TW201827590A - Modified streptomyces fungicidicus isolates and their use - Google Patents

Abstract

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

Description

Modified antifungal streptomyces (STREPTOMYCES FUNGICIDICUS) isolate and use thereof

The present invention relates to modified anti-fungal Streptomyces fungicidicus isolates, compositions containing such isolates, and a method for biologically synthesizing persistent bacteriocins (persistin) using these isolates and compositions . The invention further relates to a modified antifungal Streptomyces isolate, which biosynthesizes persistent bacteriocin and promotes the production of durable mycotoxins in a more efficient manner.

The persistent bactericidin (also known as persistent mycin) sold by MSD Animal Health under the name of Enradin ^® is a naturally occurring 17 amino lipopeptide peptide antibiotic that is a bacterial antifungal by a natural manufacturer that produces persistent bacteriocin Streptomyces is biosynthesized and secreted. Perseptic is a generic name for persimidin analogs and includes Persistin A, B, C and D. The peptides can be separated from the yeast solution and mycelium, which is mainly in the form of a mixture of persistent bacteriocins A and B, which are one carbon different in length of the attached lipid chains. Structurally, persistent bacteriocins can be derived from C ₁₂ or C ₁₃ 2 Z , 4 E branched chain fatty acid moieties and many non-proteinaceous amino acid residues (such as enduracididine, 4-hydroxyphenylglycine). Acid, 3,5-dichloro-4-hydroxyphenylglycine, citrulline, and ornithine) are distinguished, and this moiety is linked to the aspartic acid residue by an amidine bond. Persistent bacteriocins are shown to include Clostridium perfringens , medicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus , VRE) has a wide range of Gram-positive organisms with effective antibacterial activity in vitro and in vivo. By complexing with extracellular lipid II (precursor of bacterial cell wall), persistent bacteriocin inhibits biosynthesis of bacterial peptidoglycan cell wall. The site of lipid II complex with persistent bacteriocin is different from that recognized by vancomycin, and causes the effect of persistent bacteriocin against vancomycin-resistant organisms. To date, there is no documented cross-resistance of persistent antibiotics with any clinically used antibiotics, and there is no indication that resistance, acquired resistance or transferable resistance will develop. The absence of any known form of transferable resistance mechanism, lack of oral bioavailability, its low toxicity, and its significant antibacterial activity against Clostridium spp . Make persistent bacteriocin a key antibiotic for controlling Clostridial enteritis in poultry. Therefore, when administered in poultry feed, persistent bacteriocin inhibits the growth of bacteria that cause necrotic enteritis and growth inhibition in poultry. Persistent bacteriocin also has a zero withdrawal period. In addition, persistent bactericidin is stable in feed and pellets, can be administered to chickens at very low doses, and no residue is found in the meat or eggs of treated chickens. Over the years, a large number of antifungal Streptomyces strains have been obtained, which have been modified to enhance the efficacy of these persistent bacteriocin-producing microorganisms, such as antifungal Streptomyces B-5477 (IFO-12439, ATCC-21013) and antifungal Streptomyces B-5477-m (IFO-12440, ATCC-21014) [see, for example, US 3,577,530, US 3,786,142, and US 4,465,771]. However, there is still a great need for further improvements to these modified antifungal Streptomyces isolates to form even more effective microbicide-producing microorganisms. The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" in this application.

Therefore, the present invention provides a novel modified antifungal Streptomyces isolate that is more effective in producing persistent bactericidins. In certain embodiments, the modified antifungal Streptomyces isolate encodes one or more or all of the following: 95% or greater identity to the amino acid sequence of SEQ ID NO: 2 and Orf2798 retaining serine residues at acid position 2; Orf3866 having 95% or more identity to the amino acid sequence of SEQ ID NO: 4 and retaining threonine residues at amino acid position 124; and Orf5175 having an amino acid sequence of SEQ ID NO: 6 having 95% or greater identity and retaining a serine residue at position 91 of the amino acid; and 95% of the amino acid sequence of SEQ ID NO: 8 Orf5387 or higher identity and retaining serine residues at amino acid position 164. In certain embodiments, the modified antifungal Streptomyces isolate further: (i) comprises a nucleic acid encoding Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9 instead 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 a specific embodiment of this type, the lack of a functional Orf682 protein and / or a lack of a functional Orf4868 protein