CN101153274A - Method for improving volume of production of rifamycin - Google Patents
Method for improving volume of production of rifamycin Download PDFInfo
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Abstract
The present invention relates to a method of controlling the two-component regulatory system of bacteria, in particular to an amrE gene and/or an amkE gene or homologous genes of the amrE gene or the amkE gene, which improves the yield of antibiotics and other useful secondary metabolites.
Description
Technical field
The two component regulation systems that the present invention relates to utilize the control bacterium especially wherein amrE and/or the amkE gene or with its highly the method for homologous gene improve the method for the output of microbiotic and other useful secondary metabolite.Particularly, the present invention relates to Mediterranean Sea and intend no mycolic acids bacterium (Amycolatopsismediterranei), and use this bacterium to produce the method for rifomycin.
Background technology
Two component regulation systems (two-component systems) are a kind of important signal conduction regulation systems in the bacterial cell, and it is by responding to protein (sensor kinase) and replying adjusting protein (responseregulator) and form (Parkinson JS and Kofoid EC.Communication modules in bacterialsignaling proteins.Annu Rev Genet.1992; 26:71-112).Bacterium is at first experienced the stimulation of outer signals, under the situation that ATP exists, responds to proteinic Histidine site and carries out autophosphorylation, and then the energy-rich phosphate radical transfer on the Histidine is regulated on the proteic asparagicacid residue to replying.It is just initial or close its downstream target for modulation gene transcription (Ann M.Stock, Victoria L.Robinson and Paul N.Goudreau.Two-component signal transduction.Annu.Rev.Biochem.2000 that the replying of phosphorylation regulated protein; 69:183-215).There are afsQ1/afsQ2, cutR/cutS and absA1/absA2 etc. in the typical two component systems relevant with the microbiotic metabolism.In muta lead mycillin, AfsQ1/afsQ2 can stimulate (Ishizuka H such as generating actinomycetes C.I. Natural Red 8 and prodigiosin, Horinouchi S, Kieser HM, Hopwood DA and Beppu T.A putative two-component regulatory system involved insecondary metabolism in Streptomyces spp.J Bacteriol.1992Dec; 174 (23): 7585-94), the cutR/cutS two-component system is generation (the Chang HM of negative regulation actinomycetes C.I. Natural Red 8 (Act) then, Chen MY, Shieh YT, Bibb MJ and Chen CW.The cutRS signaltransduction system of Streptomyces lividans represses the biosynthesis of thepolyketide antibiotic actinorhodin.Mol Microbiol, 1996 Sept.; 21 (5): 1075-85).The two component systems of absA1/absA2 influence the antibiotic generation of streptomyces coelicolor by the expression of regulating the single-minded regulatory gene of microbiotic, regulation and control of overall importance have been participated in, the interruption of absA1 or absA2 gene causes the too early generation of microbiotic actinorhodin (Act) and prodigiosin (Red), so the antibiotic secondary metabolism of absA1/absA2 negative regulation (Brian P, Riggle PJ, Santos RA and Champness WC.Globalnegative regulation of Streptomyces coelicolor antibiotic synthesis mediated by anabsA-encoded putative signal transduction system.J Bacteriol.1996Jun; 178 (11): 3221-31).
Rifomycin is a subgroup of AMSA microbiotic (Ansamycins), can suppress to depend on the activity of the RNA polymerase of DNA specifically, make germ can not normally synthesize the RNA product, thereby reach antibacterial purpose, be widely used in the various infection (Lal that treatment is caused by mycobacterium (Mycobacterium), streptococcus pneumoniae (Pneumococcus) and other gram-positive microorganisms clinically, R. and Lal, S., Recent trends in rifamycin research.BioEssays 1994,16:211-216).Two classes in three class transmissible diseases of the present keypoint control of China: in the treatment of tuberculosis and the multiple complications that caused by acquired immune deficiency syndrome (AIDS), rifamycin drug all is a line medication.At present, main production rifamycin B or SV in the factory, and then synthetic on this basis Rifampin, rifapentine etc.
Mediterranean Sea is intended no mycolic acids bacterium and is used to produce rifomycin industrial.It is a kind of gram-positive microorganism that no mycolic acids bacterium (Amycolatopsis mediterranei) is intended in Mediterranean Sea.Nineteen fifty-seven, Mediterranean Sea is intended no mycolic acids bacterium and is separated near the soil sample French St.Raphael.At first, Mediterranean Sea is intended no mycolic acids bacterium and is accredited as Streptomyces mediterranei (Margalith, P., with G.Beretta.Rifomycin.XI.Taxonomic study on Streptomyces mediterranei nov.sp.Mycopathol.Mycol.Appl.1960,13:321-330).Afterwards, people such as Thiemann rename the (J.E.Thiemann into Nocardia mediterranei according to the chemical ingredients of cell walls with it, G.Zucco and G.Pelizza.A proposal forthe transfer of Streptomyces mediterranei margalith and beretta 1960 tothe genus Nocardia as Nocardia mediterranea (margalith and beretta) Comb.Nov.Arch.Microbiol.1969,67:147-155).To the eighties mid-term, people such as Lwchevalier lack branched acid according to the composition of its cell walls and features such as the phage of Nocardia and Rhodococcus is insensitive are classified as it again and intend no mycolic acids bacterium (Amycolatopsis) (Lechavalier, MP, Prauser, H., Labeda, DP and Ruan, JS.Two new genera of Nocardioformactinomycetes:Amycolata gen.nov.and Amycolatopsis gen.nov.Int.J.Syst.Bacteriol.1986,36:29-37).
It is the generation bacterium of Rifamycin Sodium that no mycolic acids bacterium (Amycolatopsis mediterranei) U-32 is intended in Mediterranean Sea.Ni Liuying etc. (1984, rifomycin synthesizes the positive correlation with the glutamine synthetase vigor, the microorganism journal, 24 (3): 217～2231) metabolic process of the synthetic Rifamycin Sodium of the no mycolic acids bacterium U-32 of Mediterranean Sea plan has been carried out deep physiological and biochemical research.
Present stage, mainly utilize physics or chemical process that industrial producing strain is carried out mutagenesis, screen by bacterial strain and obtain superior strain mutagenesis.The workload of these methods is big, the cost height, and success ratio is low, and the superior strain that obtains is often unstable, is easy to take place reverse mutation.Therefore, improving the secondary metabolite output of producing bacterial strain by new method has great significance for saving cost, enhancing competitiveness of product.
