CN110072881A - Cabicidin streptomycete isolate of modification and application thereof - Google Patents

Abstract

The present invention relates to the cabicidin streptomycete isolate of modification, the composition comprising these isolates, and using such isolate and composition in the method for biosynthesis enduracidin (enramycin).In addition, the present invention relates to the cabicidin streptomycete isolate of modification, biosynthesis enduracidin and promote the generation of enramycin in a more effective manner.

Description

Cabicidin streptomycete isolate of modification and application thereof

Cross reference to related applications

The application requires the U.S.Provisional Serial 62/ submitted on December 6th, 2016 according to 35 U.S.C. § 119 (c) 430,455 priority, the content of the provisional application is herein with it entirely through being incorporated by.

Invention field

The present invention relates to the cabicidin streptomycete of modification (Streptomyces fungicidicus) isolate, include The composition of these isolates, and using such isolate and composition with biosynthesis enduracidin (enduracidin；Enramycin (enramycin)) method.The invention further relates to the cabicidin streptomycetes of modification Isolate biosynthesis enduracidin and promotes the generation of enramycin in a more effective manner.

Background of invention

Enduracidin (also referred to as enramycin, and as Enradin^®Sold by MSD Animal Health) it is by thin (for generating the natural producer of the enduracidin) biosynthesis of bacterium cabicidin streptomycete and secrete naturally occurring 17 Rouge ester peptide (lipodepsipeptide) antibiotic of a amino acid.

Enduracidin is the common name of enduracidin analog, and including enduracidin A, B, C and D.The peptide can To separate from fermentation liquid and mycelium, mainly as enduracidin A and B, (length of its lipid chain connected is differed One carbon) mixture.In structure, the difference of enduracidin is the C connecting by amido bond with asparagicacid residue₁₂ Or C₁₃ 2Z,4EAmino acid residue (such as enduracididine of Branched fatty acid moieties and many non proteinogenics (enduracididine), 4- hydroxyphenyl, 3,5- bis- chloro- 4- hydroxyphenyls, citrulling and ornithine) In the presence of.

Enduracidin shows effective in vitro and in vivo antibacterial activity, institute for the gram-positive organism of wide spectrum State gram-positive organism include C.perfringens (Clostridium perfringens), methicillin-resistant staphylococcus Staphylococcus (methicillin-resistant Staphylococcus aureus, MRSA) and vancomycin-resistant enterococcus (vancomycin-resistant Enterococcus, VRE).Enduracidin is by the way that (bacterium is thin with extracellular lipid II The precursor of cell wall) biosynthesis compound and that inhibit bacterium peptidoglycan cell wall.Lipid II and the compound site of enduracidin Different from vancomycin identification site, and be enduracidin be directed to Vancomycin resistant organism effect the reason of. So far, the cross tolerance of the antibiotic of the enduracidin and any clinical use that do not confirm, and do not develop, it obtains Or transferable resistance evidence.There is no the transferable resistance mechanism of any known form, lack oral administration biaavailability, Hypotoxicity, and to fusobacterium kind (Clostridium spp.) significant antibacterial activity, made enduracidin become control The crucial antibiotic of clostridium property enteritis in poultry processed.

Therefore, when applying in poultry feed, enduracidin inhibition causes necrotic enteritis and growth in poultry The growth of the bacterium of inhibition.Enduracidin also has zero-time withdrawal time.In addition, enduracidin is steady in feed and particle It is fixed, it can be applied with low-down dosage to chicken, and do not cause to find residue in the meat and egg from processed chicken.

For many years, many cabicidin streptomycete bacterial strains have been obtained, have been had been modified to improve these and generate persistently The efficiency of the microorganism of bacteriocidin, the microorganism such as cabicidin streptomycete B-5477 (IFO-12439, ATCC- 21013) and cabicidin streptomycete B-5477-m (IFO-12440, ATCC-21014) [see, for example, U.S. 3,577, 530, U.S. 3,786,142 and U.S. 4,465,771].However, still there is the cabicidin strepto- for being further improved such modification Bacterium isolate with generate even more effectively generate enduracidin microorganism significant demand.

Herein cited any bibliography is not necessarily to be construed as recognizing that such bibliography can be used as the " existing of the application Technology " obtains.

Summary of the invention

Therefore, the present invention provides the cabicidin streptomycete isolates for the new modification for more effectively generating enduracidin. In certain embodiments, the cabicidin streptomycete isolate coding of the modification is below one or more or whole: Orf2798 has 95% or higher identity with the amino acid sequence of SEQ ID NO:2, and protects in amino acid position 2 Stay serine residue；Orf3866 has 95% or higher identity with the amino acid sequence of SEQ ID NO:4, and Amino acid position 124 retains threonine residues；Orf5175 has 95% or higher with the amino acid sequence of SEQ ID NO:6 Identity, and amino acid position 91 retain serine residue；And Orf5387, the amino acid sequence with SEQ ID NO:8 Column have 95% or higher identity, and retain serine residue in amino acid position 164.In certain embodiments, The cabicidin streptomycete isolate of the modification is further, nucleic acid (i) comprising coding Orf4755, and it includes SEQ ID The nucleotide sequence of NO:9, instead of the nucleotide sequence of SEQ ID NO:23；And/or (ii) lacks functionality Orf682 albumen； And/or (iii) lacks functionality Orf4868 albumen.In such specific embodiment, lack functionality Orf682 Albumen and/or shortage functionality Orf4868 albumen are due in the gene of coding Orf682 albumen and/or Orf4868 albumen Frameshift mutation.

In a particular embodiment, the modification cabicidin streptomycete isolate coding it is below one or more or All: Orf2798 has 98% or higher identity with the amino acid sequence of SEQ ID NO:2, and in amino acid position Set 2 reservation serine residues；Orf3866 has 98% or higher identity with the amino acid sequence of SEQ ID NO:4, And retain threonine residues in amino acid position 124；Orf5175 has 98% with the amino acid sequence of SEQ ID NO:6 Or higher identity, and retain serine residue in amino acid position 91；And Orf5387, the ammonia with SEQ ID NO:8 Base acid sequence has 98% or higher identity, and retains serine residue in amino acid position 164.In certain embodiment party In case, the cabicidin streptomycete isolate of the modification is further, nucleic acid (i) comprising coding Orf4755, and it includes SEQ The nucleotide sequence of ID NO:9, instead of the nucleotide sequence of SEQ ID NO:23；And/or (ii) lacks functionality Orf682 egg It is white；And/or (iii) lacks functionality Orf4868 albumen.In such specific embodiment, lack functional Orf682 albumen and/or shortage functionality Orf4868 albumen are the bases due to coding Orf682 albumen and/or Orf4868 albumen Frameshift mutation because in.