is due to a frameshift mutation in a gene encoding the Orf682 protein and / or Orf4868 protein. In a specific embodiment, the modified antifungal Streptomyces isolate encodes one or more or all of the following: 98% or greater identity to the amino acid sequence of SEQ ID NO: 2 and Orf2798 retaining serine residues at position 2; Orf3866 with 98% or greater identity to the amino acid sequence of SEQ ID NO: 4 and retaining threonine residues at amino acid position 124; and SEQ with Orf5175 having an amino acid sequence of ID NO: 6 having 98% or higher identity and retaining a serine residue at position 91 of the amino acid; and 98% or more of the amino acid sequence of SEQ ID NO: 8 Orf5387 which is more consistent and retains serine residues at amino acid position 164. In certain embodiments, the modified antifungal Streptomyces isolate further: (i) comprises a nucleic acid encoding Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9 instead 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 a specific embodiment of this type, the lack of a functional Orf682 protein and / or a lack of a functional Orf4868 protein is due to a frameshift mutation in a gene encoding the Orf682 protein and / or Orf4868 protein. In yet other embodiments, the modified antifungal Streptomyces isolate encodes one or more or all of the following: 99% or greater identity to the amino acid sequence of SEQ ID NO: 2 and Orf2798 which retains serine residues at acid position 2; Orf3866 which has 99% or more identity with the amino acid sequence of SEQ ID NO: 4 and retains threonine residues at amino acid position 124; and Orf5175 having an amino acid sequence of SEQ ID NO: 6 having 99% or higher identity and retaining a serine residue at position 91 of the amino acid; and 99% of the amino acid sequence of SEQ ID NO: 8 Orf5387 or higher identity and retaining serine residues at amino acid position 164. In certain embodiments, the modified antifungal Streptomyces isolate further: (i) comprises a nucleic acid encoding Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9 instead 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 specific examples of this type, the lack of a functional Orf682 protein and / or a lack of a functional Orf4868 protein is due to a frameshift mutation in a gene encoding the Orf682 protein and / or Orf4868 protein. In yet other embodiments, the modified antifungal Streptomyces isolate encodes one or more or all of the following: Orf2798 comprising the amino acid sequence of SEQ ID NO: 2, or the amino group comprising SEQ ID NO: 4 Orf3866 of the acid sequence, Orf5175 of the amino acid sequence of SEQ ID NO: 6 and Orf5387 of the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the modified antifungal Streptomyces isolate further: (i) comprises a nucleic acid encoding Orf4755 comprising the nucleotide sequence of SEQ ID NO: 9 instead 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 a specific embodiment of this type, the lack of a functional Orf682 protein and / or a lack of a functional Orf4868 protein is due to a frameshift mutation in a gene encoding the Orf682 protein and / or Orf4868 protein. In related embodiments, the modified antifungal Streptomyces: (i) comprises a nucleic acid encoding Orf4755 comprising a nucleotide sequence of SEQ ID NO: 9 instead of a nucleotide sequence of SEQ ID NO: 23; and / or ( ii) lacks a functional Orf682 protein; and / or (iii) lacks a functional Orf4868 protein. In a specific embodiment of this type, the lack of a functional Orf682 protein and / or a lack of a functional Orf4868 protein is due to a frameshift mutation in a gene encoding the Orf682 protein and / or Orf4868 protein. In a specific embodiment, the modified antifungal Streptomyces isolate comprises the immunogenicity and / or physical and / or genetic characteristics of a modified antifungal Streptomyces isolate with ATCC accession number PTA-122342. In a more specific embodiment, the modified antifungal Streptomyces is an isolate having ATCC accession number PTA-122342, or a progeny and / or derivative of the isolate having ATCC accession number PTA-122342. In a related aspect, all modified antifungal streptomyces of the present invention are also provided in the form of an isolated modified antifungal streptomyces isolate. Primers having the following nucleotide sequences are also included in the present invention: 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 NO: 37 and 38; SEQ ID NO: 39 and 40; SEQ ID NO: 41 and 42; SEQ ID NO: 43 and 44; SEQ ID NO: 45 and 46; SEQ ID NO: 47 and 48; SEQ ID NO: 49 and 50; and / or any combination thereof. In addition, a set containing one or more of these primer sets is also part of the invention. In addition, the use of these primers in the assay to identify the antifungal Streptomyces genome of the present invention is also included in the present invention. These and other aspects of the invention will be better understood by reference to the drawings, implementations, and examples.

