Novel Streptomyces sporosporus strains and novel antibiotics against bacteria and fungi

Abstract

Disclosed is a novel Streptomyces sporosporus strain SC263 produced by UV mutagenesis, the produced antibiotics exhibit increased thermal stability, which exhibit antibiotic activity against bacteria and fungi. Antibiotics produced by SC263 and compositions thereof are also provided for the treatment of bacterial and fungal infections. The antibiotics have a very high molecular weight, which is suitable for topical administration.

Description

[0001] Refer to related applications

[0002] This non-provisional application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 343,298, filed on May 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a novel Streptomyces sporogenes strain SC263 and an antibiotic produced by the strain SC263. The present invention also relates to a composition containing an antibiotic for combating bacteria and fungi. Background Art

[0004] The development of antimicrobial resistance in pathogenic bacteria and fungi has led to a global health emergency[1]. According to the World Health Organization, diseases caused by drug-resistant pathogens cause more than 700,000 deaths each year in more than 120 countries[2]. There is an urgent need for new antibiotics or antifungal compounds from natural sources.

[0005] Immunocompromised patients are susceptible to multidrug-resistant Candida[3] and Aspergillus[4]. Almost all clinical antifungal drugs have a molecular weight of less than 2000 Da[5]. These commonly used antifungal drugs, including amphotericin B (for intravenous injection), flucytosine (for oral and intravenous injection), and itraconazole (for oral and intravenous injection), may exhibit serious side effects such as nephrotoxicity, hepatotoxicity, bone marrow suppression, and heart failure, which limit the use of these drugs[6-8]. Less toxic drugs, such as natamycin produced by Streptomyces species, are considered relatively safe and can be used for topical application (such as eye drops), but are almost insoluble in water and therefore have low bioavailability when used orally[9].

[0006] Streptomyces, which is commonly found in fertile soil, is a major producer of natural products. It produces nearly 8,000 natural products, accounting for about 45% of microbial sources

[10] . About two-thirds of clinically useful antibiotics are produced by Streptomyces

[11] , including many important drugs such as streptomycin, neomycin, kanamycin, rapamycin, chloramphenicol, vancomycin, etc.

[12] . It is estimated that this genus has about 100,000 antibiotics, of which only 3% have been discovered

[13] . For decades, people have been exploring Streptomyces to find new and useful antibiotics [14,15].

[0007] There is a need to develop novel strains that produce antibiotics with enhanced antibiotic efficacy. Summary of the invention

[0008] Disclosed herein is a novel Streptomyces sporogenes strain SC263, which produces antibiotic peptides exhibiting increased thermostability.

[0009] The present invention also provides an antibiotic produced by SC263, named AF1, which shows antibiotic activity against at least six fungi and two Gram-positive bacteria, and has an apparent molecular weight greater than 5 kDa, particularly greater than 100 kDa, which is unusual in antibiotics. The use of AF1 is also disclosed herein, particularly for the preparation of an antifungal composition.

[0010] In one aspect, the present invention provides a novel Streptomyces sporogenes strain SC263 that produces an antibiotic with increased thermostability.

[0011] In the present invention, the sporophycetes strain SC263 is deposited in the Leibniz-Institut of the German Microorganism Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), which is an international depository institution (IDA) under the Budapest Treaty of the World Intellectual Property Organization. Its accession number is DSM 34277.

[0012] In one embodiment of the present invention, the strain SC263 has a DNA sequence as shown in SEQ ID NO.:1.

[0013] In another embodiment of the present invention, the strain SC263 has a DNA sequence as shown in SEQ ID NO.:2.

[0014] In another embodiment of the present invention, the strain SC263 has a DNA sequence as shown in SEQ ID NO.:3.

[0015] In another aspect, the present invention provides a composition comprising strain SC263.

[0016] In another aspect, the present invention provides an antibiotic AF1 produced by strain SC263 and concentrated by membrane filtration, with an apparent molecular weight greater than 5 kDa, specifically greater than 100 kDa.

