WO2013180699A1 - Biosynthesis of paenibacillin - Google Patents

Abstract

[00129] The disclosure also provides the biosynthetic machinery including isolated proteins, isolated polynucleotides, vectors, host cells, and methods for production of the antimicrobial agents described herein.

Description

BIOSYNTHESIS OF PAENIBACILLIN

Sequence Listing

[0001] A computer readable copy of the sequence listing has been submitted with the application (9015_SeqList.ST25.txt, created on May 30, 2012, and 72,678 bytes in size) and is incorporated herein by reference.

Background

[0002] There is a shortage of new potent antimicrobial agents needed to combat the increase in emergent and increasingly virulent pathogens. Human pathogens are becoming increasingly resistant to the various classes of antibiotic agents that are in use, which can render these antibiotics ineffective in curing diseases. Similarly, there is shortage of antimicrobial preservatives suitable for food applications. This coincides with the increase in incidence and severity of food-transmitted diseases.

[0003] Accordingly, there is a perpetual need for new antimicrobial agents owing to the inevitable development of antibiotic resistance that follows the introduction of antimicrobials to the healthcare industry (e.g., hospitals, clinics, etc.), as well as the agriculture and food industry. Multidrug-resistant bacteria, including Staphylococcus aureus and Mycobacterium tuberculosis, are on the rise. It is estimated that methicillin-resistant S. aureus (MRSA) causes -19,000 deaths each year in the United States (Fischbach and Walsh, (2009) Science

325: 1089-1093). In the food industry, nisin has been widely used as a natural food

preservative worldwide for decades; but nisin resistant L. monocytogenes has been reported recently (Collins, B., et al, (2010) Antimicrob. Agents Chemother. 54:4658-4663; Gravesen, A., et al., (2002) Appl. Environ. Microbiol. 68:756-764). Therefore, new antimicrobials are needed to combat these bacterial pathogens.

[0004] Lantibiotics have gained attention for both food and potential clinical use in recent years (Breukink and De Kruijff (2006) Nat. Rev. Drug Discov. 5:321-323; Cotter, P. D., et al, (2005) Nat. Rev. Microbiol. 3:777-788; van Heel, A. J., et al, (2011) Expert Opin. Drug Metab. Toxicol. 1-6). Lantibiotics are ribosomally synthesized lanthionine-containing peptides produced by Gram-positive bacteria. Lantibiotics are encoded by a structural gene (lanA) which encodes a prepropeptide, consisting of an N-terminal leader peptide and a C-terminal propeptide. Serine and threonine residues in the propeptide undergo dehydration and cyclization, resulting in the characteristic lanthionine and methyl lanthionine bridges in lantibiotics (Chatterjee, C, et al, (2005) Chem. Rev. 105:633-684). In type AI lantibiotics (e.g. nisin), lantibiotic dehydratase (LanB) and cyclase (LanC) are responsible for dehydration and cyclization, respectively. Additional genes such as those involved in leader peptide removal, export, regulation and self-resistance are usually found in lantibiotic gene clusters (Chatterjee, C, et al. 2005).

[0005] Paenibacillin is a recently identified lantibiotic produced by and purified from Paenibacillus polymyxa OSY-DF (see, US 2011/0245152; and He, Z., et al, (2007) Appl. Environ. Microbiol. 73: 168-178). The chemical structure of paenibacillin has been determined by NMR and MS/MS analyses (US 2011/0245152; He, Z., et al, (2008) FEBS Lett. 582:2787- 2792). Synthetic routes for producing complex lantibiotics such as paenibacillin have proven difficult and as such identification of the biosynthetic machinery is highly desirable for the large-scale production of such molecules (e.g., expression vectors, recombinant cells, etc.). Further, identifying the biosynthetic pathway of paenibacillin would not only enable its large- scale production and commercial use, but would also enable the development of new lantibiotic variants based on its structure and that can exhibit improved antimicrobial properties.

Summary

[0006] In an aspect, the disclosure relates to an isolated polynucleotide comprising a sequence encoding a polypeptide having at least 80% amino acid identity to at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments the isolated polynucleotide comprises a sequence encoding a polypeptide having at least 90% amino acid identity to at least one PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments, the isolated

polynucleotide comprises a sequence encoding at least one polypeptide of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

[0007] In another aspect, the disclosure relates to an isolated polynucleotide comprising a sequence having at least 80% identity to at least one of paenA (SEQ ID ^~NO X), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paeni (SEQ ID NO:9), paenT (SEQ ID NO: 11), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO:19), or paenN (SEQ ID NO:21). In some embodiments the isolated

polynucleotide comprises a sequence having at least 90% identity to at least one of paenA (SEQ ID NO: l\ paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paeni (SEQ ID ^0:9), paenT (SEQ ID NO: 11), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21). In some embodiments the isolated polynucleotide comprises at least one sequence of paenA (SEQ ID NO: l\ paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paeni (SEQ ID ^0:9), paenT (SEQ ID NO: 11), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21). In some embodiments the vector comprises the paen gene cluster (SEQ ID NO:23).

[0008] In embodiments of the above aspects, the polynucleotide can comprise a cDNA sequence. In some embodiments, the polynucleotide can encode a polypeptide that exhibits the same activity as at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paeni (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments the polynucleotide comprises a sequence encoding for at least one of a dehydratase, a cyclase, and a protease. In some embodiments the isolated polynucleotide further comprises a sequence encoding for a dehydratase, a cyclase, and a protease.

[0009] In some embodiments the polynucleotide can be operably connected to a promoter sequence. In some embodiments the polynucleotide can further comprise an enhancer sequence. In some embodiments, the polynucleotide sequence and the promoter and/or enhancer sequence are suitably not natively associated with each other (e.g., are not associated in a naturally-occurring organism).

[0010] In another aspect, the disclosure provides a vector comprising an isolated polynucleotide comprising a sequence encoding a polypeptide having at least 80%> amino acid identity to at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments the vector comprises a polynucleotide comprising a sequence encoding a polypeptide having at least 90% amino acid identity to at least one PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments, the vector comprises a polynucleotide comprising a sequence encoding at least one polypeptide of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments the vector comprises a polynucleotide comprising a sequence having at least 80% identity to at least one of paenA (SEQ ID NO: l\ paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenl (SEQ ID NO:9\ paenT (SEQ ID NO: 1 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21). In some embodiments the vector comprises a polynucleotide comprising a sequence having at least 90% identity to at least one of paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenl (SEQ ID NO :9), paenT (SEQ ID NO: l 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21). In some embodiments the vector comprises a polynucleotide comprising at least one sequence of paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenl (SEQ ID NO:9), paenT (SEQ ID NO: l 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21). In some embodiments the vector comprises polynucleotide sequences comprising at least paenA (SEQ ID NO: \), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), and paenC (SEQ ID NO:7). In some embodiments the vector comprises the paen gene cluster (SEQ ID NO:23). In the embodiments relating to vectors, including expression vectors, the disclosure encompasses a collection of multiple vectors that can include polynucleotides of sequences that encode one or more of the various paen- (or agr-) related sequences. Such vectors can be co-transformed with each other as to allow for co-expression of the paen/agr sequences and the recombinant synthesis of paenibacillin. [0011] In another aspect, the disclosure relates to an isolated polypeptide comprising a sequence having at least 80% amino acid identity to any one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments, the polypeptide has at least 90% amino acid identity to any one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22). In some embodiments the polypeptide comprises a sequence selected from the group PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

[0012] In another aspect, the disclosure relates to a recombinant cell comprising a polynucleotide, a vector, or a polypeptide of any of the various aspects and embodiments disclosed herein. In some embodiments the recombinant cell comprises a prokaryotic cell. In some embodiments, the recombinant cell comprises a bacterial cell selected from the genus groups consisting of Paenibacillus, Bacillus, Streptomyces, Escherichia and Pseudomonas.

[0013] In a further aspect, the disclosure relates to a method of modifying production of paenibacillin in Paenibacillus polymyxa OSY-DF comprising introducing into Paenibacillus polymyxa OSY-DF a polynucleotide or a vector of any of the aspects and embodiments disclosed herein.

[0014] In another aspect, the disclosure relates to a method for the biosynthetic production of paenibacillin or an analog thereof, comprising growing a recombinant cell under conditions that allow synthesis of paenibacillin or a paenibacillin analog, wherein the recombinant cell comprises polynucleotides encoding proteins, PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), and PaenC (SEQ ID NO: 8), or homologs thereof, wherein the polynucleotides are operably connected to a promoter. In some embodiments the recombinant cell further comprises polynucleotides encoding at least one protein selected from PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22), or homologs thereof. [0015] In further aspects, the disclosure provides paenibacillin and compositions comprising paenibacillin produced by the various methods disclosed herein, and methods for the use thereof, as well as kits, as further described herein.

[0016] The disclosure provides for a number of additional aspects and embodiments that will be apparent to one of skill in the art in light of description and drawings that follow.

