for producing the NRPS described herein, which method comprises culturing an NPRS-transformed host cell under conditions that provide for expression of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4).
The present invention further provides a method of producing a cyclic peptide synthesized by of the NRPS comprised of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4), which peptide is an antibiotic. In a preferred embodiment, the antibiotic is AC98.
Also provided by the present invention are methods of modifying the adenylation domains of NRPS in order to produce an antibiotic having a modified peptide core, and a method for evaluating the structural regions of the modified peptide.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1A–B depict the chemical structures of the lipoglycopeptide antibiotic AC98.
FIG. 2 shows a representation of the NRPS complex from this Streptomyces hygroscopicus strain NS17 that is demonstrated to be the minimal biosynthetic machinery responsible for the biosynthesis of the peptide core of AC98.
FIG. 3 is a pictorial representation of the biosynthesis of the AC98 peptide core by the novel NRPS described herein.
FIGS. 4A–E depicts the two-dimensional representation of the binding pockets of adenylation domains within modules of the NRPS of the invention. Amino acid residues 235, 236, 239, 278, 299 & 301, are those that determine the specificity of the binding pocket. FIG. 4A shows the serine-specific binding pocket of the adenylation domain of module 1 within MppA (SEQ ID NO:2). FIG. 4B shows the glycine-specific binding pocket of the adenylation domain of module 2 within MppA (SEQ ID NO:2). FIG. 4C shows the phenylalanine-specific binding pocket of the adenylation domain of module 3 within MppA (SEQ ID NO:2). FIG. 4D shows the tyrosine-specific binding pocket of the adenylation domain of module 1 within MppB (SEQ ID NO:4). FIG. 4E shows the cyclo-arginine-specific binding pocket of the adenylation domain of module 2 within MppB (SEQ ID NO:4).



