CN101724672A - Method for constructing lidamycin high-yield strain through genetic manipulation for biosynthetic controlling gene - Google Patents

Abstract

The invention relates to a method for constructing a lidamycin high-yield strain through the genetic manipulation for a biosynthetic controlling gene, in particular to a method for improving the performance of a lidamycin production strain for producing lidamycin, which comprises the step of improving the expression of one or more genes selected from sgcR1, sgcR2 and sgcR3 in the lidamycin production strain so as to obtain a recombination strain with the improved lidamycin yield.

Description

By the genetic manipulation of biosynthetic controlling gene being made up the method for lidamycin (LDM) superior strain

Invention field

The present invention relates to the regulatory gene sgcR1 in the biological synthesis gene cluster of lidamycin (LDM) among the styreptomyces globispotus strain C-1027 (Streptomyces globisporus C-1027), sgcR2, sgcR3 (styreptomyces globispotus strain C-1027 regulatory gene 1,2,3) reach by regulatory gene sgcR1 the formed regulated and control network of sgcR2 and sgcR3.The invention still further relates to by improving gene copy number and utilizing the strong promoter of constitutive expression that positive regulating gene was carried out expressing to improve the method for lidamycin (LDM) output.The invention still further relates to the recombinant bacterial strain that obtains by these methods.

Background technology

Lidamycin (LDM) (lidamycin has another name called C-1027) is produced [1] by the styreptomyces globispotus strain C-1027 that finds in the soil of Qianjiang, China Hubei.Lidamycin (LDM) is the strongest material of finding up to now of anti-tumor activity, and is stronger more than 10000 times [2] than Zorubicin commonly used clinically.Through new drug research and the exploitation in surplus ten years, lidamycin (LDM) entered the clinical II phase and has studied [3] at present, is expected to develop into the new and effective antitumor drug with China's independent intellectual property right.

Lidamycin (LDM) belongs to enediyne class microbiotic, is made up of small molecules chromophoric group and apoprotein, and chromophoric group has ring-type enediyne structure, is its active centre that tumour cell is had extremely strong lethal effect.Enediyne class microbiotic generally is divided into nonatomic ring (as neocarzinostatin (neocarzinostatin, NCS), maduropeptin (MDP) and lidamycin (LDM)) and ten-ring (as calicheamicins, esperamicin and dynemicins) two classes.The antibiotic mechanism of action uniqueness of enediyne class combines back enediyne core and resets with DNA, cause into the ring aromizing and form a temporary benzenoid diradical compound, and unsettled intermediate product impels dna single chain or double-strand break [4,5].Enediyne class microbiotic is because of its distinctive molecular structure, the mechanism of action of extremely strong antitumor action and novelty is at chemistry, biology and medical field all get most of the attention, except that lidamycin (LDM) is carrying out the clinical study in China, 1997 neocarzinostatin poly (styrene-co-maleic acid) conjugate (trade(brand)name SMANCS) in Japan listing [6], be used for surplus the clinical therapy of tumor ten year in Japan, and calendar year 2001 Calicheamicin-CD33 antibody coupling matter (trade(brand)name Mylotarg) becomes first is used for oncotherapy by the FDA approval monoclonal antibody targeted drug [7].Lidamycin (LDM) also can be used as " bullet " because its intensive biological activity can develop into new and effective antitumor drug, makes targeted drug miniaturization, high efficiency, and wide prospect has been showed in its research and application.Though the anti-tumor activity of lidamycin (LDM) is extremely strong, its chromophoric group structural stability is poor, fermentation yield is low, is unfavorable for the research and development of large-scale production and targeted drug.Though it is traditional breeding method is effective within the specific limits, directed, making up the superior strain of lidamycin (LDM) reasoningly need be to the deep parsing of its biosynthesizing mechanism.

The lidamycin (LDM) biological synthesis gene cluster has obtained clone (GenBank accession number AY048670).About 75 kb of lidamycin (LDM) biological synthesis gene cluster, totally 56 genes.Infer that its chromophoric biosynthetic process is at first synthetic four structural units (aminopyridine sugar, β-tyrosine, nitrogen oxa-naphthoic acid and chromophoric group enediyne core), and then fit together [8].But up to the present, the biosynthesizing mechanism of lidamycin (LDM) only obtains preliminary parsing, and research mainly concentrates on infers [9,10 to the biological function explore of biosynthetic enzyme in its biological synthesis gene cluster and biosynthesizing step, 11,12,13,14,15,16], still have many details to remain to be furtherd investigate, especially its biosynthetic controlling Study on Mechanism is not appeared in the newspapers as yet.

Streptomycete can produce a large amount of secondary metabolites, has significant values in medicine and agriculture production, and about 70% known microbiotic is produced by streptomycete.The biosynthetic molecular regulation of most antibiotics often is among a regulated and control network complexity, multistage level.Usually the biosynthetic regulation and control of microbiotic are mainly carried out three levels: approach specificity (pathway-specific) regulation and control, pleiotropy (pleiotropic) regulation and control and (global) of overall importance regulation and control [17].Include an approach specificity regulatory gene in most of known streptomycete antibiotic biological synthesis gene clusters, as the special activating transcription factor actII-ORF4[18 of the approach of actinorhodin, 19], the special activating transcription factor StrR[20 of Streptomycin sulphate approach, 21] etc., the gene in the microbiotic biological synthesis gene cluster at place is carried out special, meticulous transcriptional control.Along with the clone and the order-checking of increasing microbiotic biological synthesis gene cluster, find to have a plurality of approach specificity regulatory genes in some bigger microbiotic biological synthesis gene clusters, as daunorubicin [22,23,24,25,26,27], tylosin [28,29,30,31,32,33], rapamycin [34] etc.After the biological synthesis gene cluster of lidamycin (LDM) is cloned, the biological synthesis gene cluster of Calicheamicin (AF497482), neocarzinostatin (AY117439) and MDP (AY271660) also obtains to resolve successively, has all found the regulatory gene more than 3 in these 3 antibiotic biological synthesis gene clusters of enediyne class.