Brief Description Of The Drawings

[0017] Figure 1 depicts a representation of the structure of paenibacillin (SEQ ID NO:34), where the encircled Ac is Acetyl, and— S— indicates a thioether bridge.

[0018] Figure 2 depicts a schematic of the genes involved in paenibacillin biosynthesis, as well as the sequences for each identified gene. (A) Depiction of organization of open reading frames (ORFs) in a 11.7-kb DNA fragment (SEQ ID NO:23) for paenibacillin biosynthesis. (B) paenAfPaenA (SEQ ID NO: 1/2). (C) paenP/VaenP (SEQ ID NO:3/4). (D) paenB/VaenB (SEQ ID NO:5/6). (E) paenC/VaenC (SEQ ID NO:7/8). (F) paenlfPaenl (SEQ ID NO:9/10). (G) paenT/PaenT (SEQ ID NO: 11/12). (H) agrB/AgrB (SEQ ID NO: 13/14). (I) agrD/AgrO (SEQ ID NO: 15/16). (J) agrC/AgrC (SEQ ID NO: 17/18). (K) agrA/AgxA (SEQ ID

NO: 19/20). (L) paenNfPaenN (SEQ ID NO:21/22). (M) paen gene cluster (SEQ ID:23).

[0019] Figure 3 depicts the structural gene of paenibacillin (paenA, SEQ ID NO: 1) and 117-bp preceding noncoding sequence (SEQ ID NO:35). Underlined nucleotides are putative promoter (-35 and -10 element) and ribosome binding site (RBS). The deduced leader peptide sequences of PaenA (SEQ ID NO:2) are shaded.

[0020] Figure 4 depicts an alignment of paenibacillin with related lantibiotic

prepropeptides. BtlA (SEQ ID NO:36) and Bt2A (SEQ ID NO:37), putative lantibiotics in Bacillus thuringinesis str. TO 1001; PaenA (SEQ ID NO:2) paenibacillin precursor; BsnA (SEQ ID NO:38), putative lantibiotic in Bacillus subtilis Bsn5; ElkA (SEQ ID NO:39), epilancin K7; ElxA (SEQ ID NO:40), epilanicin 15X; EciA (SEQ ID NO:41), epicidin 280.

[0021] Figure 5 depicts the proposed post-translational modification of paenibacillin. PaenB, lantibiotic dehydratase; PaenC, lantibiotic cyclase; PaenP, peptidase; and subsequent acetylation by PaenN, acetylase.

[0022] Figure 6 depicts an alignment of putative signal precursor AgrD (SEQ ID NO: 16) from Paenibacillus polymyxa OSY-DF with AgrD (ZP 00237849.1; SEQ ID NO:42) from Bacillus and AgrD (AAL65845.1; SEQ ID NO:43) from Staphylococcus. Underlined amino acids may form the activating ligand.

[0023] Figure 7 depicts a proposed model of paenibacillin production and regulation

[0024] Figure 8 compares the positions of paenibacillin gene cluster integration in the genomes of Paenibacillus polymyxa SC2 a paenibacillin non-producing strain and

Paenibacillus polymyxa OSY-DF a paenibacillin producing strain.

[0025] Figure 9 MALDI-MS analysis of paenibacillin production of OSY-DF wild type and ApaenB mutant strains cultured for 36 h. (A) MutantB; paenibacillin was not detected in the culture broth. (B) Wildtype strain; peak (arrow) [M+H]⁺=2984.54 and peak

[M+Na]⁺=3006.57 corresponding to paenibacillin and its sodium adduct ion.

Detailed Description

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0027] Unless otherwise indicated, all numbers that are used to express an absolute or relative quantity and/or range as used herein are to be understood as allowing for being inclusive in all instances of values that result is insubstantial differences from the recited numbers (e.g., inclusive of the values that are "about" the recited value). Accordingly, unless otherwise indicated, the numerical values and ranges set forth herein can relate to

approximations that may vary depending on the desired properties sought to be obtained in a particular aspect or embodiment. Notwithstanding that the numerical ranges and parameters setting forth the scope of the disclosure are approximations, the recited numerical values provided in the specific examples are reported as precisely as possible. As one of skill in the art appreciates, any numerical values inherently contain certain errors necessarily resulting from error found in their respective measurements. It also will be understood that any numerical range recited herein includes any and all values from the lower value to the upper value. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10%> to 30%>, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0028] In a broad sense, the disclosure provides isolated polynucleotides, isolated polypeptides, nucleic acid constructs, vectors, and recombinant cells for paenibacillin biosynthesis. The inventors have identified, cloned, characterized, and expressed the biosynthetic machinery (gene sequences and encoded functional proteins) for the antimicrobial polypeptide, paenibacillin. Accordingly, the disclosure provides for isolated polynucleotide sequences, including the paenibacillin gene cluster (designated as paen), individual polynucleotide of the paen gene cluster that encode proteins involved in the biosynthesis of paenibacillin, isolated proteins and polypeptides and functional equivalents thereof, vectors, recombinant cells, and methods for the recombinant biosynthesis of paenibacillin.

[0029] Paenibacillin is a well characterized member of the class of antimicrobial polypeptides known as "lantibiotics." Lantibiotics are group I bacteriocins that are

synthesized and post-translationally modified by Gram-positive bacteria. These modifications generate dehydrated amino acids, for example, α,β-didehydroalanine (Dha) and α,β- didehydrobutyric acid (Dhb) and thioether bridges of lanthionine (Lan) and β- methyllanthionine (MeLan), as well as some other less frequently encountered modifications. These modified residues may stabilize molecular conformations that are essential for the antimicrobial activity of lantibiotics and their resistance to proteases of the producing bacterial strains.

[0030] Suitably, lantibiotics such as paenibacillin exhibit antimicrobial activity against microbes other than the producer (e.g., bactericidal activity against bacteria), generally by forming pores in cell membrane resulting in efflux of cellular components. Pores are generally formed when lantibiotics bind unspecifically to bacteria cell membrane, a wide-spread property among antimicrobial polypeptides. However, some lantibiotics specifically target Lipid II, the precursor in cell wall synthesis, leading to pore formation. Microbes may not develop very robust resistance to lipid II-targeting lantibiotics relative to antibiotics that target a single enzyme involved in cell wall assembly. Accordingly, lantibiotics present a valuable source of antimicrobial agents against microbial strains that exhibit multi-drug resistance as well as resistance to active agents such as vancomycin.

[0031] As used herein, "antimicrobial agent," an agent that "exhibits antimicrobial activity," or an agent that "affects microbial activity" means a compound that slows or stops growth and/or proliferation, slows or stops the rate of growth and/or proliferation, or stuns, inactivates, or kills a microbe. Antimicrobial agents can encompass the terms antibiotics, antibacterials (e.g., bactericidal or bacteriostatic agents), antivirals (e.g., virucidal agents), antifungals (e.g., fungicidal or fungistatic agents), mold-inhibiting agents, anthelminthics (e.g., vermifuge or vermicidal agents), antiparasitics, and the like. For purposes of the disclosure, antimicrobial activity may be determined according to any procedure that is described herein or that is otherwise known in the art.

[0032] As used herein "paenibacillin" relates to a polypeptide that exhibits at least one antimicrobial property, and which comprises the structure depicted in FIG.l (SEQ ID NO: 34). In some embodiments the disclosure provides for active fragments, homologs, derivatives of paenibacillin. In some embodiments, the active paenibacillin fragments, homologs, and/or derivatives have one or more of the following modifications: (i) a thioether bridge of lanthionine (Lan) between the amino acids in positions 1 1 and 15, and/or positions 25 and 29; (ii) a thioether bridge of β-methyllanthionine (MeLan between a pair of amino acids in positions 17 and 20, a pair in positions 19 and 22, and a pair in positions 23 and 26; (iii) an acetylated amino acid in the N-terminal; (iv) dehydration of one or more serines to dehydro- alanine (Dha); (v) dehydration of one or more threonines to dehydro-butyrine (Dhb); (vi) a Dhb-Dhb tandem, or a combination thereof. Additional variants fall within the scope of the disclosure. The structure, function, and chemistry of individual amino acids are well known to those of skill in the art, and thus, one of skill will be able to identify particular variants that comprise conservative amino acid substitutions for the amino acid sequence.

[0033] "PaenA" as used herein relates to an amino acid sequence of a paenibacillin precursor, or the polynucleotide sequence ("paenA") encoding a paenibacillin precursor amino acid sequence. In some embodiments PaenA can relate to an amino acid sequence comprising SEQ ID NO:2, any fragment of SEQ ID NO:2 that can be processed (e.g., via a peptidase or protease to remove an amino acid leader sequence) to generate active paenibacillin (SEQ ID NO:34), as well as any polypeptide intermediates (e.g., comprising one or more dehydrated amino acid residues, one or more thioether bridge, etc.) in the biosynthesis of paenibacillin (SEQ ID NO:34).