Summary of the invention
One aspect of the present invention relates to a kind of method for preparing microbiotic or the further bacterial strain that improves of other useful secondary metabolite output, this method comprises: to comprising amrE and amkE gene or operate with two component regulation systems of its height homologous gene in the original strain of this bacterial strain, make described amrE and/or amkE gene or with its height homologous gene inactivation, the bacterial strain that obtains suddenling change, this mutant strain are microbiotic or the further bacterial strain that improves of other useful secondary metabolite output.
In a preferred embodiment, described method also comprises step: cultivate described mutant strain, measure it and produce antibiotic ability, filter out the bacterial strain that microbiotic output further improves.
In a preferred embodiment, described bacterial strain be selected from Mediterranean Sea intend no mycolic acids bacterium (Amycolatopsismediterranei) or have similar metabolic regulation mechanism and comprise amrE and/or the amkE gene or with its microorganism strains of two component regulation systems of homologous gene highly.
In a preferred embodiment, with described amrE and/or amkE gene or with its height homologous gene knockout, the bacterial strain that obtains suddenling change.
In another preferred embodiment, described microbiotic is a rifomycin.In preferred embodiment, described bacterial strain is that no mycolic acids bacterium (Amycolatopsis mediterranei) is intended in Mediterranean Sea, and described rifomycin is a Rifamycin Sodium.
The present invention relates to a kind of bacterial strain that adopts method of the present invention to make on the other hand, it be selected from Mediterranean Sea intend no mycolic acids bacterium (Amycolatopsis mediterranei) or have similar metabolic regulation mechanism and comprise amrE and/or the amkE gene or with its microorganism strains of two component regulation systems of homologous gene highly, wherein, this bacterial strain comprises a pair of component regulation system, amrE gene in this regulation system and/or amkE gene or with its height homologous gene inactivation.
In a preferred embodiment, this bacterial strain is the bacterial strain that no mycolic acids bacterium (Amycolatopsismediterranei) is intended in Mediterranean Sea, and amrE gene in its pair component regulator control system and/or amkE gene be inactivation.
Another aspect of the invention also relates to the antibiotic method of a kind of production, and this method comprises:
(a) cultivate the bacterial strain that adopts the inventive method to obtain; With
(b) from substratum, collect the microbiotic that described bacterial strain produces.
In a preferred embodiment, described microbiotic is a rifomycin.In preferred embodiment, this rifomycin is a Rifamycin Sodium.
In another preferred embodiment, described bacterial strain is that no mycolic acids bacterium is intended in Mediterranean Sea.
The invention still further relates to a kind of dna molecular, it contains 456-1139 position and/or 1178-2533 position nucleotide sequence shown in the SEQ ID NO:1, and the purposes of this dna molecular in the middle of further bacterial strain that improves of preparation microbiotic output and raising microbiotic output.
Description of drawings
Fig. 1 shows dot blot screening positive clone from the no mycolic acids bacterium U-32 of Mediterranean Sea plan.Figure 1A represents first round screening U-32 mixing library, obtains being numbered 2,10,21 and 63 4 positive hybridization points; Figure 1B shows that No. 63 mixing plasmid that first round screening is obtained carries out second and take turns screening, obtains being numbered 50 recombinant cloning." positive control " refers to positive control among Figure 1B.
Fig. 2 show Mediterranean Sea intend the no total DNA of mycolic acids bacterium U-32 and plasmid pLR6350 respectively through SacI and BglII enzyme cut with probe hybridization after the gained result, left side figure be an EB dyeing picture behind the electrophoresis, right side figure is that Southern is hybridized picture behind the commentaries on classics film.Wherein, numbering " 1 " is a λ DNA/HindIII marker, and numbering " 2 " is pLR6350/SacI; Numbering " 3 " is the total DNA/SacI of U-32.
Fig. 3 A and 3B show amrE and amkE gene nucleotide complete sequence and amino acid sequence corresponding, and wherein the underscore mark is the possible RBS site of amrE gene and amkE gene.
Fig. 4 A and 4B show the comparison of AmrE and AmkE and homologous protein aminoacid sequence.Wherein the comparative sequences among Fig. 4 A is intended no mycolic acids bacterium U-32 (AmrE), Streptomycescoelicolor (AfsQ1 from: Mediterranean Sea respectively, BAA01502), Mycobacterrium tuberculosis (MtrA, AAK47686), intestinal bacteria Escherichia coli (OmpR1, NP_417864), Bacillus subtilis (PhoP, CAA47908).Among Fig. 4 B relatively sequence respectively from: no mycolic acids bacterium U-32 (AmkE), Stretomyces coelicolor (AfsQ2BAA01503), Mycobacterium tuberculosis (MtrB are intended in Mediterranean Sea, AAB07807), intestinal bacteria (Envz, P0AEJ4), Bacillus subtilis (PhoR, P23545).
Fig. 5 shows that PCR checking amrE and amkE interrupt mutant strain gained result, wherein use checking primer AmE-cf and AmE-ca amplification.
Fig. 6 shows the output of the Rifamycin Sodium that U32, U32-101 and U32-107 fermented 5 days in the seed nitrate culture-medium.
Fig. 7 shows the output of the Rifamycin Sodium that U32, U32-101 fermented 5 days in fermention medium.
Fig. 8 shows the output of the Rifamycin Sodium that U32-109, U32-110 fermented 5 days in the seed nitrate culture-medium.
Embodiment
One aspect of the present invention relates to a kind of method that improves this bacterial strain production secondary metabolite product output by the two component regulation systems in the regulation and control bacterial isolates, this method comprises two component regulation systems of destroying this bacterial strain, make its inactivation, obtain the bacterial strain of sudden change, utilize the bacterial strain of this sudden change to produce secondary metabolite, realize the raising of output.
The invention particularly relates to the method that improves the output of rifomycin by two component regulation systems of the no mycolic acids bacterium of regulation and control Mediterranean Sea plan.This pair component regulation system comprises amrE and amkE gene, destroys wherein arbitrary gene or both, and the bacterial strain that is obtained can both improve the output of rifomycin greatly.
The invention still further relates to by control and have similar metabolic regulation mechanism and comprise described amrE and amkE gene or improve the method for relevant secondary metabolite output with two component regulation systems of the microorganism strains of its height homologous gene.It is identical or similar that similar metabolic regulation mechanism refers to that the regulatory mechanism of amrE/amkE pair of component regulation systems in the no mycolic acids bacterium is intended in mechanism and the Mediterranean Sea of its regulation and control secondary metabolism, thereby can improve the output of secondary metabolite by one of them gene of inactivation or two genes.
" height homology " refers to that in this article its protein sequence compares with AmrE or the proteinic sequence of AmkE, have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or above sequence homogeny, and all should contain conservative functional domain.Can adopt tool and method known in the art to measure the sequence homogeny, for example, utilize the BLAST instrument on the NCBI website (for example, b12seq) to carry out the comparison of two protein sequences.