DETAILED DESCRIPTION OF THE INVENTION
The following definitions are The following definitions are provided for the full understanding of terms and abbreviations used in this specification.
The abbreviations in the specification correspond to units of measure, techniques, properties or compounds as follows: “min” means minutes, “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmole” means millimole(s), “kb” means kilobase, “bp” means base pair(s), and “IU” means International Units. “Polymerase chain reaction” is abbreviated PCR; “Reverse transcriptase polymerase chain reaction” is abbreviated RT-PCR; “Estrogen receptor” is abbreviated ER; “DNA binding domain” is abbreviated DBD; “Ligand binding domain” is abbreviated LBD; “Untranslated region” is abbreviated UTR; “Sodium dodecyl sulfate” is abbreviated SDS; and “High Pressure Liquid Chromatography” is abbreviated HPLC.
Streptomyces hygroscopicus NS17 is a terrestrial actinomycete which produces a novel lipoglycopeptide antibiotic complex (AC98; See FIG. 1). This strain has been deposited with the Agricultural Research Service Culture Collection, 1815 North University St., Peoria, Ill. 61604, Deposit No. NRRL 30439. This antibiotic has been shown to be active against Gram-positive pathogens including, but not limited to, vancomycin resistant enterococci (VRE), methicillin resistant Staphlococcus aureus (MRSA) and Streptococcus pneumoniae. The present invention is based on the isolation of the genes encoding a novel NRPS complex from this Streptomyces strain that is demonstrated to be the minimal biosynthetic machinery responsible for the biosynthesis of the peptide core of AC98 (see FIG. 2).
A number of open reading frames (ORFs), that are predicted to play a role in the biosynthesis of AC98, have been isolated and characterized by sequence analysis. Sequence comparisons of specific ORFs indicate that the proteins that are encoded by the ORFs are tailoring enzymes that are involved in such modifications of the peptide core as glycosylation, methylation and acylation. Other ORFs putatively encode enzymes that may be involved in resistance. A detailed description of the NRPS and its function in biosynthesis of the AC98 peptide core is presented in FIG. 3. The genes required to make the NRPS and the necessary tailoring enzymes are localized to the chromosome of the producing microbe.
NRPS
The NRPS enzymes are generally composed of modules where a minimal module contains three domains, an adenylation domain, a thiolation domain, and a condensation domain.
The adenylation domain is typically about 60 kDa. The main function of this domain is to select and activate a specific amino acid as an aminoacyl adenylate. Based on its function, the adenylation domain regulates the sequence of the peptide being produced. Once charged (as an amino acyl adenylate moiety), the amino acid is transferred to a thiolation domain (peptidyl carrying center).
The second domain is the thiolation domain, also referred to as a peptidyl carrier protein. This domain is typically 8–10 kDa and contains a serine residue that is post-translationally modified with a 4-phosphopantetheine group. This group acts as an acceptor for the aminoacyl adenylate moiety on the amino acid. A nucleophilic reaction leads to the release of the aminoacyl adenylate and conjugation of the amino acid to thiolation domain via a thioester bond.
The third domain is the condensation domain. This domain is typically about 50–60 kDa in size. The main function of this domain is to catalyze the formation of a peptide bond between two amino acids. In this reaction an upstream tethered peptidyl group is translocated to the downstream aminoacyl-s-Ppant and linked to the amino acid by peptide bond formation.
This minimal module for chain extension is typically repeated within a synthetase. Additionally, and typically, a co-linear relationship exists between the number of modules present and the number of amino acids in the final product with the order of the modules in the synthetase determining the order of the amino acids in the peptide. This 1:1 relationship, with every amino acid in the product having one module within the enzyme, is referred to as the co-linearity rule. Examples have been found that violate this rule, and in such cases, the NRPS contains more modules than one would expect based on the number of amino acids incorporated in the peptide product (Challis et al., Chem. Biol. 2000; 7:211–24). In some cases the minimal module also is supplemented with additional domains (epimerization, N- or C-methylation, or cyclization domain), with their position in the synthetase determining the substrate upon which they can act. In addition, it has been observed that NRPSs contain inter-domain spacers or linker regions. It has been proposed that these spacers may play a critical role in communication between domains, modules, and even entire synthetases.
There are highly conserved motifs in the catalytic domains of peptide synthetases including: 10 conserved motifs in the adenylation domain; 1 conserved motif in the thiolation domain; 7 conserved motifs in the condensation domain; 1 conserved motif in the thioesterase domain; 7 conserved motifs in the epimerization domains; and 3 conserved motifs in the N-methylation domains. These are detailed in Marahiel et al., Chemical Rev. 1997; 97:2651–73. In addition to modifications such as epimerization, methylation and cyclization during peptide synthesis, post-translational modifications including methylation, hydroxylation, oxidative cross-linking and glycosylation can occur (Walsh et al., Curr. Opin. Chem. Biol. 2001; 5:525–34).
In the present invention, a biosynthetic pathway containing the genes for a NRPS from Streptomyces hygroscopicus NS17 has been isolated and characterized (SEQ ID NO:1). The NRPS exists as two separate components that have been termed MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4). These components both are involved in the synthesis of the core of AC98.
MppA (SEQ ID NO:2) is composed of three minimal modules, where each module is comprised of an adenylation, thiolation, and condensation domain. MppA (SEQ ID NO:2) conjugates a serine amino acid to a glycine amino acid to produce a peptide. This peptide is then conjugated (through the glycine) to a phenylalanine amino acid. Each amino acid is incorporated into the peptide chain by a unique module. In one embodiment, MppA (SEQ ID NO:2) is about 295 kDa. In another embodiment, MppA (SEQ ID NO:2) is about 2747 amino acids in length. In one embodiment, MppA has an amino acid sequence as depicted in SEQ ID NO:2. In another embodiment, the MppA protein (SEQ ID NO:2) is encoded by a nucleic acid sequence as depicted in SEQ ID NO:3. After addition of the phenylalanine, the peptide chain is then transferred to the MppB (SEQ ID NO:4) component.
The specificity of each AC98 adenylation domain in the NRPS of the present invention was predicted based on the method described in Challis et al., Chem. Biol. 2000; 7:211–24. Amino acid residues 235, 236, 239, 278, 299 & 301 lining the binding pocket of each adenylation domain were found to define domain specificity (the adenylation domains of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4) modules are depicted in FIG. 4) and, in turn, the order of amino acid incorporation into the growing AC98 peptide chain (See FIG. 3).
MppB (SEQ ID NO:4) is composed of 21⁄2 modules and two epimerization domains. In other words, MppB (SEQ ID NO:4) is comprised of 2 complete minimal modules (as described above for MppA (SEQ ID NO:2)) and an additional condensation and thiolation domain (which constitutes the 1⁄2 module). The peptide chain synthesized by MppA (SEQ ID NO:2) is transferred to MppB (SEQ ID NO:4) where a tyrosine amino acid is added to the chain. Prior to the condensation domain, an epimerization enzyme alters the chirality of the tyrosine residue from an L-amino acid to a D-amino acid. The peptide chain is then transferred to a module where a first cycloarginine moiety is added to the peptide. The module which incorporates the first cycloarginine moiety into the peptide is then reused to incorporate a second cycloarginine moiety. A second epimerization domain then alters the chirality of the second cycloarginine from an L-amino acid to a D-amino acid. The terminal module of MppB (SEQ ID NO:4) is unique in that there is only one adenylation domain used for the addition of two cycloarginine residues to the peptide core.
In one embodiment, MppB (SEQ ID NO:4) is about 394 kDa. In another embodiment, MppB (SEQ ID NO:4) is about 3668 amino acids in length. In one embodiment, mppB has an amino acid sequence as depicted in SEQ ID NO:4. In another embodiment, the MppB (SEQ ID NO:4) protein is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 5. After epimerization, the peptide sequence is then modified by tailoring enzymes including, but not limited to, glycosylation enzymes, methylation enzymes and acylation enzymes.
Tailoring Enzymes
After production of the core of the peptide, the sequence may then be modified by additional enzymes which are herein termed “tailoring enzymes”. These enzymes alter the amino acids in the compound without altering the number or the specific amino acids present within the compound. Such tailoring enzymes may include, but are not limited to, arginine cyclase, an O-mannosyltransferase, a phenylalanine C-methyltransferase, a first isovaleryl transferase, and a second isovaleryl transferase.
In the present invention, these tailoring enzymes have been determined to be ORFs present on the AC98 biosynthetic gene cluster and have been termed ORF1–ORF24 (with SEQ ID NOs as described in Table 1). Sequence comparison of these ORFs with homologs provide preliminary information about the function of the enzymes. Table 1 below provides a correlation between the ORF, its location within SEQ ID NO: 1, and its proposed function.
The present invention permits specific changes to be made to the ORFs that encode the tailoring enzymes, either by site directed mutagenesis or replacement, to genetically modify the peptide core. The modifications may be made in a rational manner to improve the biological activity of the antibiotic produced by the bacterial strain or to direct synthesis of compounds that are structurally related to AC98. The invention also allows for the ORFs encoding tailoring enzymes to be isolated and used for biotransformation experiments to produce enzymes to modify and possibly improve other useful compounds.
The determination of the entire biosynthetic pathway of AC98 also enables one of ordinary skill in the art to clone and express the pathway into a heterologous organism. Any organism may be used; preferably a bacterial strain is used. The choice of organism is dependent upon the needs of the skilled artisan. For example, a strain that is amenable to genetic manipulation may be used in order to facilitate modification and production of AC98.
The present invention advantageously permits specific changes to be made to individual modules of NRPS, either by site directed mutagenesis or replacement, to genetically modify the peptide core. Additionally, the NRPS modules can be used to modify other NRPSs that direct the synthesis of other useful peptides through module swapping. For example, the module in NRPS that incorporates tyrosine into the peptide core of the antibiotic may be modified so as to incorporate a serine in its place.