[0034] "PaenP" as used herein relates to an amino acid sequence encoding a putative peptidase, or a polynucleotide sequence ("paenP") encoding a putative peptidase that can cleave a paenibacillin precursor sequence (e.g., PaenA). In some embodiments, PaenP comprises 324 amino acids (SEQ ID NO:4) with a theoretical mass of about 36.0 kDa. In some embodiments PaenP comprises sequence similarity to other proteases or peptidases that are active in lantibiotic biosynthesis (e.g., subtilisin-like serine protease LanP, such as PepP (CAA90024.1), elkP (CAA60861.1) and NisP (CAA80420.1). In some embodiments, PaenP comprises conserved catalytic triad residues (Asp43, His 102 and Ser290) as well as the oxyanion hole Asnl85 that are the characteristic of PaenP proteases. In some embodiments, PaenP may be a cytoplasmic polypeptide. In some alternative embodiments PaenP can comprise an N-terminal sec-signal sequence and C-terminal cell wall anchor sequence (LPXTGX). In some embodiments PaenP can be modified so that paenibacillin production can occur within the cell, while it is crossing the cell membrane or within the periplasmic space, or once it is outside of the cell.

[0035] "PaenB" as used herein relates to a dehydratase (e.g., a lantibiotic dehydratase), or a polynucleotide sequence ("paenB") encoding a dehydratase. In some embodiments PaenB comprises 1027 amino acids (e.g., SEQ ID NO:6) with a theoretical molecular weight of about 119.0 kDa. In some embodiments PaenB can dehydrate serine and threonine residues in a paenibacillin propeptide (e.g., PaenA) to produce unsaturated didehydroalanine (Dha) and didehydrobutyrine (Dhb) residues, respectively. In some embodiments PaenB shares homology with lantibiotic dehydratases (LanB), PepB (CAA90025.1), EpiB (CAA44253.1), EciB (CAA74350.1), SpaB (AAA22779.1) and NisB (CAA48381.1).

[0036] "PaenC" as used herein relates to a cyclase (e.g., a lantibiotic cyclase), or a polynucleotide sequence ("paenC") encoding a cyclase. In some embodiments PaenC comprises 423 amino acids (e.g., SEQ ID NO:8) with a theoretical mass of about 47.3 kDa. In some embodiments, PaenC comprises sequence homology to lantibiotic cyclases (LanC) such as, for example, PepC (CAA90026.1), EciC (CAA74351.1), EpiC (CAA44254.1), SpaC (AAB91588.1) and NisC (CAA48383.1). In some embodiments PaenC comprises a catalytic site for binding Zn(II) comprising cysteine, histidine, and aspartate amino acid residues. In some embodiments PaenC comprises a sequence that allows for cyclization of a paenibacillin precursor peptide (e.g., PaenA) and can facilitate formation of thioether linkages between amino acids such as, for example, cysteine and Dha/Dhb. In some embodiments, enzymes that are known and capable of catalyzing protein/peptide disulfide bond formation (e.g., thiol- disulfide oxidoreductases) can be used to facilitate thioether bond formation, and belong to the thioredoxin enzyme superfamily.

[0037] "Paenl" as used herein relates to a putative self-immunity protein, or a

polynucleotide sequence ("paenF') encoding a self-immunity protein. In some embodiments Paenl comprises 185 amino acids (e.g., SEQ ID NO: 10) with a theoretical mass of about 21.0 kDa. In some embodiments, Paenl comprises membrane spanning helices and may be integrated with the cell membrane, or associated with a cell membrane protein and may facilitate the export of paenibacillin.

[0038] "PaenT" as used herein relates to a putative ATP -binding cassette (ABC) transporter protein, or a polynucleotide sequence ^paenV) encoding an ABC transporter protein. In some embodiments PaenT comprises 597 amino acids (e.g., SEQ ID NO: 12) with a theoretical molecular weight of 67.2 kDa. In some embodiments, PaenT may function to export the processed paenibacillin to the extracellular medium. In some embodiments the structure of PaenT can comprise membrane spanning helices in the N-terminal domain, while the C-terminal domain can comprise an ATP -binding site (and hydrolysis site) that provides the energy for paenibacillin transportation.

[0039] "AgrB" as used herein relates to an accessory gene regulator (agr) protein, or a polynucleotide sequence ("agrB") encoding an accessory gene regulator protein. In some embodiments AgrB comprises 137 amino acids (e.g. SEQ ID NO: 14) with a theoretical molecular weight of 13.7 kDa. In some embodiments AgrB comprises membrane spanning helices and may be involved in processing a signal peptide AgrD (SEQ ID NO: 16).

[0040] "AgrD" as used herein relates to a signal peptide precursor in an agr regulatory system, or a polynucleotide sequence ("agrD") encoding a signal peptide precursor. In some embodiments AgrD comprises 57 amino acids (e.g. SEQ ID NO: 16) with a theoretical molecular weight of 6.7kDa. In some embodiments AgrD is processed by AgrB for production of the activating ligand, a 7-9 amino acid peptide with a thiolactone ring.

[0041] "AgrC" as used herein relates to a histidine kinase protein present in the quorum sensing machinery, or a polynucleotide sequence "agrC encoding a histidine kinase. In some embodiments AgrC comprises 442 amino acids (e.g. SEQ ID NO: 18) with a theoretical molecular weight of 50.9 kDa. In some embodiments AgrC is a transmembrane signal receptor which senses the presence of signal peptides outside the cell and self-phosphorylates. The phosphate is transferred to a response regulator AgrA, which activates the transcription of relevant genes.

[0042] "AgrA" as used here relates to response regulator protein in the quorum sensing machinery, or polynucleotide sequence "agrA" encoding a response regulator. In some embodiments AgrA comprises 244 amino acids (e.g. SEQ ID NO:20) with a theoretical molecular weight of 28.4 kDa. In some embodiments AgrA accepts the phosphate group from AgrC and activates the transcription of relevant genes.

[0043] "PaenN" as used herein relates to a protein that can acetylate the N-terminal amino acid of a polypeptide sequence, or a polynucleotide sequence ("paenN") encoding such a protein. In some embodiments PaenN comprises 256 amino acids (e.g., SEQ ID NO:22) with a theoretical mass of 28.7 kDa. In some embodiments PaenN can relate to any protein that has activity for acetylating the N-terminal amino acid of a polypeptide sequence (e.g., TraX protein family (pfam05857), which acetylates the N-terminal alanine residue of F-pilin).

[0044] In some embodiments of the disclosure, the function of the various Paen

polypeptides disclosed above can be supplemented or provided by alternative proteins (e.g., homologous proteins from other bacterial strains) or synthetic chemical techniques that provide the same function/activity. For purposes of illustration of one non-limiting example of a chemical synthetic route, a variety of procedures for producing lanthionine are known and include, for example, sulfur extrusion from cysteine (see, Harpp and Gleason (1971) J. Org. Chem. 36:73-80), ring opening of serine β-lactone (see, Shao, H., et al. (1995) J. Org. Chem. 60:2956-2957), and hetero-conjugate addition of cysteine to dehydroalanine (see, Probert, J.M., et al. (1996) Tetrahedron Lett. 37: 1101-1104). According to the literature, however, the sulfur extrusion method is the only pathway that has been successful in the total synthesis of a lantibiotic (e.g., generating lanthionine). Accordingly, some embodiments of the disclosure can provide a method comprising the partial biosynthesis of paenibacillin (e.g., generating a pro-polypeptide comprising PaenA) and further steps that include isolating the partially synthesized paenibacillin from the cell, and performing one or more additional synthetic steps (e.g., cleaving a leader polypeptide or a fusion polypeptide, dehydrating one or more amino acids, forming one or more thioether bonds (i.e., cyclizing paenibacillin), and/or acetylating the paenibacillin).

[0045] The term "identity" when used herein reference to a sequence (e.g., "percent identity") refers to the number of elements (i.e., amino acids or nucleic acid residues) in a sequence that are identical within a defined length of two optimally aligned DNA, RNA or protein segments. To calculate the "percent identity", the number of identical elements is divided by the total number of elements in the defined length of the aligned segments and multiplied by 100. When percentage of identity is used in reference to proteins it is understood that certain amino acid residues may not be identical but are nonetheless conservative amino acid substitutions that reflect substitutions of amino acid residues with similar chemical properties (e.g., acidic or basic, hydrophobic, hydrophilic, hydrogen bond donor or acceptor residues). Such substitutions may not change the functional properties of the molecule.

Consequently, the percent identity of protein sequences can be increased to account for conservative substitutions. One of skill can use any number of the bioanalytical software packages and applications that are well known in the art can be used to determine a sequence alignment and/or the identity of one sequence to another sequence. For example, the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for

Biotechnology Information website (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. For amino acid sequences, a software package such as the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).