AmrE and amkE gene are two genes that the inventor finds in the process that the genomic dna of Mediterranean Sea being intended no mycolic acids bacterium is studied.Utilize the design of OmpR family protein conserved amino acid sequence to obtain the degenerate pcr primer, intending the total chromosomal DNA of no mycolic acids bacterium U-32 with Mediterranean Sea is template, and amplification obtains the dna fragmentation of one section 390bp.With this dna fragmentation is probe, and the gene library of screening U-32 is carried out enzyme to positive colony that obtains and cut the hybridization analysis with Southern, and measured the fragment of about 3.2kb.The result that the dna sequence dna that records is analyzed shows, exists two to read frame in the sequence, and the inventor is respectively with its called after AmrE and AmkE (SEQ ID NO:1).By carrying out homology relatively with known protein, the product of prediction AmrE and AmkE belongs to replying in two component modulin systems respectively and regulates albumen and induction histidine kinase, and comprises conservative amino acid or aminoacid sequence and consistent functional zone.
Respectively amrE gene among the U32 and amkE gene have been carried out gene disruption, made its inactivation, found that the output that gene interrupts the Rifamycin Sodium of mutant strain all has by a relatively large margin raising with respect to wild-type U32.Further this pair component has been carried out overexpression in U32, found that the output of the Rifamycin Sodium of expression strain had significantly reduction with respect to the U32 contrast of having changeed empty plasmid.Infer this generation of amrE and amkE thus to two component regulator control systems negative regulation Rifamycin Sodium in U32.
By AmrE protein sequence and GenBank data are relatively found, the microorganism of many species also contains the very high modulin of homology, wherein mostly be and reply adjusting albumen in two components, as ZP_00574191 (the blue gram of fluorine Bordetella, Frankia sp.EAN1pec), NP_824658 (deinsectization streptomycete, Streptomyces avermitilis MA-4680), CAB89435 (streptomyces coelicolor, Streptomycescoelicolor A3 (2)), CAB90924 (Streptomyces coelicolor A3 (2)) or the like (sequence data is from GenBank).Similarity according to sequence can be inferred: these and ArmE have replying of high homology to regulate albumen and also with the regulation and control of microorganism secondary metabolism very confidential relation are arranged.
Simultaneously, known result of study shows that the regulation and control of the secondary metabolism of many microorganisms are similar.For example, people such as Jiao Ruishen finds to add 0.8% KNO in substratum
3Can make the output of rifomycin SV SV improve (Jiao Ruishen more than 1.7 times, Chen Yumei, Wu Meng Gan and Gu Weiling, the research I. nitrate that the metabolism of Nocardia intermedien (Nocardiamediterranei) composite force pluramycin is regulated is to the promoter action of Nocardia intermedien composite force pluramycin SV, the plant physiology journal, 1979,5 (4): 395-402), find again that afterwards nitrate also has very big promoter action (Jiao Ruishen etc. to the biosynthesizing of lincomycin, nitrate, magnesium salts is to the biosynthetic promoter action of lincomycin, journal of biological chemistry, 1997,13 (6): 709-714).In addition, at rifamycin B (El-Tayeb, O.M.1, Salama, A.A.2, Hussein, M.M.M.2 and El-Sedawy, H.F.Optimization of industrial production of rifamycin B byAmycolatopsis mediterranei.I.The role of colony morphology and nitrogen sourcesin productivity.African Journal of Biotechnology.2004,3 (5): 266-272.), Lie Weidu mycin (Zhou Xiaoyun and Wang Huizhong, the Lie Weidu mycin produces the research that bacterium M814 produces the Lie Weidu mycin. the journal .1995 of Zhejiang Polytechnical University, 23 (1): 68-72), azalomycin B (Jiang Shu and Huang Weiyi, streptomyces hygroscopicus NND-52-C bacterial strain produces azalomycin B Optimizing Conditions of Fermentation, biological processing, 2004,2 (1): 53-57) etc. in the antibiotic biosynthetic process, the interpolation of nitrate also can increase substantially antibiotic output.This also guards in these have the microorganism of similar secondary metabolism regulatory mechanism the control methods of two component system to estimate to intend no mycolic acids bacterium amrE/amkE in Mediterranean Sea.
Therefore, same or analogous secondary metabolism regulatory mechanism is arranged and comprise microorganism with amrE/amkE pair of component height homologous genes for intending no mycolic acids bacterium, can improve the output of thalline secondary metabolite by interruption and amrE and/or amkE homologous gene with Mediterranean Sea.
In the present invention, " inactivation ", comprise and gene is knocked out fully and make this gene not possess activity fully, also comprise this gene is suddenlyd change, thereby make this gene activity compare decline at least 30%, 40%, 50%, 60%, 70%, 80% or more with original strain or when not undergoing mutation.Sudden change can be to make this genetically deficient, sudden change or insert some Nucleotide, for example lack 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or the Nucleotide of arbitrary proportion therebetween, make this gene activity descend more than at least 30%; Sudden change comprises that then the part Nucleotide that makes in this gene (as 1-100,1-50,1-20 or the like) undergos mutation, thereby makes it actively descend 30% or more; Insert then comprise insert that 1-100 is individual or more, Nucleotide such as 1-50,1-20, make the active decline 30% or more of this mutator gene simultaneously.Certainly, said mutation is not limited to listed content, can adopt other method known in the art to make described gene inactivation.
The present invention's used " original strain " refers to that it comprises amrE and/or amkE gene or is not operated with two component regulation systems of its height homologous gene, described amrE and/or amkE gene or with its height homologous gene bacterial strain of inactivation not, it can be natural bacterial strain, also can be the bacterial strain that uses in present industry or the laboratory.
Can adopt the known method of state of the art genes involved to be knocked out or makes it undergo mutation.For example, can utilize the method for dna homology reorganization, make up genes involved and interrupt carrier, goal gene is knocked out (Ding Xiaoming etc. utilize homologous recombination to set up Mediterranean Sea and intend the chromosomal gene substitution/interrupt system of no mycolic acids bacterium U32, biotechnology journal, 2002,18 (4): 431-437).
Also can adopt whether inactivation of gene that the known method of state of the art judges sudden change, for example check goal gene whether to suddenly change with Southern hybridization or with the method for pcr amplification; Whether the gene that the phenotype that utilize to detect mutant strain changes the method for (as, the change of production of Rifamycin Sodium) or judges sudden change by the relevant physiological biochemical test inactivation.
