CROSS-REFERENCE TOO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(c) of provisional application U.S. Serial No. 62/430,455 filed on December 6, 2016, the contents of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to modified Streptomyces fungicidicus isolates, to compositions comprising these isolates, and to methods of using such isolates and compositions to biologically synthesize enduracidin (enramycin). The present invention further relates to modified Streptomyces fungicidicus isolates that biologically synthesize enduracidin and facilitate the production of enramycin in a more efficient manner.

BACKGROUND OF THE INVENTION

Enduracidin, also known as enramycin, and sold as Enradin ^® by MSD Animal Health, is a naturally-occurring 17 amino acid lipodepsipeptide antibiotic that is bio synthesized and excreted by the bacterium Streptomyces fungicidicus, a natural manufacturer for producing enduracidin.

Enduracidin is the generic name of enduracidin analogs and includes enduracidin A, B, C and D. The peptide can be isolated from the fermentation broth and mycelia primarily as a mixture of enduracidins A and B, which differ by one carbon in the length of the attached lipid chain. Structurally, enduracidins are distinguished by a C ₁₂ or C ₁₃ 2Z,4E branched fatty acid moiety attached by an amide linkage to an aspartic acid residue, and the presence of numerous nonproteinogenic amino acid residues such as enduracididine, 4-hydroxyphenylglycine, 3,5- dichloro-4-hydroxyphenylglycine, citrulline, and ornithine.

Enduracidin exhibits potent in vitro and in vivo antibacterial activity against a wide spectrum of Gram-positive organisms, including Clostridium perfringens, methicillin-resistant Staphylococcus aureus (MRS A) and vancomycin-resistant Enterococcus (VRE). Enduracidin inhibits bacterial peptidoglycan cell wall biosynthesis by complexing with extracellular Lipid II, a precursor for the bacterial cell wall. The site of Lipid II complexation with enduracidin is distinct from that recognized by vancomycin and accounts for the action of enduracidin against vancomycin-resistant organisms. To date, there is no documented cross-resistance of enduracidin with any clinically-used antibiotic and no evidence of developed, acquired, or transferrable resistance. The absence of any known form of transferrable resistance mechanism, the lack of oral bioavailability, its low toxicity, and significant anti-bacterial activity towards Clostridium spp. have made enduracidin a key antibiotic for controlling clostridial enteritis in poultry.

Accordingly when administered in poultry feed, enduracidin inhibits the growth of the bacteria that cause both necrotic enteritis and growth depression in poultry. Enduracidin also possesses a zero time withdrawal period. Furthermore, enduracidin is stable in feed and pellets, can be administered to chickens at a very low dosage, and results in no residue being found in either meat and eggs from the treated chickens.

Through the years, a number of Streptomyces fungicidicus strains have been obtained that had been modified to improve the efficiency of these enduracidin-producing

microorganisms, e.g., Streptomyces fungicidicus B-5477 (IFO-12439, ATCC-21013) and Streptomyces fungicidicus B-5477-m (IFO-12440, ATCC-21014) [see e.g., U.S. 3,577,530, U.S. 3,786,142, and U.S. 4,465,771]. However, there remains a significant need to further improve upon such modified Streptomyces fungicidicus isolates to create even more efficient

enduracidin-producing microorganisms.

The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the instant application. SUMMARY OF THE INVENTION

Accordingly, the present invention provides new modified Streptomyces fungicidicus isolates that more efficiently produce enduracidin. In certain embodiments the modified Streptomyces fungicidicus isolate encodes one or more, or all of the following: a Orf2798 that has 95% or greater identity with the amino acid sequence of SEQ ID NO: 2, and which retains a serine residue at amino acid position 2; an Orf3866 that has 95% or greater identity with the amino acid sequence of SEQ ID NO: 4, and which retains a threonine residue at amino acid position 124; an Orf5175 that has 95% or greater identity with the amino acid sequence of SEQ ID NO: 6, and which retains a serine residue at amino acid position 91 ; and an Orf5387 that has 95% or greater identity with the amino acid sequence of SEQ ID NO: 8, and which retains a serine residue at amino acid position 164. In a certain embodiments, the modified Streptomyces fungicidicus isolate further: (i) comprises a nucleic acid that encodes an Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9, in place of the nucleotide sequence of SEQ ID NO: 23; and/or (ii) lacks a functional Orf682 protein; and/or (iii) lacks a functional Orf4868 protein. In particular embodiments of this type the lack of a functional Orf682 protein and/or the lack of a functional Orf4868 protein is due to a frame-shift mutation in a gene that encodes the Orf682 protein and/or the Orf4868 protein.