[0017] In one embodiment of the present invention, the composition comprises antibiotics having an apparent molecular weight greater than 100 kDa as measured by membrane filtration concentration, and antibiotics having an apparent molecular weight between 10 kDa and 100 kDa.

[0018] In one embodiment of the present invention, the antibiotic AF1 is effective against fungi.

[0019] In one embodiment of the present invention, the antibiotic AF1 is effective against Gram-positive bacteria.

[0020] In yet another aspect, the present invention provides an antimicrobial composition comprising an antibiotic produced by the Streptomyces sporogenes strain SC263 and a pharmaceutically acceptable carrier.

[0021] In one embodiment of the present invention, the composition is used to treat fungal infection.

[0022] In one embodiment of the present invention, the composition is used to treat bacterial infection, especially Gram-positive bacterial infection.

[0023] In one embodiment of the present invention, the composition is administered topically.

[0024] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing summary of the invention as well as the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the accompanying drawings presently preferred embodiments.

[0026] In the attached picture:

[0027] Figure 1 The morphology of SC26 is shown: (A) Colony morphology of SC26 on YM1 agar after 6 days of culture at 30°C. (B) Spore chains of SC26 under field emission scanning electron microscopy (FE-SEM). The long bar represents 1 μm.

[0028] Figure 2The antifungal properties of SC26 and its derivatives are shown. The antifungal activity of SC26 and its derivatives was examined using different fermentation times up to day 5 (A) and treating the culture broth at different pH values ​​for 24 h (B). All inhibition zones against C. albicans were measured.

[0029] Figure 3 The phylogenetic tree of the 16S ribosomal RNA of SC26 is shown. The most probable phylogenetic analysis was based on 16S rRNA gene sequences from 62 species of Streptomyces. The phylogenetic tree is drawn to scale, with branch lengths indicating the number of substitutions per site between species obtained using MEGA 11.

[0030] Figure 4 The temperature stability of bioactive compounds from SC26 and its derivatives is shown. Overnight cultures of SC26 and three of its derivatives (SC261, SC262 and SC263) were stored at 25°C, 37°C and 65°C. The antimicrobial activity against Candida albicans (A) and Staphylococcus aureus (S. aureus) (B) was measured at different time points.

[0031] Figure 5 The antibacterial and antifungal activities of SC263 are shown as follows: (A) Antibiotic activity of SC263 cultured for 6 days using the cross-streak method. 3 (B) 10 μl of SC263 culture suspension was spotted in quadruplicate on the lawn of the tested fungi and bacteria. Each plate was inoculated with 10 6 The remaining inoculated 10 5 CFU. (C) The diameter of the inhibition zone was measured in triplicate. Error bars were calculated based on quadruplicate circles for each tested microorganism. White columns represent fungi, and black columns represent Gram-positive bacteria.

[0032] Figure 6 Antibiotic activity of SC263 membrane filtrates is shown. (A) Different fractions of SC263 culture fluid were tested against Candida albicans. 1: culture fluid; 2: filtrate <100 kDa; 3: compound >100 kDa (F100 fraction). (B) The reconstituted lyophilized powder was tested for inhibition zones against different fungi and Gram-positive bacteria in duplicate spots.

[0033] Figure 7Shown are protease treatments of AF1 and F100 from SC263. (A) The antibiotic activity of AF1 and F100 treated with proteinase K (PK) or protease E (PE) was examined. The numbers in the figures represent the different treatment conditions as shown below. 1: AF1. 2: F100. 3: AF1 treated with PE or PK. 4: F100 treated with PE or PK. 5: PE or PK dissolved in sterile water. (B) The diameter of the inhibition zone was measured.

[0034] Figure 8 Shown is a genomic comparison between SC263 and S. sporogenes NBRC 15456. Biosynthetic gene clusters (BGCs) were predicted using the antiSMASH 6.0 web server and the gene clusters of the two strains are numbered sequentially at the top and bottom lines, respectively. A genomic comparison between these two strains was generated using EASYFIG v.2.2.5 and is placed in the middle.

[0035] Fig. 9 Shows the results of inhibition zone assays for several yeasts / fungi.