In other embodiments again, the cabicidin streptomycete isolate coding of the modification is below a kind of or more Kind is whole: Orf2798, has 99% or higher identity with the amino acid sequence of SEQ ID NO:2, and in amino Sour position 2 retains serine residue；Orf3866, it is same with 99% or higher with the amino acid sequence of SEQ ID NO:4 Property, and retain threonine residues in amino acid position 124；Orf5175 has with the amino acid sequence of SEQ ID NO:6 99% or higher identity, and retain serine residue in amino acid position 91；And Orf5387, with SEQ ID NO:8 Amino acid sequence have 99% or higher identity, and amino acid position 164 retain serine residue.In certain realities It applies in scheme, the cabicidin streptomycete isolate of the modification is further, (i) include the nucleic acid for encoding Orf4755, packet The nucleotide sequence of the NO:9 of ID containing SEQ, instead of the nucleotide sequence of SEQ ID NO:23；And/or (ii) lacks functionality Orf682 albumen；And/or (iii) lacks functionality Orf4868 albumen.In such specific embodiment, lack function Energy property Orf682 albumen and/or shortage functionality Orf4868 albumen are due to coding Orf682 albumen and/or Orf4868 albumen Gene in frameshift mutation.

In still other embodiments, the cabicidin streptomycete isolate coding of the modification is below a kind of or more Kind is whole: Orf2798, it includes the amino acid sequences of SEQ ID NO:2；Orf3866, it includes the ammonia of SEQ ID NO:4 Base acid sequence；Orf5175, it includes the amino acid sequences of SEQ ID NO:6；And Orf5387, it includes SEQ ID NO:8's Amino acid sequence.In certain embodiments, the cabicidin streptomycete isolate of the modification is further, (i) comprising coding The nucleic acid of Orf4755, it includes the nucleotide sequences of SEQ ID NO:9, instead of the nucleotide sequence of SEQ ID NO:23；With/ Or (ii) lacks functionality Orf682 albumen；And/or (iii) lacks functionality Orf4868 albumen.Such specific In embodiment, lacking functionality Orf682 albumen and/or lacking functionality Orf4868 albumen is due to encoding Orf682 albumen And/or the frameshift mutation in the gene of Orf4868 albumen.

In relevant embodiment, the cabicidin streptomycete of the modification: (i) includes the core of coding Orf4755 Acid, it includes the nucleotide sequences of SEQ ID NO:9, instead of the nucleotide sequence of SEQ ID NO:23；And/or (ii) lacks Functional Orf682 albumen；And/or (iii) lacks functionality Orf4868 albumen.In such specific embodiment, Lack functionality Orf682 albumen and/or lack functionality Orf4868 albumen be due to encode Orf682 albumen and/or Frameshift mutation in the gene of Orf4868 albumen.

In specific embodiments, the cabicidin streptomycete isolate of the modification includes to have ATCC deposit number The immunogenicity and/or physics and/or hereditary feature of the cabicidin streptomycete isolate of the modification of PTA-122342.More In the embodiment of body, the cabicidin streptomycete of the modification is the isolate with ATCC deposit number PTA-122342, or The offspring of isolate with ATCC deposit number PTA-122342 and/or derivative.In related fields, all modifications of the present invention Cabicidin streptomycete also as isolated modification cabicidin streptomycete isolate provide.

It further include the primer sets with following nucleotide sequence in the present invention: SEQ ID NO:29 and 30；SEQ ID NO: 31 and 32；SEQ ID NO:33 and 34；SEQ ID NO: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, being also the present invention comprising one or more groups of kits in such primer sets A part.In addition, further including identifying cabicidin streptomycete of the invention using these primers in the assay in the present invention Genome.

These and other aspects of the invention are better understood with by reference to attached drawing, detailed description and embodiment.

Brief description

Fig. 1 depicts the positioning of 11 kinds of polymorphisms on cabicidin streptomycete BM38 genome, is used as in Examples below The mutation markers of the screening of the based on PCR of description.

Detailed description of the invention

Bacterium secondary metabolites or natural products of the exploitation for business application (such as physianthropy or agricultural application) propose Such as overcome the challenge of the low-yield from the required compound of parent/wild-type organisms.In the past few decades, we are right The progress of the understanding of natural products biosynthesis has revealed that the generation of secondary metabolites can be adjusted and be controlled by several ways System.In most cases, the gene for bacterium natural products biosynthesis, including it is responsible for precursor formation, structure assembling, group Modification, self resistance and the gene of adjusting are on bacterial chromosome together with gathering after dress.The biosynthesis of natural products can be with Caused by extracellular and intracellular signal transduction molecule, the signal transducers and approach specificity regulatory factor or control are more In a kind of pleiotropism regulatory factor interaction of the generation of product.Occur these adjust genes or system it is any in Mutation can increase, and reduce or eliminate the generation of antibiotic.

Bacterial strain is improved can have cost-benefit commercial scale raw in antibiotic or other microbial secondary metabolites It plays a role in production.It can be destroyed by the targeting of specific gene, or eliminate biosynthesis way by introducing by random mutation The gene of bottleneck in diameter, to generate the mutant strain that can generate specific metabolite with increased yield.It has been proved that exchanging It saves gene and biosynthesis gene carries out genetic manipulation to cause the excess generation of specific secondary metabolites to be actinomycetes strain It is improved strong and successfully tactful.In specific aspect of the invention, the crucial coding of cabicidin streptomycete has been identified Sequence generates the enduracidin that commercial object is used for for biosynthesis and/or generation when being modified and/or eliminating Isolate with improved property.