In particular embodiments the modified Streptomyces fungicidicus isolate encodes one or more, or all of the following: a Orf2798 that has 98% or greater identity with the amino acid sequence of SEQ ID NO: 2, and which retains a serine residue at amino acid position 2;

an Orf3866 that has 98% or greater identity with the amino acid sequence of SEQ ID NO: 4 and which retains a threonine residue at amino acid position 124; an Orf5175 that has 98% or greater identity with the amino acid sequence of SEQ ID NO: 6, and which retains a serine residue at amino acid position 91 ; and an Orf5387 that has 98% or greater identity with the amino acid sequence of SEQ ID NO: 8, and which retains a serine residue at amino acid position 164. In a certain embodiments, the modified Streptomyces fungicidicus isolate further: (i) comprises a nucleic acid that encodes an Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9, in place of the nucleotide sequence of SEQ ID NO: 23; and/or (ii) lacks a functional Orf682 protein; and/or (iii) lacks a functional Orf4868 protein. In particular embodiments of this type the lack of a functional Orf682 protein and/or the lack of a functional Orf4868 protein is due to a frame-shift mutation in a gene that encodes the Orf682 protein and/or the Orf4868 protein.

In yet other embodiments the modified Streptomyces fungicidicus isolate encodes one or more, or all of the following: a Orf2798 that has 99% or greater identity with the amino acid sequence of SEQ ID NO: 2, and which retains a serine residue at amino acid position 2; an Orf3866 that has 99% or greater identity with the amino acid sequence of SEQ ID NO: 4, and which retains a threonine residue at amino acid position 124; an Orf5175 that has 99% or greater identity with the amino acid sequence of SEQ ID NO: 6, and which retains a serine residue at amino acid position 91 ; and an Orf5387 that has 99% or greater identity with the amino acid sequence of SEQ ID NO: 8, and which retains a serine residue at amino acid position 164. In a certain embodiments, the modified Streptomyces fungicidicus isolate further: (i) comprises a nucleic acid that encodes an Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9, in place of the nucleotide sequence of SEQ ID NO: 23; and/or (ii) lacks a functional Orf682 protein; and/or (iii) lacks a functional Orf4868 protein. In particular embodiments of this type the lack of a functional Orf682 protein and/or the lack of a functional Orf4868 protein is due to a frame-shift mutation in a gene that encodes the Orf682 protein and/or the Orf4868 protein.

In still other embodiments the modified Streptomyces fungicidicus isolate encodes one or more, or all of the following: a Orf2798 comprising the amino acid sequence of SEQ ID NO: 2, an Orf3866 comprising the amino acid sequence of SEQ ID NO: 4, an Orf5175 comprising the amino acid sequence of SEQ ID NO: 6, and an Orf5387 comprising the amino acid sequence of SEQ ID NO: 8. In a certain embodiments, the modified Streptomyces fungicidicus isolate further: (i) comprises a nucleic acid that encodes an Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9, in place of the nucleotide sequence of SEQ ID NO: 23; and/or (ii) lacks a functional Orf682 protein; and/or (iii) lacks a functional Orf4868 protein. In particular embodiments of this type the lack of a functional Orf682 protein and/or the lack of a functional Orf 868 protein is due to a frame-shift mutation in a gene that encodes the Orf682 protein and/or the Orf 868 protein.

In related embodiments the modified Streptomyces fungicidicus: (i) comprises a nucleic acid that encodes an Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9, in place of the nucleotide sequence of SEQ ID NO: 23; and/or (ii) lacks a functional Orf682 protein; and/or (iii) lacks a functional Orf4868 protein. In particular embodiments of this type the lack of a functional Orf682 protein and/or the lack of a functional Orf 868 protein is due to a frame-shift mutation in a gene that encodes the Orf682 protein and/or the Orf 868 protein.