[0036] Fig.10 Shows the results of the inhibition zone assay for two Gram-positive bacteria. DETAILED DESCRIPTION

[0037] The above invention contents of the present invention are further described with reference to the following examples. However, it should not be understood that the contents of the present invention are limited to the following examples, and all inventions based on the above contents of the present invention belong to the scope of the present invention.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.

[0040] The term "composition" means a product resulting from the mixing or combination of more than one active ingredient, and includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredient and a certain auxiliary agent are administered to the patient simultaneously in the form of a single entity or dosage. "Non-fixed combination" means that the active ingredient and a certain auxiliary agent are administered to the patient as separate entities simultaneously, concurrently or sequentially without specific intervening time limits, wherein such administration provides effective concentrations of both agents in the patient. The latter also applies to cocktail therapy, for example, the administration of three or more active ingredients.

[0041] The term "composition" or "pharmaceutical composition" refers to a therapeutically effective amount of an antibiotic produced by SC263 and optionally a pharmaceutically acceptable carrier.

[0042] The term "pharmaceutically acceptable" means that a drug is suitable for contact with the tissues of individuals (such as humans) to whom it is administered without excessive toxicity, irritation, allergic response or other problems or complications, and with a reasonable benefit / risk ratio. Each carrier must be compatible with the other ingredients to achieve "acceptability".

[0043] The term "carrier" means a non-toxic compound or agent that has the function of assisting cells or tissues to absorb active ingredients. The carrier is selected from, for example, excipients, adjuvants, diluents, fillers or bulking agents, granulators, coating agents, release control agents, adhesives, disintegrants, lubricants, preservatives, surfactants, antioxidants, buffers, suspending agents, thickeners, stabilizers or other carriers used in pharmaceutical compositions. Examples of carriers include, but are not limited to, polyvinyl alcohol, polyvinyl pyrrolidone (povidone), hydroxypropyl methylcellulose, poloxamer, hydroxyethyl cellulose cyclodextrin, carboxymethyl cellulose (CMC), phosphate buffered saline (PBS), water, emulsifiers (such as water-in-oil emulsifiers) or wetting agents. Tension regulators can be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerol, or any other suitable tension regulator. Various buffers and means for adjusting pH can be used as long as the resulting preparation is acceptable. Thus, buffers include acetate buffers, citrate buffers, phosphate buffers, and borate buffers. Optionally, the pH of such preparations may be adjusted using an acid or base. Similarly, acceptable antioxidants for use in the present invention include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene. Other excipient components that may be included in the preparation are chelating agents. A preferred chelating agent is edetate disodium, but other chelating agents may be used instead or in combination therewith.

[0044] The term "effective amount" or "therapeutically effective amount" means the administration of a sufficient amount of an agent or compound that will relieve to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a peptide or protein disclosed herein required to provide a clinically significant reduction in disease symptoms. In any individual case, an appropriate "effective" amount can be determined using techniques such as dose escalation studies.

[0045] The terms "treat," "treating," or "treatment" include alleviating, alleviating, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, such as arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or prophylactically and / or therapeutically arresting the symptoms of a disease or condition.

[0046] In the present invention, Streptomyces strain SC26 was isolated from a mountainous area in northern Taiwan, China, and a late-sporulating mutant was subsequently obtained from a culture of strain SC26, which was named SC263. It was unexpectedly found that the antibiotic produced by strain SC263 exhibited higher thermal stability than the antibiotic produced by strain SC26. The genome sequence of SC263 was determined, and SC263 was classified as a known species of Streptomyces sporosus by pairwise comparison between SC263 and Streptomyces sporosus NBRC15456. According to the sequencing results, the genome of SC263 contains the DNA sequence of SEQ ID NO: 1, the DNA sequence of SEQ ID NO: 2, or the DNA sequence of SEQ ID NO: 3.

[0047] The present invention provides a novel Streptomyces sporogenes strain SC263, which produces an antibiotic with increased thermostability.

[0048] The present invention also provides a composition comprising strain SC263 or an antibiotic produced by strain SC263. In the composition, strain SC263 is viable or inactivated.