As everyone knows, strain improvement is the key that improves microbiotic output always.Traditional breeding method blindness is big, and workload is big, and efficient is low.Utilize the breeding of metabolic engineering method, on the basis of metabolic analysis, design cell, and then use the instrument of modern molecular genetics to come engineered cells, can be rational, output improved significantly.The means of metabolic engineering breeding on the one hand can the reconstruction structure gene, can improve antibiotic output [35] by specific positive regulatory factor of overexpression approach or inactivation negative regulatory factor on the other hand.Regulatory gene is carried out in the transformation of metabolic engineering, the method for the positive regulatory factor of more employing overexpression, positive regulating gene often by promoting a plurality of important structure expression of gene, finally influences antibiotic output.The method of the positive regulatory factor of overexpression is the regulatory gene that imports other copy in wild strain, can with the integrative plasmid carrier, the regulatory gene of single copy is incorporated on the genome, realize overexpression, can be carrier also by self-existent multiple copied plasmid, import the regulatory gene of a plurality of copies, can also add the controlling element of promote expressing (as the strong promoter of constitutive expression etc.) by the upstream of regulatory gene in expression plasmid, positive regulating gene is farthest expressed.

The present invention is by carrying out the functional analysis of inside and outside to regulatory gene possible in the lidamycin (LDM) biological synthesis gene cluster, biosynthetic controlling gene sgcR1, the sgcR2 of lidamycin (LDM) and the function of sgcR3 have been illustrated, and the regulation relationship between sgcR1, sgcR2 and the sgcR3, by improving gene copy number and the method that the strong promoter that utilizes constitutive expression carried out expression to these three positive regulating genes, improve the output of lidamycin (LDM) on this basis.

Summary of the invention:

The present invention has carried out bioinformatic analysis to 3 ORF that are noted as biosynthetic controlling gene in the lidamycin (LDM) biological synthesis gene cluster among the GenBank.These ORF encoded protein are carried out finding behind the homology analysis sgcR1 encoded protein and known modulin StrR (the GenBank accession number CAA07385) consistence in length range is 44%, similarity is 59% (as Fig. 1 a); The modulin of a sgcR2 encoded protein and an AraC/XylS family (GenBank accession number ABP55137) consistence in length range is 50%, and similarity is 62% (as Fig. 1 b); SgcR3 encoded protein and known modulin TylR (the GenBank accession number AAF29380) consistence in length range is 33%, and similarity is 47% (as Fig. 1 c).In addition, to being all the antibiotic C-1027 of enediyne class, neocarzinostatin, the biological synthesis gene cluster of maduropeptin and calicheamicin has carried out the ORF compare of analysis, there is the homologous gene of sgcR1, sgcR2 and sgcR3 in discovery in the biological synthesis gene cluster of neocarzinostatin, the homologous gene that has sgcR1 and sgcR2 at the biological synthesis gene cluster of maduropeptin, there is the sgcR2 homologous gene in the biological synthesis gene cluster of calicheamicin, illustrates that these regulatory genes have vital role in the biosynthesizing of enediyne class microbiotic.In three nonatomic ring class enediyne gene clusters, sgcR1, sgcR2 and sgcR3 and their homologous gene all are centered around near enediyne polyketide synthase gene (PKSE) and the genes involved thereof, and also closely similar (as Fig. 2) formed in sequence in the gene, shows that the biosynthetic transcriptional control of this class microbiotic has general character.

The present invention studies the adjusting function of sgcR1, sgcR2 and sgcR3 in the gene cluster.At first, respectively regulatory gene in the gene cluster is carried out gene disruption by the homology double exchange, analyze it to the biosynthetic influence of lidamycin (LDM), find after sgcR1R2 (sgcR1 and sgcR2), sgcR2 and the sgcR3 blocking-up that all force of labor does not reach mycin, show that sgcR1, sgcR2 and sgcR3 are positive regulating gene (as Fig. 4).Structure gene sgcA1, sgcC4, sgcD6, sgcE8 carry out gene transcription level in wild strain, sgcR1R2 and sgcR3 blocking-up bacterial strain analytical results shows, the expression of these structure genes all obviously reduces (as Fig. 9) in the blocking-up bacterial strain, and sgcR1, sgcR2 and sgcR3 gene positive regulating gene and carry out the biosynthetic control of lidamycin (LDM) by the transcriptional control to the biosynthesis gene of four structural units in the lidamycin (LDM) biosynthetic pathway really is described.SgcR1, sgcR2 and three genes of sgcR3 are imported wild strain respectively carry out overexpression, the output of lidamycin (LDM) has obtained raising in various degree (as Fig. 6), and further having proved conclusively sgcR1, sgcR2 and sgcR3 is positive regulating gene.

The present invention also changes sgcR1, sgcR2 or sgcR3 gene respectively over to the blocking-up bacterial strain of different regulatory genes, cross complementary is analyzed it to the biosynthetic influence of lidamycin (LDM), the result shows the blocking-up that sgcR1, sgcR1R2 can complementary sgcR3, the blocking-up (as Fig. 7) that sgcR3 but can not complementary sgcR1R2.Respectively sgcR1, sgcR2 and the transcriptional level of sgcR3 in sgcR1R2 and sgcR3 blocking-up strain are analyzed with real-time quantitative RT-PCR, the result shows, the expression level of sgcR1 and sgcR2 all significantly reduces in the sgcR3 blocking-up strain, and the expression uninfluenced (as Fig. 8) of sgcR3 in the sgcR1R2 blocking-up strain.This result verification the cross complementary experiment, show that sgcR3 is positioned at sgcR1 and sgcR2 upstream in lidamycin (LDM) biosynthetic controlling network.Heterogenous expression and purifying SgcR3 albumen in intestinal bacteria find that with the gel blocking method SgcR3 can be combined in the promoter region of sgcR1R2, and further specifying SgcR3 is by regulating and control the expression of transcribing sgcR1 and sgcR2 (as Figure 10) of sgcR1R2.

The present invention furthers investigate the regulation relationship between them again behind the positive regulating and controlling effect of having proved conclusively sgcR1, sgcR2 and three genes of sgcR3, has determined that sgcR3 is positioned at sgcR1 and sgcR2 upstream in lidamycin (LDM) biosynthetic controlling network.On this basis, proposed that lidamycin (LDM) is produced the bacterium biosynthetic controlling gene and carried out the method that genetic manipulation improves the bacterial strain production performance.