TABLE 1







ORF Correlation
















Sequence







No. Amino
Homolog
Percent


Orf
Position (bp)
Acids
Accession No.*
Identity
Proposed Function





orf1
  77–1048
323
BAB69251
68%
Acetyltransferase



(SEQ ID

(SEQ ID NO:21)
Pfam PF00583


NO:6)


orf2
1045–2460
471
BAB69250
61%
ABC transporter


(SEQ ID

(SEQ ID NO:22)
Pfam PF01574


NO:7)


orf3
2495–3406
303
BAB69249
70%
ABC transporter


(SEQ ID NO 8)

(SEQ ID NO:23)
Pfam PF00528


orf4
3403–4293
296
BAB69248
67%
ABC transporter


(SEQ ID NO 9)

(SEQ ID NO:24)
Pfam PF00528


orf5
4359–5635
425
G75191
34%
Dolichol-phosphate


(SEQ ID

(SEQ ID NO:25)
Pfam PF00535

mannosyltransferase


NO:10)


orf6
5822–7234
470
AE007470
20%
Dolichol-phosphate


(SEQ ID

(SEQ ID NO:26)


mannose protein


NO:11)




mannosyltransferase


orf7
7293–8822
509
X91736
29%
Unknown


(SEQ ID

(SEQ ID NO:27)


NO:12)


orf8
9012–10025
337
X79146
27%
methyltransferase


(SEQ ID

(SEQ ID NO:28)
Pfam PF00891


NO:13)


orf9
29319–30638
439
Z13972
32%
D-aminoacyl hydrolase


(SEQ ID

(SEQ ID NO:29)


superfamily


NO:14)


orf10
30658–32010
450
BAB69335
29%
efflux protein


(SEQ ID

(SEQ ID NO:30)


NO:15)


orf11
32181–33407
408
AF263245
38%
isovaleryl transferase


(SEQ ID

(SEQ ID NO:31)
Pfam PF01757


NO:16)


orf12
33422–34792
456
AF263245
31%
isovaleryl transferase


(SEQ ID

(SEQ ID NO:32)
Pfam PF01757


NO:17)


orf13
34905–35930
341
AF210249
45%
enduricydidine


(SEQ ID

(SEQ ID NO:33)


synthase


NO:18)


orf14
36383–37264
293
AF110468
31%
Transaminase


(SEQ ID

(SEQ ID NO:35)


NO:34)


orf15
37264–38514
415
AE001954
30%
Transaminase


(SEQ ID

(SEQ ID NO:37)


NO:36)


orf16A
38466–39374
302
ZP_00095168
38%
hypothetical protein


(SEQ ID

(SEQ ID NO:39)


NO:38)


orf16B
39389–40375
329
NP_629045
34%
putative regulatory


(SEQ ID

(SEQ ID NO:55)


protein


NO:54)


orf17
40440–40655
 71
AL035654
69%
cda-orfX homolog


(SEQ ID

SEQ ID NO:41)


NO:40)


orf18
46384–47649
421
NP 823141.1
53%
putative secreted


(SEQ ID

(SEQ ID


protein


NO:42)

NO:43)


orf19
44182–45813
543
ZP 00058556.1
29%
hypothetical protein


(SEQ ID

(SEQ ID NO:45)


NO:44)


orf20
43248–44168
306
NP 422360.1
42%
ABC transporter


(SEQ ID

(SEQ ID NO:47)


NO:46)


orf21
42817–43245
142
NP 826991.1
38%
putative lipoprotein


(SEQ ID

(SEQ ID NO:49)


NO:48)


orf22
41586–42758
390
AAP03102.1
34%
two component sensor


(SEQ ID

(SEQ ID NO:51)


kinase


NO:50)


orf23
40773–41441
222
AAP03103.1
58%
two component


(SEQ ID

(SEQ ID NO:53)


response regulator


NO:52)


orf24
47770–48180
136
CAD18970.1
60%
putative lactone-


(SEQ ID

(SEQ ID NO:57)


dependent


NO:56)




transcriptional







regulator


mppA
10069–18309
2747 
AL035640

NRPS


(SEQ ID

(SEQ ID NO:2)


NO:3)


mppB
18309–29312
3668 
AL035640

NRPS


(SEQ ID

(SEQ ID NO:4)


NO:5)