In specific embodiments the modified Streptomyces fungicidicus isolate comprises the immunogenic and/or physical and/or genetic characteristics of a modified Streptomyces fungicidicus isolate having the ATCC deposit number PTA-122342. In more specific embodiments the modified Streptomyces fungicidicus is an isolate having the ATCC deposit number PTA-122342 or a progeny of and/or derivative of the isolate having the ATCC deposit number PTA-122342. In a related aspect, all of the modified Streptomyces fungicidicus of the present invention are also provided as isolated modified Streptomyces fungicidicus isolates.

Primer sets having the nucleotide sequences of: SEQ ID NOs: 29 and 30; SEQ ID NOs: 31 and 32; SEQ ID NOs: 33 and 34; SEQ ID NOs: 35 and 36; SEQ ID NOs: 37 and 38; SEQ ID NOs: 39 and 40; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; and/or any combination thereof, are also included in the present invention. In addition, kits comprising one or more of such sets of primers are also part of the present invention. Furthermore, using these primer(s) in an assay to identify a Streptomyces fungicidicus genome of the present invention is also included in the present invention.

These and other aspects of the present invention will be better appreciated by reference to the Drawing, Detailed Description, and Examples. BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the location of 11 polymorphisms on the S. fungicidicus BM38 genome used as mutation markers for PCR-based screening described in the examples below.

DETAILED DESCRIPTION OF THE INVENTION

Developing bacterial secondary metabolites, or natural products, for commercial applications such as human medicine or agricultural applications presents challenges such as overcoming the low yields of the desired compound from the parent/wild-type organism.

Advances in our understanding of natural product biosynthesis over the past decades have revealed that the production of secondary metabolites can be regulated and controlled in several ways. In most instances, the genes for bacterial natural product biosynthesis, including the genes responsible for precursor formation, structural assembly, post-assembly modification, self- resistance and regulation are clustered together on the bacterial chromosome. Biosynthesis of natural products may be elicited by both extracellular and intracellular signaling molecules that interact with pathway specific regulators or pleiotropic regulators that control the production of more than one product. Mutations occurring in any of these regulatory genes or systems may increase, decrease, or abolish antibiotic production.

Strain improvement can play a role in the cost effective industrial scale production of antibiotics or other microbial secondary metabolites. Mutant strains able to produce increased yields of particular metabolites can be generated through random mutations, by targeted disruption of specific genes, or by the introduction of gene(s) that eliminate bottlenecks in a biosynthesis pathway. Genetic manipulation of regulatory genes, as well as biosynthetic genes, to generate hyper-production of specific secondary metabolites has been proven to be a powerful and successful strategy of actinomycete strain improvement. In a particular aspect of the invention, key coding sequences of Streptomyces fungicidicus have been identified that when modified and/or eliminated lead to isolates that have improved properties for biosynthesizing and/or producing enduracidin for commercial purposes.

A 116 kilobasepair DNA sequence from the wild-type S. fungicidicus ATCC 21013 that harbors the enduracidin biosynthetic gene cluster and its flanking regions has been previously reported [US. 8, 188, 245, which is hereby incorporated by reference in its entirety] and is available in GenBank [accession No. DQ403252]. As indicated below, the total genome sequence of BM38-2 (ATCC NO. PTA-122342) has been determined and compared to the wild type genome. This comparative analysis has allowed the identification of at least 77 polymorphisms or mutational differences between the genomes, and the selection of seven (7) key open reading frames that relate to the improved properties of ATCC NO. PTA-122342, as shown below. Unexpectedly, however, none of these seven key open reading frames are associated with the enduracidin bio synthetic gene cluster and its flanking regions of the S.

fungicidicus chromosome. Regardless however, the modification of these seven key open reading frames allows the facile construction of new modified Streptomyces fungicidicus isolates by standard recombinant technology that have comparable or even superior properties to prior reported isolates.

The use of singular terms for convenience in the description is in no way intended to be so limiting. Thus, for example, reference to an "isolate" includes reference to one or more of such isolates, unless otherwise specified. The use of plural terms is also not intended to be limiting, unless otherwise specified.

As used herein, the term, "approximately," is used interchangeably with the term "about" and generally signifies that a value is within twenty- five percent of the indicated value, unless otherwise indicated, e.g., "about" a 4-fold increase in enduracidin production can be a 3 to 5 fold increase.