As everyone knows, strain improvement is the key that improves microbiotic output in the fermented liquid always.Conventional strain improvement method adopts " random mutation+directed screening ", be to be based upon on the basis that the secondary metabolites generation structure is handled as "black box", if the bacterial classification of the uncomprehending meta-bolites of gene of seed selection pathways metabolism and participation synthetic enzyme, it is effective adopting this method.But it is big that the shortcoming of this method is a workload, and efficient is low.The metabolic engineering breeding refers to design cell on the basis of metabolic analysis, and then uses the instrument of modern molecular genetics to come engineered cells.If the structure gene of the enzyme of known organism route of synthesis and regulatory gene just can be carried out directed strain improvement, remove rate-limiting step, thereby the integral part of amplification whole piece approach or this approach increases ultimate capacity.The means of metabolic engineering breeding on the one hand can the reconstruction structure gene, increases the copy number (comprising the amplification of whole piece approach, the amplification of segmental amplification of approach or rate-limiting step enzyme gene) of structure gene, improves the speed etc. that product is striden film output.On the other hand, from the viewpoint of regulating, metabolic engineering can be started with from many aspects and be improved the throughput of secondary metabolite, can increase antibiotic growing amount by the copy number that increases positive regulator gene, also can remove those negative regulon (repressor), so that this approach realizes constitutive expression.In addition, it is derivable that regulatory gene is become, thereby change the conditional curve that microbiotic generates.

On the function of the present invention's three positive regulating genes in further investigation lidamycin (LDM) biological synthesis gene cluster and the basis of the regulation relationship between them, utilize the copy number that increases positive regulator gene and use the method for strong promoter to come the overexpression positive regulating gene, thereby increase antibiotic output.The method of the copy number of increase positive regulator gene involved in the present invention is to point to the recombinant expression plasmid that imports three positive regulating genes of other one or more copies in the lidamycin (LDM) wild strain.This a series of recombinant expression plasmid all comprises sgcR1, sgcR2 and nucleotide sequence of one of them and the transcribing and translation initiation and termination signal of gene self at least of sgcR3 gene, and the upstream of gene contains a kind of promoter sequence at least.

SgcR1, sgcR2 that the present invention is used and sgcR3 gene coded sequence are known array [8], according to the design of the known array among GenBank primer (seeing the primer table), with the wild bacterium genomic dna of lidamycin (LDM) is template, carry out the PCR reaction, amplified production comprises the encoding sequence of gene and transcribing and translation initiation and termination signal of gene self.

The carrier that makes up recombinant expression plasmid can be the self-replicating carrier, and as the carrier that the outer entity of a kind of karyomit(e) exists, it duplicates and does not rely on THE REPLICATION OF CHROMOSOME, can exist with the form of multiple copied, for example, plasmid pKC1139.In addition, carrier also can be a kind of like this carrier, and after introducing host cell, it is incorporated in the genome and with the karyomit(e) of being integrated and duplicates, for example, and plasmid pSET152.For self-replicating, carrier can further contain make carrier can be in host cell the replication orgin of self-replicating, for example include but not limited to allow pIJ101, the pJV1 that in streptomycete, duplicates, the replication orgin of pSG5, pSGL1.

Carrier of the present invention preferably contains one or more selected markers that transformant can be screened easily.Selected marker is a kind of like this gene, and its product provides antibiotics resistance, biocide or virus resistance, heavy metal resistance or the like.The example of bacterium selected marker includes but not limited to give the mark of penbritin, kantlex, paraxin or the tetracyclin resistance of antibiotics resistance as being used for screening intestinal bacteria or is used for the thiostrepton of streptomycete screening, A Pu mycin resistance etc.

Target gene to be expressed and selected prokaryotic host cell are depended in the selection of suitable expression.Be applicable to that expression vector of the present invention includes but not limited to pKC1139, pIJ680, pIJ486/487, pIJ702, pFD666, pSGL1, pSET152, pIJ6902, pIJ8600 or their derivative, other preferred expression vector is referring to Kiese etc., and streptomycete genetics is put into practice [37].

A kind of nucleotide sequence that promoter sequence is promptly discerned by host cell for the expression of nucleotide sequence.Contain the transcriptional control sequence that mediates expression of polypeptides in the promoter sequence.Promotor can be any nucleotide sequence that shows transcriptional activity in selected host cell, comprises mutant, truncation type and heterozygous promotor, and can be outside coding and host cell homology or allogenic born of the same parents or in the born of the same parents obtains the gene of polypeptide.Include but not limited to the promotor that from following gene, obtains: aphD (aminoglycoside phosphotransferase gene) for produce guiding recombinant expression plasmid of the present invention is transcribed in the bacterium available promotor at lidamycin (LDM), tsr (thiostrepton resistant gene), ermE (erythromycin resistance gene), ssi, vsi (subtilisin inhibitor gene), STI-II (serpin gene), β-gal (beta galactosidase enzyme gene), vph (Viothenate resistant gene), the promotor ermE*p of the ermE gene after tipA (tipA gene in the shallow Streptomyces glaucoviolaceus) etc. and the sudden change.

Producing the method for introducing recombinant expression vector in the bacterium to lidamycin (LDM) can be protoplast transformation [37], electroporation [37] or bonding method [37].

The high copy number plasmid pKC1139 that adopts self-replicating in the streptomycete among the present invention is as carrier, and the promotor of the ermE*p of constitutive expression and gene self is as promotor.Import some genes or a plurality of gene among positive regulating gene sgcR1, sgcR2 and the sgcR3 respectively in wild strain, cooperate promotor and/or ermE*p with gene self, the bacterial strain output of acquisition all has raising in various degree than wild strain.Wherein dual-gene with expression sgcR1 and sgcR2 behind ermE*p, and expressing sgcR3 after self promotor, to have obtained two strain bacterial strain output the highest, called after styreptomyces globispotus strain ER1R2C and styreptomyces globispotus strain R3C, improve the output 206% and 35.7% of lidamycin (LDM) (as Figure 11 b, 11c) than wild strain respectively.