*SeqWeb TM, which uses Wisconsin [GCG] Package version 10






Methods of Modifying Bacterial Proteins
The role of the proteins encoded by mppA (SEQ ID NO:3), mppB (SEQ ID NO:5), or ORF1–ORF24 (as described in Table 1) may be evaluated using any method known in the art. For example, specific modifications to a protein sequence may be produced to alter the final product. Other non-limiting examples of studies that may be conducted with these proteins include (i) evaluation of the biological activity of a protein and (ii) manipulation of a synthetic pathway to alter the final product from bacteria. More detailed discussion of these proposed uses follows.
Genetic manipulations and expression of the proteins discussed herein may be conducted by any method known in the art. For example, the effect of point mutations may be evaluated. The mutations may be produced by any method known in the art. In one specific method the manipulations and protein expression may be conducted using a vector that comprises at least one Gram-negative and at least one Gram-positive origin of replication. The origins of replication allow for replication of the nucleic acid encoded by the vector, in either a Gram-negative or a Gram-positive cell line. In one embodiment, the vector comprises one Gram-negative and one Gram-positive origin of replication. Additionally, the vector comprises a multiple cloning site that allows for the insertion of a heterologous nucleic acid that may be replicated and transcribed by a host cell.
The most evolved mechanism of transfer of nucleic acids is conjugation. As used herein, the term “conjugation” refers to the direct transfer of nucleic acid from one prokaryotic cell to another via direct contact of cells. The origin of transfer is determined by a vector, so that both donor and recipient cells obtain copies of the vector. Transmissibility by conjugation is controlled by a set of genes in the tra region, which also has the ability to mobilize the transfer of chromosomes when the origin of transfer is integrated into them (Pansegrau et al., J. Mol. Biol., 239:623–663, 1994; Fong and Stanisich, J. Bact., 175:448–456, 1993).
Evaluation of the Biological Activity of a Protein
Evaluation of the mechanism of a protein and role the protein plays in the synthesis of a compound has traditionally been determined using sequence homology techniques. However, such techniques may not be accurate and better methods of evaluating novel proteins need to be developed. The vector described previously may be used to assess the biological activity of an unknown protein. The vector may be used to disrupt a protein, either by partial or complete removal of the gene encoding the protein, or by disruption of that gene. Evaluation of the products produced when the altered protein is present is useful in determining the function of the protein.
Manipulation of a Synthetic Pathway to Alter the Final Product
As discussed above, many compounds obtained from organisms have complex stereochemistries. These compounds are not amenable to production or manipulation by conventional synthetic methods. Therefore, new methods are needed to produce altered products.
Specific proteins within the biochemical pathway of the product may be modified to assess the activity of the compounds produced by these altered proteins and to determine which sections of the product are important for activity and function.
The present invention contemplates any method of altering any of the proteins of the present invention. More specifically, the invention contemplates any method that would insert amino acids, delete amino acids or replace amino acids in the proteins of the invention. Additionally, a whole domain in a module in MppA (SEQ ID NO:2) or MppB (SEQ ID NO:4) may be replaced. Therefore, for example, the acylation domain that incorporates tyrosine into the final product may be replaced with a domain that incorporates serine. The modifications may be performed at the nucleic acid level. These modifications are performed by standard techniques and are well known within the art.
Upon production of the nucleic acid encoding the modified protein, the protein can be expressed in a host cell. Then the host cell can be cultured under conditions that permit production of a product of the altered pathway.
Once the product is isolated, the activity of the product may be assessed using any method known in the art. The activity can be compared to the product of the non-modified biosynthetic pathway and to products produced by other modifications. Correlations may be drawn between specific alterations and activity. For example, it may be determined that an active residue at a specific position may increase activity. These types of correlations will allow one of ordinary skill to determine the most preferred product structure for specified activity.
The present invention also contemplates a method for using an intergeneric vector, described infra in the examples, to manipulate, modify, or isolate a protein involved in the synthesis of a specific product. For example, the vector of the present invention may be used to alter an enzyme which is involved in incorporation of an alanine residue into a peptide, so that a tyrosine residue is incorporated instead. The effect of this modification on peptide function may be then be evaluated for biological efficacy. In the above example, modifications to the enzyme may include, but are not limited to, removal of amino acids and/or sequences that specifically recognize alanine and/or incorporation of amino acids and/or sequences that specifically recognize tyrosine.
Therefore, in general terms, the vector of the present invention may be used to alter a gene sequence by insertion of nucleic acid sequences, deletion of nucleic acid sequences, or alteration of specific bases within a nucleic acid sequence to alter the sequence of a protein of interest; thereby producing a modified protein of interest. Preferably, the protein of interest is involved in the synthesis of a compound of interest. The method of modifying a protein comprises (i) transfecting a first bacterial cell with the vector of the present invention, (ii) culturing the first bacterial cell under conditions that allow for replication of the vector, (iii) conjugating the first bacterial cell with a second bacterial cell under conditions that allow for the direct transfer of the vector from the first bacterial cell to the second bacterial cell, and (iv) isolating the second bacterial cell transformed with the vector. In a preferred embodiment, the first cell is a Gram-negative bacterial cell and the second cell is a Gram-positive cell.
Molecular Biology
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
“Amplification” of DNA as used herein denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al., Science 1988, 239:487.
A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA—DNA, DNA-RNA and RNA—RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
A “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein). This includes single- and double-stranded molecules, i.e., DNA—DNA, DNA-RNA and RNA—RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operatively associated with other expression control sequences, including enhancer and repressor sequences.
A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
The term “gene”, also called a “structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.
A coding sequence is “under the control of” or “operatively associated with” expression control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, particularly mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.
The term “expression control sequence” refers to a promoter and any enhancer or suppression elements that combine to regulate the transcription of a coding sequence. In a preferred embodiment, the element is an origin of replication.