[0049] According to the present invention, the apparent molecular weight of the antibiotic produced by strain SC263 is greater than 100 kDa by concentration through membrane filtration.

[0050] In one embodiment, the antibiotic is used to prepare an antimicrobial composition for use against bacteria or fungi.

[0051] In one embodiment, the composition is a pharmaceutical composition that can be topically administered to the skin of a subject.

[0052] The present invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation.

[0053] Materials and Methods

[0054] Soil sample collection and separation of SC26

[0055] Soil samples were collected from a mountainous area in northern Taiwan, China, and stored in sterile bags at 4°C until use. Samples were sieved with a 1-mm metal mesh, suspended in sterile water, serially diluted, plated on YM1 agar (0.4% yeast extract, 1% malt extract), and incubated at 30°C for 5 to 7 days. Individual spore-producing colonies were picked and streaked on YM1 agar. Spores of these isolates were collected, suspended in sterile 20% glycerol, and stored at -80°C.

[0056] Morphology under electron microscope

[0057] Spore chains of SC263 were collected from 5-day cultures grown on YM1 agar coverslips, fixed using liquid nitrogen, coated with gold particles, and observed under a thermal field emission scanning electron microscope (FE-SEM) JSM-7610F (JOEL Ltd., Tokyo, Japan).

[0058] Phylogenetic analysis of 16S rRNA

[0059] The full-length 16S rRNA sequence was amplified by PCR using primer 27F and reverse primer 1492R

[25] and subjected to Sanger sequencing. The 16S rRNA sequence of SC263 was compared with the database on the BLASTN web server of the National Center for Biotechnology Information (NCBI)

[26] . The 62 top hit sequences were transferred to MEGA11 for alignment and phylogenetic tree construction [27,28].

[0060] Microbial strains and culture conditions

[0061] The fungi used in the antibiotic tests were Aspergillus niger ATCC 16878, Aspergillus niger ATCC 16404, Candida albicans ATCC 10231, Candida tropicalis ATCC 13803, N. gypseum ATCC 14683, and Saccharomyces cerevisiae BCRC22286. The two Gram-positive bacteria used were Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538. The three Gram-negative bacteria used were Escherichia coli ATCC 8739, Salmonella SL1344, and Enterobacterium enteritidis ATCC 13076.

[0062] Table M below provides information on the yeasts / fungi that were further tested.

[0063] Table M. Yeasts / fungi tested further

[0064]

[0065]

[0066] Further Gram-positive bacteria tested included Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538.

[0067] The fungi tested were grown on Sabouraud dextrose agar (SDA). The bacteria tested were grown on tryptone soy agar (TSA). All bacteria and fungi were cultured at 30°C.

[0068] Streptomyces were grown on solid YM1 agar at 30°C or in liquid YM1 broth at 30°C with shaking.

[0069] Cross-score screening for antibiotic activity

[0070] Actinomycete-like soil isolates were streaked onto YM1 agar and incubated at 30°C for 24 h. 1 μl of 10 3 colony forming units (CFU) of the tested bacteria or fungi ( Figure 5 A) and continued to culture for four days

[29] .

[0071] Inhibition zone assay for antibiotic activity

[30]

[0072] All the items containing 10 5 Inoculum of 10 CFU (except for Aspergillus niger, Aspergillus niger and Neisseria gypseum, each of which contained 10 6 10 μl of liquid containing antibiotic (AF1) was spotted. For the culture supernatant, the culture was grown at 30°C for 24 hours and centrifuged at 12000g for 5 minutes. The supernatant was collected and used for inhibition test. For the lyophilized powder, 0.1 g was dissolved in 1 ml of sterile water. The culture plate was cultured at 30°C for 5 days.

[0073] Membrane filtration concentration of SC263

[0074] The molecular weight of AF1 was determined using 10 kDa and 100 kDa centrifugal filter units (AMICON). Two liters of SC263 culture supernatant was filtered through a 100 kDa mPES membrane (#D04-100 kDa, Repligen-Spectrumlabs) until the volume was reduced to 500 ml. The filtered and retained fractions were tested for antibiotic activity.