[0046] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

[0047] Polynucleotides and Polypeptide Sequences [0048] In one aspect, the disclosure provides an isolated polynucleotide encoding a polypeptide having at least 80%, 85%, 90%, 95%, or greater (e.g., 96%, 97%, 98%, or 99%) amino acid identity to at least one protein selected from PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22). In some embodiments, the

polynucleotide encodes at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

[0049] In some embodiments, the polynucleotide comprises a sequence that has at least 80%, 85%, 90%, 95%, or greater (e.g., 96%, 97%, 98%, or 99%) identity to at least one polynucleotide selected from paenA (SEQ ID NO: l) paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO ), paenl (SEQ ID NO :9), paenT (SEQ ID NO:l l), agrB(SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), and paenN (SEQ ID NO:21). In some embodiments, the polynucleotide comprises at least one of paenA (SEQ ID NO: l) paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenl (SEQ ID NO:9\ paenT (SEQ ID NO: 11), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), and paenN (SEQ ID NO:21).

[0050] In some embodiments the polynucleotide comprises a sequence that comprises four essential genes for paenibacillin biosynthesis (e.g. paenA (SEQ ID NO: 1), paenP (SEQ ID NO:3) , paenB (SEQ ID NO:5), and paenC (SEQ ID NO:7)) In some embodiments, the polynucleotide comprises a sequence having at least 80%>, 85%>, 90%>, 95%>, or greater (e.g., 96%, 97%, 98%, or 99%) identity to paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), and paenC (SEQ ID NO:7).

[0051] In some embodiments the polynucleotide comprises the entire paen gene cluster (SEQ ID NO:23). In various embodiments the polynucleotide comprises a cDNA sequence of any of the polynucleotide sequences disclosed herein.

[0052] In another embodiment, polynucleotide sequences encoding one or more specific polypeptides in the paenibacillin biosynthetic pathway can be replaced with polynucleotide sequences encoding analogous polypeptides, or modules or domains from other distinct but related polypeptides, such as those herein described or otherwise known in the art. In some embodiments such proteins can be a native protein to a recombinant host cell. Accordingly, in some embodiments, genetically engineered bacteria expressing such sequences can be used to develop bacterial strains capable of synthesizing paenibacillin or analogs thereof.

[0053] In another aspect, the disclosure relates to an isolated polypeptide having at least 80%, 85%, 90%, 95%, or greater (e.g., 96%, 97%, 98%, or 99%) identity to PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22), and having the corresponding activity PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22), respectively. In some embodiments, the polypeptide comprises at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

[0054] As discussed herein, the disclosure also provides for one or more of the sequences PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22) to be modified (e.g., post-translational modification) or genetically manipulated to alter the specificity or activity of the encoded protein. For example, the coding sequences could be modified by site-directed mutagenesis or random mutagenesis to make specific substitutions of one or more amino acids. Such modifications can also be used to optimize or otherwise modify the biosynthetic production of paenibacillin in a particular recombinant host cell (e.g., wherein one or more of the Paen polypeptides has diminished, or no, activity in a particular host cell). The structure, function, and chemistry of individual amino acids are well known to those of skill in the art. Amino acids as described herein can include alpha-amino acids of the general formula H₂NCHRCOOH, where R is an amino acid side chain comprising an organic substituent, as well as uniquely structured amino acids such as, for example, proline. Amino acids include, for example, isoleucine, leucine, alanine, asparagine, glutamine, lysine, aspartic acid, glutamic acid, methionine, cysteine, phenylalanine, threonine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, histidine, norleucine, ornithine, taurine,

selenocysteine, selenomethionine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, hypusine, citrulline, 3-aminopropanoic acid, aminobutryic acid (alpha, beta, and gamma) diaminobutyric acid, and the like. Accordingly, the term "amino acid side chain" refers to the various organic substituent groups (e.g., "R" in H₂NCHRCOOH) that differentiate one amino acid from another. A "derivative" of an amino acid side chain refers to an amino acid side chain that has been modified structurally (e.g., through chemical reaction to form new species, covalent linkage to another molecule, etc.).

[0055] In some embodiments, homologs of the proteins encoded by the paen gene cluster include, but are not limited to, proteins that share at least about 40%, 50%>, 60%>, 70%> or more amino acid similarity and/or 25%, 35%, 45%, 55% or more amino acid identity and catalyzing analogous reactions. Homologs may share specific domains within the proteins, or include conservative amino acid substitutions, as discussed herein.

[0056] Vectors and Nucleic Acid Constructs

[0057] In an aspect, the disclosure provides for nucleic acid constructs comprising a polynucleotide sequence as described herein operably linked to one or more control sequences that direct the expression of the polynucleotide in a suitable host cell under conditions compatible with the control sequences. In some embodiments, the nucleic acid constructs can comprise more than one of the polynucleotide sequences disclosed herein.

[0058] In another aspect, the disclosure provides recombinant constructs and vectors comprising a polynucleotide disclosed herein operably linked to a promoter. Promoters may be any promoter active in the cell and capable of driving gene expression. Promoters include constitutive and inducible promoters. In some embodiments a single promoter can drive the expression of one or more of the paen sequences (e.g., when a single nucleotide is transcribed as a polycistronic mRNA, or when multiple nucleotides are under the control of the same promoter). A variety of suitable promoters are known to those of skill in the art. Suitably the promoter is not the promoter natively associated with the polynucleotide. A vector comprising one or more of the polynucleotides or the polynucleotides operably connected to a promoter are also provided. Suitable vectors include, but are not limited to, a plasmid, a cosmid, a transposon, a virus, a phage, a BAC, a YAC or any other vectors known to those of skill in the art or which may be subsequently developed.

[0059] A polynucleotide sequence as disclosed herein may be manipulated in a variety of ways to provide for expression of the polypeptide for which it encodes. Manipulation of the nucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing recombinant DNA methods are well known in the art.

[0060] The control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of the nucleotide sequence.

Typically a promoter sequence contains transcriptional control sequences which ultimately mediate the expression of the polypeptide encoded by the polynucleotide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0061] Non-limiting examples of suitable promoters for directing the transcription of the polynucleotide constructs described herein in a recombinant bacterial host cell include the promoters obtained from phage T7 RNA polymerase gene, the E. coli lac operon,

Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, prokaryotic beta-lactamase gene, as well as the tac promoter. Further promoters are known in the art (Molecular cloning: a laboratory manual, Sambrook and Russell, CSHL Press, 2001).

Similarly, a number of suitable promoters for directing the transcription of the nucleic acid constructs disclosed herein in other expression systems (e.g., fungal host cells, yeast host cells, etc.) are known in the art.

[0062] In some embodiments, the control sequence can comprise a suitable transcription terminator sequence that is recognized by a recombinant host cell to terminate transcription. Suitably, the terminator sequence is operably linked to the 3' terminus of the polynucleotide sequence encoding a polypeptide. Any terminator which is functional in the host cell of choice may be used.

[0063] A control sequence may also comprise a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into a particular region of a cell such as, for example, the secretory pathway. In some embodiments the 5' end of the polynucleotide coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be advantageous or even required where the polynucleotide coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. Some embodiments provide for any signal peptide coding region that directs a paenibacillin polypeptide or pro-polypeptide (e.g., PaenA) into the secretory pathway of a host cell of choice. Such signal peptide coding regions for bacterial host cells, yeast host cells, other host cells are known in the art (see, for example Simonen and Palva, (1993) Microbiological Reviews (57)109-137; Romanos et al, (1992), Yeast (8)423-488.).

[0064] In some embodiments, the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a

polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0065] It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Such regulatory sequences can allow for advantageous timing for the ultimate production of paenibacillin in a recombinant system. For example, if the recombinant host cell exhibits sensitivity to the antimicrobial action of paenibacillin, expression of one or more of the Paen polypeptides can be inhibited in order to delay a synthetic step in the paenibacillin biosynthetic pathway. Non-limiting examples of regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used.

Similar control sequences are known in a number of other expression systems. It will be appreciated by one of skill in the art that various embodiments of the disclosure allow for one or more than one of the polynucleotides disclosed herein to be under control (e.g., "operably connected to") a single control sequence such as, for example, an enhancer, promoter, regulator or termination sequence. Embodiments of the disclosure also provide for one or more of the polynucleotides disclosed herein to be under control of two or more different control sequences allowing for the precise control of expression of specific polynucleotide sequences. [0066] Expression Vectors

[0067] The disclosure also relates to recombinant expression vectors comprising a polynucleotide or nucleic acid construct as disclosed herein. The various polynucleotide and control sequences described herein may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide sequence encoding one or more polypeptides at such sites. Alternatively, the polynucleotide sequence may be expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0068] The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced, and is well within the knowledge of one of ordinary skill in the art. The vectors may be linear or closed circular plasmids.

[0069] The vector may be an autonomously replicating vector (i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0070] In some embodiments, the vectors may contain one or more selectable markers which permit easy selection of successfully transformed cells that harbor the vector. Selectable markers are known in the art and can include a gene that provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. A number of non-limiting examples of bacterial selectable markers are known in the art. [0071] In some embodiments the vectors may contain one or more elements that permit stable integration of the vector into the recombinant host cell genome or autonomous replication of the vector in the cell independent of the genome. A number of strategies and sequences are known in the art for the integration of a vector into a host cell genome (e.g., by homologous or non-homologous recombination). More than one copy of a nucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleotide sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. The procedures that can be used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known in the art (see, e.g., Sambrook et al, 1989, supra).