As used herein one amino acid sequence is 100% "identical" to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50%> of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.

As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using the default parameters. As used herein, an organism that comprises a "lack of a functional" polypeptide or "lacks a functional" polypeptide (e.g., an Orf682) is an organism that does not express that polypeptide and/or expresses a modified polypeptide that has at most 10% of the natural biological function of the corresponding wild type polypeptide e.g., a truncated enzyme that has an enzymatic activity (e.g., enzymatic efficiency, i.e., Vmax/Km) for its natural substrate that is at most 10% of that determined for the corresponding wild type enzyme under the same standard assay conditions. In particular embodiments, a "lack of a functional" polypeptide in an organism is equivalent to the specific biological function of that polypeptide being absent.

As used herein, an open reading frame that has been "nulled" has been modified by e.g., an in- frame-deletion, frame-shifting, insertion, and/or point mutation, such that an organism that comprises an open reading frame that has been "nulled" either does not express the polypeptide encoded by the corresponding wild type open reading frame at all and/or expresses a modified polypeptide that has at most 10% of the natural biological function of the corresponding wild type polypeptide. In particular embodiments, there is an absence of the specific biological function of the polypeptide encoded by the corresponding wild type open reading frame in an organism that comprises an open reading frame, which has been "nulled".

As used herein a "gene cluster" is a set of genetic elements grouped together on the chromosome, the protein products of which have a related function, such as forming a natural product biosynthetic pathway.

A conservative substitution is an amino acid substitution that generally does not substantially alter the activity (specificity or binding affinity) of the molecule. Typically conservative amino acid substitutions involve substitutions of one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). The following table shows exemplar conservative amino acid substitutions:

Preparation of Modified Streptomyces fungicidicus

Suitable methods and materials for the practice of the disclosed embodiments are described below. In addition, any appropriate method or technique well known to the ordinarily skilled artisan can be used in the performance of the disclosed embodiments. Some

conventional methods and techniques applicable to the present disclosure are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999; and Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A.: Practical Streptomyces genetics, John Innes Centre, Norwich

Research Park, Colney, Norwich NR4 &UH, England, 2000.

Recombinant Streptomyces fungicidicus expression plasmid vectors can be prepared for use in making the modified Streptomyces fungicidicus isolates of the present invention. In some embodiments, an engineered recombinant Streptomyces fungicidicus vector comprises at least one selected open reading frame of Streptomyces fungicidicus. In certain embodiments, an engineered recombinant Streptomyces fungicidicus vector comprises at least one selected open reading frame of Streptomyces fungicidicus expressed under the control of a promoter. In other embodiments, the promoter is a strong constitutive Streptomyces promoter that results in the enhanced production of enduracidin when the vector is expressed in a strain of Streptomyces fungicidicus. In some embodiments, the open reading frame is operatively linked to a heterologous promoter instead of its own native promoter. For example, it may be operatively linked to a constitutive promoter, such as a strong constitutive expression promoter or an inducible promoter. In specific embodiments, the strong constitutive promoter is ermE*p from the erythromycin producer, Saccharopolyspora erythraea. In others, the inducible promoter is the thiostrepton inducible promoter, tipA. In yet other embodiments, the P(m¾4)-NitR system [Herai et al, Proc Natl Acad Sci U S A., 101(39):14031-14035 (2004)] or the Streptomycete promoter SF14 is employed. In still others, a native promoter of the apramycin resistant gene amRp is employed. In yet others, VhrdB, Vtc _P 83o, and/or Fneos are employed. In certain embodiments, the engineered recombinant vector comprises an open reading frame orf2798 comprising the nucleotide sequence of SEQ ID NO: 1 and/or an open reading frame orf682, which has been nulled.

Accordingly, recombinant Streptomyces fungicidicus strains can be constructed that are capable of producing enhanced enduracidin yields as compared to a wild-type Streptomyces fungicidicus strain. In certain embodiments, an engineered recombinant Streptomyces fungicidicus strain comprises at least one selected open reading frame from Streptomyces fungicidicus introduced onto the chromosome and expressed under the control of a promoter, such as a strong constitutive Streptomyces promoter, that results in the enhanced production of enduracidin in the engineered strain. In other embodiments, the expression of the introduced open reading frame in the Streptomyces fungicidicus is driven by a heterologous promoter instead of its own native promoter. For example, it may be operatively linked to a constitutive promoter, such as a strong constitutive expression promoter or an inducible promoter. In some embodiments, the strong constitutive promoter is ermE*p. In other embodiments, the inducible promoter is tipA. In some examples, the P(m ^' t4)-NitR system [see, above] or the SF14 promoter is employed. In still other embodiments, the constitutive expression promoter is amRp. In yet other embodiments, VhrdB, Vtc _P 83o, and/or Fneos promoters are employed.