Description of drawings

Fig. 1: SgcR1, SgcR2 and SgcR3 homology compare of analysis, wherein, Fig. 1 a is that the consensus sequence of SgcR1, MdpR1, NcsR6 and StrR compares; Fig. 1 b is that the consensus sequence of SgcR2, CalR7, MdpR2, NcsR5 compares; Fig. 1 c is that the consensus sequence of SgcR3, NcsR7, TyR compares.

Fig. 2: the biological synthesis gene cluster ORF compare of analysis of nonatomic ring class enediyne microbiotic C-1027, neocarzinostatin and maduropeptin.

Fig. 3: Fig. 3 a is the blocking-up strain construction synoptic diagram of sgcR3, and Fig. 3 b, 3c are respectively the Southern blot results of hybridization of making probe with the AB arm.

The blocking-up bacterial strain bacteriostatic activity of Fig. 4: sgcR1, sgcR2 and sgcR3 detects.

Fig. 5: Fig. 5 a is the structure synoptic diagram of crossing expression plasmid of sgcR1, sgcR2 and sgcR3,

Fig. 5 b has added sgcR1, the sgcR2 of strong promoter ermE*p and the expression plasmid structure synoptic diagram of sgcR3, and Fig. 5 c cuts check analysis figure for the enzyme of each plasmid.

Swimming lane M: λ DNA EcoRI+HindIII marker (2,1226bp, 5,418bp, 4,973bp, 4,268bp, 3,520bp, 2,027bp, 1,904bp, 1,584bp, 1,375bp, 947bp, 831bp, 564bp); Swimming lane 1:pKCR3/EcoI+XbaI; The pcr amplified fragment of swimming lane 2:sgcR3 and self promoter region; Swimming lane 3:pKCER1R2/BamHI+EcoRI; Swimming lane 4:pKCER1/BamHI+EcoRI; Swimming lane 5:pKCR1R2/BamHI+EcoRI; Swimming lane 6:pKCR1/BamHI+EcoRI; Swimming lane 7:pKCER2/BamHI+EcoRI.

The bacteriostatic activity of crossing expression strain of Fig. 6: sgcR1, sgcR2 and sgcR3 detects.

The bacteriostatic activity of the cross complementary bacterial strain of Fig. 7: sgcR1, sgcR2 and sgcR3 detects.

Fig. 8: sgcR1, sgcR2 and the sgcR3 expression in wild strain, R1R2KO and R3KO blocking-up bacterial strain.

Fig. 9: sgcA1, sgcC4, sgcD6 and the sgcE8 expression in wild strain, R1R2KO and R3KO blocking-up bacterial strain.

Figure 10: gel blocking method analysis SgcR3 albumen combines with sgcR1 and sgcR2 promoter region.Swimming lane 1, sgcR1 and sgcR2 promoter fragment; Swimming lane 2-6, and sgcR1 and sgcR2 promoter fragment+SgcR3 albumen (123ng, 153ng, 307ng, 615ng, 1230ng); Swimming lane 7, sgcR1 and sgcR2 promoter fragment+SgcR3 albumen (307ng)+200 times unlabelled sgcR1 and sgcR2 promoter fragment.

Figure 11: the HPLC of superior strain output analyzes.

Embodiment

Embodiment 1sgcR1, sgcR2 and sgcR3 gene coded sequence and acquisition approach

The nucleotide coding sequence of sgcR1, sgcR2 and sgcR3 gene known (GenBank accession number AY048670), the present invention extracts the genomic dna of the wild bacterium of lidamycin (LDM) by the method [37] of streptomycete operational manual, and as template design primer amplification sgcR1, sgcR2 and sgcR3 nucleotide sequence, primer sees Table 2.The pcr amplification condition is: 95 ℃ 5 minutes; 95 ℃ 50 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 29 circulations; 72 ℃ 4 minutes.Amplification gained fragment cloning checks order (worker company is given birth in Shanghai) in commercial carrier pGEM-T (U.S. Promage company), and is in full accord during the result shows resulting fragment nucleotide sequence and reports.The gene coded sequence of sgcR1, sgcR2 and sgcR3 is illustrated respectively in SEQ IDNo.1, among SEQ ID No.2 and the SEQ ID No.3.

The structure and the lidamycin (LDM) output thereof of embodiment 2. blocking-up bacterial strains detect

Adopt the method for homologous recombination double exchange, gene function is blocked.Design comprises two dna fragmentations (being called forearm and postbrachium) of goal gene upstream and downstream respectively, and PCR method amplifies two arms, and (the PCR condition is generally: 95 ℃ 5 minutes; 95 ℃ 50 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 29 circulations; 72 ℃ 4 minutes).With thiostrepton resistant gene (thiostrepton resistance gene, tsr) insert between two arms, and be cloned into the suicide plasmid pOJ260 that is used for conjugal transfer or the multiple clone site of temperature sensitive type matter pKC1139, be converted among the intestinal bacteria ET12567/pUZ8002, conjugal transfer is to styreptomyces globispotus strain C-1027, two arms can be recombinated with homologous sequence in the wild strain genome, reorganization back thiostrepton resistant gene is replaced the gene between the two arm homologous regions in the genome, obtain to take place the zygote of double exchange with the antibiotics resistance screening, it is the goal gene mutant strain, gene function is blocked, and the blocking-up bacterial strain is called after styreptomyces globispotus strain R2KO after Southern blot checking, R1R2KO and R3KO.

R3KO is the structure of three blocking-up of example explanation strain with sgcR3 blocking-up strain styreptomyces globispotus strain:

Adopt the method amplification of PCR to be used for two arm WAB and WCD of double exchange, the base scope of WAB and WCD is 76,800bp～78, and 205bp and 78,713bp～80,151bp is positioned at the both sides (primer sequence is seen the primer table) of sgcR3 gene.With the total DNA of wild strain is template, and the Pfu archaeal dna polymerase carries out the PCR reaction, draws EcoRI and BglII for two sections at WAB, and BglII and HindIII are introduced in the WCD two ends.