The DNA sequence dna of 116 kilobase pair from wild type cabicidin streptomycete ATCC 21013 is had reported before this, It contains enduracidin biological synthesis gene cluster and its flanking region [US. 8,188,245, herein with it entirely through drawing With being incorporated to], and can be obtained in GenBank [registration number DQ403252].As follows, it has been determined that BM38-2 Total genome sequence of (ATCC NO. PTA-122342) is simultaneously compared with wild type gene group.The comparative analysis allows to reflect It makes at least 77 kinds of polymorphisms or the mutation differences between genome, and selects and change with ATCC NO. PTA-122342 Into a crucial open reading frame in relevant seven (7) of property, as follows.Unexpectedly, however, this seven crucial openings are read The enduracidin biological synthesis gene cluster and its flanking region phase of none in frame and the cabicidin streptomycete chromosome It closes.However, anyway, the modification of this seven crucial open reading frame allows easily to construct by standard recombinant techniques newly Modification cabicidin streptomycete isolate, have property suitable or even superior with the isolate being previously reported.

It is restrictive for facilitating singular references used in the description to be not intended to.Thus, for example, unless otherwise saying It is bright, otherwise refer to that " isolate " includes referring to one or more such isolates.Unless otherwise stated, plural term makes With being also not intended as restrictive.

As used herein, term " about " is interchangeably used with term " about ", and usually expression value is in indicated value Within 25%, unless otherwise indicated, for example, 4 times of enduracidin yield increase " about " can be 3 to 5 times of increase.

As used herein, when the amino acid residue of two sequences is identical, an amino acid sequence and second amino acid Sequence 100% " identical ".Therefore, when 50% amino acid residue of two amino acid sequences is identical, amino acid sequence with Second amino acid sequence 50% " identical ".Sequence compares in given protein (such as the portion of protein or the polypeptide compared Point) carry out on the continuous amino acid residue section that is included.In specific embodiments, it is contemplated that otherwise can change two ammonia The missing or insertion of the selection of corresponding relationship between base acid sequence.

As used herein, C, MacVector can be used in nucleotide and amino acid sequence identity percentage (MacVector, Inc. Cary, NC 27519)、Vector NTI (Informax, Inc. MD)、Oxford Molecular Group PLC (1996) and Clustal W algorithm, it is true using comparison default parameters and identity default parameters It is fixed.These commercially available programs can also be used for determining sequence similarity using same or similar default parameters.Alternatively, can be with Using default filter condition under Advanced Blast search for, for example, using GCG (Genetics Computer Group, The procedure manual of GCG software package, the 7th edition, Madison, Wisconsin) pileup program, use default parameters.

As used herein, the biology comprising " lacking functional " polypeptide or " lacking functional " polypeptide (for example, Orf682) Body is the organism that do not express the polypeptide and/or express modified polypeptide, and the modified polypeptide has corresponding wild polypeptide extremely More 10% natural biological function, for example, having the enzymatic activity (for example, enzymatic efficiency, i.e. Vmax/Km) to its natural substrate Truncated enzyme, the enzymatic activity be under identical standard determination condition to corresponding wild-type enzyme measurement enzymatic activity extremely More 10%.In a particular embodiment, polypeptide " is lacked functional " in organism and is equal to the particular biological there is no the polypeptide Function.

As used herein, by the open reading frame of engineering noise for example, by in-frame deletion, frameshit, insertion and/or Point mutation is modified, so that being opened comprising not expressed completely by the organism of the open reading frame of engineering noise by corresponding wild type The polypeptide and/or expression of putting reading frame codes have the modification of at most 10% native biological function of corresponding wild polypeptide Polypeptide.In a particular embodiment, comprising by the organism of the open reading frame of engineering noise, there is no by corresponding wild The special biological of the polypeptide of type open reading frame coding.

As used herein, " gene cluster " is the one group of genetic elements to flock together on chromosome, protein product With relevant function, such as form natural products biosynthesis pathway.

Conservative substitution is amino acid substitution, does not significantly change the activity (specificity or binding affinity) of molecule usually. In general, conserved amino acid substitution be related to an amino acid substitution another with similar chemical character (such as charge or hydrophobic Property) amino acid.Following table shows Exemplary conservative's amino acid substitution:

Original Residue	Conservative substitution	Original Residue	Conservative substitution
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln；Glu
				Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr
				Cys	Ser	Ser	Thr
Gln	Asn	Thr	Ser
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu

The preparation of the cabicidin streptomycete of modification

The suitable method and material for implementing disclosed embodiment is described below.In addition, those of ordinary skill is ripe Any method or technique appropriate known can be used for implementing disclosed embodiment.Some conventional methods suitable for the disclosure With technical description in such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989；Sambrook et al.,Molecular Cloning: A Laboratory Manual, the 3rd edition, Cold Spring Harbor Press, 2001；Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and extremely It 2000 years revises and enlarges)；Ausubel et al.,Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, the 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, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A.:Practical Streptomyces genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 &UH, England, 2000。

The cabicidin of modification of the invention can be used to prepare with preparation and reorganization cabicidin streptomycete expression plasmid carrier Streptomycete isolate.In some embodiments, the recombination cabicidin Streptomyces vector of engineering includes cabicidin strepto- The selected open reading frame of at least one of bacterium.In certain embodiments, the recombination cabicidin Streptomyces vector packet of engineering It is contained at least one selected open reading frame of the lower cabicidin streptomycete expressed of promoter control.In other embodiments In, the promoter is strong composing type Streptomyces promoter, when the carrier is expressed in the bacterial strain of cabicidin streptomycete, The promoter causes the yield of enduracidin to increase.In some embodiments, the open reading frame operationally connects It is connected to the allogeneic promoter of the natural promoter instead of its own.For example, it can be operatively attached to constitutive promoter, Such as strong composition type expression promoter or inducible promoter.In a particular embodiment, the strong constitutive promoter comes From erythromycin producer red saccharopolyspora (Saccharopolyspora erythraea)ermE*p.In other embodiment party In case, the inducible promoter is thiostrepton inducible promotertipA.In other embodiments again, P is used (nitA)-NitR system [Herai et al.,Proc Natl Acad Sci U S A., 101(39):14031-14035 Or Streptomyces promoter SF14 (2004)].In still other embodiments, the natural starting of apramycin resistance gene is used SonamRp.In other embodiments again, P is used _hrdB , P _tcp830 And/or Pneos.In certain embodiments, the work The recombinant vector of journey includes: the open reading frame orf2798 of the nucleotide sequence comprising SEQ ID NO:1, and/or by nothing The open reading frame orf682 of effect.