Those skilled in the art can adopt the disclosed knowledge screening of prior art deactivated strain.For example when making up the interruption carrier, introduce resistant gene, interrupt genes involved with interrupting carrier.The substratum that utilization contains resistance comes the screening-gene deactivated strain, and with the method for above-mentioned checking gene inactivation the bacterial strain that is screened is verified.The method that the mensuration bacterial strain is produced antibiotic ability also is that this area is known, for example, mensuration to sharp pluramycin SV, can measure biological value (Jiao Ruishen with the inhibition zone laboratory method, Chen Yumei, Wu Meng Gan, Gu Weiling, the research I. nitrate that the metabolism of Nocardia intermedien (Nocardia mediterranei) composite force pluramycin is regulated is to the promoter action of Nocardia intermedien composite force pluramycin SV, the plant physiology journal, 1979,5 (4): 395-402) or adopt document (Wang W, Zhang W, Chen H, Chiao J, Zhao G, JiangW.Molecular and biochemical characterization of a novel two-component signaltransduction system, amrA-amkA, involved in rifamycin SV production inAmycolatopsis mediterranei U32.Arch.Microbiol 2002,178:376-386) described method is measured its chemical titer.
The present invention also relates to a kind of bacterial strain of novelty on the other hand, and this bacterial strain contains a pair of component regulation system, and one or two gene in this regulation system is inactivation, wherein, this gene be amrE gene, amkE gene or with its height homologous gene.This bacterial strain can be selected from Mediterranean Sea intend no mycolic acids bacterium (Amycolatopsis mediterranei) or have similar metabolic regulation mechanism and comprise amrE and the amkE gene or with its microorganism strains of two component regulation systems of homologous gene highly.The present invention also relates to the method that improves secondary metabolite output on the other hand, and this method comprises cultivates bacterial strain of the present invention, collects the secondary metabolite that is produced then from substratum.Secondary metabolite can be microbiotic etc., preferably rifomycin.
Can under the conventional condition of using in this area, cultivate bacterial strain of the present invention.For example, the acid of branch bacillus is intended not having in Mediterranean Sea can cultivate (Wang W with Ben Shi solid or liquid nutrient medium at 26 ℃-30 ℃, Zhang W, ChenH, Chiao J, Zhao G, Jiang W.Molecular and biochemical characterization of anovel two-component signal transduction system, amrA-amkA, involved inrifamycin SV production in Amycolatopsis mediterranei U32.Arch.Microbiol2002,178:376-386).Method reference literature (Jiao Ruishen, Chen Yumei, Wu Menggan, the Gu Weiling of no mycolic acids bacterium fermentation intended in Mediterranean Sea, the research I. nitrate that the metabolism of Nocardia intermedien (Nocardia mediterranei) composite force pluramycin is regulated is to the promoter action of Nocardia intermedien composite force pluramycin SV, the plant physiology journal, 1979,5 (4): 395-402).The method of collecting also is that this area is known, for example collects tunning with the filter paper filtering fermented liquid.
The invention still further relates to a kind of contain described amrE gene and/or amkE gene or with the dna sequence dna of its height homologous gene.Described height homology refers to that homologous gene should contain conservative functional domain on protein level, and should have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or above sequence homogeny.Below will set forth the present invention in the mode of embodiment.Should be understood that the present invention is not limited only to following specific embodiment.Those skilled in the art can make suitable modification, change to the present invention, and these modifications and change are all within the scope of the present invention.In the present invention, relating to the operation of DNA can be with reference to " molecular cloning experiment guide " (third edition); But the extraction reference (D.A.Hopwood that relates to the total DNA of karyomit(e), M.J.Bibb, K.F.Chater et al.Genetic Manipulationof Srteptomyces.A Laboratory Manual.England:John Innes Foundation Press, 1985.); No mycolic acids bacterium U-32 genome cosmid library cosmid carrier pLAFR3 (Staskawicz B is intended in Mediterranean Sea, Dahlbeck D, Keen N, Napoli C (1987) Molecular characterizationof cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv.Glycinea.J Bacteriol 169:5789-5794) makes up, its building process is referring to document (Zhang W, LiL, Jiang W, Zhao G, Yang Y, Chiao J (2000) .A novel transmembraneserine/threonine protein kinase gene from a rifamycin SV-producing Amycolatopsismediterranei U32.Eur J Biochem 267:3744-3752).The sequence measurement of DNA is the method for this area routine, also can provide test by commercial company; The method of fermentation can be referring to document (Jiao Ruishen, Chen Yumei, Wu Menggan, Gu Weiling, the research I. nitrate that the metabolism of Nocardia intermedien (Nocardia mediterranei) composite force pluramycin is regulated is to the promoter action of Nocardia intermedien composite force pluramycin SV, the plant physiology journal, 1979,5 (4): 395-402); And the measuring method of Rifamycin Sodium chemical titer can be referring to document (Wang W, Zhang W, Chen H, Chiao J, Zhao G, Jiang W.Molecularand biochemical characterization of a novel two-component signal transductionsystem, amrA-amkA, involved in rifamycin SV production in Amycolatopsismediterranei U32.Arch.Microbiol 2002,178:376-386).
It is molecule Microbiological Lab of plant physiology ecological Studies institute preservation (Li Guolong, Yang Yunliu, Jiao Ruishen that no mycolic acids bacterium U32 is intended in the Mediterranean Sea that uses in experiment, the gene clone of no mycolic acids bacterium U-32 nitrate reductase is intended in Mediterranean Sea, the biotechnology journal, 1994,10 (3): 200-205; With Zhang W, YangL, Jiang W, Zhao G, Yang Y, Chiao J.Molecular analysis and heterologousexpression of the gene encoding methylmalonyl-coenzyme A mutase fromrifamycin SV-producing strain Amycolatopsis mediterranei U32.Appl BiochemBiotechnol.1999Dec; 82 (3): 209-25).Cultivation Ben Shi substratum (the Wang W of no mycolic acids bacterium U32 is intended in Mediterranean Sea, Zhang W, Chen H, Chiao J, Zhao G, Jiang W.Molecular andbiochemical characterization of a novel two-component signal transduction system, amrA-amkA, involved in rifamycin SV production in Amycolatopsis mediterraneiU32.Arch.Microbiol 2002,178:376-386), intestinal bacteria cultivate and to use the LB substratum.The used seed culture medium of no mycolic acids bacterium fermentation is intended in Mediterranean Sea, the prescription of fermention medium is seen (Jiao Ruishen, Chen Yumei, Wu Menggan, Gu Weiling, the research I. nitrate that the metabolism of Nocardia intermedien (Nocardia mediterranei) composite force pluramycin is regulated is to the promoter action of Nocardia intermedien composite force pluramycin SV, the plant physiology journal, 1979,5 (4): 395-402); The seed nitrate culture-medium is to add KNO in seed culture medium
3To final concentration be 0.8%.Adding antibiotic final concentration in the substratum is: penbritin 100 μ g/ml, kantlex 50 μ g/ml, A Baila (aparamycin) 30 μ g/ml, erythromycin 200 μ g/ml.