[0075] UV mutagenesis of SC26

[0076] UV mutagenesis of Streptomyces was performed according to the method of Kumar

[31] . Surviving colonies were streaked onto new culture plates and late spore mutants were screened.

[0077] Processing enzymes

[0078] For the culture supernatant of SC263 or F100, proteinase K and protease E were added to a concentration of 0.04 mg / mL and incubated at 37°C for 1 hour. 1 mg / mL of RNaseA or DNaseI was treated and incubated at 37°C for 0.5 hour. 10 mg / mL of pancreatic enzymes from porcine pancreas (Sigma-Aldrich) (which consisted of amylase, trypsin, lipase, ribonuclease and protease) were added and incubated at 37°C for 1 hour.

[0079] Genomic DNA purification

[0080] SC263 was grown in YM1 at 30°C for 3 days. Mycelia were harvested by centrifugation and washed with 10% sucrose. Genomic DNA was extracted according to the method of Kutchma et al.

[32] . The purified genomic DNA was stored in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) at -20°C.

[0081] Whole genome sequencing

[0082] The genome DNA was processed according to the experimental protocol of PacBio CLR. The sequencing format was SMRTcell sequencing performed via the Sequel IIe platform. SMRT Link (version 10.1.0.119588) was used to assemble the sequence fragments. The QV score of the three fragment contigs produced was at least 91.39. Fragment contigs with scores lower than 90 were removed.

[0083] Genome analysis

[0084] The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate the SC263 genome and the results were stored in the NCBI database with the accession number JAKGSF000000000. OrthoANI was used to perform average nucleotide identity (ANI) analysis

[22] . Easyfig was used to perform whole genome similarity comparison

[33] . AntiSMASH6.0 was used to mine the existing biosynthetic gene clusters

[23] .

[0085] result

[0086] Example 1 Isolation of antibiotic-producing SC26

[0087] A total of six soil samples from the mountainous area of ​​northern Taiwan were screened for microorganisms on YM1 agar. Among the colonies grown from these samples, isolate SC26 showed actinomycete-like morphology, including gray spores ( Figure 1 A). Field emission scanning electron microscopy (FE-SEM) of SC26 showed mycelium and spiral spore chains ( Figure 1 B), which is characteristic of Streptomyces.

[0088] The culture supernatant of SC26 showed antibiotic activity against the fungus Candida albicans ATCC 10231 ( Figure 2 A). The 16S rRNA sequence of SC26 was determined. Phylogenetic analysis showed that among the 62 Streptomyces species compared, SC26 16S rRNA was similar to Streptomyces sporogenes NBRC 15456 and LMG 20313, Streptomyces xanthophaeus NBRC 12829 and NRRL B-5414, Streptomyces nojiriensis NBRC 13794 and LMG 20094 ( Figure 3 ) belong to the same evolutionary clade.

[0089] Example 2 Isolation and Characterization of UV-Induced Derivatives

[0090] The antibiotic produced by SC26 (designated AF1) is heat labile and is destroyed within 3-5 hours at 65°C ( Figure 4A, 4B). UV mutagenesis of SC26 was performed to identify strains that produce relatively thermostable AF1. Three mutant strains (SC261, SC262, and SC263) were selected because of their delayed sporulation (2 days compared to SC26). These mutants showed no differences in colony morphology or the time course of AF1 production from SC26 ( Figure 2 A).

[0091] The thermostability analysis of AF1 produced by these mutants against Candida albicans and Staphylococcus aureus ATCC 6538 was performed ( Figure 4 ). AF1 produced by SC262 showed similar thermal instability as AF1 produced by SC26, while AF1 produced by SC261 and SC263 were stable against Candida albicans for up to 192 hours and against Staphylococcus aureus for up to 144 hours at 25°C. SC263 was selected for further detailed studies.

[0092] The pH stability test was performed on AF1 of SC26 and its derivatives. AF1 of SC26 was relatively unstable at pH 3 and pH 10 compared with its derivatives ( Figure 5 B) AF1 of SC263 is relatively stable at pH 2 to 12, but weakened at pH 13 to 14.