In some embodiments, the engineered strain comprises an open reading frame orf3866 from Streptomyces fungicidicus. In particular embodiments of this type, the open reading frame orf3866 is operatively linked to a heterologous promoter. For example, it can be linked to a strong constitutive promoter such as ermE*p. In other examples, the open reading frame orf3866 is operatively linked to promoter tipA, SF14, amRp, VhrdB, Vtc _P 83o, and/or Fneos.

In other embodiments, the engineered strain encodes an altered open reading frame orf4868. The open reading frame orf4868 can be nulled by insertional disruption, in- frame deletion, frame-shifting and/or point mutation. In some examples, the open reading frame orf4868 is nulled by an in- frame deletion. In general, any internal in- frame deletion over orf4868 should result in a nulled function of orf4868 due to its incompleteness. In related embodiments, the engineered strain involves two, three, four, five, six, seven or more open reading frames of Streptomyces fungicidicus.

In certain embodiments, the modified Streptomyces fungicidicus isolate is derived from a wild type parent strain, such as, but not limited to, Streptomyces fungicidicus American Tissue Culture Company (ATCC) No. 21013. In other embodiments, the engineered strain of

Streptomyces fungicidicus is derived from the conventional mutant strains, such as, but not limited to Streptomyces fungicidicus ATCC 31729, Streptomyces fungicidicus ATCC 31730 and Streptomyces fungicidicus ATCC 31731.

In certain embodiments, enhanced production of enduracidin is an at least 1.2 fold increase, such as at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least a 3 fold, at least a 3.5 fold, at least a 4 fold, at least a 4.5 fold increase, including, but not limited to a 1.2 to 10 fold increase, a 1.2 to 4.6 fold increase, and about a 2 to 5 fold increase in enduracidin production as compared to the wild type Streptomyces fungicidicus strain.

In certain embodiments, the modified Streptomyces fungicidicus may be constructed by integration of a recombinant plasmid comprising at least one enduracidin production enhancing open reading frame into the chromosome of a parent strain of Streptomyces fungicidicus. The integrative conjugal vector may have, or may be engineered to have, a strong constitutive Streptomyces promoter. In some embodiments, the plasmid may lack a streptomycete replicon and may be integrated into the chromosome by site-specific single crossover homologous recombination. In other embodiments, the plasmid may be present as a free plasmid. In some embodiments, a conjugal vector may be engineered in which the plasmid insert carries a partially or completely deleted gene of interest, and its flanking regions, that may be integrated into the chromosome after double crossover homologous recombination to generate an in-frame deletion mutant.

Production of Enduracidin from Recombinant Strains of Streptomyces fungicidicus

The recombinant strains of Streptomyces fungicidicus provided by the present disclosure provide for methods of producing enhanced levels of enduracidin. This technical advance in the art allows for significant cost savings associated with the production of enduracidin. In certain embodiments, the method of producing enduracidin comprises culturing a disclosed recombinant strain of Streptomyces fungicidicus under conditions sufficient for producing enduracidin. In other embodiments, the method further comprises isolating the enduracidin from the culture medium following culturing. In still other embodiments, the method further comprises determining the antibacterial activity of the produced enduracidin, such as by HPLC analysis or bioassay using the S. aureus ATCC 29213 or Bacillis subtilis ATCC 6633 as indicating microorganisms .

In some embodiments, enduracidin is produced by a disclosed Streptomyces fungicidicus strain by utilizing fermentation conditions as previously described for the production of enduracidin [Higashide et ah, J. Antibiot. 21 : 126-137 (1968)]. After production, the

compounds can be purified and/or analyzed including HPLC analysis. Methods of producing enduracidin and harvesting this compound from growth medium can be found in U.S. 4,465,771, which is hereby incorporated by reference in its entirety.