The terms “vector”, “cloning vector” and “expression vector” refer to the vehicle by which DNA can be introduced into a host cell, resulting in expression of the introduced sequence. In one embodiment, vectors comprise a promoter and one or more control elements (e.g., enhancer elements) that are heterologous to the introduced DNA but are recognized and used by the host cell. In another embodiment, the sequence that is introduced into the vector retains its natural promoter that may be recognized and expressed by the host cell (Bormann et al., J. Bacteriol 1996; 178:1216–1218).
An “intergeneric vector” is a vector that permits intergeneric conjugation, i.e., utilizes a system of passing DNA from E. coli to actinomycetes directly (Keiser, T. et al., Practical Streptomyces Genetics (2000) John Innes Foundation, John Innes Centre (England)). Intergeneric conjugation has fewer manipulations than transformation.
Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A “cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Vector constructs may be produced using conventional molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or secreted. The term “intracellular” means something that is inside a cell. The term “extracellular” means something that is outside a cell. A substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
The term “transfection” means the introduction of a foreign nucleic acid into a cell. The term “transformation” means the introduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cells genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
The term “host cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays that are described infra. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal). For the present invention, host cells include but are not limited to Streptomyces species and E. Coli. 
The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. In a specific embodiment, the host cell of the present invention is a Gram-negative or Gram-positive bacteria. These bacteria include, but are not limited to, E. coli and Streptomyces species. An example of a Streptomyces species that may be used includes, but is not limited to, Streptomyces hygroscopicus. 
The term “heterologous” refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. For example, the present invention includes chimeric DNA molecules that comprise a DNA sequence and a heterologous DNA sequence which is not part of the DNA sequence. In this context, the heterologous DNA sequence refers to an DNA sequence that is not naturally located within the NRPS sequence. Alternatively, the heterologous DNA sequence may be naturally located within the NRPS sequence, but is found at a location in the NRPS sequence where it does not naturally occur. A heterologous expression regulatory element is such an element is operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed.
The terms “mutant” and “mutation” mean any detectable change in genetic material, e.g. DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g. protein or enzyme) expressed by a modified gene or DNA sequence.
The term “variant” may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant. Two specific types of variants are “sequence-conservative variants”, a polynucleotide sequence where a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position, and “function-conservative variants”, where a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide. Amino acids with similar properties are well known in the art. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Clustal Method, wherein similarity is based on the algorithms available in MEGALIGN. A “function-conservative variant” also includes a polypeptide or enzyme which has at least 60% amino acid identity as determined by BLAST or FASTA alignments, preferably at least 75%, more preferably at least 85%, and most preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.
As used herein, the terms “homologous” and “homology” refer to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., Cell 50:667, 1987). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.
Accordingly, the term “sequence similarity” refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
In a specific embodiment, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 80%, and most preferably at least about 90% or 95% of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. An example of such a sequence is an allelic or species variant of the specific genes of the invention. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.
Similarly, in a particular embodiment, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 80% of the amino acids are identical, or greater than about 90% are similar. Preferably, the amino acids are functionally identical. Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 10, Madison, Wis.) pileup program, or any of the programs described above (BLAST, FASTA, etc.).
A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm (melting temperature) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SCC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6×SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50–9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7–11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
In a specific embodiment, the term “standard hybridization conditions” refers to a Tm of 55° C., and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60° C.; in a more preferred embodiment, the Tm is 65° C. In a specific embodiment, “high stringency” refers to hybridization and/or washing conditions at 68° C. in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
Suitable hybridization conditions for oligonucleotides (e.g., for oligonucleotide probes or primers) are typically somewhat different than for full-length nucleic acids (e.g., full-length cDNA), because of the oligonucleotides' lower melting temperature. Because the melting temperature of oligonucleotides will depend on the length of the oligonucleotide sequences involved, suitable hybridization temperatures will vary depending upon the oligoncucleotide molecules used. Exemplary temperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligoncucleotides), 55° C. (for 20-base oligonucleotides) and 60° C. (for 23-base oligonucleotides). Exemplary suitable hybridization conditions for oligonucleotides include washing in 6×SSC/0.05% sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.
EXAMPLES
The present invention will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.
Example 1
Identification and Cloning of the Non-Ribosomal Peptide Synthetase Complex Responsible for Antibiotic Production (AC98) in S. hygroscopicus 
Methods
Isolation of genomic DNA from S. hygroscopicus. Streptomyces hygroscopicus strain designated NS17 was cultured by inoculation of 25 ml of sterile tryptone soya broth (TSB) (Oxoid, Ogdensberg, N.Y.) prepared by combining 30 g of TSB in 1 L of distilled water) with 100 μl of a frozen glycerol stock of NS17. Cultures were grown at 28° C. while shaking at 200 rpm for 2 days. Cells were harvested by centrifugation at 3000×g for 10 min, followed by resuspension of the pelleted cells in 2 ml lysis buffer (2% Triton X-200, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mm EDTA) and vortexing. After vortexing, 2 ml of phenol/chloroform/isoamyl alcohol (25/24/1 v/v) was added and the suspension was vortexed again for about 1 min to ensure lysis. The sample was then centrifuged for 5 min at 3000×g and the aqueous phase was added to 2 volumes of 95% ethanol to precipitate the genomic DNA. The precipitate was collected by centrifugation or by spooling, washed once with 70% ethanol, and air dried. DNA was resuspended in 100 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
Isolation of a peptide synthetase probe and Southern hybridization. Degenerate PCR primers were designed based on the highly conserved core motifs of peptide synthetase adenylation domains A3 and A8 (Marahiel et al., 1997).