Therefore, recombination cabicidin streptomycete bacterial strain can be constructed, compared with wild type cabicidin streptomycete bacterial strain, Increased enduracidin yield can be generated.In certain embodiments, the recombination cabicidin streptomycete bacterial strain of engineering Comprising at least one selected open reading frame from cabicidin streptomycete, it is introduced on chromosome and in the control of promoter The lower expression of system, for example strong composing type Streptomyces promoter of the promoter, this leads to the production for being engineered enduracidin in bacterial strain Amount increases.In other embodiments, expression of the open reading frame being introduced into cabicidin streptomycete is by replacing its own Natural promoter allogeneic promoter driving.For example, it can be operatively attached to constitutive promoter, such as strong composition Type expresses promoter or inducible promoter.In some embodiments, the strong constitutive promoter isermE*p.At other In embodiment, the inducible promoter istipA.In some instances, using P (nitA)-NitR system [seeing above] Or SF14 promoter.In still other embodiments, the composition type expression promoter isamRp.In again other embodiment party In case, P is used _hrdB 、P _tcp830 And/or PneosPromoter.

In some embodiments, the engineering bacterial strain includes the open reading frame from cabicidin streptomycete orf3866.In such specific embodiment, the open reading frame orf3866 is operably connected to heterologous open Mover.For example, it may be coupled to strong constitutive promoter, such asermE*p.In other instances, the open reading frame Orf3866 is operably connected to promotertipA、SF14、amRp、P _hrdB 、P _tcp830 And/or Pneos。

In other embodiments, the open reading frame orf4868 that the engineering bacterial strain coding changes.Open reading frame Orf4868 can be deactivated by insertion destruction, in-frame deletion, frameshit and/or point mutation.In some instances, opening is read Frame orf4868 is deactivated by in-frame deletion.In general, any internal in-frame deletion on orf4868 should all cause Invalid function of the orf4868 due to its imperfection.In relevant embodiment, engineering bacterial strain is related to cabicidin strepto- Two of bacterium, three, four, five, six, seven or more open reading frame.

In certain embodiments, the cabicidin streptomycete isolate of the modification is originated from wild-type parent bacterial strain, example Such as, but not limited to, cabicidin streptomycete, American type culture collection (American Tissue Culture Company, ATCC) number 21013.In other embodiments, the engineering bacterial strain of cabicidin streptomycete is originated from conventional prominent Become bacterial strain, such as, but not limited to cabicidin streptomycete ATCC 31729, cabicidin streptomycete ATCC 31730 and antifungal Plain streptomycete ATCC 31731.

In certain embodiments, the yield increase of enduracidin is and wild type cabicidin streptomycete bacterial strain phase Than, at least 1.2 times of the yield increase of enduracidin, such as increase at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, At least 3.5 times, at least 4 times, at least 4.5 times including but not limited to increase by 1.2 to 10 times, increase by 1.2 to 4.6 times, and increase About 2 to 5 times.

In certain embodiments, can by will include at least one increase enduracidin yield open reading frame The recombinant plasmid parent strain that is integrated into cabicidin streptomycete chromosome in, to construct the cabicidin strepto- of modification Bacterium.The engagement carrier of integration can have, or can have through engineering, strong composing type Streptomyces promoter.In some realities It applies in scheme, plasmid can lack streptomycete replicon, and can be arrived by locus specificity single crossover homologous recombination and integration In chromosome.In other embodiments, plasmid can be used as free plasmid presence.In some embodiments, it can will connect Carrier engineering is closed, wherein plasmid inserts carry the target gene that partially or completely lacks and its flanking region, can be It is integrated into after double crossing over homologous recombination in chromosome to generate in-frame deletion mutant.

Enduracidin is produced from recombination cabicidin streptomycete bacterial strain

The recombination cabicidin streptomycete bacterial strain that the disclosure provides provides the method for generating the enduracidin of increase level.This This technological progress permission significant cost savings relevant to enduracidin yield in field.In certain embodiments, raw The method for producing enduracidin includes that disclosed recombination cabicidin chain is cultivated under conditions of being enough to generate enduracidin Fungal strain.In other embodiments, the method also includes separating enduracidin from culture medium after incubation.Still In other embodiments, the method also includes the antibacterial activities of determining generated enduracidin, such as pass through HPLC is analyzed or is used staphylococcus aureus ATCC 29213 or bacillus subtilis ATCC 6633 as indicator microoraganism Bioassay.

In some embodiments, by utilizing as previously described for generating the fermentation condition of enduracidin, by open Cabicidin streptomycete bacterial strain generate enduracidin [Higashide et al.,J. Antibiot. 21:126-137 (1968)].After a birth, it can purify and/or analysis of compounds, including HPLC analysis.Generate enduracidin and from life The method that long culture medium harvests this compound can be seen in U.S. 4,465,771, herein with its entirely through reference simultaneously Enter.

In a particular embodiment, cabicidin streptomycete isolate disclosed by the invention is trained in tryptic soy broth It supports in base (TSB) and cultivates (such as cultivating 48 hours at 225rpm and 30 DEG C) on the oscillator, and be subsequently transferred to persistently kill Rhzomorph generates culture medium (EPM, the following table 1), continues for some time for continuously fermenting, for example, at least 5 days and up to 11 days, wraps It includes 5,6,7,8,9,10 or 11 days and continuously ferments.In a more particular embodiment, wild type is carried out in Fermentation It is generated with the enduracidin of derivative strain.