[0072] In some embodiments, the one or more of the various paen nucleotide sequences can be included in a single vector such as, for example an expression vector. In some embodiments the paen nucleotide sequences (e.g., one, two, three, four, etc.) can be incorporated into multiple versions of the same vector such as, for example, an expression vector, or different vectors such as, for example, different expression vectors. Accordingly, embodiments of the disclosure relate to a collection of vectors that each comprises at least one paen nucleotide sequence, which allows for the transformation of a selected vector or selected group of vectors (e.g., expression vector(s) into a host cell.

[0073] Host Cells

[0074] In an aspect the disclosure relates to a recombinant host cell comprising the polynucleotide or nucleic acid construct (i.e., vector) which are advantageously used in the recombinant production of the polypeptides. As noted above, a vector comprising a polynucleotide can be introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The host cell may be a unicellular microorganism (a prokaryote) or a non-unicellular microorganism (a eukaryote).

[0075] In some embodiments, the host cell comprises a bacterial cell such as gram-positive bacteria that does not ordinarily synthesize paenibacillin or analogs thereof. As disclosed herein, bacteria that do not natively possess the paen biosynthetic gene cluster, for example, Paenibacillus strains other than Paenibacillus polymyxa OSY-DF, Bacillus, Streptomyces, Escherichia or lactic acid bacteria (LAB), may be genetically modified to express polypeptides having at least 80%, 85%, 90%, 95% or greater amino acid identity to one or more of the various Paen sequences disclosed herein. In some embodiments the polypeptide includes at least one PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrA (SEQ ID NO: 18), AgrC (SEQ ID NO:20), and PaenN (SEQ ID NO:22). In some embodiments the bacterial cell comprises a gram-positive bacterial cell and can include a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaternum, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus. In some embodiments the bacterial cell comprises a gram-negative bacterial cell such as E. coli and Pseudomonas spp.. In some embodiments, the host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell. In some embodiments, the fungal host cell is a yeast cell such as, for example, a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

[0076] Techniques for the introduction of a vector into a bacterial host cell are well known in the art and may include, for example, protoplast transformation, use of competent cells, electroporation, or conjugation.

[0077] Methods of Production

[0078] In an aspect, the disclosure provides a method for producing a polypeptide, (e.g., paenibacillin or a paenibacillin precursor polypeptide), wherein the method comprises (a) cultivating a host cell under conditions that allow for production of the polypeptide; and optionally (b) purifying/isolating the polypeptide.

[0079] Typically cells are cultivated in a nutrient medium suitable for production of paenibacillin using common techniques known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. Any suitable nutrient medium (e.g., a medium comprising carbon and nitrogen sources, inorganic salts, etc.) can be used to cultivate the cells using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., Sambrook, et al, or as provided by the American Type Culture Collection). In embodiments wherein the polypeptide is secreted from the cell into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates or as inclusion bodies.

[0080] The resulting paenibacillin may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0081] The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromato focusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0082] Uses

[0083] While the disclosure is generally drawn to methods for the biosynthesis of paenibacillin, as well as the related polynucleotide and polypeptide sequences, vectors, and recombinant cells, it will be appreciated that the paenibacillin produced by the methods disclosed herein can be used in any setting that is exposed to contamination by microbes (e.g., bacteria, fungi, yeast or algae) including those applications disclosed in U.S. Published Patent Application No. 2011/0245152, which is incorporated herein by reference. Similarly, the paenibacillin provided by the methods disclosed herein can be formulated for use in any particular application in which an antimicrobial effect is desired.

[0084] Some non-limiting examples relating to the use of paenibacillin include aqueous systems such as cooling water systems, laundry rinse water, oil systems such as cutting oils, lubricants, and oil fields. It can also be used in the protection of wood, latex, adhesive, glue, paper, cardboard, textile, leather, plastics, and caulking. Paenibacillin can be incorporated in methods for producing animal feed as well as human food and beverage production, processing, and packaging. It can be used to preserve food, beverages, and cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products, or enzyme formulations. Paenibacillin may by useful as a disinfectant, e.g., in the treatment of acne, infections in the eye or the mouth, skin infections; in antiperspirants or deodorants; in foot bath salts; for cleaning and disinfection of contact lenses, hard surfaces, teeth (oral care), wounds, bruises and the like. Paenibacillin may also be useful for cleaning, disinfecting or inhibiting microbial growth on any hard surface, such as surfaces of process equipment used in, for example, dairies, chemical or pharmaceutical process plants, water sanitation systems, oil processing plants, paper pulp processing plants, water treatment plants, and cooling towers. Paenibacillin can also be used and formulated as a composition such as, for example a pharmaceutical composition or as a medicament. In such embodiments, the pharmaceutical composition is used to control or combat the proliferation of microorganisms, such as fungal organisms or bacteria, (e.g., gram-positive bacteria) and is provided in amounts effective for providing antimicrobial effect.

[0085] The examples that follow are provided only for the purpose of illustration of some of the aspects and embodiments that are provided by the above disclosure and should not be interpreted to be limiting to the claims.

Examples

[0086] Materials and Methods

[0087] DNA manipulations, genome library construction, and DNA sequencing

[0088] General DNA manipulations, including plasmid preparation, restriction enzyme digestion, agarose gel electrophoresis, subcloning, and bacterial transformation, were done according to standard protocols (Molecular cloning: a laboratory manual, Sambrook and Russell, CSHL Press, 2001) or manufacturer's instructions.

[0089] Strains and growth conditions

[0090] Strain Bacillus subtilis 1A771 (Example 5) was obtained in Bacillus Genetic Stock Center (BGSC, Columbus, OH). Other strains were obtained from the culture collection of The Ohio State University food safety laboratory. P. polymyxa OSY-DF was grown in tryptic soy broth (Becton Dickinson, Sparks, MD) supplemented with 0.6% yeast extract (TSBYE) at 30°C with agitation at 200 rpm. Escherichia coli DH5a was cultured in Luria-Bertani (LB) broth (Becton Dickinson) or on LB agar at 37°C. When appropriate, LB media were supplemented with ampicillin (100 μ /ml). Indicators strains L. innocua ATCC 33090 and B. subtilis lA771were grown in LB medium at 37°C.

[0091] Example 1. Identification of the Paenibacillin Structural Gene paenA by PCR

[0092] Genomic DNA of strain OSY-DF was purified using a DNA isolation kit (DNeasy Blood & Tissue kit; QIAGEN, Valencia, CA). The structural gene encoding paenibacillin prepropeptide was amplified by PCR. Forward primers (PaenAFl, PaenAF2 or PaenAF3) were designed based on the conserved "FDLD" motif in the leader peptide of type AI lantibiotics (See, FIG. 4; Chatterjee, C, et al, (2005) Chem. Rev. 105:633-684). The reverse primer PaenAR was based on the DNA sequences encoding the C-terminus of a putative paenibacillin homologue BtlA in Bacillus thuringiensis (FIG.4). See, Table 1.

[0093] Table 1. Listing of primers

Primer Oligonucleotide Sequences SEQ ID Name NO.

PaenAFl 5 ' - ATGAAT AAAGAATT ATTTG ATTTAGATATT-3 ' 24

PaenAF2 5 ' - ATGAAT AAAGAATT ATTTG ATTTAGATATT-3 ' 25

PaenAF3 5 ' -GAATT ATTTGATTT AAACCTAAAC AAA-3 ' 26

PaenAR 5 ' -CTT AC AATT AGAGC ATG ANCC AGTAC A-3 ' 27

Walkupl 5 ' -CGCTCGTCCTATGGCTACGATGTACTG-3 ' 28

Walkup2 5 '-CCTATGGCTACGATGTACTGGATCATG-3 ' 29

Walkdnl 5 ' - ACGTGATTTAGGGGTACC ACTGAAATC-3 ' 30

Walkdn2 5 ' - ATGAAAGTAGACC AAATGTTTGACCTT-3 ' 31

API 5^*-GTAATACGACTCACTATAGGGC-3^* 32

[0094] PCR amplification was performed using a Taq DNA polymerase kit (QIAGEN) under the following conditions: the reaction mixture (50μ1) was subjected to an initial denaturation at 94°C for 3 min, followed by 35 cycles, including 1 min at 94°C, 1 min at 59°C and 30 seconds at 72°C. A final extension was carried out at 72°C for 10 min. The amplified PCR product from each reaction was purified using a Qiaquick gel extraction kit (QIAGEN), ligated to the pGEM-T Easy vector (Promega, Madison, WI) and introduced into TOP 10 competent E. coli cells (Invitrogen, Carlsbad, CA) by heat shock at 42°C for 30 seconds. The recombinant plasmid carrying the paenibacillin structural gene was isolated from overnight culture of TOP 10 cells using QIAprep Spin Miniprep kit (QIAGEN). Resultant plasmid DNA was sequenced using a 3730 DNA Analyzer (Applied Biosystems, Foster city, CA) at the Plant-Microbe Genomics Facility at the Ohio State University (Columbus, OH).