forward
5′-ACG/CTCG/CGGCT/ACGCACCGGCCIGCCG/CAAG-3′
(SEQ ID NO: 19)



primer


reverse
5′ AGCTCG/CAT/CG/CCGG/CTAGCCG/CCGG/CAT/CCTTG/CACCTG-3′
(SEQ ID NO: 20)


primer


G/C or T/A or T/C denote either base at that position






NS17 genomic DNA was used as a template to synthesize a fragment of about 800 bp in length by PCR using a Perkin Elmer DNA Thermal Cycler 480 (Boston, Mass.-30 cycles: 95° C.-1 min, 55° C.-1 min, 72° C.-1 min). This fragment was subjected to end sequencing using an Applied Biosystems, Inc. ABI3700 sequencer (Foster City, Calif.) to determine that it corresponded to a portion of peptide synthetase adenylation domain, and used to as a probe in Southern hybridization of NS17 genomic DNA under standard conditions (Sambrook et al., 1989).

Identification of a functional NS17 peptide synthetase module. A 3 kb fragment containing a putative peptide synthetase module identified from the Southern hybridization was sequenced as described above for confirmation, and used in a biosynthetic assay to determine whether the putative peptide synthetase module was part of the AC98 biosynthetic cluster. Specifically, the method described under Example 2, below, was used to insertionally inactivate the putative peptide synthetase, which was then used to replace the endogenous peptide synthetase in S. hygroscopicus NS17, by homologous recombination. If the 3 kb fragment was part of the AC98 biosynthetic gene cluster, replacement of the endogenous gene with the insertionally inactivated 3 kb fragment would inhibit antibiotic production if the peptide synthetase encoded by 3 kb fragment is part of the AC98 biosynthetic cluster.
To evaluate antibiotic production, samples were removed from 50 ml cultures NS17 carrying the disrupted gene. Cultures were grown at 28° C. in PharmaMedia (Chrysalis PharmaMedia, NJ:10 g/L PharmaMedia, 5 g/L CaCO3, 40 g/L glucose) and were analyzed by HPLC. 20 μl aliquots were loaded onto a Waters 4 mm×50 mm YMC ods-a-columm (Milford, Mass.) and eluted with a gradient of 10% acetonitrile/90% TFA (20%) in water to 34% acetonitrile/66% TFA in water over 15 minutes. AC98 related compounds were detected by UV-DAD at 226 nm. Chromatograms were compared to chromatograms of samples taken from a similarly treated culture of the parental strain.
Preparation and Screening of an NS17 Cosmid Library. Genomic DNA isolated from NS17 as described above was used for the construction of a cosmid library. Optimal conditions for partial digestion of the DNA by restriction enzymes, to produce DNA fragments of about 35 kb, was determined using published techniques (Sambrook et al, 1989). The digested DNA fragments were dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs, Beverly, Mass.) according to the protocol provided by the manufacturer, and ligated into the commercial vector, pWE 15 (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions. Packaging of the ligated mixture was accomplished using Gigapack III XL packaging extract (Stratagene), and the resulting library was titered and amplified according to the manufacturer's instructions.
The cosmid library was screened using the 3 kb peptide synthetase fragment, identified as described above, according to standard colony hybridization protocols (Sambrook et al., 1989). One cosmid, designated pNWA117, was selected for further study.
Cosmid analysis and identification of ORFs 1–13 (SEQ ID NOs:6–18). Cosmid pNWA117 was digested with EcoRI, subjected to agarose gel electrophoresis and used in a Southern hybridization with the 3 kb fragment, identified as described above, as a probe. Following confirmation that the pNWA117 contained the 3 kb fragment, the cosmid was sequenced (MWG Biotech, Highpoint, N.C.).
Nucleotide BLAST analysis (SeqWebTM, which uses Wisconsin [GCG]Package version 10) was performed to identify individual ORFs and their putative function, according to their homology with known sequences. Results are presented in Table 1.
Cosmid analysis and identification of ORFs 14–24. Genomic DNA downstream of pNWA117 was isolated from a cosmid library by using a fragment of DNA from ORF12 of the analyzed sequence to select cosmids containing stretches of genomic DNA encoding that region of AC98 biosynthetic pathway. This process is commonly referred to as chromosomal walking. One such cosmid, pNWA105, was selected after restriction analysis indicated that it contained approximately 12 Kb of DNA downstream of ORF13. Nucleotide BLAST analysis of sequence data obtained was performed to identify twelve complete ORFs (ORF14 (SEQ ID NO:34), ORF15 (SEQ ID NO:36), ORF16A (SEQ ID NO:38) and ORF16B (SEQ ID NO:54), ORF17 (SEQ ID NO:40), ORF18 (SEQ ID NO:42), ORF19 (SEQ ID NO:44), ORF20 (SEQ ID NO:46), ORF21 (SEQ ID NO:48), ORF22 (SEQ ID NO:50), ORF23 (SEQ ID NO:52), and ORF24 (SEQ ID NO:56)), and their putative function in AC98 biosynthesis, according to their homology with known sequences. Results are presented in Table 1.
Results
Isolation of an NRPS in NC17 responsible for the production of AC98. Results from the experiments described above demonstrate that cosmid pNWA117 contains the genes encoding a NRPS required for the synthesis of the peptide core of the novel antibiotic complex AC98, which is produced by the terrestrial actinomycete Streptomyces hygroscopicus. pNWA117 also contains additional ORFs proposed to be involved in the synthesis of the AC98 complex. PNWA105 contains at least 4 additional ORFs that are proposed to be involved in AC98 biosynthesis. The NRPS complex exists as two separate components, MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4). MppA (SEQ ID NO:2) is encoded within bp 10069 and 18309 of the sequence listed in SEQ ID. NO: 1, and is comprised of about 2747 amino acids (SEQ ID NO: 2). MppB (SEQ ID NO:4) is encoded within bp 18309 and 29312 of the sequence listed in SEQ ID NO: 1, and is comprised of about 3668 amino acids (SEQ ID NO: 3). Additional description and characterization of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4) is described infra, under the heading DETAILED DESCRIPTION.
Table 1 lists the 24 ORFs and corresponding SEQ ID NO's that were identified and determined to be tailoring enzymes involved in the production of the protein core of AC98 (column 1). Column 2 lists the bp position of each ORF according to the sequence contained within cosmid pNWA117 (SEQ ID NO: 1), along with the number of the amino acids encoded by each ORF (column 3). Column 4 identifies the public sequence with which each ORF is most homologous, according to BLAST analysis, and column 5 lists the proposed function of each polypeptide encoded by the individual ORFs based on the sequence homology.
Example 2
Preparation of an Intergeneric Vector
Materials
DNA restriction and modification enzymes and T4 DNA ligase were obtained from New England Biolabs. Plasmid DNA was isolated using commercial kits (Qiagen) and DNA fragments were purified using commercial kits (Tetra Link International). Competent E. coli cells were obtained from Stratagene. All were used according to manufacturer's specifications and with buffers and reagents supplied by the manufacturer. Streptomyces chromosomal DNA was prepared according to published protocols (Keisser et al. Practical Streptomyces Genetics, John Innes Centre, Norwich, England, 2000). Antibiotics were purchased from Sigma.
Methods