The restriction enzyme that uses in the experiment, T4DNA ligase enzyme, RNase A, Taq enzyme, Klenow enzyme, 1kb DNA ladder are MBI company product, high-fidelity DNA polymerase KOD-plus is a ToYoBo company product, [a-32P]-dCTP is Beijing inferior brightness biotech firm product, and nylon membrane (Hybond N+) is an Amersham company product.
Embodiment 1: the preparation of the required dna probe of the two component systems of clone
Regulate in the protein at present known replying, OmpR (e. coli dna is conjugated protein) studies very deep bacterium modulin (Parkinson JS and Kofoid EC.Communication modules inbacterial signaling proteins.Annu Rev Genet.1992; 26:71-112), the three-dimensional structure that obtains by X-ray crystalline diffraction shows that its C-end has the DNA calmodulin binding domain CaM of very typical alpha-helix-ring-alpha-helix.The OmpR proteinoid by with the combining of the promoter region of gene, strengthen the functional transcription of RNA polymerase, thereby reach the effect that the regulation and control corresponding gene is transcribed.(nearly N end is GLEVGADD according to the conservative amino acid sequence of OmpR class family protein, nearly C end is TVRGVGY) (Wray LVJr, Fisher SH.The Streptomyces coelicolor glnR gene encodes a protein similar toother bacterial response regulators.Gene.1993Aug 16; 130 (1): 145-50), designed and synthesized two PCR primer: AmE-DPN (SEQ ID NO:3) and AmE-DPC (SEQ ID NO:4).The total DNA that intends no mycolic acids bacterium U-32 with Mediterranean Sea is that about 400bp DNA product that template amplification obtains is connected with pGEM-T carrier (Promega).Sequencing result (SEQ ID NO:2) shows, the aminoacid sequence and the OmpR family protein of the dna sequence encoding that the clone obtains have higher homology, the HLH DNA land that comprises OmpR family protein C end, and several sections conservative aminoacid sequences, prove that this dna fragmentation can be used as to intend among the no mycolic acids bacterium U-32 screening in Mediterranean Sea and reply the proteic probe of adjusting.
Embodiment 2: intend screening amrE and amkE gene the no mycolic acids bacterium U-32 genomic library from Mediterranean Sea
Owing to have OmpR proteinoid gene in the bacillus coli DH 5 alpha (Gibco-BRL), use bacterial colony in situ hybridization to be difficult to be sieved to correct positive colony, so adopt spot DNA hybridizing method to screen.46-50 bacterium colony is divided into one group extracts the mixing plasmid, extracted 80 groups altogether and mixed plasmids (being equivalent to 3800 recombinant plasmids).Utilize 96 hole dot hybridization sample injectors, the mixing plasmid after the sex change is put on the nylon membrane, the dna probe hybridization that makes with embodiment 1 then.Select the strict film condition of washing, the result obtains 4 positive hybridization points on film, numbering is respectively 2,20,21,63 (seeing Figure 1A), extract phase is for the plasmid of 50 bacterium colonies of No. 63, carry out second and take turns screening by hybridization, obtain being numbered 50 recombinant cloning (seeing Figure 1B), its recombinant plasmid called after pLR6350.Further dna molecule hybridize shows that the pLR6350 after identical restriction enzyme is cut has identical hybrid belt with the total DNA of the karyomit(e) of U-32, proves that the external source fragment among the pLR6350 derives from the U-32 (see figure 2) really.
Embodiment 3: Mediterranean Sea is intended no mycolic acids bacterium U-32amrE and the order-checking of amkE gene order and is analyzed
Plasmid pLR6350 uses BglII, StuI, SacI respectively, PstI, XhoI enzyme carry out that enzyme is cut, electrophoresis, Southern shift, and hybridize with the dna probe that embodiment 1 makes, the result shows BglII, StuI, SacI, PstI, the cutting of XhoI enzyme does not produce 2.5kb, 12kb, 2.0kb, the single hybrid belt of 1.2kb and 4.6kb.Respectively the PstI endonuclease bamhi of the BglII endonuclease bamhi of 2.5kb and 2.0kb is cloned into the BamHI and the PstI site of pBlueScriptIIKS carrier (Stratagene), obtains plasmid pKS2.5B and pKS2.0P.The dna sequence dna (see figure 3) that plasmid pKS2.5B and pKS2.0P are checked order and obtain 3238bp altogether.
Sequential analysis shows, on the dna segment of the 3.2kb that has checked order, has two closely linked entire reading frame framves (ORF).ORF1 is initial from the 456th ATG, end at the 1139th TGA, total length 684bp, coding one 228 amino acid whose protein (AmrE), (G+C) percentage composition is 73.3%, the higher characteristics of G+C percentage composition that meet actinomycetes DNA, codon the 3rd bit base are that the content of G or C is 92%, meet the codon preference characteristics of actinomycetes gene.At 447～451bp place, there is a possible ribosome bind site (RBS): GGAGC.Protein sequence and GenBank data comparative result show, it with two component regulator control systems in typically reply and regulate albumen very high similarity is arranged.Asp-14, Asp-57 and Lys-107 also are high conservative at other in proteinoid among the AmrE, and wherein Asp-57 is a phosphorylation site.At the proteic C-terminal of AmrE, has typical DNA calmodulin binding domain CaM (HLH) (seeing Fig. 4 A).
ORF2 is initial from the 1178th GTG, end at 2533 TGA, total length 1356bp, coding one 452 amino acid whose protein (AmkE), (G+C) percentage composition is 75.9%, the higher characteristics of G+C percentage composition that meet actinomycetes DNA, codon the 3rd bit base are that the content of G or C is 94.2%, meet actinomycetes gene codon preference characteristics.At 1167～1171bp place, there is possible RBS site a: GGATC.Protein sequence and GenBank data comparative result show, it with two component regulator control systems in typical histidine protein matter kinases very high similarity is arranged.The primary structure prediction shows, the terminal homology of the N-of AmkE is poor, and its C-is terminal the same with many histidine protein matter kinases, has very conservative several structural domains (seeing Fig. 4 B): H, N, G1, F and G2 district, wherein H contains histidine residues in the district, is the self-phosphorylation site; Glycine residue is rich in G1 and G2 district, is the Nucleotide calmodulin binding domain CaM.The N-end of AmkE has two typical hydrophobic regions, may be to stride the film district.