This application claims priority from US Provisional Application No. 62 / 430,455, filed on December 6, 2016, in accordance with 35 USC § 119 (c), the contents of which are incorporated herein by reference in their entirety. There are challenges in developing bacterial secondary metabolites or natural products for commercial applications such as human pharmaceuticals or agricultural applications, such as overcoming low yields of desired compounds from parental / wild-type organisms. Advances in our understanding of the biological synthesis of natural products over the past decades have shown that the production of secondary metabolites can be regulated and controlled in several ways. In most cases, genes used for the biological synthesis of bacterial natural products, including genes responsible for precursor formation, structural assembly, post-assembly modification, resistance, and self-regulation, are clustered together on the bacterial chromosome. The biological synthesis of natural products can be caused by extracellular and intracellular signaling molecules that interact with pathway-specific regulators or pleiotropic regulators that control the production of more than one product. Mutations in any of these regulatory genes or systems can increase, decrease, or eliminate antibiotic production. Strain improvement can play a role in cost-effective industrial-scale production of antibiotics or other microbial secondary metabolites. Mutant strains capable of producing enhanced yields of specific metabolites can be generated by random mutations, by targeting disruption of specific genes, or by introducing genes that eliminate bottlenecks in biologically synthetic pathways. Gene manipulation to regulate genes and to synthesize genes biologically to produce high yields of specific secondary metabolites has proven to be a powerful and successful strategy for the improvement of actinomycete strains. In a particular aspect of the invention, key coding sequences for antifungal Streptomyces have been identified which, when modified and / or eliminated, produce a biosynthetic and / or probiotic-resistant biocide for commercial purposes. Isolates with improved characteristics. A 116-kilobase pair DNA sequence from wild-type antifungal Streptomyces ATCC 21013 containing a biocide biosynthetic gene cluster and its flanking regions has previously been reported [US. 8,188,245, which is incorporated herein by reference in its entirety Medium], and it can be obtained in [GenBank 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. As shown below, this comparative analysis allows the identification of at least 77 polymorphisms or mutation differences between genomes and the selection of seven (7) key open reading frames for improved characteristics of ATCC No. PTA-122342. Unexpectedly, none of these seven key open reading frames were related to the biosynthetic gene cluster and flanking regions of the persistent fungicidin against the fungal Streptomyces chromosome. In any case, with standard recombination techniques, these seven key open reading frame modifications make it easy to construct new modified antifungal Streptomyces isolates that have comparable or even superior characteristics to previously reported isolates. The use of singular terms in the description for convenience is by no means intended to be so limited. Thus, for example, unless stated otherwise, reference to "isolate" includes reference to one or more of such isolates. Unless otherwise stated, the use of plural terms is also not intended to be limiting. As used herein, the term "about" is used interchangeably with the term "about" unless otherwise indicated, and usually indicates a value within 25 percent of the indicated value, for example, an increase in persistent bacteriocin production "about" A 4-fold increase can be 3 to 5 times. As used herein, when the amino acid residues of two sequences are identical, one amino acid sequence is 100% "consistent" with the second amino acid sequence. Therefore, when 50% of the amino acid residues of the two amino acid sequences are identical, one amino acid sequence is 50% "consistent" with the second amino acid sequence. Sequence comparisons are made within consecutive blocks of amino acid residues contained by a given protein (eg, a protein or a portion of a polypeptide being compared). In a particular embodiment, considered are selected deletions or insertions that may otherwise alter the correspondence between two amino acid sequences. As used herein, C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc.) with aligned preset parameters and consensus preset parameters can be used for percent identity of nucleotide and amino acid sequences. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm. These commercially available programs can also be used to determine sequence similarity using the same or similar preset parameters. Alternatively, you can use Advanced Blast search under preset filtering conditions, for example, using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) stacking program using preset parameters. As used herein, an organism containing a "lack of a functional / lacks a functional" polypeptide (e.g., Orf682) is an organism that does not express the polypeptide and / or exhibits a modified polypeptide, and the modified polypeptide has a corresponding Up to 10% of the natural biological functions of wild-type polypeptides, such as truncated enzymes, which have enzymatic activity (eg, enzymatic hydrolysis efficiency, that is, Vmax / Km) for their natural substrates are tested against the corresponding wild species under the same standard test conditions Up to 10% of the activity measured by type I enzymes. In certain embodiments, a "lack of functionality" polypeptide in an organism is equivalent to the absence