pNWA200 vector preparation. A purified PstI fragment containing oriT from the R plasmid, RP4, was ligated to pFD666 (Denis & Brzezinski, Gene, 111:115, 1992), which was then linearized by digestion with Pst1 and dephosphorylated with calf intestinal phosphatase. This ligation mixture was transformed into competent XL-10 E. coli cells (Stratagene) following manufacturer's directions. The transformed cells were then plated onto nutrient agar plates containing 50 μg/ml kanamycin and incubated at 37° C. for 1 day. The incubation resulted in about 150 colonies. The colonies were replica plated onto a second kanamycin containing agar plate covered by a positively charged nylon filter, and after 6 hours incubation, the nylon filter containing the embedded colonies was treated with 0.5M NaOH (in 1M NaCl) to lyse the bacteria and denature their DNA according to standard Southern blotting procedures (Southern et al., J Mol. Biol., 98:503, 1975). The nylon filter was probed with a radioactively labeled 0.76 kb PstI fragment and one colony was selected on the basis of its hybridizing signal. The recombinant plasmid was then extracted from a fresh culture of the original hybridizing colony. Digestion of the plasmid with PstI produced two DNA fragments which electrophoresed to positions of 5.25 kb and 0.76 kb, corresponding to linear pFD666 (5.25 kb) and the 0.76 kb oriT containing Pst1 fragment. This recombinant vector replicated stably in E coli strains and did not show genetic rearrangement upon repeated subculturing and further isolation.
Example 3
Methods for the Modification of the NRPS AC98 Peptide Core
Based on the sequence data of MppA (SEQ ID NO:2) and MppB (SEQ ID NO:4) described above, and available data defining the critical binding pocket features, i.e., amino acid residues in the adenylation domain that determine the specificity of the amino acid that is accepted by the domain, those skilled in the art will be able to modify any of the adenylation domains of the NRPS and change the primary amino acid sequence in the peptide core of AC98, thus, modifying the properties of the molecule. This Example provides two methods for modifying the peptide core.
Preparation of an engineered bacterial strain that produces AC98. Preparation of an AC98-producing host strain for use for the production of modified AC98 described by the methods below, is done according to the following steps:
1. Clone a fragment of the adenylation domain from and NRPS of choice (e.g., using E. coli).
2. Insert an antibiotic resistance determinant, such as apramycin, within the cloned fragment from step 1, above. This insertion inactivates adenylation domain after it is inserted into a host genome.
3. Clone the construct in an intergeneric conjugation vector, such as pNWA200, which contains a second antibiotic resistance determinant, such as kanamycin.
4. Introduce the vector containing the cloned construct into an AC98-producing strain.
5. Select for a strain that has the construct integrated into the homologous region of the host's genomic DNA by screening for conjugates that are apramycin-resistant and kanamycin-sensitive.
6. Confirm integration of the apramycin resistance determinant at the desired location by southern hybridization and fermentation.
Once the proper host strain is selected, the insertionally inactivated module in the host genome can be replaced by either the same module that has been modified by site-directed mutagenesis, or by a module that has an adenylation domain that specifies acceptance of a different amino acid.
Method 1
Modification of the adenylation domain for the production of modified AC98. The cloned module of choice, e.g., the adenylation domain, is modified by introducing nucleotide changes that will alter the codons that specify the incorporation of the amino acids critical to the biding pocket. The nucleotide changes can be introduced using any method known in the art, such as site-directed mutagenesis. For example, changing the triplet codon for Ser239 in the tyrosine binding pocket of MppB (SEQ ID NO:4) from TCC to TGG, and changing the triplet codon for Valine299 from GTC to ATC, would change the amino acids at those sites to tryptophan and isoleucine, respectively. According to published data, one would expect that these changes would alter the specificity of the module from tyrosine to phenylalanine (Stachelhaus et al., Chemistry and Biol. 1999; 6:493–505).
The mutated module is then introduced, by conjugation, into a host strain that has been modified, as outlined above, to be resistant to an antibiotic, such as apramycin. Selection for the presence of a recombinant strain is two-fold. First, exconjugates are selected that are able to grown in the presence of kanamycin and apramycin. These strains represent those that have taken up the vector construct. Second, clones are selected that have lost resistance to both kanamycin and apramycin. This indicates that the susceptible clones have lost the apramycin resistance due to homologous recombination between the insertionally inactivated host tyrosine domain and the site-directed mutant version of the tyrosine domain. This loss of resistance further indicates that the vector is no longer in the host strain, and the mutated version of the tyrosine domain (i.e., the one specific for phenylalanine) replaced the insertionally inactivated tyrosine domain of the host (engineered as described above). Southern hybridization is then performed to confirm the insertion of the correct construct. Fermentation of the strain will result in the production of modified AC98 products where the cyclic peptide core contains phenylalanine in place of tyrosine.
Method 2
Module replacement or swapping for the production of modified AC98. For this method, two regions of approximately 1 kb flanking the module selected for replacement are cloned by PCR (the “arms”). The two arms are engineered so that the ends closest to the module (i.e., the 3′ end on one arm and the 5′ arm on the other) have appropriate restriction sites for subsequent insertion of the module in the correct orientation between the flanking arms. Next, the module of choice for insertion from another peptide synthetase (e.g., the threonine module from the CDA NRPS of S. coelicolor) is amplified by PCR. This PCR product is also engineered to contain the appropriate restriction sites compatible with those present in the cloned arms, in order for insertion between the two arms. After ligation of the arms and the module, the construct is transferred to the intergeneric conjugation vector, and introduced into a antibiotic-resistant host strain (e.g., the apramycin-resistant strain as described above). Selection of the conjugates is then performed as described above for the mutated module method. Those strains expressing the threonine module from the CDA NRPS instead of the tyrosine module of the AC98 NRPS are identified as being the result of homologous recombination between the arms of the vector and the homologous regions on the host NRPS that flank the insertionally inactivated tyrosine module. Production of the modified AC98, where the cyclic peptide core contains threonine, is achieved by fermentation.
Appropriate steps should be taken to ensure maintenance of the integrity of the ORFs during the processes described above. For example, sequencing of all PCR products is preferred to confirm that no inadvertent mutations are introduced into the sequences that will be used for cloning.
In addition or as an alternative to the peptide synthetase module of the NRPS, tailoring enzymes, such as those indicated in Table 1, may also be modified according to these methods in order to produce antibiotic molecules having a modified peptide core. As one example, inactivation of a methyltransferase enzyme will result in an antibiotic lacking specific methyl groups, which then may be evaluated for improved antibiotic activity.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that values are approximate, and are provided for description.
Patents, patent applications, publications, procedures, and the like are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.