Embodiment 4: to the research of U-32amrE and the two component gene functions of amkE
Interruption to amrE gene among the U-32 and amkE gene
This intends function among the no mycolic acids bacterium U-32 to two component genes in Mediterranean Sea for further research amrE and amkE, and we have carried out the gene interruption to amrE and amkE respectively with the method for homologous recombination.
With the total karyomit(e) of U32 is template, is primer with AmE-bf (SEQ ID NO:5), AmE-ba (SEQ IDNO:6), obtains the dna fragmentation of about 2.9kb with high-fidelity DNA polymerase KOD-plus (ToyoBo) amplification.With the fragment cloning that obtains EcoRV site, obtain plasmid pAmr-1 to pMD18-T (TakaRa).Plasmid pAmr-1 is cut with the XhoI enzyme and put down with Klenow enzyme benefit, again with from plasmid pBCAm (Ding Xiaoming etc., utilize homologous recombination to set up Mediterranean Sea and intend the chromosomal gene substitution/interrupt system of no mycolic acids bacterium U32. the biotechnology journal, 2002,18 (4): 431-437) upward downcut the 1.5kb dna fragmentation that contains the A Baila resistant gene and be connected, obtain the interruption plasmid pAmr-2 of amkE gene with the SmaI enzyme.Plasmid pAmr-1 is cut with the BglII enzyme, be connected with the 1.5kb dna fragmentation that the cutting-out of BamHI enzyme contains the A Baila resistant gene again with from plasmid pBCAm, obtain the interruption plasmid pAmr-3 of amrE gene.
According to document (Ding Xiaoming etc. utilize homologous recombination to set up Mediterranean Sea and intend the chromosomal gene substitution/interrupt system of no mycolic acids bacterium U32, the biotechnology journal, 2002,18 (4): method prepares the electric shock competence of U32 thalline 431-437).Be inserted in immediately on ice the interruption plasmid pAmr-2 of amkE gene heated abundant sex change in 10 minutes in boiling water after, electric shock transforms the U32 competence then, and adds in Ben Shi on the flat board of A Baila resistance and screen transformant.Under 28 ℃,, will grow the mutant strain that the amkE gene interrupts on the flat board through 4-5 days cultivation.The method of interrupting the amrE gene with the interruption plasmid pAmr-3 of amrE gene is identical with the method for interruption amkE.
Be used for making up 2.9kb dna segment outside design a pair of checking primer AmE-cf (SEQ ID NO:7) and the AmE-ca (SEQ ID NO:8) that interrupts carrier, possible mutant strain is carried out the PCR checking, and the U32 bacterial strain is together carried out pcr amplification as negative contrast.
The result of checking shows that the mutant strain of amrE and amkE all can amplify the band of a treaty 4.5kb, and contrast U32 has amplified the band (see figure 5) of a treaty 3.0kb, proved that amrE gene and amkE gene have obtained successful interruption respectively, obtained Mediterranean Sea and intended no mycolic acids bacterium U-32amrE mutant strain U32-101 and the no mycolic acids bacterium U-32amkE mutant strain U32-107 of Mediterranean Sea plan.
Intending no mycolic acids bacterium U32 in contrast with Mediterranean Sea, is that indicator has been cooked the inhibition zone experiment with Sarcina lutea (Sarcina lutea).Found that no matter be on the Ben Shi substratum, still add 0.8%KNO at Ben Shi
3Substratum on, the antibacterial circle diameter of amrE mutant strain (U32-101) and amkE mutant strain (U32-107) is all compared and is gone out manyly according to U32 greatly, and the bacteriostatic diameter of U32-101 and U32-107 two strain bacterium is similar.
Select amrE mutant strain (U32-101) and amkE mutant strain (U32-107), use the seed nitrate culture-medium to carry out fermenting experiment.Through 5 days fermentation, the output of the Rifamycin Sodium of U32-101 was 368 ± 61, and the output of the Rifamycin Sodium of U32-107 is 302 ± 67, and the output of the Rifamycin Sodium of contrast U32 is 132 ± 23 (see figure 6)s.The result of fermentation shows that the output of the Rifamycin Sodium of U32-101 and U32-107 all has significantly with respect to contrast U32 to be increased, and wherein the output of the Rifamycin Sodium of U32-101 is high slightly.
Select U32 and U32-101 further with the fermention medium fermentation, through 5 days fermentation, the output of the Rifamycin Sodium of U32 was 1424 ± 187, and the output of the Rifamycin Sodium of U32-101 is 2062 ± 250 (see figure 7)s.
The two component systems of amrE and amkE overexpression in U32
Plasmid pULVK2A (Kumar CV, Coque JJ, Martin JF.Efficient Transformationof the Cephamycin C Producer Nocardia lactamdurans and Development of Shuttleand Promoter-Probe Cloning Vectors.Appl Environ Microbiol.1994Nov; 60 (11): 4086-4093) be the plasmid of can in U32, duplicating of using always and stable existence.On the basis of pULVK2A plasmid, made up the pDXM4 plasmid that contains erythromycin resistance gene.With the total karyomit(e) of U32 is template, is primer with AmE-bf and AmE-ba, amplifies the dna fragmentation that contains complete amrE and amkE gene and promoter region thereof with high-fidelity KOD-plus archaeal dna polymerase.The dna fragmentation of this 2.9kb is cloned into the EcoRV site of pDXM4 plasmid vector, obtains overexpression carrier pVK2.9AmE.
Use Mediterranean Sea to intend no mycolic acids bacterium U32 and make the electric shock competence.Respectively pDXM4, pVK2.9AmE are transformed the U32 competence,, on the flat board of Ben Shi interpolation erythromycin, screen transformant 28 ℃ of cultivations.Through about 7 days cultivation, can see transformant on the flat board.Plasmid in the extracting transformant and checking are correct transformant difference called after U32-109 and U32-110.
U32-109 and U32-110 are fermented, and method is the same, except add erythromycin in substratum.Through 5 days fermentation, in the seed nitrate culture-medium, the output of the Rifamycin Sodium of U32-109 was 257 ± 39, and the output of the Rifamycin Sodium of U32-110 is 15 ± 10 (see figure 8)s.Owing to added erythromycin, under experimental conditions, U32-109 and U32-110 all can not detect the generation of Rifamycin Sodium in fermention medium.