In particular embodiments a disclosed Streptomyces fungicidicus isolate of the present invention is cultured in tryptic soy broth (TSB) on a shaker (such as at 225 rpm and 30°C for 48 hours) and then transferred to a enduracidin production medium (EPM, Table 1 below) for a period of time for continuous fermentation, such as for at least five days and up to eleven days, including 5, 6, 7, 8, 9, 10 or 11 days of continuous fermentation. In more particular

embodiments, the production of enduracidin by the wild-type and derivative strains is conducted in automatic fermenters.

TABLE 1

TABLE 2

SEQUENCE LISTING

AA is an amino acid sequence; NA is a nuc eic acid sequence.

BIOLOGICAL DEPOSIT

Cultures of the following biological material have been deposited with the following international depository: American Type Culture Collection (ATCC) 10801 University

Boulevard, Manassas, Va. 20110-2209, U.S.A., under conditions that satisfy the requirements of the Budapest Treaty.

Organism Accession No. Date of Deposit

Streptomyces fungicidicus PTA-122342 August 5, 2015

(BM38-2)

The present invention may be better understood by reference to the following non- limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES

EXAMPLE 1

MODIFIED STREPTOMYCES FUNGICIDICUS ISOLATE

Production of Streptomyces fungicidicus Biomass for use in Enduracidin Biosynthesis

The fermentation of Streptomyces fungicidicus can be completed in deep tank sanitary design industrial fermenters with systems to monitor and control pH, temperature, oxygen, aeration, and agitation. Each fermented batch of S. fungicidicus is initiated from a characterized and controlled working seed stock of the production seed stored in a secure location and held in low temperature environment. The fermentation process occurs in three stages, followed by further processing downstream:

Stage I:

Working seed cultures, containing 10 ⁷ -10 ¹⁰ spores/mL can be used to start a

fermentation batch. One-to- five vials of frozen seed are retrieved from low temperature storage and thawed either naturally on benchtop, or placed in a water bath at 28-32°C until the contents are thawed. The thawed culture(s) are aseptically transferred into 800- 1000 mL of sterile water held at room temperature and gently mixed to re-suspend the culture. Stage II:

The re-suspended culture is aseptically transferred into seed medium. The seed medium is composed of glucose (0.5 g/L), Dextrin (2.5 g/L), corn steep liquor (1.0-4.0 mL/L), soybean powder (3.0 g/L), ammonium sulfate (0.25 g/L), mono-potassium phosphate (0.13-0.54 g/L), ferrous sulfate (0.00-0.5 g/L), potassium hydroxide (0.13 mL/L), precipitated calcium carbonate (1.5 g/L), silicone-based de-foaming agent (0.1 mL/L), and water, q. s. The medium is sterilized at 125°C for 45 minutes and then cooled to 28°C. The volume of medium is adjusted using sterile water to the desired working volume. The pH is adjusted to 6.6-6.8. The operating parameters of the seed scale up cycle include: incubation temperature of 28°C ± 2°C, an internal pressure of 0.5-1.5 kg/cm ² , an aeration rate of 2-4 Nm ³ /min, and an agitation rate of approximately 80 rpm, depending upon the size and configuration of the vessel. The pH, oxygen consumption, and viscosity are monitored, but not controlled. The culture is grown for 50-60 hours before transfer into the main production fermenter. The viscosity at the time of transfer should range from 350-600 cps, and the pH should be <6.0, and there should be an increase in oxygen consumption. The seed culture is aseptically transferred into the main fermentation medium to complete the fermentation cycle. Stage III:

The main Production Fermenter medium composition includes: corn flour (13.0-15.0 w/v%), corn gluten meal (3.0-6.0 w/v%), cotton seed flour (0.3 w/v%), corn steep liquor (0-0.6 v/v%), sodium chloride (0.3 w/v%), ammonium sulfate (0.25-0.6 w/v%), lactic acid (0-0.5 v/v%), zinc chloride (0.01 w/v%), ferrous sulfate (0.0-0.02 w/v%), potassium hydroxide (0.20-0.5 v/v%), calcium sulfate (0.0-0.5 w/v%), precipitated calcium carbonate (0.5 w/v%), alpha amylase (0.056 w/v%), potassium hydroxide (0.05 v/v%), soybean oil (0.5-2.0 v/v%), de-foaming agent, and water, q. s. The ingredients are added according to the order listed. Water is added to the ingredients up through alpha amylase, then the resulting composition is heated to 80°C for 15 minutes to allow the enzyme to break down the complex carbohydrates. The remaining ingredients are then added, the pH is adjusted to pH 6.6-6.8, and water is added to q. s. The media is sterilized at 125°C for 45 minutes, cooled to 28°C, and water is added to q. s. to the desired working volume.