The PCR product of forearm WAB and postbrachium WCD uses the Taq archaeal dna polymerase to add dATP at the product afterbody after reclaiming purifying through glue, adds the A product and links to each other with pGEM-T respectively, connects product and is converted into E.coli TG1 competent cell respectively.The recombinant plasmid that obtains carries out enzyme and cuts checking, and correct back is to inserting sequencing fragment in the recombinant plasmid, and the sequence of reporting among sequencing result and the GenBank is in full accord.

Plasmid pUC18 reclaims big fragment behind EcoRI and HindIII double digestion, be connected with postbrachium WCD fragment with forearm WAB, obtains recombinant plasmid pUC18WABCD.Plasmid pUCWABCD and reclaims tsr fragment (1.0kb) after the BclI enzyme is cut pIJ680 and is connected after the BglII enzyme is cut, and obtains recombinant plasmid pUCWABtsrWCD.Plasmid pOJ260 is the suicide plasmid that has the A Pu mycin resistant gene, and no streptomycete replicon can import in the streptomycete by the mode of conjugal transfer, could exist in the streptomycete host after having only plasmid and genome generation homologous recombination.POJ260 reclaims big fragment behind EcoRI and HindIII double digestion, be connected with postbrachium WCD fragment with the forearm WAB, the tsr that downcut from plasmid pUCWABtsrWCD, and (Fig. 3 a) to obtain recombinant plasmid pOJR3KO.

Plasmid pOJR3KO is transformed to E.coli ET12567/pUZ8002 earlier, imports in the streptomycete by conjugal transfer (method of conjugal transfer sees streptomycete operational manual [37] for details) again.Utilize antibiotics resistance screening zygote, single cross may take place and change in the clone that can grow in the flat board that contains thiostrepton and A Pu mycin simultaneously; If can only grow containing on the flat board of thiostrepton, in the flat board that contains the A Pu mycin, can not grow, then double exchange may take place.

Extract the correct zygote genomic dna of resistance checking that obtains, use different endonuclease digestions respectively, and carry out Southern blot checking with different probes.The AB arm is cut with the BamHI enzyme as probe, and wild strain should be hybridized and obtained 4, and 176bp band (Fig. 3 b swimming lane 1), mutant strain should be hybridized and obtained 4, and 668bp is hybridized band (Fig. 3 b swimming lane 2).SgcR3 genetically deficient part is as probe, cut with the EcoRV enzyme, wild strain should be hybridized and obtained 5,339bp band (Fig. 3 c swimming lane 1), blocked mutant amixia band (Fig. 3 c swimming lane 3) is cut with the BclI enzyme, and wild strain should be hybridized and obtained 9,774bp band (Fig. 3 c swimming lane 2), blocked mutant amixia band (Fig. 3 c swimming lane 4).Southern blot results of hybridization is consistent with expection, blocking-up strain called after styreptomyces globispotus strain R3KO.

SgcR2 blocking-up strain R2KO and sgcR1, construction process and the R3KO basically identical of the dual-gene blocking-up strain of sgcR2 R1R2KO.

Used and constructed bacterial strain and plasmid see Table 1 among the present invention, and used PCR primer sees Table 2.

Table 1: bacterial strain and plasmid tabulation

Table 2: primer tabulation

Each bacterial strain is cultivated 7-8 days in the inclined-plane after, dig down the inclined-plane that carries disease germs of about 2 * 1cm size, be seeded in the fermention medium, 28 ℃ of shaking culture 48 hours with inoculating needle.Inoculum size with 2～10% is seeded in the second order fermentation substratum, 28 ℃ of shaking culture 72～168 hours.Get fermented liquid centrifugal (3,000rpm, 5min), supernatant carries out bacteriostatic activity and detects.With Bacillus subtilus (CMCC (B) 63501) gemma suspension, the inoculum size with 0.4% is seeded to and detects in the substratum.In flat board, add the germ-carrying detection substratum of 15mL, after solidifying the Oxford cup is placed on the substratum gently.Fermented liquid supernatant 180 μ l are added in the cup of Oxford, and the liquid level in each glass is consistent, 37 ℃ of overnight incubation, the production of observation inhibition zone.

The result shows that styreptomyces globispotus strain R2KO, R1R2KO and R3KO fermented liquid do not have bacteriostatic activity, does not produce lidamycin (LDM) (as Fig. 4).

The expression study excessively of embodiment 3.sgcR1, sgcR2 and sgcR3

The design primer, total DNA is a template with the wild bacterium of lidamycin (LDM), the PCR method amplification comprises the upstream promoter zone of sgcR1, sgcR2 and sgcR3 gene nucleotide series and gene self and introduces restriction enzyme site at the fragment two ends that primer sees Table 2.The fragment that amplification obtains at first is cloned in the pGEM-T carrier, determine through checking order correct after, be cloned into the multiple clone site of pKC1139, obtain to be used for plasmid pKCR1, the pKCR1R2, the pKCR3 that express, plasmid construction figure sees Fig. 5 a.Simultaneously, the sgcR1 that will increase, sgcR2 and sgcR1R2 fragment and strong promoter ermE*p (450bp[37]) fragment is cloned into the multiple clone site of pKC1139 jointly again, and place strong promoter ermE*p downstream, obtain plasmid pKCER1, pKCER2 and pKCER1R2.Plasmid construction figure sees Fig. 5 b.

Plasmid pKCER2 (as Fig. 6 b), pKCR1 (as Fig. 6 c), pKCER1 (as Fig. 6 d), pKCR1R2 (as Fig. 6 e), pKCER1R2 (as Fig. 6 f) and pKCR3 (as Fig. 6 g) are imported in the lidamycin (LDM) wild strain by conjugal transfer, cut checking back (seeing Fig. 5 c) by enzyme, carry out fermenting experiment and bacteriostatic activity and detect.The result shows, the expression strain of crossing of sgcR1, sgcR2 and sgcR3 all (a) has improved the output of lidamycin (LDM) as Fig. 6 to some extent than wild strain.Further having proved conclusively sgcR1, sgcR2 and sgcR3 thus is positive regulating gene.