Table 1

Enduracidin generates the composition (pH 6.7) of culture medium (EPM)

Ingredient	Concentration (%)	Ingredient	Concentration (%)
				Soluble starch	1.5	KH2PO4	0.05
Glucose	1.0	(NH4)2SO4	0.15
				Corn flour	2.5	CaCO3	0.5
Corn protein powder	2.0	Lactose	0.5
				Corn leaching solution	0.25	ZnCl2	0.005
Sodium chloride	0.25	Chicken fat	0.7
				NaH2PO4	1.3

Table 2: sequence table

SEQ ID NO:	Open reading frame	Type	SEQ ID NO:	Open reading frame	Type
							ATCC No. PTA-122342			WT B-5477
1	Orf2798	NA	15	Orf2798	NA
						2	Orf2798	AA	16	Orf2798	AA
3	Orf3866	NA	17	Orf3866	NA
						4	Orf3866	AA	18	Orf3866	AA
5	Orf 5175	NA	19	Orf 5175	NA
						6	Orf 5175	AA	20	Orf 5175	AA
7	Orf5387	NA	21	Orf5387	NA
						8	Orf5387	AA	22	Orf5387	AA
9	Orf4755	NA	23	Orf4755	NA
						10	Orf4755	AA	24	Orf4755	AA
11	Orf682	NA	25	Orf682	NA
						12	Orf682	AA	26	Orf682	AA
13	Orf4868	NA	27	Orf4868	NA
						14	Orf4868	AA	28	Orf4868	AA

AA is amino acid sequence；NA is nucleic acid sequence.

Biological deposits

The culture of following biomaterial is deposited in following international accession under conditions of meeting budapest treaty requirement Mechanism: American type culture collection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A。

The present invention may be better understood by reference to following non-limiting embodiments, provide the embodiment as this hair Bright example.Following embodiment is presented so that the preferred embodiments of the invention are more fully explained.However, they should never be solved It is interpreted as limiting broad range of the invention.

Embodiment

Embodiment 1

The cabicidin streptomycete isolate of modification

The production of cabicidin streptomycete biomass for enduracidin biosynthesis

The fermentation of cabicidin streptomycete can be completed in deep trouth hygienic design industrial fermentation tank, and the fermentor has monitoring With control pH, temperature, oxygen, ventilation and stirring system.Each batch fermentation of cabicidin streptomycete, which originates in, to be stored in The characterization of home and the production seed being maintained in low temperature environment and controlled work seed stock object.Fermentation process point three A stage carries out, and is then further downstream processing:

First stage: it can be used containing 10⁷-10¹⁰The work inoculum of a spore/mL is to start batch fermentation.From low One to five bottle freezing seed and natural thaw on the table are taken out in temperature storage, or be placed in 28-32 DEG C of water-bath up to Content thaws.The culture of defrosting is transferred aseptically in the 800-1000mL sterile water kept at room temperature, and is gently mixed It closes so that culture is resuspended.

Second stage: the culture of resuspension is transferred aseptically in seed culture medium.Seed culture medium is made of following: Portugal Grape sugar (0.5 g/L), dextrin (2.5 g/L), corn leaching solution (1.0-4.0 mL/L), soy 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), winnofil (1.5 g/L), defoaming agent (0.1 mL/L) and suitable water based on silicone.By culture medium at 125 DEG C Sterilizing 45 minutes, and it is subsequently cooled to 28 DEG C.The volume of culture medium is adjusted to required working volume using sterile water.By pH It is adjusted to 6.6-6.8.The operating parameter in seed expansion period includes: 28 DEG C ± 2 DEG C of cultivation temperature, 0.5-1.5 kg/cm²'s Internal pressure, 2-4 Nm³The Ventilation Rate of/min, and the stirring rate of about 80 rpm depend on the size and structure of container It makes.PH, oxygen consumption and viscosity are monitored, but without control.It grows culture 50-60 hours, is then transferred into main life It produces in fermentor.It is 350-600 cps that viscosity when transfer, which answers range, and pH answers≤6.0, and should have oxygen consumption and increase Add.Inoculum is transferred aseptically in main fermentation base to complete fermentation period.

Phase III: the culture media composition of main production fermentor includes: corn flour (13.0-15.0 w/v%), beautiful Rice gluten powder (3.0-6.0 w/v%), cotton seed meal (0.3 w/v%), corn leaching solution (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%), winnofil (0.5 w/v%), alpha amylase (0.056 w/v%), potassium hydroxide (0.05 v/v%), soybean oil (0.5-2.0 v/v%), Defoaming agent and suitable water.The ingredient adds in the order listed.The ingredient is added water to, up to alpha amylase, then will Resulting composition is heated to 80 DEG C, continues 15 minutes to allow enzyme to decompose complicated carbohydrate.Then, it adds remaining PH is adjusted to pH 6.6-6.8, and adds suitable water by ingredient.Culture medium is sterilized 45 minutes at 125 DEG C, is cooled to 28 DEG C, and suitable water is added to desired working volume.

Content from seed fermentation tank is transferred in main fermentation base, and fermentor is set as following item Part: 28 DEG C of temperature, 40 Nm of Ventilation Rate³/ min, 0.5 kg/cm of internal pressure², and stirring rate equivalent is to be set as about 1.85 kW/m³.From fermentation period after 2 hours, by the way that dissolved oxygen is set as 12.75 ppm, ventilation is increased into 50 Nm³/ Min, and internal pressure is increased into 0.7 kg/cm², and change operating condition.Then, adjust Ventilation Rate, internal pressure and Stirring rate is the determinant of rate limit to ensure dissolved oxygen not.When viscosity increases to the limited point of dissolved oxygen, Xiang Pei It supports object and adds sterile water.It is careful to control foam to prevent from polluting or flow out during whole cycle.It is big after oxygen demand increase About 3 hours, start to control pH.Following parameter: pH is controlled and/or monitored in entire fermentation period, is ventilated, dissolved oxygen, CO₂, Viscosity, purity, mixing speed, internal pressure and residual sugar.Until bacterial growth stops, pH is maintained at 6.8, but then allows it Natural trend is until harvest.Typical fermentation period is 220-300 hours.When bioassay effect is greater than 5,000 μ g/L, pH liter To pH 7.5 or higher, viscosity is reduced, and when oxygen demand stops, can harvesting culture.By the way that culture is heated to 70 DEG C, hold Make inactivation of bacteria to harvest fermentation material, and harvest liquid is then cooled to 25-28 DEG C within continuous 30 minutes.

Downstream processing: removing water from biomass, and biomass is dry, is then configured to pre-composition.