The contents from the seed fermenter are transferred into the main fermentation medium and the fermenter is set to the following conditions: temperature 28°C, aeration rate 40 Nm ³ /min, internal pressure 0.5 kg/cm ² , and the agitation rate equivalent is set to about 1.85 kW/m ³ . The operating conditions are changed after two hours from the start of fermentation cycle by setting the dissolved oxygen to 12.75 ppm, increasing aeration to 50 Nm ³ /min, and increasing the internal pressure to 0.7 kg/cm ² . The aeration rate, internal pressure, and agitation rates are adjusted thereafter to ensure that the dissolved oxygen is not a rate limiting determinate. Sterile water is added to the culture when the viscosity increases to a point in which the dissolved oxygen is restricted. Throughout the cycle foaming is carefully controlled to prevent contamination or outflow.

Approximately 3 hours after oxygen demand increases the control of the pH is begun. The following parameters are controlled and/or monitored throughout the fermentation cycle: pH, aeration, dissolved oxygen, C0 ₂ , viscosity, purity, agitation speed, internal pressure, and residual sugar. Until the bacteria growth ceases the pH is maintained at 6.8, but then it is allowed to change naturally until harvest. The typical fermentation cycle is 220-300 hours. The culture is ready to be harvested when the bioassay potency is greater than 5,000 μg/L, the pH rises to pH 7.5 or higher, the viscosity decreases, and the oxygen demand ceases. The fermentation is harvested by heating the culture to 70°C for 30 minutes to inactivate the bacteria, and then the harvest fluids are cooled to 25°-28°C.

Downstream Processing: Water is removed from the biomass, the biomass dried, then formulated into premix.

EXAMPLE 2

EFFECT OF OPTIMIZING STREPTOMYCES FUNGICIDICUS ENDURACIDIN PRODUCTION

Yield improvements were made through the years by treatments of the parent strain (as listed in Table 3). Strain BM38-2 (PTA-122342) produces the highest enduracidin yields. The strain was optimized by treating GAB-453 (ATCC 31729) using a series of cultural and physical treatments.

1. ATCC 21013: Streptomyces fungicidicus Original wild Strain B-5477, deposited by Takeda

2. ATCC 21014: mutant derived by y-irradiation of B-5477, strain designated as B-5477w, deposited by Takeda

3. ATCC 31729: mutant derived by UV-irradiation of B- 5477, strain designated as GAB-453, deposited by Takeda

4. ATCC 31730: mutant obtained by growing B-5477 on agar plates containing w-fluoro-DL- tyrosine (MFT); mutant designated as Emt-36-3, deposited by Takeda

5. ATCC 31731 : double mutant obtained by subjecting GAB-453 first to N-methyl-N'-nitro- N- nitrosoquanidine, then w-fluoro-DL-tyrosine (MFT), resulting in mutant strain designation as Emt 2-140., deposited by Takeda

6. ATCC 21388: Closely related strain to Streptomyces fungicidicus (S. macrosporeus), deposited by Squibb and Sons.

In terms of highest to lowest enramycin biosynthesis: ATCC No. PTA-122342>ATCC 31731>ATCC 31730ATCC 31729>ATCC 21013 and ATCC 21014.

Notably, there is greater than a two-fold enhancement of enduracidin obtained when ATCC PTA-122342 is compared to the next most productive isolate and greater than 12-fold enhancement of enduracidin obtained when ATCC PTA-122342 is compared to Parent Strain, wild type.

EXAMPLE 3

GENOMIC ANALYSIS OF STREPTOMYCES FUNGICIDICUS ISOLATES

A comparative genomic analysis was performed between Streptomyces fungicidicus wild type strain, ATCC 21013 (B-5477) and a derivative strain of the present invention, BM38-2, which included the regions around the enduracidin (enramycin) biosynthesis gene cluster.