of a specific biological function of the polypeptide. As used herein, open reading frames that have been "ineffective" have been modified by, for example, in-frame deletions, frame shifts, insertions, and / or point mutations, so that organisms containing open reading frames that have been "ineffective" are completely ineffective A modified polypeptide that exhibits a polypeptide encoded by the corresponding wild-type open reading frame and / or exhibits up to 10% of the natural biological function of the corresponding wild-type polypeptide. In a specific embodiment, there is no specific biological function of a polypeptide encoded by a corresponding wild-type open reading frame in an organism that contains an open reading frame that has been "ineffective". As used herein, a "gene cluster" is a group of genetic elements grouped together on a chromosome, whose protein products have related functions, such as forming a biologically synthetic pathway for natural products. Conservative substitutions are amino acid substitutions that generally do not substantially change the molecular activity (specificity or binding affinity). Conservative amino acid substitutions typically involve the replacement of one amino acid with another amino acid having similar chemical properties (eg, charge or hydrophobicity). The following table shows exemplary conservative amino acid substitutions: Preparation of Modified Antifungal Streptomyces Suitable methods and materials for implementing the disclosed embodiments are described below. In addition, any suitable method or technique familiar to those skilled in the art can be used to implement the disclosed embodiments. Some conventional methods and techniques suitable for the present invention are described below, for example, Sambrook et al., Molecular Cloning : A Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning : A Laboratory Manual , 3rd Edition, Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology , Greene Publishing Associates, 1992 (and 2000 Supplement); Ausubel et al., Short Protocols in Molecular Biology : A Compendium of Methods from Current Protocols in Molecular Biology , 4th edition, 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, MJ, Buttner, MJ, Chater, KF, and Hopwood, DA: Practical Streptomyces genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 & UH, England, 2000. Recombinant antifungal Streptomyces performance plastid vectors can be prepared for use in preparing the modified antifungal Streptomyces isolates of the present invention. In some embodiments, the engineered recombinant antifungal Streptomyces vector comprises at least one selected open reading frame of the antifungal Streptomyces. In certain embodiments, the engineered recombinant antifungal Streptomyces vector comprises at least one selected open reading frame of an antifungal Streptomyces that is under the control of a promoter. In other embodiments, the promoter is a potent constitutive streptomyces promoter, which results in increased yield of persistent bactericidin when the vector is expressed in a strain resistant to Streptomyces. In some embodiments, the open reading frame is operably linked to a heterologous promoter rather than its own native promoter. For example, it is operably linked to a constitutive promoter, such as a potent constitutively expressing promoter or an inducible promoter. In a specific embodiment, the strong constitutive promoter is ermE ^* p from a erythromycin producer ( Saccharopolyspora erythraea ). In other embodiments, the inducible promoter is thiostrepton-inducible promoter tipA . In yet other embodiments, the P ( nitA ) -NitR system [Herai et al., Proc Natl Acad Sci US A., 101 (39): 14031-14035 (2004 ) ] or the Streptomyces promoter SF14 is used. In yet other embodiments, the natural promoter of the apramycin resistance gene amR p is used. In still other embodiments, P _hrdB , P _tcp830 and / or P neos are used . In certain embodiments, the engineered recombinant vector comprises an open reading frame orf2798 containing the nucleotide sequence of SEQ ID NO: 1 and / or an open reading frame orf682 that has been rendered ineffective. Therefore, a recombinant antifungal Streptomyces strain can be constructed which is capable of producing an increased yield of persistent bactericidin compared to a wild type antifungal Streptomyces strain. In certain embodiments, the engineered recombinant antifungal Streptomyces strain comprises at least one selected open reading frame from an antifungal Streptomyces that is introduced onto a chromosome and is in a constitutive manner such as Performance under the control of the promoter of the Streptomyces promoter results in increased yields of persistent bactericidin in engineered strains. In other embodiments, the heterologous promoter rather than its own native promoter drives the performance of the open reading frame introduced into the antifungal Streptomyces. For example, it is operably linked to a constitutive promoter, such as a potent constitutively expressing 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 ( nitA ) -NitR system [see above] or the SF14 promoter is used. In yet other embodiments, the constitutive expression promoter is amR p. In yet other embodiments, _PhrdB , _Ptcp830, and / or P neos promoters are used. In some embodiments, the engineered strain comprises an open reading frame orf3866 from an antifungal Streptomyces. In a specific embodiment of this type, the open reading frame orf3866 is operably 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 the promoters tipA , SF14, amR p, _PhrdB , _Ptcp830, and / or Phos . In other embodiments, the engineered strain encodes an open reading frame orf4868 with a change. The open reading frame orf4868 can be rendered ineffective by insertional destruction, in-frame