[0093] Example 3 Antimicrobial activity of AF1 from SC263

[0094] AF1 of SC263 showed the same antibiotic spectrum as SC26, i.e., it was effective against two fungi (Aspergillus niger ATCC16404 and Candida albicans) and two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis ATCC 6051), but not against three Gram-negative bacteria (Escherichia coli ATCC 8739, Salmonella SL1344 and Enterobacterium enteritidis ATCC 13076) ( Figure 5 A).

[0095] In addition, AF1 of SC263 also inhibited the growth of four other fungi, namely Aspergillus niger, Candida tropicalis, Neisseria gypseum and Saccharomyces cerevisiae ( Figure 5 B). The largest inhibition zone appeared in Aspergillus niger, with an average diameter of 32.0 mm, while the smallest inhibition zone appeared in P. gypseum, with an average diameter of 7.1 mm ( Figure 5 C).

[0096] The inhibition zones produced by AF1 on both Gram-positive bacteria were opaque ( Figure 5B), which contains microcolonies that are able to grow when transferred to a new plate. In contrast, the inhibition zone on the fungal plate contains no viable cells. This suggests that AF1's activity against both Gram-positive bacteria is bacteriostatic, rather than bactericidal.

[0097] Fig. 9 showed more yeast / fungus test results, while Fig.10 Results of tests for two Gram-positive bacteria are shown.

[0098] Example 4 Characterization of SC263 antibiotic by membrane filtration

[0099] To assess the size of AF1 in SC263, the inventors filtered the culture fluid through 10-kDa and 100-kDa pore size membranes. The active compound was not present in the filtrate, but was present in the >100 kDa fraction (designated "F100"; Figure 6 A). When lyophilized and reconstituted, F100 exhibited the same antibiotic spectrum as the culture supernatant ( Figure 6 B).

[0100] The antibiotics in SC26 culture medium produced the same sizing results. The larger apparent size of AF1 distinguishes it from most known antibiotics, most of which are small metabolites

[16] . Most known antifungal antibiotics have a molecular weight of less than 2000 Da [5].

[0101] Many large antifungal natural products are proteins [17,18]. Treatment of SC263 culture medium and F100 with proteinase K or protease E did not eliminate the antibiotic activity ( Figure 7 ), which indicates that AF1 is not a protein. Treatment with DNaseI, RNaseA or pancreatin (which consists of amylase, trypsin, lipase, ribonuclease and protease) also did not eliminate the antibiotic activity of AF1 (data not shown).

[0102] Example 5 Genome sequence of SC263

[0103] In order to identify SC263 taxonomically and to study the secondary metabolic system of SC263, the whole genome sequence of SC263 was determined. Three fragment contigs were generated: Ctg1 (7,201,269 bp), Ctg2 (996,243 bp) and Ctg3 (20,182 bp) (Table 1). The total GC content was 71.92%.

[0104] Table 1. Genome characteristics of SC263

[0105]

[0106] A total of 7,370 genes and 137 pseudogenes were annotated. 91 RNAs were predicted, including 7 rRNAs, 73 tRNAs, and 3 non-coding RNAs. The chromosome replication origin oriC is located in the center between the dnaA and dnaN genes on Ctg1 (positions 3,676,752-3,677,627) [19-21].

[0107] Four genomes on the same 16S rRNA clade were used for pairwise comparison with SC263. The average nucleotide identity (ANI)

[22] values ​​for S. sporogenes NBRC 15456, S. noaheri NBRC 13794, S. flavus NBRC 12829 and NRRL B-5414 were 99.57, 92.88, 86.57 and 86.47, respectively. Therefore, the inventors classified SC263 as S. sporogenes. Whole genome comparison showed that SC263 had relatively high synteny with NBRC 15456.

[0108] Example 6: Genome sequence of SC263

[0109] To evaluate the biosynthetic potential of secondary metabolites in SC263, the inventors used the antiSMASH web server

[23] to predict possible biosynthetic gene clusters (BGCs) on its genome. Ctg1 contained 21 predicted BGCs, Ctg2 contained 7 BGCs, and Ctg3 contained no BGCs (Table 2). The predicted secondary metabolites produced by these BGCs were all less than 2,000 Da.

