Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• 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.
• 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.
• 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.
• enduracidin when administered in poultry feed, 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.
• Streptomyces fungicidicus B-5477 IFO-12439, ATCC-21013
• Streptomyces fungicidicus B-5477-m IFO-12440, ATCC-21014
• U.S. 3,577,530, U.S. 3,786,142, and U.S. 4,465,771 there remains a significant need to further improve upon such modified Streptomyces fungicidicus isolates to create even more efficient
• the present invention provides new modified Streptomyces fungicidicus isolates that more efficiently produce enduracidin.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the modified Streptomyces fungicidicus 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.
• 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.
• 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.
• 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.
• all of the modified Streptomyces fungicidicus of the present invention are also provided as isolated modified Streptomyces fungicidicus isolates.
• kits comprising one or more of such sets of primers are also part of the present invention.
• using these primer(s) in an assay to identify a Streptomyces fungicidicus genome of the present invention is also included in the present invention.
• 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.
• 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.
• 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].
• 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.
• 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.
• 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.
• 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.
• GCG Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin
• an organism that comprises a "lack of a functional" polypeptide or "lacks a functional" polypeptide 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.
• a "lack of a functional" polypeptide in an organism is equivalent to the specific biological function of that polypeptide being absent.
• 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.
• 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.
• 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:
• Recombinant Streptomyces fungicidicus expression plasmid vectors can be prepared for use in making the modified Streptomyces fungicidicus isolates of the present invention.
• an engineered recombinant Streptomyces fungicidicus vector comprises at least one selected open reading frame of Streptomyces fungicidicus.
• an engineered recombinant Streptomyces fungicidicus vector comprises at least one selected open reading frame of Streptomyces fungicidicus expressed under the control of a promoter.
• 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.
• the open reading frame is operatively linked to a heterologous promoter instead of its own native promoter.
• it may be operatively linked to a constitutive promoter, such as a strong constitutive expression promoter or an inducible promoter.
• the strong constitutive promoter is ermE*p from the erythromycin producer, Saccharopolyspora erythraea.
• the inducible promoter is the thiostrepton inducible promoter, tipA.
• the P(m3 ⁇ 44)-NitR system [Herai et al, Proc Natl Acad Sci U S A., 101(39):14031-14035 (2004)] or the Streptomycete promoter SF14 is employed.
• a native promoter of the apramycin resistant gene amRp is employed.
• VhrdB, Vtc P 83o, and/or Fneos are employed.
• 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.
• recombinant Streptomyces fungicidicus strains can be constructed that are capable of producing enhanced enduracidin yields as compared to a wild-type Streptomyces fungicidicus strain.
• 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.
• the expression of the introduced open reading frame in the Streptomyces fungicidicus is driven by a heterologous promoter instead of its own native promoter.
• a constitutive promoter such as a strong constitutive expression promoter or an inducible promoter.
• the strong constitutive promoter is ermE*p.
• the inducible promoter is tipA.
• the P(m ' t4)-NitR system [see, above] or the SF14 promoter is employed.
• the constitutive expression promoter is amRp.
• VhrdB, Vtc P 83o, and/or Fneos promoters are employed.
• the engineered strain comprises an open reading frame orf3866 from Streptomyces fungicidicus.
• the open reading frame orf3866 is operatively linked to a heterologous promoter.
• it can be linked to a strong constitutive promoter such as ermE*p.
• the open reading frame orf3866 is operatively linked to promoter tipA, SF14, amRp, VhrdB, Vtc P 83o, and/or Fneos.
• 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.
• the open reading frame orf4868 is nulled by an in- frame deletion.
• any internal in- frame deletion over orf4868 should result in a nulled function of orf4868 due to its incompleteness.
• the engineered strain involves two, three, four, five, six, seven or more open reading frames of Streptomyces fungicidicus.
• 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.
• ATCC Streptomyces fungicidicus American Tissue Culture Company
• 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.
• 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.
• 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.
• 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.
• 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.
• the recombinant strains of Streptomyces fungicidicus 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.
• the method of producing enduracidin comprises culturing a disclosed recombinant strain of Streptomyces fungicidicus under conditions sufficient for producing enduracidin.
• the method further comprises isolating the enduracidin from the culture medium following culturing.
• 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 .
• 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
• 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.
• TLB tryptic soy broth
• EPM enduracidin production medium
• the production of enduracidin by the wild-type and derivative strains is conducted in automatic fermenters.
• AA is an amino acid sequence
• NA is a nuc eic acid sequence
• ATCC American Type Culture Collection
• 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:
• Working seed cultures containing 10 7 -10 10 spores/mL can be used to start a
• 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 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.
• 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.
• Downstream Processing Water is removed from the biomass, the biomass dried, then formulated into premix.
• 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.
• ATCC 21013 Streptomyces fungicidicus Original wild Strain B-5477, deposited by Takeda
• ATCC 21014 mutant derived by y-irradiation of B-5477, strain designated as B-5477w, deposited by Takeda
• ATCC 31729 mutant derived by UV-irradiation of B- 5477, strain designated as GAB-453, deposited by Takeda
• 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
• 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
• ATCC 21388 Closely related strain to Streptomyces fungicidicus (S. macrosporeus), deposited by Squibb and Sons.
• 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.
• 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.
• 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.
• the S. fungicidicus ATCC No. PTA-122342 industrial strain was developed through repeated rounds of mutagenesis followed by selection of high enramycin producing mutants.
• 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.
• 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.
• 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.]

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