deletions, frame shifts, and / or point mutations. In some examples, the open reading frame orf4868 is ineffective due to in-frame deletion. In general, any internal frame deletion in orf4868 due to its incompleteness should result in invalid function of orf4868. In a related embodiment, the engineered strain comprises two, three, four, five, six, seven or more open reading frames of the antifungal Streptomyces. In certain embodiments, the modified antifungal Streptomyces isolate is derived from a wild-type parental strain, such as, but not limited to, antifungal Streptomyces American Tissue Culture Company (ATCC) No. 21013. In other embodiments, the engineered strains of antifungal Streptomyces are derived from conventional mutant strains such as, but not limited to, antifungal Streptomyces ATCC 31729, antifungal Streptomyces ATCC 31730, and antifungal Streptomyces ATCC 31731. In certain embodiments, the increased yield of persistent bactericidin is increased by at least 1.2 times, such as by at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times more than that of wild-type antifungal Streptomyces strains Times, at least 3.5 times, at least 4 times, at least 4.5 times, including but not limited to an increase of 1.2 to 10 times, an increase of 1.2 to 4.6 times, and an increase of about 2 to 5 times. In certain embodiments, the modified antifungal streptomyces can be constructed by integrating a recombinant plastid containing at least one open reading frame that increases the yield of persistent fungicidin into the chromosome of a parental strain of antifungal streptomyces. The integrated junction vector can have or be engineered to have a strong constitutive Streptomyces promoter. In some embodiments, the plastids may lack streptomyces replicons and may be integrated into the chromosome by site-specific single exchange homologous recombination. In other embodiments, the plastids can exist as free plastids. In some embodiments, the conjugation vector can be engineered, in which the plastid insert carries a related gene that is partially or completely deleted, and its flanking regions, which can be integrated into the chromosome after double-exchange homologous recombination to generate in-frame Deletion mutation. Production of persistent bactericidin from recombinant strains of antifungal streptomyces Provided by the present invention are recombinant strains of antifungal streptomyces that provide methods for producing increased persistent bacteriocin content. This technological advancement in this technology has resulted in significant cost savings associated with the production of persistent bacteriocins. In certain embodiments, a method of producing a persistent bactericidin comprises culturing the disclosed recombinant strain of antifungal Streptomyces under conditions sufficient to produce the persistent bactericidin. In other embodiments, the method further comprises isolating the persistent bactericidin from the culture medium after the culturing. In still other embodiments, the method further comprises determining the antibacterial activity of the produced persistent bacteriocin, such as by HPLC analysis or using Staphylococcus aureus ATCC 29213 or Bacillis subtilis ATCC 6633 as a biological test for the indicator microorganism . In some embodiments, the persistent spinosyn using Po by fermentation as previously disclosed by the conditions of the antifungal Streptomyces strains [Higashide et al., J Antibiot lasting for generating the described spinosyns 21: 126-137 ( 1968)]. After production, the compounds can be purified and / or analyzed, including HPLC analysis. Methods for producing persistent bacteriocins from growth media and collecting this compound can be found in US 4,465,771, which is incorporated herein by reference in its entirety. In a specific embodiment, the disclosed antifungal Streptomyces isolates are applied to a tryptic soy broth (TSB) on a shaker, such as at 225 rpm for 30 hours at 30 ° C. Cultivate and then transfer to enduracidin production medium (EPM, Table 1 below) for a period of time for continuous fermentation, such as at least five days and up to eleven days, including continuous fermentation 5, 6, 7, 8, 9, 10 or 11 days. In a more specific embodiment, the production of persistent biocides by wild-type and derived strains is performed in an automatic fermentation tank. TABLE 1 Persistence spinosyn production medium (EPM) Composition (pH 6. 7) Table 2 Sequence Listing AA is an amino acid sequence; NA is a nucleic acid sequence. Biological deposits Subject to the requirements of the Budapest Treaty, cultures of the following biological materials have been deposited in the following international depositories: American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209, USA. Biological Accession No. Date of deposit Streptomyces antifungal PTA-122342 2015 dated. 5 years. 8 Day (BM38-2) reference to the following non-limiting examples of the present invention may be better understood, these examples illustrate the present invention as provided. In order to more fully illustrate the preferred embodiments of the present invention, the following examples are presented. However, it should not be construed as limiting the broad scope of the invention. EXAMPLES Example 1 Modified antifungal Streptomyces isolates are used for biosynthesis of biocides of Streptomyces antifungal. Biomass production of antifungal Streptomyces can be used to monitor and control pH, temperature, oxygen, The deep tank hygienic design of the aeration and stirring system is completed in the industrial fermentation tank. Each fermented batch of antifungal Streptomyces is caused by a characteristic and controlled stock of working strains of production strains that are stored in a safe location and in a low temperature