The research of the regulation relationship of embodiment 4.sgcR1, sgcR2 and sgcR3

The cross complementary of sgcR1, sgcR2 and sgcR3

Plasmid pKCR3 is imported among the styreptomyces globispotus strain R1R2KO by conjugal transfer, plasmid pKCER1, pKCR1R2, pKCER1R2 are imported among the styreptomyces globispotus strain R3KO by conjugal transfer, after cutting checking by PCR checking and enzyme, carry out the detection of fermenting experiment and bacteriostatic activity.The result shows that pKCR3 imports among the R1R2KO, the generation that can not recover lidamycin (LDM), and the importing of pKCER1, pKCR1R2 and pKCER1R2 has recovered the generation of lidamycin (LDM) among the R3KO (as Fig. 7).The function of the complementary sgcR3 of sgcR1, sgcR1R2 energy shows that sgcR1, sgcR2 are in the downstream position of sgcR3 in the cascade regulation and control.

Gene expression research in the blocking-up bacterial strain

Respectively regulatory gene sgcR1, sgcR2 and the transcriptional level of sgcR3 in R1R2KO and R3KO bacterial strain are analyzed with real-time quantitative RT-PCR.The result shows, sgcR1, sgcR2 transcriptional level in the R3KO bacterial strain obviously reduces than wild strain, and sgcR3 transcriptional level and wild strain suitable (as Fig. 8) in the R1R2KO bacterial strain, verified the cross complementary result of experiment, show sgcR3 blockage effects the expression of sgcR1 and sgcR2, sgcR3 is positioned at sgcR1 and sgcR2 upstream in lidamycin (LDM) biosynthetic controlling network.

Analytical structure gene sgcA1, sgcC4, sgcD6 and sgcE8 be gene transcription level in wild strain, R1R2KO, R3KO blocking-up bacterial strain, the result shows, the expression of these structure genes all obviously reduces (as Fig. 9) in the blocking-up bacterial strain, illustrates that sgcR1, sgcR2 and sgcR3 gene are by the transcriptional control of the biosynthesis gene of four structural units in the lidamycin (LDM) biosynthetic pathway being controlled the biosynthesizing of lidamycin (LDM).

The gel blocking method is analyzed the proteic target sequence of SgcR3

Genomic dna with the lidamycin (LDM) wild strain is a template, and the encoding sequence of amplification sgcR3 is cloned into plasmid pET-16b, and enzyme is cut checking back transformed into escherichia coli BL21 (DE3).Recombinant bacterial strain is 37 ℃ of overnight incubation in the LB substratum, and 1% culture is inoculated in the fresh LB substratum and continues to be cultured to OD ₆₀₀0.6 IPTG induces after 6 hours and collects thalline.Expression product SgcR3 albumen merges ten Histidines at the N end, and with the His tag purification column purifying of GE company, dialysis, desalination, cold dried back are in-80 ℃ of preservations after the expression in intestinal bacteria.

Design primer amplification sgcR1R2 upstream promoter district about 450bp fragment is as probe, carries out gel retardation assasy with the test kit of PIERCE company.The result probe migration band (as Figure 10) occurs after showing electrophoresis, shows that SgcR3 albumen can be incorporated into the promoter region of sgcR1R2 in external specificity.

By above experimental result as can be known, the function of the complementary sgcR3 of sgcR1, sgcR1R2 energy, sgcR3 is positioned at sgcR1 and sgcR2 upstream in lidamycin (LDM) biosynthetic controlling network, the expression of may command sgcR1 and sgcR2, SgcR3 albumen is by regulating and control the expression of sgcR1 and sgcR2 in conjunction with the promoter region of sgcR1R2.

The HPLC of embodiment 5. superior strains analyzes

Picking out bacterial strain that two strain lidamycin (LDM) output significantly improve from the bacterial strain of above-mentioned structure carries out HPLC and analyzes.Adopt strong promoter and high copy number plasmid, in wild strain, import positive regulatory factor sgcR1 and sgcR2, called after styreptomyces globispotus strain ER1R2C.Adopt self promotor and high copy number plasmid sgcR3 to be imported wild strain, called after styreptomyces globispotus strain R3C.These two bacterial strains are fermented, and separation and Extraction lidamycin (LDM) chromophoric group, its output analyzed with the HPLC method.

The fermented liquid of each bacterial strain is centrifugal, and (3,000rpm 5min) gets supernatant, 4 ℃ of 100% ammonium sulfate saturation ratio precipitation, transfers to pH4.0 with 0.1N HCl, standing over night.The centrifuging and taking precipitation, with 0.1M pH8.0 potassium phosphate buffer dissolution precipitation, ethyl acetate extracting, vacuum volatilizes, and is dissolved in methyl alcohol.HPLC analysis Sweden Kromasil C-18 reversed-phase column (5 μ m, 150 * 4.6mm, Bohus, SE), moving phase is that (50: 50, v/v), flow velocity was that 1.0ml/min detects at UV absorption value 350nm to 20mM potassiumphosphate (pH 6.86)/acetonitrile.

The result shows that ER1R2C (as Figure 11 b) and R3C (as Figure 11 c) improve the output 206% and 35.7% of lidamycin (LDM) respectively than former strain.

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Sequence table

＜110〉biotechnology research institute

＜120〉by the genetic manipulation of biosynthetic controlling gene being made up the method for lidamycin (LDM) superior strain