A total of 77 DNA sequence variations were identified differentiating the effect of physical and cultural manipulations of the parent B-5477 strain. The information gained from the genome analysis permitted rapid and definitive strain comparison by selecting 1 1

representative variations located across the BM38-2 genome as mutation markers (see, Figure 1). PCR primers were designed for each of the mutation markers that would amplify DNA fragments containing the mutation sites for subsequent sequencing and comparison (see, Table 4)·

The PCR primers targeting the marker regions were used to analyze five (5) enramycin-producing strains plus one ( 1 ) closely related strain available through ATCC including wild-type and mutants deposited by Takeda, and compared to BM38-2 strain. Table 5 summarizes the findings and shows the DNA signature at the 11 mutational markers.

TABLE 4

PCR PRIMER SETS (WITH CORRESPONDING SEQ ID NOs:) USED TO AMPLIFY 11 MUTATION MARKERS LOCATED ON THE S. FUNGICIDICUS BM38 GENOME

Tables 5A-5B identify genetic differences between the parent strain (ATCC 21013 B- 5477) and earlier reported strains. Most of these earlier reported strains are derivative strains of ATCC 21013 B-5477 that were obtained through cultural and/or physical manipulations. The most dramatic genetic differences were found for BM38-2 (ATCC No. PTA-122342). As is readily apparent, the primers of Table 4 also can be used to unequivocally identify BM38-2 (PTA- 122342) from other Streptomyces fungicidicus strains and/or closely related Streptomyces species.

EXAMPLE 4

ANALYSIS OF SELECTED MUTATIONS IN STREPTOMYCES FUNGICIDICUS BM38-2

The S. fungicidicus ATCC No. PTA-122342 industrial strain was developed through repeated rounds of mutagenesis followed by selection of high enramycin producing mutants. To gain insight into mutations introduced into ATCC No. PTA-122342 that may contribute to the elevated yields of enramycin, the total genome sequence of ATCC No. PTA-122342 was determined and compared with that of its wild-type S. fungicidicus predecessor. This comparative analysis identified at least 77 polymorphisms, or mutational differences, between the two genomes. Surprisingly, only one difference was detected in the region of the chromosome harboring the enramycin biosynthesis gene cluster. This difference was a single nucleotide change in the endC gene. The mutation of nucleotide 6,260,317 from a C to T in the endC gene, results in the change of a CTC codon to a CTT codon, a silent mutation inasmuch as both are codons for leucine. Therefore, this mutation is unlikely to play a significant role in the observed increase in enramycin yield in BM38-2. The absence of other mutations within the enramycin gene cluster indicates that chromosomal changes responsible for the increase in yield of enramycin in BM38-2 may reside in pleiotropic (non-pathway specific) regulatory elements or global regulatory genes located elsewhere in the genome.

Several response regulators in actinomycetes have been shown to effect natural product biosynthesis in more than one pathway. A key example is the absAlA2 locus, found in the CDA gene cluster of S. coelicolor, which encodes a two-component signal transduction system similar to that found in the enramycin biosynthesis gene cluster of S. fungicidicus. The phosphorylated form of AbsA2 has been shown to inhibit antibiotic production by directly interfering with the expression of pathway-specific regulators of the CDA, actinorhodin and undecylprodigiosin biosynthetic gene clusters. Mutations that inhibit AbsA2 kinase activity thereby enhance antibiotic production. Another example of pleiotropic regulation is found in S. clavuligerus where ccaR, a gene found within the cephamycin C cluster, encodes a regulatory protein that controls both cephamycin C and clavulanic acid production.

Mutations in the ATCC No. PTA-122342 genome that may have the greatest likelihood of being related to increases in enramycin yields could be those occurring in genes predicted by bioinformatic analysis to encode regulatory products, including those that may have pleiotropic regulatory properties. Examples are provided below of putative regulatory genes identified in the S. fungicidicus BM38-2 genome that have mutational differences compared to the wild- type S. fungicidicus strain. [The mutation differences in each of the following examples are highlighted by a missing asterisk and the different/missing/inserted nucleotides are in bold.]

It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, provided to describe nucleic acids and polypeptides according to the invention are approximate within conventional measurement variations.