environment. The fermentation process is carried out in three stages, followed by further processing downstream: Phase I: A working strain culture containing 10 ⁷ -10 ¹⁰ spores / mL can be used to start the fermentation batch. One to five frozen bacteria vials are taken from the low temperature storage bin and naturally thawed on a workbench or placed in a water bath at 28-32 ° C until the contents are thawed. The thawed culture is aseptically transferred to 800-1000 mL of sterile water stored at room temperature, and gently mixed until the culture is resuspended. Phase II: Aseptically transfer the resuspended culture to the seed culture medium. The bacteria culture medium consists of the following: glucose (0.5 g / L), dextrin (2.5 g / L), corn pulp (1.0-4.0 mL / L), soybean meal (3.0 g / L), ammonium sulfate ( 0.25 g / L), monopotassium 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 defoamer (0.1 mL / L) and appropriate amount of water. The medium was sterilized at 125 ° C for 45 minutes, and then cooled to 28 ° C. Use sterile water to adjust the volume of the medium to the required working volume. Adjust the pH to 6.6-6.8. The operating parameters of the bacteria scale expansion cycle include: depending on the size and configuration of the container, a cultivation temperature of 28 ° C ± 2 ° C, an internal pressure of 0.5-1.5 kg / cm ² and an inflation rate of 2-4 Nm ³ / min And a stirring rate of about 80 rpm. Monitor but do not control pH, oxygen consumption and viscosity. The culture is allowed to grow for 50-60 hours and then transferred to the main production fermentation tank. The viscosity during transfer should be in the range of 350-600 cps, the pH should be ≤6.0, and the oxygen consumption should be increased. The strain culture is aseptically transferred to the main fermentation medium to complete the fermentation cycle. Phase III: The main composition of the fermentation tank culture medium includes: corn meal (13.0-15.0 w / v%), corn protein meal (3.0-6.0 w / v%), cottonseed meal (0.3 w / v%), corn pulp (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%), a defoamer and an appropriate amount of water. Add ingredients in the order listed. Water was added to each ingredient until the alpha amylase, and the resulting composition was then heated to 80 ° C for 15 minutes to allow the enzyme to decompose complex carbohydrates. Then add the remaining ingredients, adjust the pH to pH 6.6-6.8, and add an appropriate amount of water. The medium was sterilized at 125 ° C for 45 minutes, cooled to 28 ° C, and an appropriate amount of water was added to the required working volume. The contents of the yeast fermentation tank were transferred to the main fermentation medium, and the fermentation tank was set to the following conditions: the temperature was 28 ° C, the aeration rate was 40 Nm ³ / min, and the internal pressure was 0.5 kg / cm ² And the stirring rate equivalent is set to about 1.85 kW / m ³ . The operating conditions were changed two hours after the start of the fermentation cycle: the dissolved oxygen was set to 12.75 ppm, the aeration was increased to 50 Nm ³ / min, and the internal pressure was increased to 0.7 kg / cm ² . Thereafter, the aeration rate, internal pressure, and agitation rate were adjusted to ensure that dissolved oxygen was not a determining factor for the rate limit. When the viscosity increased to the point where the dissolved oxygen was limited, sterile water was added to the culture. Throughout the cycle, foaming is carefully controlled to prevent contamination or outflow. The control of pH is started approximately 3 hours after the oxygen demand increases. The following parameters are controlled and / or monitored throughout the fermentation cycle: pH, aeration, dissolved oxygen, CO ₂ , viscosity, purity, stirring speed, internal pressure and residual sugar. Until bacterial growth stopped, the pH was maintained at 6.8, but then allowed to change naturally until collected. A typical fermentation cycle is 220-300 hours. When the bioassay titer is above 5,000 µg / L, the pH rises to pH 7.5 or higher, the viscosity decreases, and oxygen demand ceases, the culture is ready to be collected. The yeast was collected by heating the culture to 70 ° C for 30 minutes to deactivate the bacteria, and then the collected fluid was cooled to 25 ° C-28 ° C. Downstream processing: The water is removed from the biomass, the biomass is dried, and then formulated into a premix. Example 2 Optimizing the effect of persistent fungicidin against antifungal streptomyces. Yields have been improved over the years by treating parent strains (as listed in Table 3). Strain BM38-2 (PTA-122342) produced the highest persistent bactericidin yield. The strain was optimized by treating GAB-453 (ATCC 31729) with a series of culture and physical treatments. Table 3: Yield improvement of antifungal Streptomyces B - 54577 References: Patents 4,465,771; APR 14, 1984, Nogami, et al. Takeda Chemical Industries, Ltd., Osaka, Japan. References: Patent 3,577,530, May 4, 1971, directed to ATCC 21388. 