Embodiment 2

Optimize the effect that the enduracidin of cabicidin streptomycete generates

For many years, obtaining yield by the processing of parent strain improves (listed by such as table 3).Bacterial strain BM38-2 (PTA- 122342) highest enduracidin yield is generated.By using a series of cultures and physical treatment, to GAB-453 (ATCC 31729) it is handled and optimizes bacterial strain.

Table 3:

The yield improvement of cabicidin streptomycete B-54577

No. ATCC	Bacterial strain	Processing	Yield μ g/mL
				ATCC 21013 (is disclosed) before this	B-5477	Parent strain, wild type	730
ATCC 21014 (is disclosed) before this	B-5477m	Radiation	-
				ATCC 31729 (is disclosed) before this	GAB-453	UV- radiation	1290
ATCC 31730 (is disclosed) before this	Emt 36-3	mFluoro- DL- tyrosine	2560
				ATCC 31731 (is disclosed) before this	Emt 2-140	N-methyl-N'-nitro-N-nitrosoguanidine andmFluoro- DL- tyrosine	4360
PTA-122342 (undisclosed before this)	BM38-2	Repeatedly culture and physical treatment	9690

Bibliography: patent 4,465,771；14 days, Nogami et al., Takeda Chemical April in 1984 Industries, Ltd., Osaka, Japan；

Bibliography: patent 3,577,530；On May 4th, 1971, for ATCC 21388.

1. ATCC 21013: the original wild strain B-5477 of cabicidin streptomycete, by Takeda preservation；

2. ATCC 21014: by B-5477-mutant that derives of radiation, be named as B-5477mBacterial strain, by Takeda preservation；

3. ATCC 31729: the mutant derived is radiated by the UV- of B-5477, is named as the bacterial strain of GAB-453, by Takeda preservation；

4. ATCC 31730: by containing B-5477mIt grows and obtains on the agar plate of fluoro- DL- tyrosine (MFT) Mutant；It is named as the mutant of Emt-36-3, by Takeda preservation；

5. ATCC 31731: by making GAB-453 successively receive N-methyl-N'-nitro-N-nitrosoguanidine, the fluoro- DL- junket ammonia of m- The double-mutant of sour (MFT) processing and acquisition, generates the mutant strain for being named as Emt 2-140, by Takeda preservation；

6. ATCC 21388: with cabicidin streptomycete closely related bacterial strain (megaspore streptomycete (S. macrosporeus)), by Squibb and Sons preservation.

For being up to minimum enramycin biosynthesis:

ATCC number: 31729 > ATCC of PTA-122342 > ATCC 31731 > ATCC, 31730 > ATCC, 21013 He ATCC 21014。

It is worth noting that, when the ATCC PTA-122342 isolate high with yield second to be compared, acquisition There is the increase greater than 2 times in enduracidin, and when ATCC PTA-122342 to be compared with wild-type parent bacterial strain, There is the increase greater than 12 times in the enduracidin of acquisition.

Embodiment 3: the genome analysis of cabicidin streptomycete isolate

Between cabicidin streptomycete wild-type strain ATCC 21013 (B-5477) and derivative strain BM38-2 of the invention The icp gene group analysis of progress comprising the region around enduracidin (enramycin) biological synthesis gene cluster.

77 kinds of mutant dna sequences in total are identified, the physics of parent strain B-5477 and the influence of culture operation are identified. By selecting 11 be located on BM38-2 genome representative variations as mutation markers, the information obtained from genome analysis Allow the Strain comparison quickly and determined (referring to Fig. 1).PCR primer is designed to each mutation markers, amplification is contained into mutation The DNA fragmentation in site for subsequent sequencing and compares (referring to table 4).

Using the PCR primer of targeting marked region, analysis generates the bacterium of enramycin by available five (5) kind of ATCC Closely related bacterial strain is planted in strain and one (1), including the wild type and mutant by Takeda preservation, and with BM38-2 bacterial strain into Row compares.Table 5 summarizes result of study, and shows the DNA characteristics at 11 mutation markers.

Table 4

PCR primer group for expanding 11 mutation markers being positioned on cabicidin streptomycete BM38 genome is (and corresponding SEQ ID NO :)

Mutation markers number#

SEQ ID NO:

WT†Sequence

BM38-2 sequence

Primer

Primer SEQ

It is expected that size*(bp)

1

29

G

A

SF1F

GGACGATGCCATCAAAACCG

737

30

SF1R

ACTGCTTGATCACCGAGGTG

2

31

AC

TT

SF2F

CTGTTCAACCTGACCGGGAA

954

32

SF2R

GAAGACCACGACCTCGATGG

3

33

G

A

SF3F

GGGCAGGAGTTCTTGAGGAC

864

34

SF3R

CCACGTCAAGAACCTGGTGA

4

35

GA

AT

SF4F

GGCAAGTCGACCCTCATCAA

428

36

SF4R

ATGACCGAGAACAGCGTCTC

5

37

A

G

SF5F

CGCATCTGTTCCAGGCAATG

459

38

SF5R

TCATCGTGGCGCATAGGTAC

6

39

G

A

SF6F

TGTGGGGATGTCCTTCGGAA

173

40

SF6R

CGACGATGCCAAGAGACTGT

7

41

AGGGCG

CGGGCC

SF7F

GGACGATGCCATCAAAACCG

685

42

SF7R

ACTGCTTGATCACCGAGGTG

8

43

AT

GA

SF8F

TGCTGCTGTCGATCATGGAG

686

44

SF8R

AGGAAGGGCTGGTAGGTCAT

9

45

G

C

SF9F

CGAGTCCGAAGCAGAAGGAG

745

46

SF9R

GGGTCGGCAACATCTTCCTC

10

47

C

T

SF10F

GAGCACACGAGGACTGAGAG

976

48

SF10R

GTACCCCCTCATAGCGCTTC

11

49

C

T

SF11F

AGAAGTACGGGATTCGGGGA

1000

50

SF11R

ACTGCTTGATCACCGAGGTG

^#Mutation markers number；^*Expected PCR product size；^†Wild-type strain (WT): ATCC 21013.