The relative U32 of output of the Rifamycin Sodium of the mutant strain U32-101 that the amrE gene interrupts has raising by a relatively large margin.When overexpression amrE and amkE in U32 pair of component system, the output of crossing the Rifamycin Sodium of expression strain U32-110 reduces significantly with respect to control strain U32-109.Interrupt and cross the data of expressing two aspects and can reach a conclusion from gene: no mycolic acids bacterium U-32 generation Rifamycin Sodium is intended in the two component negative genes regulation and control of amrE and amkE Mediterranean Sea.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉method of raising volume of production of rifamycin
<130>065775
<160>8
<170>PatentIn?version?3.3
<210>1
<211>3238
<212>DNA
＜213〉no mycolic acids bacterium (Amycolatopsis mediterranei) is intended in Mediterranean Sea
<400>1
ctgcagaagc?ggatcaacgg?cggccccgag?gtcgccggtt?cggtcgccaa?cctcaaggcg 60
cgggcccagg?accagcgcgc?cgccgggcac?gaggaccgcg?cgaagaagct?cgacgaccgc 120
gccaccaagc?gccagggcaa?gctcggcgag?ctgaacacgg?ccaagcagaa?gctcgacgcg 180
ttcgcgtccg?cgcactgcaa?gccggccaag?tgaggggcgc?gagggtcgcg?gcggcggtcg 240
gggccgccgc?ggtcatcgcg?ctcggctgct?cggcgtgccg?cgacaacctg?atgggttcgc 300
cctccgggga?cgcgcccgcg?ccgtcgtcgg?tgtcgtcgtc?ggagctgagc?gggatcgagt 360
cgacactgaa?cggcatcgag?tcggacatga?acagcgatgg?ctcgccctga?ccggctagcg 420
tctcccctct?gaacacggcg?gagacgggag?caggcatggg?gtcaccgggg?cgcggtctcg 480
tcctcgtggt?cgaggacgag?gccgcgatcg?ccgagctggc?ggcgctctac?ctcaagcgtg 540
acggcttcgg?tgtgcacgtc?gaggccgacg?gcggccgggc?cctggacgcc?gtccggcgcc 600
tcaagccggt?ggcgatcgtg?ctcgacatcg?gactgtccgg?aatggacggc?atcgagatct 660
gcaaggcgtt?gcgcgcggcc?ggggactgga?cgccggtgct?gttcgtcacc?gcccgcgacg 720
acgagctcga?ccggctgctg?ggcctggaga?tcggcgcgga?cgactacctc?accaagccgt 780
tcagcccgcg?cgagctggcg?gctcgggtcc?ggacggtgct?gcgccgggcc?gccggggtgg 840
cgccggccgc?ggagacgttc?gccgtgggcg?gcgcgcgcgt?cgacgtgctt?tcccggcggg 900
cgtgggcggg?tgacgccgag?atagtgctga?cgtccacgga?gttcgacctc?ctcacgcacc 960
tgctccggca?cccggggcag?gtgctttcgc?gcgaccagct?gctgagtgcc?gtgtggggct 1020
acgccgcggc?ggcgggcacg?cgcacggtgg?acgtccacgt?ggctcagctg?cgcgcgaaac 1080
tcggcgcgtc?gagtccgatt?cggacggtgc?ggggcatcgg?gtacgcggcg?gatccgtcgt 1140
gaagttccgg?gggacgctgg?ccgggcggat?cacgctggtg?tgcctggccg?tcgcgggcat 1200
cgcggtgatc?gtggcggggc?tggtcgcctc?ccggctgatc?cggaccacgg?cggacaacgt 1260
gctgcagtcg?acgctggccg?cgcaggccga?cgtcgtcgcg?tcgcaattgg?acgaaacggg 1320
catcggcaac?cggctgggcg?tcggcaaggt?ggccgacgtg?gtgcgcggcc?agggcatctc 1380
ggtggtggtc?cgccgcgcag?gcgggcaggc?gctcggggac?ggcgtcgcgg?tccaggccgc 1440
cgggaagctc?gggctggacc?gcaaccactc?ggggcgcgtc?acggtggcgg?ggtcgcagta 1500
cctggtcgag?acgcgggtgg?tgggcgcacg?cggagcggcg?ttcgcgctgg?tcctgcccgc 1560
gcgcaacgca?caagcgacgc?aacgggcact?ggtgcgcaac?atcttgctgg?cgctcgggat 1620
cgggctgctg?gtggcggccc?tggcgggctt?cgtctcgggc?cggctgctgg?gccgtccgct 1680
gcgccgggcc?gcgctggccg?cgggatcgct?gcgggcggga?cgtcgcgacg?tgcgggtgcc 1740
ggtgcagggg?ccccgcgagg?tggcggaggt?ggccgggtcg?ctgaactcgc?tggccgacgc 1800
gctggccgtc?agcgaggcgc?ggcagcggga?gttcctgctg?tcggtgtcgc?acgagctgcg 1860
gacgccgctg?accgccgtga?cgggcttcgc?cgaggcgatc?gcggacggcg?tcgccacggg 1920
gccggacgcc?gtccgggccg?ggcagacgat?ccagcgcgag?gcggtgcggc?tcgagcggct 1980
ggtcacggac?ctgctggagc?tggcccgcct?gggcgcggac?gagttccggc?tggacatcgc 2040
ggtgctggac?ctggctgcgt?tggtccgcga?ctgtgcggag?gtgtggcagc?tgcggtgcgg 2100
ccgggaggac?gtccggctct?cggtttcggc?tcctgccggg?ccggttccgg?tgctggcgga 2160
cgcgcggcgg?ctgcaccagg?tggtcgacgg?cttggcggag?aacgccctgc?gcgtcacgcc 2220
cgcgggggcc?ccgatggtgt?tctcgctgtc?ggtgtccgat?tcggcggctt?cgctggcggt 2280
ccgcgacggc?ggcccgggcc?tggcgccgga?ggactacccg?gtggcgttcg?agcgaggggt 2340
cctgaacgcc?cgctaccgcg?accgccgtcc?ggtcggctcg?ggcatcggcc?tggccctggt 2400
ccacgccctg?gtcacccgga?tgggcgggac?attgacggcc?ggcccggccc?ccgaaggcgg 2460
cgcggcgttc?acgatcacgt?tcccgctggc?cgcgtcgacg?gccacggtcc?cgccccccgc 2520
cgctgacctg?ggctgacgac?cgccggcgag?ctgaaccggc?gatctcgcac?ggccgaccgc 2580
accggcgtcc?acggccactc?gtccgtaact?tggtgtcccc?gatccgcgac?agaaacgctg 2640
gagtgatgcg?tttcaaccct?gcgatggtgt?tcaaggtcga?cagcatccgg?cgaaccgaat 2700
ttccatgcac?gaacggtgtg?acgggagtgc?ggaatcgatg?gcggaacgag?gacggcacga 2760
cgactcggag?aatgcgggtt?tcgagcggga?cagcgacctg?gtccgggcct?acgaacgagg 2820
aacgtcgatc?ggcgaactcg?ccgagcggac?cggcttgagc?tacacgtgga?tgcgcagacg 2880
gttgatcaac?gccggggcgg?agctccggcc?ccggccggtc?gcgaaggcct?gcccggtgcc 2940
gatcgagcag?ctggcggacg?agtaccgcgc?cggcgccagc?attctccagc?tcgccaagac 3000
gcacggcctc?tactacaaac?gggtccgcga?actgctgctc?gatcacgaag?tgccattgcg 3060
cccctcgact?cgcgccatac?ccgaaacctg?actcgattgt?ggcccaggtc?accgattttt 3120
ttcctcctgt?gcagttgcac?gggagtgttc?gtgctttcga?gtggaaattc?ccgtgctcat 3180
cgcggagcga?gcccgatcgg?ccggtagaca?gagaagttcg?ggacttgcgg?gggagatc 3238
<210>2
<211>382
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉probe sequence
<400>2
gcctggagat?cggcgcggac?gactacctca?ccaagccgtt?cagcccgcgc?gagctggcgg 60
ctcgggtccg?gacggtgctg?cgccgggccg?ccggggtggc?gccggccgcg?gagacgttcg 120
ccgtgggcgg?cgcgcgcgtc?gacgtgcttt?cccggcgggc?gtgggcgggt?gacgccgaga 180
tagtgctgac?gtccacggag?ttcgaccttc?tcacgcacct?gctccggcac?ccggggcagg 240
tgctttcgcg?cgaccagctg?ctgagtgccg?tgtggggcta?cgccgcggcg?gcgggcacgc 300
gcacggtgga?cgtccacgtg?gctcagctgc?gcgcgaaact?cggcgcgtcg?agtccgattc 360
ggacggtgcg?gggcatcggg?ta 382
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
aagaattcga?ggtgggcgcs?gacgac 26
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