1. ATCC 21013: the initial wild strain of antifungal Streptomyces B-5477, deposited by Takeda 2. ATCC 21014: a mutant obtained from the radiation of B-5477, known as B-5477 m , deposited by Takeda 3 ATCC 31729: A mutant obtained by UV-radiation of B-5477, a strain called GAB-453, deposited by Takeda 4. ATCC 31730: By using m -fluoro-DL-tyrosine (MFT) Mutant obtained by growing B-5477 on agar plates; a mutant called Emt-36-3, deposited by Takeda 5. ATCC 31731: by subjecting GAB-453 to N-methyl-N'-nitrogen first -N-nitrosoguanidine then undergoes a double mutant obtained with m -fluoro-DL-tyrosine (MFT), resulting in a mutant strain called Emt 2-140, deposited by Takeda 6. ATCC 21388: with antifungal chain mold (megaspore Streptomyces, S. macrosporeus) is closely related to the strain, registered by the Squibb and Sons. In terms of biosynthesis of highest to lowest persistent mycins: ATCC No. PTA-122342> ATCC 31731> ATCC 31730> ATCC 31729> ATCC 21013 and ATCC 21014. Notably, when ATCC PTA-122342 was compared with the next highest yield isolate, the persistent bactericidin was more than doubled, and when ATCC PTA-122342 was compared with the parent strain, wild type, the obtained Persistent bacteriocin increased more than 12 times. Example 3 Genomic analysis of an antifungal Streptomyces isolate. Comparative genomic analysis was performed between an antifungal Streptomyces wild-type strain ATCC 21013 (B-5477) and a derivative strain BM38-2 of the present invention. Biocide (persistin) biosynthesizes the area around the gene cluster. A total of 77 DNA sequence variations have been identified to distinguish the role of physical and cultural manipulations of parental B-5477 strains. By selecting 11 representative mutations located throughout the BM38-2 genome as mutation markers (see Figure 1), the information obtained from the genomic analysis allows for rapid and definitive strain comparisons. PCR primers are designed for each mutation marker, which will amplify a DNA fragment containing the mutation site for subsequent sequencing and comparison (see Table 4). PCR primers targeted to the marker region were used to analyze five (5) strains producing duramycin plus one (1) closely related strains available through ATCC, including wild-type and Takeda-registered mutants, and BM38 -2 strain compared. Table 5 summarizes the results and shows the DNA tags at 11 mutation markers. Table 4 PCR primer sets for amplifying 11 mutation markers located on the genome of antifungal Streptomyces BM38 ( with corresponding SEQ ID NO :) ^# Mutation tag ID; ^{* †} desired PCR product size wild type strain (WT): ATCC 21013 and ATCC of Table 5A PTA - No. 122,342 as compared to the antifungal Storage of mutant strains of Streptomyces Marker WT = matches parent wild type. M = Match variant sequence PTA-122342 * MEGASPORES Table 5B Streptomyces strain closely related to the antifungal Streptomyces and BM38 - 2 shared or used as a marker for comparison of mutant PCR Table 5A-5B identifies genetic differences between the parental strain (ATCC 21013 B-5477) and previously reported strains. Most of these previously reported strains are derived strains of ATCC 21013 B-5477, which are obtained through culture and / or physical manipulations. BM38-2 (ATCC No. PTA-122342) shows the most significant genetic difference. As apparent, the primers of Table 4 can also be used to clearly identify BM38-2 (PTA-122342) from other antifungal Streptomyces strains and / or closely related Streptomyces species. Example 4 Antifungal Streptomyces BM38 - Analysis of mutations in the two antifungal selected Streptomyces strain ATCC No. PTA-122342 Department of Industrial through repeated rounds of mutagenesis followed by selected mutants will produce a high persistence of neomycin generated . To further understand the mutations introduced in ATCC No. PTA-122342 that may promote increased yield of persistent mycin, the total genomic sequence of ATCC No. PTA-122342 was determined and compared with the sequence of its wild-type antifungal Streptomyces leader. This comparative analysis identified at least 77 polymorphisms or mutation differences between the two genomes. Surprisingly, only one difference was detected in the region of chromosomes with biosynthetic clusters of genes that persisted with persistin. This difference is a single nucleotide change in the endC gene. Mutation of nucleotides 6,260,317 from C to T in the endC gene causes the CTC codon to become the CTT codon. Because both are codons of leucine, it is a silent mutation. Therefore, this mutation is unlikely to play an important role in the observed increase in persistent mycinin production in BM38-2. The presence of other mutations within the persistin gene cluster indicates that the chromosomal changes responsible for increasing the yield of persistin in BM38-2 may be due to pleiotropic (non-path specific) regulatory elements or global regulation located elsewhere in the genome gene. Several response regulators in actinomycetes have been shown to affect the biosynthesis of natural products in more than one pathway. Examples absA1A2 key locus, which is found in Streptomyces coelicolor (S. Coelicolor) the CDA gene cluster which encodes a neomycin found in biological persistence similar synthesis gene cluster of Streptomyces antifungal system of two-component Sub-signal Transduction System. The phosphorylated form of AbsA2 has been shown to inhibit antibiotics by directly interfering with the performance of pathway-specific regulators of biological clusters of gene clusters that directly interfere with CDA, actinorhodin, and undecylprodigiosin produce. Inhibition of mutations in AbsA2 kinase activity further increases antibiotic yield. Another example of a pleiotropic regulator of Streptomyces clavuligerus found in (S. Clavuligerus), wherein CCAR, the gene encodes a visible within cephamycin (cephamycin) C cluster regulatory proteins that control cephamycin and clavulanic C Acid (clavulanic acid) yield. Mutations in the ATCC PTA-122342 genome (which may have the greatest probability associated with increased yields of persistent mycin) are mutations present in genes predicted by bioinformatics analysis to encode regulatory products, including pleiotropic Mutations in regulatory characteristics. Examples of putative regulatory genes identified in the genome of antifungal Streptomyces BM38-2 with mutational differences compared to wild-type antifungal Streptomyces strains are provided below. [The mutation differences for each of the examples below are