[0095] Multiple sequence alignment showed that paenibacillin shows high sequence similarity to lantibiotic epilancin 15X (P86047.1) at the N-terminal region, and resembles the putative lantibiotic BtlA (ZP 04136593.1) at the C-terminal residues (FIG.4). To identify the paenibacillin structural gene, PCR was performed using a forward primer (PaenAFl, PaenAF2 or PaenAF3) and a reverse primer PaenAR. The resultant PCR products were sequenced after cloning in a plasmid vector and the deduced prepropeptide sequence from DNA sequence matched exactly the primary structure of paenibacillin determined by MS/MS and NMR. PaenA is 53 -amino acid prepropeptide of paenibacillin with a leader peptide from residues -1 to -23 (FIG.4). As depicted in FIG.4, alignment of PaenA with other lantibiotics, such as epilancin K7, epilanicin 15X and epicidin 280 revealed a conserved PQ cleave site and a conserved FDLD motif in paenibacillin leader peptide, which may be required for inducing modification, export and cleavage of leader peptide.

[0096] Example 2. Identification of paenB and paenP by Genome Walking

[0097] Genome walking is a known technique for identifying unknown genomic sequences adjacent to known sequences. The unknown DNA sequences flanking paenA were amplified by PCR using a GENOMEWALKER™ universal kit (Clontech, Mountain View, CA) according to the manufacturer's instruction with some modifications. Briefly, genomic DNAs of OSY-DF were digested individually with 4 restriction enzymes (Dra I, EcoR V, Pvu II and Stu I) at 37°C overnight. The resulting blunt-ended DNA fragments were purified using Qiaquick spin columns (QIAGEN) and ligated to the GENOMEWALKER™ adaptors at 16°C overnight. PCR-based DNA genome walking was performed using a paenA gene specific primer (Walkupl, Walkup2, Walkdnl or Walkdn2) and a universal primer API derived from the adaptor sequence.

[0098] PCR amplification was carried out using an Advantage^® 2 PCR Kit (Clontech) under the following two-step cycle parameters: 94°C for 25 seconds and 72°C for 3 min (7 cycles), 94°C for 25 seconds and 65°C for 3 min (32 cycles), followed by a final extension at 72°C for 7 min. Selected PCR product was purified using a Qiaquick gel extraction kit (QIAGEN) and sequenced at the Plant-Microbe Genomics Facility at the Ohio State

University. Predicting protein functions from new DNA sequences was performed using the BLASTX program against NCBI protein database. The adjacent DNA sequence flanking paenA was obtained by a PCR-based genome walking method. Two ORFs encoding putative lantibiotic dehydratase (PaenB) and subtilisin-like serine peptidase (PaenP) were identified in the downstream region of paenA (FIG.2A).

[0099] PaenB was identified as a protein having 1027 amino acid residues and a theoretical molecular weight of 119.0 kDa. PaenB is a homolog of lantibiotic dehydratases (LanB) and shares the highest sequence similarity to LanB-like proteins from B. thuringinesis T01001 (ZP 04136675, 31% identity), B. thuringinesis IBL200 (ZP 04075567.1, 31% identity) and B. subtilis Bsn5 (YP 004206153.1, 29%> identity), whose genome encodes paenibacillin-like lantibiotics but their structures have not yet been determined experimentally. Homo logs of PaenB from well-studied lantibiotics include PepB (CAA90025.1), EpiB

(CAA44253.1), EciB (CAA74350.1), SpaB (AAA22779.1) and NisB (CAA48381.1). From this data, it is expected that PaenB is responsible for dehydration of serine and threonine residues in the paenibacillin propeptide to produce unsaturated didehydroalanine (Dha) and didehydrobutyrine (Dhb) residues, respectively.

[00100] PaenP was identified as a protein having 324 amino acid residues and a theoretical molecular weight of 36.0 kDa. PaenP has a high degree of sequence similarity to subtilisin- like serine proteases (LanP) such as PepP (CAA90024.1), elkP (CAA60861.1) and NisP (CAA80420.1). PaenP contains the conserved catalytic triad residues (Asp43, Hisl02 and Ser290) as well as the oxyanion hole Asnl85, which are the characteristics of this protease family (van der Meer, J. R., et al, (1993) J. Bacteriol. 175:2578-2588). However, PaenP lacks the N-terminal sec-signal sequence and C-terminal cell wall anchor sequence (LPXTGX) found in NisP, suggesting that PaenP may be located inside the cytoplasm (Sahl and Bierbaum (1998) Annu. Rev. Microbiol. 52:41-79). From this data, it is expected that PaenP functions as a peptidase removing the leader peptide of PaenA within the cytoplasm and generating the mature paenibacillin inside the cell.

[00101] Example 3. Identification of the paen Gene Cluster by Whole Genome

Sequencing

[00102] RNase-treated genomic DNA in Tris-Cl (10 mM, pH 8.5) buffer was used for library construction and whole genome sequencing using the next-generation sequencing technology. Briefly, a paired-end library of OSY-DF DNA was prepared using a TRUSEQ™ DNA sample preparation kit (Illumina, San Diego, CA) according to the manufacture's instruction. The constructed library was sequenced (2x76 cycles) in a flow cell lane using the Illumina Genome Analyzer II at the Molecular and Cellular Imaging Center at the Ohio State University. De novo assembly of the P. polymyxa genome was performed using CLC

Genomics Workbench 4.7.2 (CLCBio, Cambridge, MA) on a desktop computer with 4 GB random access memory (RAM). Open reading frames (ORFs) of the assembled contigs were analyzed by Artemis (Rutherford, K., et al, (2000) Bioinformatics 16:944-945) and the protein function was predicted by searching for homo logs using BLASTP at NCBI database.

[00103] The above analysis determined that the OSY-DF draft genome comprises 5.70 megabases (Mb) in 139 contigs over 200 bp. The paenibacillin gene cluster was identified in a large contig of 203 kb by BLAST search of paenA in the draft genome using CLC Genomics Workbench. The identified biosynthetic cluster (Genbank accession number: JQ728481) covered 1 1.7 kb DNA fragment and contained 1 1 putative ORFs (FIG.2A). The length of each ORF, nearest homolog, identity percentage and the proposed function are shown in Table 2.

[00104] Table 2. ORFs identified in the paenibacillin gene cluster.

ORF Name, Proposed Length Homologs % Identity

PaenC, lantibiotic 423 YP 004206154. 1 . lanthionine 33% (396) cyclase synthetase C-like protein [Bacillus

regulator protein [Lactobacillus plantarum)

II P v :n Pⁱ ilaln e X P 05346 1 56. protein TraX 34" ,, ( 252 ) ORF Name, Proposed Length Homologs % Identity

[00105] Modification enzymes PaenB, PaenC and PaenN

[00106] As noted above, PaenB was identified as a dehydratase.

[00107] PaenC was identified as a protein having 423 amino acids and a theoretical molecular weight of 47.3 kDa. PaenC is homologous to lantibiotic cyclases (LanC) such as PepC (CAA90026.1), EciC (CAA74351.1), EpiC (CAA44254.1), SpaC (AAB91588.1) and NisC (CAA48383.1). Nisin cyclase (NisC) is a zinc metalloprotein with a metal ligand and an acid-base catalytic site (Li and van der Donk. (2007) J. Biol. Chem. 282:21169-21175; Li, B., et al, (2006) Science 311: 1464-1467). The zinc ligands (Cys 286, Cys335 and His336) and the active site residues (Asp 153 and His216) were found in paenibacillin cyclase PaenC. From this data, it is expected that PaenC functions as a cyclase via a catalytic mechanism similar to NisC and forms the thioether linkages between the cysteine and Dha/Dhb residues in paenibacillin.

[00108] As FIG.l depicts, the N-terminal amino acid of paenibacillin contains an acetyl group (He, Z., et al, (2008) FEBS Lett. 582:2787-2792). The post-translational modifications of paenibacillin are shown in FIG.5. PaenN was identified as a protein having 256 amino acids and a theoretical mass of 28.7 kDa. PaenN has high sequence homology with the TraX protein family (pfam05857), which is linked to the acetylation of the N-terminal alanine of F- pilin subunits (Marchler-Bauer, A., et al, (2011) Nucleic Acids Res. 39:D225-229; Moore, D., et al, (1993) J. Bacteriol. 175: 1375-1383), as structurally determined by NMR (Frost, L., et al., (1984) J. Bacteriol. 160:395). From this data, it is expected that PaenN functions to acetylate the N-terminal alanine after cleavage of the leader peptide from the paenibacillin polypeptide.

[00109] Peptide processing (PaenP), export (PaenT) and self-immunity (Paenl)

[00110] As noted above, PaenP was identified as a peptidase responsible for removing the leader peptide of PaenA to provide the paenibacillin polypeptide.