Table 5A

Compared with ATCC number PTA-122342, the mutation markers of the cabicidin streptomycete bacterial strain of preservation are analyzed

Mutation markers Number

ATCC 21013 B- 5477 parents

ATCC 21014 B- 5477m

ATCC 21388*

ATCC 31729

ATCC 31730

ATCC 31731

PTA- 122342

1

WT

WT

WT

WT

WT

WT

M

2

WT

WT

WT

WT

WT

WT

M

3

WT

WT

WT

M

WT

M

M

4

WT

WT

WT

WT

WT

WT

M

5

WT

WT

WT

M

M

M

M

6

WT

WT

WT

M

M

M

M

7

WT

WT

WT

WT

WT

WT

M

8

WT

WT

WT

WT

WT

WT

M

9

WT

WT

WT

WT

WT

WT

M

10

WT

M

M

M

M

WT

M

11

WT

WT

WT

WT

WT

WT

M

WT=matching parental wildtype.M=matching PTA-122342 mutant nucleotide sequence；

* the megaspore streptomycete bacterial strain closely related with cabicidin streptomycete.

Table 5B

Mutation compared with BM38-2 is shared or is used for PCR as label

Nucleotides number

21013

31729

31730

31731

BM38-2

Mutation markers number

565,966

WT

M

WT

M

M

4

607,443

WT

M

M

M

M

608,938

WT

M

M

M

M

890,965

WT

M

WT

M

M

2,285,216

WT

WT

WT

WT

M

2,387,796

WT

WT

WT

WT

M

7

2,387,801

WT

WT

WT

WT

M

7

2,659,327

WT

M

M

M

M

2,900,485

WT

M

M

M

M

2,963,171

WT

WT

WT

WT

M

1

3,147,522

WT

M

M

M

M

3,164,639

WT

WT

WT

WT

M

8

3,358,400

WT

M

M

M

M

3,391,492

WT

M

M

M

M

3,428,632

WT

WT

WT

M

M

3,556,837

WT

M

M

M

M

3,607,398

WT

M

M

M

M

3,651,914

WT

M

M

M

M

3,743,503

WT

M

M

M

M

3,823,662

WT

M

M

M

M

4,046,322

WT

WT

WT

M

M

4,158,534

WT

M

M

M

M

4,225,769

WT

M

M

M

M

4,281,871

WT

M

M

M

M

4,294,357

WT

M

M

M

M

4,310,521

WT

M

M

M

M

10

4,449,891

WT

WT

WT

WT

M

6

4,450,937

WT

M

M

M

M

4,458,773

WT

M

M

M

M

4,462,128

WT

M

M

M

M

4,478,416

WT

M

M

M

M

4,954,366

WT

WT

WT

M

M

4,989,329

WT

M

M

M

M

5,259,346

WT

WT

WT

WT

M

5,340,994

WT

M

M

M

M

5

5,349,716

WT

M

WT

M

M

3

5,757,177

WT

WT

WT

WT

M

11

5,906,501

WT

WT

WT

WT

M

2

5,947,737

WT

M

M

M

M

6,140,913

WT

M

M

M

M

6,260,317

WT

M

M

M

M

6,703,003

WT

M

M

M

M

6,706,989

WT

M

M

M

M

7,321,611

WT

WT

WT

WT

M

9

WT=matching parental wildtype.M=matching PTA-122342 mutant nucleotide sequence.

It is poor that table 5A-5B identifies the heredity between parent strain (21013 B-5477 of ATCC) and the bacterial strain of reporting It is different.Most of in the bacterial strain of these reportings are 21013 B-5477 of ATCC obtained by culture and/or physical operations Derivative strain.For BM38-2 (ATCC number PTA-122342), most significant hereditary difference is found.It is readily apparent that The primer of table 4 can also be used for clearly from other cabicidin streptomycete bacterial strains and/or closely related streptomyces kind, identification BM38-2 (PTA-122342) out.

Embodiment 4

The analysis of mutation is selected in cabicidin streptomycete BM38-2

By the mutagenesis of round repeatedly, enramycin high productive mutant is then selected, and develops cabicidin streptomycete ATCC Number PTA-122342 industrial strain.Facilitate enramycin to understand the possibility being introduced into ATCC number PTA-122342 in depth Yield improve mutation, it is determined that total genome sequence of ATCC number PTA-122342, and with its wild type cabicidin chain Total genome sequence of mould predecessor is compared.The comparative analysis identify between two genomes at least 77 kinds of polymorphisms or Mutation differences.Surprisingly, only detecting a difference in the chromosomal region containing enramycin biological synthesis gene cluster It is different.This species diversity isendCSingle nucleotide acid variation in gene.endCNucleotide 6,260,317 in gene is prominent from C to T Become, causes CTC codon to become CTT codon, this is a kind of silent mutation, because the two is all the codon of leucine.Cause This, this mutation is less likely to play remarkable effect in the increase of enramycin yield in the BM38-2 observed.Lasting There is no other mutation to show to cause the increased chromosome variation of enramycin yield in BM38-2 may deposit in mycin gene cluster It is to be located in pleiotropism (the non-approach specificity) regulating element or the global regulation gene of other positions in genome.

The biology conjunction that natural products is influenced in more than one approach has been displayed in a variety of response regulation factors in actinomyces At.One critical instance be streptomyces coelicolor (S. coelicolor) CDA gene cluster in findabsA1A2Gene Seat, coding are similar to the bi-component signal transduction found in the enramycin biological synthesis gene cluster of cabicidin streptomycete System.It has been shown that the AbsA2 of phosphorylation form passes through direct interference CDA, actinorhodin and undecylprodigiosin biology The expression of the approach specificity regulatory factor of synthetic gene cluster, and inhibit the generation of antibiotic.Inhibit the prominent of AbsA2 kinase activity Become to enhance the generation of antibiotic.Pleiotropism adjust another example be found in Streptomyces clavuligerus (S. clavuligerus) in, wherein the gene found in cephamycin C clusterccaRBoth coding-control cephamycin C and clavulanic acid The regulatory protein of generation.