[0110] Table 2. Biosynthetic gene clusters of Streptomyces sporogenes SC263 and NBRC 15456

[0111]

[0112]

[0113] The BGC on the SC263 genome showed high similarity to the BGC on the NBRC 15456 genome ( Figure 8 Compared with the NBRC 15456 genome, the SC263 genome contains three additional BGCs: meoabyssomicin / abyssomicin (regions 1-12), ectoine (regions 1-19), and class I lanthipeptides (regions 1-20).

[0114] Interestingly, another closely related strain, Streptomyces sporogenes RDS28, isolated from Saudi Arabia, also exhibits antifungal activity against a number of plant pathogens.

[24] The nature of this antibiotic is unknown.

[0115] In summary, Streptomyces sporogenes SC26 isolated from northern Taiwan, China, produces an antibiotic (AF1) against six fungi and two Gram-positive bacteria. AF1 from mutant SC263 isolated from SC26 exhibits increased thermostability. AF1 produced by these strains is unusual due to its apparent molecular weight and its dual antifungal and antibacterial activity. AF1 is a novel antibiotic that is resistant to proteolytic digestion.

[0116] The genome sequence of SC263 contains 28 predicted BGCs, none of which appear to be involved in the production of secondary metabolites larger than 2000 Da. Antibiotics with very high molecular weights have difficulty penetrating the skin barrier and may be safer for topical administration

[34] .

[0117] All publications, patents, and patent documents cited above are incorporated herein by reference as if individually incorporated by reference.

[0118] The invention has been described with reference to various specific and preferred embodiments and techniques. However, those skilled in the art will appreciate that many changes and modifications may be made without departing from the spirit and scope of the invention.

[0119] References

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Claims

1. A Streptomyces sporogenes strain SC263, which produces an antibiotic with increased thermostability.

2. A composition comprising the sporogenous Streptomyces strain SC263 as claimed in claim 1.

3. The composition of claim 2, wherein the Streptomyces sporogenes strain SC263 is viable.

4. The composition of claim 2, wherein the Streptomyces sporogenes strain SC263 is inactivated.

5. An antibiotic produced by the Streptomyces sporogenes strain SC263 as claimed in claim 1, wherein the antibiotic has increased thermostability.

6. The antibiotic as claimed in claim 5, wherein the apparent molecular weight of the antibiotic measured by membrane filtration concentration is greater than 5 kDa.

7. The antibiotic as claimed in claim 6, wherein the apparent molecular weight of the antibiotic measured by membrane filtration concentration is greater than 100 kDa.

8. The antibiotic of claim 5, comprising concentration by membrane filtration, wherein the apparent molecular weight of the antibiotic is greater than 100 kDa, and the apparent molecular weight of the antibiotic is between 10 kDa and 100 kDa.

9. The antibiotic of claim 5, wherein the antibiotic is effective against fungi.

10. The antibiotic of claim 5, wherein the antibiotic is effective against Gram-positive bacteria.

11. An antimicrobial composition or a pharmaceutical composition comprising the antibiotic according to claim 5 and a pharmaceutically acceptable carrier.

12. An antimicrobial composition or a pharmaceutical composition comprising the strain SC263 as claimed in claim 1 and a pharmaceutically acceptable carrier.

13. The composition or pharmaceutical composition according to claim 11, which is effective for treating fungal infection.

14. The composition or pharmaceutical composition according to claim 11, which is effective for treating bacterial infection.

15. The composition or pharmaceutical composition according to claim 12, which is effective for treating fungal infection.

16. The composition or pharmaceutical composition according to claim 12, which is effective for treating bacterial infection.

17. The composition or pharmaceutical composition of claim 14, which is effective against Gram-positive bacteria.

18. The composition or pharmaceutical composition of claim 16, which is effective against Gram-positive bacteria.

19. The composition or pharmaceutical composition of claim 11, which is administered topically.

20. The composition or pharmaceutical composition of claim 12, which is administered topically.

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