[00111] PaenT was identified as a protein having 597 amino acids and a theoretical molecular weight of 67.2 kDa. The N-terminal domain of PaenT contains six membrane spanning helices as predicted by TMHMM server 2.0 (Emanuelsson, O., et al, (2007) Nat Protoc. 2:953-971), where the C-terminal domain contains an ATP-binding site as determined by conserved domain analysis (Marchler-Bauer, A., et al, (2011)), with ATP hydrolysis providing the energy for peptide transportation (Sahl and Bierbaum (1998) Annu. Rev.

Microbiol. 52:41-79). From this data, it is expected that PaenT functions as an ATP-binding cassette (ABC) transporter that exports processed (mature) paenibacillin from the cell into the extracellular medium.

[00112] PaenI was identified as a protein having 185 amino acids and a theoretical molecular weight of 21.0 kDa. PaenI shows sequence similarity to a putative permease (YP 004206177.1) in Bacillus subtilis Bsn5 and a multidrug-efflux transporter (CAK02436) in Bartonella tribocorum. PaenI contains 5 membrane spanning helices as predicted by TMHMM server 2.0 (Emanuelsson, O., et al., (2007)), indicating that PaenI may be integrated to or associated with a cell membrane protein. From this data, it is expected that PaenI is a self-immunity protein that may facilitate the export of paenibacillin.

[00113] Quorum sensing system encoded by paenibacillin gene cluster

[00114] The paenibacillin gene cluster contains an accessory gene regulator (agr)-like locus whose gene products may assemble a quorum sensing system. The well-studied

Staphylococcal Agr system consists of a typical two-component signaling module (AgrC and AgrA), and AgrB and AgrD that are essential for production of the activating ligand, a 7-9 residue peptide with a thiolactone ring (Novick and Geisinger, (2008) Annu. Rev. Genet. 42:541-564). Putative AgrB in OSY-DF contains 137 amino acids with a theoretical mass of 13.7 kDa. AgrB in OSY-DF is a transmenbrane protein with 4 membrane spanning helices as predicted by TMHMM server 2.0 (Emanuelsson, O., et al, (2007)) and may be involved in processing the signal peptide AgrD. Putative AgrD in OSY-DF is a 57 amino acid (6.7 kDa) polypeptide and shows sequence similarity to other autoinducing peptides (AIPs) in Bacillus and Staphylococcus (FIG.5). Putative AgrC and AgrA in OSY-DF show sequence homology with histidine kinase and response regulator, respectively. In addition, a 117-bp of noncoding sequence preceding paenA presumably contains the promoter of the paenibacillin operon. The putative promoter was predicted with BPROM (available at the Softberry, Inc. website) and the predicted -10 and -35 elements are shown in FIG.3. The activated response regulator AgrA may activate the putative promoter and trigger the production of paenibacillin.

[00115] The production of lantibiotics is regulated and it typically occurs during late exponential phase or early stationary phase. For instance, nisin production is regulated by a typical two-component system which consists of a histidine kinase (NisK, CAA80467.1) and a response regulator (NisR, CAA80466.1) (Chatterjee, C, et al, (2005) Chem. Rev. 105:633- 684). In S. epidermidis, transcription of epidermin genes is controlled by a response regulator EpiQ (CAA44256.1) while leader peptide cleavage by EpiP (CAA44257.1) is regulated by an Agr system (Kies, S., et al, (2003) Peptides 24:329-338). However, epidermin production genes and the agr genes are not located in the same gene cluster. In P. polymyxa OSY-DF, an agr-like locus was found in the paenibacillin gene cluster whose products may regulate paenibacillin production by quorum sensing. This is the first complete agr system, to the best of our knowledge, found in a bacteriocin biosynthetic gene cluster.

[00116] Example 4. Anti-bacterial activity of paenibacillin

[00117] The activity of paenibacillin against several strains of Gram-positive bacteria is summarized in Table 3. The data is reported as minimum inhibitory concentrations (MIC) as an average of three replicates with (standard deviation) for the antimicrobials paenibacillin, nisin, vancomycin, and oxacillin. MIC refers to the lowest concentration of an antimicrobial that resulted in no visible growth of bacterial cells. MICs were determined according to the CLSI broth microdilution method (see, Clinical and Laboratory Standards Institute (CLSI). 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. M77-A8. CLSI, Wayne, PA.). Briefly, each antimicrobial was dissolved in water and diluted to appropriate concentration with the medium intended for each tested strain. Aliquots (50μ1) of serially-diluted antimicrobial was dispensed into wells of a 96-well plate; an equal amount of 1/10 diluted overnight bacterial culture was added to wells. Plates were incubated at 35°C for 24h. Cell growth after incubation was examined and determined using a microtiter plate reader at 600nm.

[00118] Table 3. Minimum inhibitory concentration (MIC)

MIC of MIC of MIC of MIC of

Strains Media paenibacillin Nisin Vancomycin Oxacillin fag/ml) fag/ml) fag/ml) fag/ml)

E. faecalis OSU48 MH broth 13.3 (4.62) 8.00 (0) 2.00 (0) 32.0 (0)

B. cereus 14579 MH broth 3.33 (1.15) 8.00 (0) 2.00 (0) 32.0 (0)

B. cereus 1 1778 MH broth 16.0 (0) 8.00 (0) 1.00 (0) 64.0 (0)

B. subtilis OSU494 MH broth 4.00 (0) 8.00 (0) 0.500 (0) 0.250 (0)

L. innocua MH broth

ATCC33090 +5% lysed 1.00 (0) 16.0 (0) 1.00 (0) 4.00 (0) horse blood

L. monocytogenes MH broth

ScottA +5% lysed 1.00 (0) 16.0 (0) 1.00 (0) 4.00 (0) horse blood

Streptococcus MH broth

agalactiae +5% lysed 26.7 (9.24) 16.0 (0) 1.00 (0) 42.7 (18.4) horse blood

M. smegmatis 7H9 broth 8.00 (0) 8.00 (0) 8.00 (0) >64.00 ^a Cation adjusted Mue ler-Hinton broth (Difco); Average of three replicates; ^c Standard deviation

[00119] The data demonstrates that paenibacillin provides for broad range antimicrobial activity at MICs that are comparable to other lantibiotics (nisin) as well as other broad- spectrum antibiotic compounds (vancomycin and oxacillin).

[00120] Example 5. Disruption of lantibiotic dehydratase gene paenB

[00121] For the disruption of paenB gene, a 541 bp DNA fragment internal to lantibiotic dehydratase gene (paenB) was amplified from strain OSY-DF by PCR using primers

PMuDelBEcoF and PMuDelBBamR (See, Table 4). The PCR product was purified by spin column (QIAquick gel extraction kit, QIAGEN, Valencia, CA) and double digested with EcoRI and BamHI. The digested PCR product was cloned into the plasmid pMUTIN4 (Vagner, V.,et al, 1998. Microbiology. 144:3097-3104.) between the EcoRI and BamHI sites. The resulting inactivation vector pMUTIN4_DelB was used to transform P. polymyxa OSY- DF.

[00122] Competent cells of P. polymyxa OSY-DF were prepared by a method as described previously (Murray and Aronstein. 2008. J. Microbiol. Meth. 75: 325-328) with some modifications. Briefly, strain OSY-DF was grown in MYPGP media at 30°C with shaking at 200 rpm to an optical density at 600 nm (OD600) of 0.3-0.4. Subsequently, the cells were washed 3 times with ice-cold electroporation buffer EB (0.625M sucrose with ImM MgCl₂) and were resuspended in 1/200 initial culture volume of cold buffer EB. Electroporation was performed with a 0.2 cm cuvette in a Gene Pulser apparatus connected to a pulse controller (Bio-Rad, Richmond, CA) at the following conditions: 8.5 kV/cm, 200Ω and 25μΕϋ.

Approximate four hundred nanograms of plasmid pMUTIN4_DelB were added to an aliquot (50μ1) of competent cells for transformation. Immediately after the application of pulse, the cells were recovered with 1ml of MYPGP media at 30°C with shaking at 200rpm for 3 h. The presumptive mutants were selected on Tryptic soy agar (TSA) plate with 0^g/ml

erythromycin at 30°C for 2-3 days. Colony PCR was performed using pMutin4 specific primers, pMUTIN4_EryF and pMUTIN4_EryR (Table 1), to detect the plasmid sequence in selected presumptive mutants. Gene disruption due to plasmid integration was further confirmed by PCR using two primer sets (PMuDelBEcoF and LacZRl; PMUTIN-1 and PMuDelBBamR, see Table 4), of which one primer is specific to plasmid pMutin4 and the other one is derived from the target gene.