<130>IDC08068

<160>43

<170>PatentIn?version?3.2

<210>1

<211>1080

<212>DNA

<213>sgcR1

<400>1

gtgaaaagtg?actctgccca?acgcgccgtg?gagcgatcac?gccgtgtcgt?acggatcgat 60

gaactcattc?ccgccgattc?cccgcgcctg?aacggaatcg?atcgttccca?tgtgcagcgc 120

ctcgcgaccg?tgtacgcgtc?cctgccgccg?gtcctggtgc?accgcccgac?catgcgggtc 180

gtcgacggca?tgcaccgcat?cggcgcggcc?cgcctgaagg?ggctggacac?ggtcgaggtc 240

accttcttcg?agggcgccga?ggagcaggtg?ttcctgcgtt?ccgtcgcggc?gaacatcacc 300

aacggcctgc?cgttgtcggt?ggccgaccgc?aagaccgccg?cggcccgcat?tctggcctcc 360

cacccgaccc?tgtccgaccg?cgcggtcgcc?gcacacgtcg?gcctcgacgc?caagaccgtg 420

gcgggggtac?ggacgtgttc?agccgcgggt?tctccgctgc?tgaacatgcg?caccggggcg 480

gacggccgcg?tccacccgtt?ggaccgcacc?gccgaacgcc?tgcacgcggc?cgcgctgctg 540

acccaggacc?cgggactccc?gttgcgctcc?gtcgtcgagc?agacggggct?gtcgctgggc 600

acggcccacg?acgtccgccg?tcggctgctg?cggggcgagg?acccggtccc?gcagaaccgg 660

cagagcgcga?tgctggagcc?gggactcgcc?ccgcagaaga?aggcgacggc?caagccgccc 720

gtcggcccgg?ccgcccgtcc?ggtcccgaag?gtgccgcccg?ccgtcgccgg?caggccgccg 780

gtgtcaccgc?ggtcccgggc?cccgctggag?gcgctgcgca?agctctccaa?cgacccctcc 840

ctgcgccact?ccgaccaggg?gcgcgaactc?atgcgctggc?tgcacaaccg?gttcgtcgtc 900

gacgaggcgt?ggcgccggcg?cgcggacgcg?gtcccggccc?actgcgtcga?ctcgatggcg 960

gagctggcgc?agcactgctc?ggacgcctgg?caccggttcg?ccgaggagat?ggttcggcgc 1020

cggcacagcg?ccgcggccga?cggctccgga?ctccgcacga?ctcagccaac?tcgccgttga 1080

<210>2

<211>855

<212>DNA

<213>sgcR2

<400>2

atgaccacga?acaccatcga?ggacgcggtc?cgccgggtcg?tcgagtacat?gcacgtcaac 60

ctgggtcaga?acctcacgat?cgatgacatg?gcgcgcacgg?cgatgttcag?caagttccat 120

ttcacccgca?tcttccgcga?agtcaccggt?acctctcccg?ggcgtttcct?gtccgcctta 180

cggattcagg?aggccaagag?acttctcgtg?cacactgcac?tcagtgtggc?cgatatcagc 240

agtcaggtcg?gctacagcag?tgtcggtact?ttcagttctc?gcttcaaggc?ctgtgtgggg 300

ctttccccga?gcgcctatcg?cgacttcggc?ggggtgcagc?cgggttttcc?ctccgccgcg 360

gcccgtctca?ctcccaccgc?gcacaatccc?tccgtgcgcg?gccgcattca?ctccgccccg 420

ggtgacaggc?ccggaaggat?cttcgtgggc?ctgttccccg?gcaggatgcg?ccagggccgc 480

ccggcgcgct?ggaccgtcat?ggagagtccc?ggggccttcg?agctccggga?cgtgcccgtg 540

ggcacctggc?acatcctggt?ccactccttc?cccgccggac?accggccgca?ccagctcgac 600

tccgaaccgc?tgttgctcgg?gcacagcgga?ccgctcgtgg?tgcaccccgg?tgccctgctc 660

cggccggcgg?acatcctcct?gcgcgcggtg?gacgccctcg?atccaccggt?cctgctggcc 720

cacttcgcgc?tggagagccg?cctcacctcg?ccgtactcac?cgtcatcggt?agccctccgc 780

gcatccgcag?ggagagcatg?ggttcggcaa?ccgcccggtg?tccggcgacg?gtacgcagat 840

cgagatcgcg?ggtga 855

<210>3

<211>1188

<212>DNA

<213>sgcR3

<400>3

atggcggcac?acgacgtgag?gcacgcgcac?ctgccggtct?ccgtacttgc?cgatcagatc 60

ttcgggcacc?tgccgagggc?cgatcagagg?gcgtgggccc?aggcctacct?gacggggctg 120

ctgaccaccg?acggcaagaa?gtcgatcaga?cggatggccg?ccaccgtctc?ccgttcgccc 180

accgcgtcgc?agtcgatgca?ccagttcctc?aatgccagcc?cctgggactg?ggccccggca 240

cgtgccgagc?tgatgagatg?ggtcgagcag?cagctcgatc?cgcgcgcctg?ggtcctgtcg 300

gtggcggtgt?tacgcaagcg?gggagatcac?tcctgcgggg?tgcaccggcg?cttcgtcccc 360

gccaccggac?gctccgtcaa?ctgccagctc?gcggtgggtg?cgttcctctc?caccggcgcg 420

acggccgtgc?ccgtccactg?ggggctcctg?ctgcccgggg?cctgggtcga?ggaccatgcg 480

cgccgcgcac?gggcccgcat?ccccgaatcg?gtcggccagc?gcagcgtcga?gcagcatgcg 540

ctggacctcg?tcgacgcgct?gtgcgggagc?acgcaactga?gtccccggcc?ggtggtggcc 600

gatctgagtc?atcattccgg?ggtcatctcg?ctggtgcgcg?gactcaccgc?caggggacgc 660

gagttcgtcg?tgtcactgcc?gggccggacc?ccggtggtcc?cggtggggag?aacgacggcg 720

cgcggcgcgt?actccgcgaa?cctgcccgcc?cccgtggagg?cacagcgcct?cttcggcctc 780

aagcacgccg?ggcatctcag?acccggcgcc?catgatgggc?acggcggcgc?cgatcgcacc 840

gccacgatga?ccacgctggt?gcggctgccg?caggtgcggc?tggcacgtca?cacgccgcac 900

gccacgtacc?ggatcttcgg?tatgccgtcc?tactcggggc?cccgctccgc?gcggatctgg 960

ctgaccaaca?tgacgcagcg?gcggaccgac?gaactgctgg?cgctggccgg?gctccagcac 1020

cgggcgggca?ccgcggtgca?gtcgctggag?gacgacttcg?gtctgctcga?cttcgaggga 1080

cggtcctttc?ccggctggca?tcaccacatg?actctcgtgt?cggcggcttc?ggcgtacaac 1140

ctgctggagc?tgtccggccg?ccggcccggc?tcccaggagg?ggagctga 1188

<210>4

<211>24

<212>DNA

＜213〉primer

<400>4

cggaatcccc?cctcgctcat?tgtc 24

<210>5

<211>25

<212>DNA

＜213〉primer

<400>5

cgtgatcacg?atggtgttcg?tggtc 25

<210>6

<211>22

<212>DNA

＜213〉primer

<400>6