There may be maximum likelihood relevant to the increase of enramycin yield in ATCC number PTA-122342 genome Mutation can be by bioinformatic analysis predict coding adjust product (including that may have pleiotropism control characteristic Those) gene in occur those of mutation.Provided hereinafter what is identified in cabicidin streptomycete BM38-2 genome to push away It sets the tone the example for saving gene, there are mutation differences compared with wild type cabicidin streptomycete bacterial strain.It is [each in following instance In a, mutation differences are highlighted by lacking asterisk, and difference/missing/insertion nucleotide is highlighted with runic].

Sequence compares

Regulatory protein in orf682: predictive coding TetR family contains helix turn helix (HTH) DNA integrated structure Domain.Additional G is inserted into in poly- track (homopolymeric track), induces frameshit.

The gene product of > WT_682 translation

The gene product of > BM38.2_682 translation

。

DNA combination transcription regulaton factor in orf2798: predictive coding MurR/RpiR family.It includes Helix-turn- Spiral (HTH) and sugared isomerase (SIS) structural domain.The mutation of G to A causes Gly to become Ser.

The gene product of > WT_2798 translation

The gene product of > BM38.2_2798 translation

。

DNA combining response regulatory factor in orf3866: predictive coding OmpR family.It includes that (it connects REC structural domain Receive the signal of sensor gametophyte in two-component system) and streptomycete antibiotic regulatory protein (SARP) it is characteristic Winged-helix (wHTH) structural domain.The mutation of G to A causes Ala to become Thr.

The gene product of > WT_3866 translation

The gene product of > BM38.2_3866 translation

。

Orf4755: predictive coding RNA polymerase σ -70 factor, σ-E family.Mutation (the CGT-of T to G in Arg codon > CGG) it is silent mutation.

The gene product of > WT_4755 translation

The gene product of > BM38.2_4755 translation

。

Orf4868: predictive coding transcript regutation protein.It includes that REC signal receives structural domain, receives and comes from bi-component The signal of sensor gametophyte in system.Missing (missing of C) in tandem repetitive sequence leads to frameshift mutation.

The gene product of > WT_4868 translation

The gene product of > BM38.2_4868 translation

。

Orf5175: predictive coding histidine kinase transcript regutation protein.There is REC signal to receive structural domain for it, receive The signal of sensor gametophyte in two-component system.The mutation of C to T causes Pro to become Ser.

The gene product of > WT_5175 translation

The gene product of > BM38.2_5175 translation

。

Transcription regulaton factor in orf5387: predictive coding MerR family.It has HTH DNA binding structural domain and REC Structural domain, the REC structural domain receive the signal of the sensor gametophyte in two-component system.The mutation of A to C causes Tyr becomes Ser.

The gene product of > WT_5387 translation

The gene product of > BM38.2_5387 translation

。

It should be understood that provide for describe all base sizes or the amino acid size of nucleic acid and polypeptide according to the present invention with And all molecular weight or molecular mass values are approximate in general measure variation.

PCT/RO/134 table

Claims (15)

1. the cabicidin streptomycete isolate of modification encodes Orf2798 albumen, the Orf2798 albumen and SEQ ID The amino acid sequence of NO:2 has 95% or higher amino acid sequence identity；

Wherein the amino acid sequence of the Orf2798 albumen includes serine residue in amino acid position 2；With

It is true with killing for the modification wherein compared with the enduracidin yield obtained with wild type cabicidin streptomycete bacterial strain The enduracidin yield that rhzomorph streptomycete obtains increases.

2. the cabicidin streptomycete isolate of the modification of claim 1, wherein the Orf2798 albumen is by SEQ ID NO:1 It is nucleotide sequence coded.

3. the cabicidin streptomycete isolate of the modification of claims 1 or 2 further encodes Orf3866 albumen, described The amino acid sequence of Orf3866 albumen and SEQ ID NO:4 have 95% or higher amino acid sequence identity；It is wherein described The amino acid sequence of Orf3866 albumen includes threonine residues in amino acid position 124.

4. the cabicidin streptomycete isolate of the modification of claim 3, wherein the Orf3866 albumen is by SEQ ID NO:3 It is nucleotide sequence coded.

5. the cabicidin streptomycete isolate of the modification of claim 1,2,3 or 4 further encodes Orf5175 albumen, institute The amino acid sequence for stating Orf5175 albumen and SEQ ID NO:6 has 95% or higher amino acid sequence identity；Wherein institute The amino acid sequence for stating Orf5175 albumen includes serine residue in amino acid position 91.

6. the cabicidin streptomycete isolate of the modification of claim 5, wherein the Orf5175 albumen is by SEQ ID NO:5 It is nucleotide sequence coded.

7. the cabicidin streptomycete isolate of the modification of claim 1,2,3,4,5 or 6 further encodes Orf5387 egg White, the amino acid sequence of the Orf5387 albumen and SEQ ID NO:8 have 95% or higher amino acid sequence identity； Wherein the amino acid sequence of the Orf5387 albumen includes serine residue in amino acid position 164.

8. the cabicidin streptomycete isolate of the modification of claim 7, wherein the Orf5387 albumen is by SEQ ID NO:7 It is nucleotide sequence coded.

9. the cabicidin streptomycete isolate of the modification of claim 1,2,3,4,5,6,7 or 8, further includes SEQ ID The nucleotide sequence of NO:9, but do not include the nucleotide sequence of SEQ ID NO:23.

10. the cabicidin streptomycete isolate of the modification of claim 1,2,3,4,5,6,7,8 or 9, not encoding function Orf682 albumen.

11. the cabicidin streptomycete isolate of the modification of claim 10, wherein lack functionality Orf682 albumen be due to The cabicidin streptomycete isolate of the modification includes frameshift mutation in the gene of coding Orf682 albumen.

12. the cabicidin streptomycete isolate of the modification of claim 1,2,3,4,5,6,7,8,9,10 or 11, does not encode Functional Orf4868 albumen.

13. the cabicidin streptomycete isolate of the modification of claim 12, wherein lack functionality Orf4868 albumen be due to The cabicidin streptomycete isolate of the modification includes frameshift mutation in the gene of coding Orf4868 albumen.

14. the method for producing enduracidin comprising under conditions of being enough to produce enduracidin, cultivate in the medium The cabicidin streptomycete isolate of the modification of claim 1,2,3,4,5,6,7,8,9,10,11,12 or 13.

15. the method for claim 14 further includes separating enduracidin from the culture medium.