[00123] The antimicrobial activities of wild type strain OSY-DF or mutants were determined using a microtiter plate assay. An aliquot (100 μΐ) of overnight culture of the tested strains was used to inoculate 15ml LB broth in a 50 ml Falcon tube (BD Biosciences, Bedford, MA). Cells were grown at 30°C with shaking at 200 rpm for 36 h. Samples withdrawn at 24 and 36 h were passed through a 0.22μιη filter and were used for antimicrobial tests. Two susceptible indicators, Listeria innocua ATCC 33090 and Bacillus subtilis 1A771 were used in the tests. Briefly, aliquots (100 μΐ) of 1/10 diluted overnight culture of each indicator were mixed with an equal amount of filtrated culture samples in wells of a 96-well plate. The mixture in the plate was incubated at 37°C for 9h and the optical density at 600nm was measured using a microplate reader (Molecular Devices Corp., Menlo Park, CA).

[00124] MALDI-TOF MS analysis was performed on a mass spectrometer (Bruker Reflex III time-of-flight, Bruker Daltonics Inc., Billerica, MA) to detect the production of

paenibacillin from the wildtype and mutant strains. Briefly, a sample of the filtrated culture (at 36 h) was mixed with a matrix at a ratio of 1 :5. The matrix is a-cyano-4-hydroxy cinnamic acid, prepared as a saturated solution in 50% acetonitrile with 0.1% TFA in water. The mixture was then spotted (Ιμΐ) on the target plate and allowed to air dry. The instrument was operated in reflection-positive ion mode at an accelerating voltage of 28 kV. The N₂ laser was operated at the minimum threshold level required to generate signal and minimize dissociation. [00125] Table 4. Primers for paenB gene inactivation

[00126] Example 6. Purification of paenibacillin from Paenibacillus polymyxa OSY-DF

[00127] The host cells P. polymyxa OSY-DF was cultivated in a rich medium and the resulting paenibacillin was recovered by chromatography techniques. For paenibacillin production, 5 ml of P. polymyxa OSY-DF overnight culture was used to inoculate a 2-liter flask containing 1000 ml tryptic soy broth (Becton Dickinson, Sparks, MD) supplemented with 0.6% yeast extract (TSBYE) broth. The flask was incubated at 30°C for 38h in a shaker (New Brunswick Scientific, Edison, NJ) at 200 rpm. The resulting paenibacillin was partially purified using a two-step chromatography procedure. Firstly, fermentation broth containing paenibacillin was centrifuged to remove the cells. The resulting supernatant was adjusted to pH 6.5 by adding 1.0 N NaOH. Then the supernatant was loaded onto an ion exchange column packed with Macro-prep High S strong cation support resin/matrix (Bio-Rad). The column was washed with 50mM phosphate buffer (pH 6.5) to remove the non-binding components from the fermentation broth. Then the column was eluted with increasing concentration of NaCl (0.3M, 0.5M and 1.0M). The active fractions were desalted by solid phase extraction using Ci₈ Sep-Pak cartridges (Waters, Milford, MA). In the final preparative step,

paenibacillin was eluted from the cartridges by 70% acetonitrile and freeze dried.

[00128] P. polymyxa OSY-DF coproduces two antimicrobial compounds: polymyxin El and paenibacillin. The two-step procedure described above can separate paenibacillin from polymyxin El and many other components. Paenibacillin elutes at low salt concentration (0.3 M and 0.5 M) from cation affinity support matrix while polymyxin El elutes at a higher salt concentration (e.g. 1.0 M). The recovery rate of paenibacillin was 6.4%. If desired, the resulting paenibacillin from this procedure can be further purified using reverse-phase HPLC. REFERENCES

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Claims

CLAIMS What is claimed is:

1. An isolated polynucleotide comprising a sequence encoding a polypeptide having at least 80% amino acid identity to at least one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

2. The isolated polynucleotide of claim 1, wherein the polypeptide has at least 90% amino acid identity to at least one PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

3. An isolated polynucleotide comprising a sequence encoding at least one polypeptide of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

4. An isolated polynucleotide comprising a sequence having at least 80% identity to at least one of paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenI (SEQ ID NO:9), paenT (SEQ ID NO: l 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21).

5. An isolated polynucleotide comprising a sequence having at least 90% identity to at least one of paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenI (SEQ ID NO:9), paenT (SEQ ID NO: l 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21).

6. An isolated polynucleotide comprising a sequence having 100%) identity to at least one of paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), paenC (SEQ ID NO:7), paenI (SEQ ID NO:9), paenT (SEQ ID NO: l 1), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), or paenN (SEQ ID NO:21).

7. An isolated polynucleotide comprising paenA (SEQ ID NO: l).

8. The isolated polynucleotide of claim 7, wherein the polynucleotide further comprises a sequence encoding for at least one of a dehydratase, a cyclase, and a protease.

9. The isolated polynucleotide of claim 7, wherein the polynucleotide further comprises a sequence encoding for a dehydratase, a cyclase, and a protease.

10. The isolated polynucleotide of claim 9, wherein the dehydratase comprises PaenB (SEQ ID NO:6), the cyclase comprises PaenC (SEQ ID NO:8), and a protease comprises PaenP (SEQ ID NO:4).

11. The isolated polynucleotide of claim 10 comprising paenA (SEQ ID NO: 1), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), and paenC (SEQ ID NO:7).

12. The isolated polynucleotide of claim 11 further comprising paenl (SEQ ID NO:9), paenT (SEQ ID NO: l l), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19) and paenN (SEQ ID NO:21).

13. The polynucleotide of any of claims 1-12, wherein the sequence is operably connected to a promoter.

14. The polynucleotide of claim 13, wherein the promoter is a prokaryotic promoter.

15. The polynucleotide of claim 13, wherein the promoter is a eukaryotic promoter.

16. The polynucleotide of claim 13, further comprising an enhancer.

17. A vector comprising the polynucleotide of any of claims 1-16.

18. An isolated polypeptide comprising a sequence having at least 80% amino acid identity to any one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO: 8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

19. The isolated polypeptide of claim 1, wherein the polypeptide has at least 90% amino acid identity to any one of PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), Paenl (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), or PaenN (SEQ ID NO:22).

20. An isolated polypeptide comprising a sequence selected from the group PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

21. A recombinant cell comprising a polynucleotide of any of claims 1-16 or the vector of claim 17.

22. A recombinant cell comprising a polypeptide of any of claims 18-20.

23. The recombinant cell of claim 20 or 21, wherein the cell comprises a prokaryotic cell.

24. The recombinant cell of claim 23, wherein the prokaryotic cell comprises a bacterial cell from the genus selected from the group consisting of Paenibacillus, Bacillus, Streptomyces, Escherichia, Pseudomonas, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus.

25. A method of modifying production of paenibacillin in Paenibacillus polymyxa OSY-DF comprising introducing a polynucleotide of any of claims 13-16, or the vector of claim 17, into Paenibacillus polymyxa OSY-DF.

26. The method of claim 25, wherein the polynucleotide comprises a sequence encoding PaenA (SEQ ID NO:2), PaenB (SEQ ID NO:6), PaenC (SEQ ID NO:8), and PaenP (SEQ ID NO:4).

27. The method of claim 25, wherein the polynucleotide further comprises a sequence encoding at least one of PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

28. A method of producing paenibacillin or an analog thereof, comprising growing a recombinant cell under conditions that allow synthesis of paenibacillin or a paenibacillin analog, wherein the recombinant cell comprises polynucleotides encoding proteins, PaenA (SEQ ID NO:2), PaenP (SEQ ID NO:4), PaenB (SEQ ID NO:6), and PaenC (SEQ ID NO:8), or a homolog thereof, wherein the polynucleotides are operably connected to a promoter.

29. The method of claim 28, wherein the recombinant cell further comprises polynucleotides encoding at least one protein selected from PaenI (SEQ ID NO: 10), PaenT (SEQ ID NO: 12), AgrB (SEQ ID NO: 14), AgrD (SEQ ID NO: 16), AgrC (SEQ ID NO: 18), AgrA (SEQ ID NO:20), and PaenN (SEQ ID NO:22).

30. The method of claim 28 or claim 29, wherein the cell is a bacterium of a genus selected from the group consisting of Paenibacillius, Bacillus, Streptomyces, Escherichia, Pseudomonas Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus.

31. The method of claim 28, wherein the polynucleotides comprise paenA (SEQ ID NO: l), paenP (SEQ ID NO:3), paenB (SEQ ID NO:5), and paenC (SEQ ID NO:7).

32. The method of claim 31, wherein the polynucleotides further comprise at least one of paeni (SEQ ID NO:9), paenT (SEQ ID NO:l l), agrB (SEQ ID NO: 13), agrD (SEQ ID NO: 15), agrC (SEQ ID NO: 17), agrA (SEQ ID NO: 19), and paenN (SEQ ID NO:21).

33. An isolated polynucleotide comprising SEQ ID NO:23.

34. A method of producing paenibacillin comprising:

introducing a polynucleotide comprising SEQ ID NO:23 into a bacterial cell, wherein the polynucleotide is optionally operably connected to a promoter; and

growing the bacterial cell under conditions that allow the production of paenibacillin.

35. The method according to any of claims 25-32 or 34 wherein the method further comprises isolating the paenibacillin.