## Claims

1. An isolated nucleic acid having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 4;
(b) a nucleic acid sequence that hybridizes to a nucleotide sequence encoding a polypeptide having the nucleotide sequence of SEQ ID NO: 5, said hybridization being performed under the following stringent conditions: 68° C. in 0.2×SSC; or 42° C. in 50% formamide; wherein the nucleotide sequence encodes an MppB protein capable of forming an NRPS complex with the MppA protein of SEQ ID NO:2; and
(c) an isolated nucleic acid fragment having a nucleotide sequence complementary to the full length of nucleotide sequence of (a), or (b).

2. An isolated nucleic acid of claim 1, wherein the nucleic acid has a nucleotide sequence of SEQ ID NO: 5.

3. An isolated nucleic acid according to claim 1, wherein the nucleic acid has a nucleotide sequence that is complementary to the sequence of SEQ ID NO: 5.

4. An isolated host cell genetically modified to express the nucleic acid of claim 1.

5. An isolated nucleic acid of SEQ ID NO: 5, or the full-length complement thereof.

6. A chimeric nucleic acid construct comprising a nucleic acid of any one of claims 1, 2, 3, or 5, wherein said nucleic acid is operatively associated with an expression control sequence.

7. An expression vector comprising the nucleic acid of any one of claims 1, 2, 3, or 5, wherein the nucleic acid is operatively associated with an expression control sequence.

8. An isolated host cell genetically modified to express the nucleic acid of any one of claims 1, 2, 3, or 5.

9. An isolated host cell comprising the expression vector of claim 7.

10. An expression vector comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleic acid sequence is operatively associated with an expression control sequence.

11. An expression vector comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleic acid sequence is operatively associated with an expression control sequence, and further comprising a second nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, wherein said second nucleic acid sequence is operatively associated with an expression control sequence.

12. The expression vector of claim 11, further comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 21–33, 35, 37, 39, 41, 43, 45, 46, 49, 51, 53, 55, or 57, wherein said nucleic acid sequence is operatively associated with an expression control sequence.

13. An isolated host cell comprising the expression vector of claim 11.

14. The host cell of claim 13, further comprising nucleic acid sequences having nucleotide sequences encoding polypeptides having amino acid sequences as set forth in SEQ ID NOs: 21–33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57.

15. The host cell of claim 14, wherein the nucleic acids have nucleotide sequences depicted in SEQ ID NOs: 6–18, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56.

16. A method for producing NRPS, which method comprises isolating NRPS produced by the host cell of claim 14, wherein NRPS is comprised of MppA of SEQ ID NO:2 and MppB of SEQ ID NO:4, and wherein the host cell has been cultured under conditions that provide for expression of MppA of SEQ ID NO:2 and MppB of SEQ ID NO:4.

17. An isolated host cell genetically modified to express the nucleic acid having nucleotide sequence as depicted in SEQ ID NO. 1.