highlighted with missing asterisks, and the different / missing / inserted nucleotide lines are shown in bold. ] Sequence comparison orf682 : predicted to encode a regulatory protein in the TetR family containing a helix-turn-helix (HTH) DNA-binding domain. The extra G insertion is in a homopolymeric orbit that induces frame shifting. ＞ WT_682 Translated Gene Product ＞ BM38.2_682 translated gene product orf2798 : predicts to encode DNA- binding transcriptional regulators in the MurR / RpiR family . It contains a helix-turn-helix (HTH) and a sugar isomerase (SIS) domain. G to A mutation causes Gly to become Ser. ＞ WT_2798 Translated Gene Product ＞ BM38.2_2798 Translated gene product orf3866 : predicted to encode a DNA binding response regulator in the OmpR family . It contains the REC domain, which receives signals from the two-component system of Streptomyces antibiotic regulatory protein (SARP) and the sensor partner in the winged helix (wHTH) domain feature. G to A mutation causes Ala to become Thr. ＞ WT_3866 Translated Gene Product ＞ BM38.2_3866 Translated gene product orf4755 : predicts to encode the RNA polymerase σ - 70 factor in the σ - E family . The T to G mutation (CG T- > CG G ) in the A Arg codon is a silent mutation. ＞ WT_4755 Translated Gene Product ＞ BM38.2_4755 Translated gene product orf4868 : predicted to encode a transcription regulator protein . It contains a REC signal receiving domain that receives signals from a sensing partner in a two component system. Deletions in tandem repeats (deletion C ) result in frameshift mutations. ＞ WT_4868 Translated Gene Product ＞ BM38.2_4868 Translated gene product orf5175 : predicted to encode a histidine kinase transcription regulator protein . It has a REC signal receiving domain that receives signals from a sensing partner in a two component system. The C to T mutation causes Pro to become Ser. ＞ WT_5175 Translated Gene Product ＞ BM38.2_5175 Translated gene product orf5387 : predicted to encode transcription regulators in the MerR family . It has an HTH DNA binding domain and a REC domain, and it receives signals from a sensory partner in a two-component system. A to C mutations cause Tyr to Ser. ＞ WT_5387 Translated Gene Product ＞ BM38.2_5387 Translated gene product It should 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 present invention are approximated within conventional measurement deviations.

Figure 1 depicts the location of 11 polymorphisms on the genome of Streptomyces antifungal BM38 used as a mutation marker based on PCR screening described in the examples below.

US United States; American Type Culture Collection (ATCC); 2015/08/05; PTA-122342

Claims (15)

A modified Streptomyces fungicidicus isolate, encoding an Orf2798 protein having an amino acid sequence identity of 95% or higher with the amino acid sequence of SEQ ID NO: 2; wherein the Orf2798 protein is The amino acid sequence contains a serine residue at amino acid position 2; and wherein the modified antifungal is compared with the yield of enduracidin obtained by a wild-type antifungal Streptomyces strain Streptomyces achieve increased production of persistent bactericidin. The modified antifungal Streptomyces isolate according to claim 1, wherein the Orf2798 protein is encoded by the nucleotide sequence of SEQ ID NO: 1. The modified antifungal Streptomyces isolate of claim 1 or 2, further encoding an Orf3866 protein having an amino acid sequence identity of 95% or higher with the amino acid sequence of SEQ ID NO: 4; wherein the The amino acid sequence of the Orf3866 protein contains a threonine residue at amino acid position 124. The modified antifungal Streptomyces isolate of claim 3, wherein the Orf3866 protein is encoded by the nucleotide sequence of SEQ ID NO: 3. The modified antifungal Streptomyces isolate of claim 4, further encoding an Orf5175 protein having an amino acid sequence identity of 95% or higher with the amino acid sequence of SEQ ID NO: 6; wherein the Orf5175 protein The amino acid sequence includes a serine residue at amino acid position 91. The modified antifungal Streptomyces isolate of claim 5, wherein the Orf5175 protein is encoded by the nucleotide sequence of SEQ ID NO: 5. The modified antifungal Streptomyces isolate of claim 6, further encoding an Orf5387 protein having an amino acid sequence identity of 95% or higher with the amino acid sequence of SEQ ID NO: 8; wherein the Orf5387 protein The amino acid sequence contains a serine residue at amino acid position 164. The modified antifungal Streptomyces isolate of claim 7, wherein the Orf5387 protein is encoded by the nucleotide sequence of SEQ ID NO: 7. The modified antifungal Streptomyces isolate of claim 8, further comprising the nucleotide sequence of SEQ ID NO: 9 but excluding the nucleotide sequence of SEQ ID NO: 23. The modified antifungal Streptomyces isolate of claim 9, which does not encode a functional Orf682 protein. The modified antifungal Streptomyces isolate of claim 10, wherein the lack of functional Orf682 protein is due to the modified Antifungal Streptomyces isolate including a frameshift mutation in the gene encoding the Orf682 protein. The modified antifungal Streptomyces isolate of claim 9, which does not encode a functional Orf4868 protein. The modified antifungal Streptomyces isolate of claim 12, wherein the lack of functional Orf4868 protein is due to the modified Antifungal Streptomyces isolate including a frameshift mutation in the gene encoding the Orf4868 protein. A method for producing a persistent bactericidin, the method comprising culturing a modified antifungal Streptomyces isolate according to any one of claims 1 to 13 in a medium under conditions sufficient to produce the durable bactericidin. The method of claim 14, further comprising isolating the persistent bacteriocin from the medium.