atctagagta?gccgacgccs?cgcac 25
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
tgaacacggc?caagcaga 18
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
gccacaatcg?agtcaggt 18
<210>7
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
tgcagaagcg?gatcaacg 18
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
tgcaactgca?caggagga 18
Claims (10)
1. method for preparing the bacterial strain that microbiotic or other useful secondary metabolite output further improves, this method comprises:
To comprising amrE and amkE gene or operate in the original strain of described bacterial strain with two component regulation systems of its height homologous gene, make described amrE and/or amkE gene or with its height homologous gene inactivation, the bacterial strain that obtains suddenling change, this mutant strain are microbiotic or the further bacterial strain that improves of other useful secondary metabolite output.
2. the method for claim 1 is characterized in that, described method also comprises step: cultivate described mutant strain, measure it and produce antibiotic ability, filter out the bacterial strain that microbiotic output further improves.
3. the method for claim 1, it is characterized in that, described bacterial strain be selected from Mediterranean Sea intend no mycolic acids bacterium (Amycolatopsis mediterranei) or have similar metabolic regulation mechanism and comprise amrE and/or the amkE gene or with its microorganism strains of two component regulation systems of homologous gene highly.
4. the method for claim 1 is characterized in that, with described amrE and/or amkE gene or with its height homologous gene knockout, the bacterial strain that obtains suddenling change.
5. bacterial strain that adopts the described method of claim 1 to make, it be selected from Mediterranean Sea intend no mycolic acids bacterium (Amycolatopsis mediterranei) or have similar metabolic regulation mechanism and comprise amrE and/or the amkE gene or with its microorganism strains of two component regulation systems of homologous gene highly, wherein, this bacterial strain comprises a pair of component regulation system, amrE gene in this regulation system and/or amkE gene or with its height homologous gene inactivation.
6. bacterial strain as claimed in claim 5 is characterized in that, described bacterial strain is the bacterial strain that no mycolic acids bacterium (Amycolatopsis mediterranei) is intended in Mediterranean Sea.
7. produce antibiotic method for one kind, it is characterized in that described method comprises:
(a) cultivate the described bacterial strain of claim 5; With
(b) from culture, collect the microbiotic that described bacterial strain produces.
8. method as claimed in claim 7 is characterized in that described microbiotic is a rifomycin.
9. method as claimed in claim 7 is characterized in that, described bacterial strain is that no mycolic acids bacterium is intended in Mediterranean Sea.
10. dna molecular, it contains 456-1139 position and/or 1178-2533 position nucleotide sequence shown in the SEQ ID NO:1.
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Cited By (5)
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| CN102286398A (en) * | 2010-06-21 | 2011-12-21 | 中国科学院上海生命科学研究院 | Method for producing high-activity and high-purity rifamycin SV |
| CN105886421A (en) * | 2015-07-16 | 2016-08-24 | 中山大学 | Amycolatopsis sp. YIM M13163 and method for preparing efrotomycin pivalate by adopting same |
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| CN1242052C (en) * | 1998-07-16 | 2006-02-15 | 布里斯托尔-迈尔斯斯奎布公司 | Biological pure culture for Nocardia |
| US7309484B2 (en) * | 2003-06-23 | 2007-12-18 | Cornell Research Foundation, Inc. | Compositions and methods for screening antibacterial compounds |
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| CN102286398A (en) * | 2010-06-21 | 2011-12-21 | 中国科学院上海生命科学研究院 | Method for producing high-activity and high-purity rifamycin SV |
| WO2011160573A1 (en) * | 2010-06-21 | 2011-12-29 | 中国科学院上海生命科学研究院 | Method for producing rifamycin sv with high activity and high purity |
| CN105886421A (en) * | 2015-07-16 | 2016-08-24 | 中山大学 | Amycolatopsis sp. YIM M13163 and method for preparing efrotomycin pivalate by adopting same |
| CN105886421B (en) * | 2015-07-16 | 2019-04-02 | 中山大学 | One plant is intended without mycolic acids bacterium and the method for preparing special penta Efrotomycin using the bacterium |
| CN108424946A (en) * | 2018-01-30 | 2018-08-21 | 华东理工大学 | The method for improving oxytetracycline yield based on afsQ1Q2-like bi-component regulating systems |
| CN108424946B (en) * | 2018-01-30 | 2021-10-15 | 华东理工大学 | Method for improving terramycin yield based on afsQ1Q2-like two-component regulation system |
| CN108504594A (en) * | 2018-03-29 | 2018-09-07 | 云南大学 | One plant of quasi- application without mycolic acids bacterium and its in preparing anti-notoginseng root rot agent |
| CN112608965A (en) * | 2021-01-15 | 2021-04-06 | 华东理工大学 | Culture medium for producing bleomycin E by using deep sea streptomycete and preparation method thereof |
| CN112608965B (en) * | 2021-01-15 | 2022-08-05 | 华东理工大学 | Culture medium for producing bleomycin E by using deep sea streptomycete and preparation method thereof |
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