cgtgatcact?gctggcccac?tt 22

<210>7

<211>22

<212>DNA

＜213〉primer

<400>7

gcaagcttgc?ggacgaagga?ag 22

<210>8

<211>22

<212>DNA

＜213〉primer

<400>8

atgaattcgg?tccagggttt?cg 22

<210>9

<211>21

<212>DNA

＜213〉primer

<400>9

ttgatcagcg?ttgggcagag?t 21

<210>10

<211>22

<212>DNA

＜213〉primer

<400>10

cgtgatcact?gctggcccac?tt 22

<210>11

<211>22

<212>DNA

＜213〉primer

<400>11

gcaagcttgc?ggacgaagga?ag 22

<210>12

<211>28

<212>DNA

＜213〉primer

<400>12

gagaattcac?cacatggaac?atctgctg 28

<210>13

<211>24

<212>DNA

＜213〉primer

<400>13

gaagatctgg?gagcacgcaa?ctga 24

<210>14

<211>29

<212>DNA

＜213〉primer

<400>14

gaagatctct?gatcggcaag?tacggagac 29

<210>15

<211>29

<212>DNA

＜213〉primer

<400>15

gaaagcttgg?tgacagttcc?gaggaggtt 29

<210>16

<211>26

<212>DNA

＜213〉primer

<400>16

atggatcccc?ctcgctcatt?gtcgtc 26

<210>17

<211>26

<212>DNA

＜213〉primer

<400>17

atgaattcca?ccgtaactcc?ctgtcg 26

<210>18

<211>22

<212>DNA

＜213〉primer

<400>18

attgatcagc?cgaggagatg?gt 22

<210>19

<211>21

<212>DNA

＜213〉primer

<400>19

gctctagaca?acgcgatgtg?g 21

<210>20

<211>26

<212>DNA

＜213〉primer

<400>20

atggatccgc?ccctcgctca?ttgtcg 26

<210>21

<211>28

<212>DNA

＜213〉primer

<400>21

atgaattccg?gatcggatgt?gctggtct 28

<210>22

<211>28

<212>DNA

＜213〉primer

<400>22

cagaattctc?tcctacgccc?tgctcatc 28

<210>23

<211>26

<212>DNA

＜213〉primer

<400>23

actctagaag?aaccgctcgg?cctgtc 26

<210>24

<211>24

<212>DNA

＜213〉primer

<400>24

gcatatggcg?gcacacgacg?tgag 24

<210>25

<211>25

<212>DNA

＜213〉primer

<400>25

aggatcccag?ctcccctcct?gggag 25

<210>26

<211>20

<212>DNA

＜213〉primer

<400>26

tggtcgaggt?catcaacaag 20

<210>27

<211>20

<212>DNA

＜213〉primer

<400>27

tggacctcga?tgaccttctc 20

<210>28

<211>20

<212>DNA

＜213〉primer

<400>28

cctgatgccg?agccgattgt 20

<210>29

<211>20

<212>DNA

＜213〉primer

<400>29

ggggcatttt?gccgagttcc 20

<210>30

<211>22

<212>DNA

＜213〉primer

<400>30

gaaaagtgac?tctgcccaac?gc 22

<210>31

<211>20

<212>DNA

＜213〉primer

<400>31

cgctgcacat?gggaacgatc 20

<210>32

<211>21

<212>DNA

＜213〉primer

<400>32

accacgaaca?ccatcgagga?c 21

<210>33

<211>20

<212>DNA

＜213〉primer

<400>33

cggaagatgc?gggtgaaatg 20

<210>34

<211>20

<212>DNA

＜213〉primer

<400>34

gtgggcgaac?gggagacggt 20

<210>35

<211>20

<212>DNA

＜213〉primer

<400>35

tgccgagggc?cgatcagagg 20

<210>36

<211>22

<212>DNA

＜213〉primer

<400>36

cagaacatat?tcaaggactg?gc 22

<210>37

<211>21

<212>DNA

＜213〉primer

<400>37

cacttgtact?gtcgggtggt?t 21

<210>38

<211>21

<212>DNA

＜213〉primer

<400>38

ctgtggctac?ggacgagatt?g 21

<210>39

<211>21

<212>DNA

＜213〉primer

<400>39

ccgaacagaa?catccccatc?t 21

<210>40

<211>19

<212>DNA

＜213〉primer

<400>40

tgcgaagacg?agcaacagg 19

<210>41

<211>20

<212>DNA

＜213〉primer

<400>41

caccatcagt?gggacgaagc 20

<210>42

<211>18

<212>DNA

＜213〉primer

<400>42

cgatggctat?gcggctca 18

<210>43

<211>18

<212>DNA

＜213〉primer

<400>43

aggacggcac?ctgcttcc 18

Claims (10)

1. improve lidamycin (LDM) and produce the method that bacterial strain is produced the output of lidamycin (LDM), it comprises making and is selected from sgcR1 that the expression of one or more gene in sgcR2 and the sgcR3 gene in lidamycin (LDM) production strain improves.

2. the method for claim 1, it comprises improves the expression in lidamycin (LDM) production strain of sgcR1 gene and sgcR2 gene.

3. the method for claim 1, it comprises improves the expression of sgcR3 gene in lidamycin (LDM) production strain.

4. the method for claim 1, it comprise be selected from sgcR1, the carrier that one or more gene in sgcR2 and the sgcR3 gene is operably connected imports to realizes in the lidamycin (LDM) production strain that described expression improves.

5. the process of claim 1 wherein that the plasmid pKCR3 that will contain the plasmid pKCER1R2 of sgcR1 gene and sgcR2 gene or contain sgcR3 imports to by conjugal transfer realizes in the lidamycin (LDM) production strain that described expression improves.

6. be selected from sgcR1, one or more gene in sgcR2 and the sgcR3 gene is used for improving lidamycin (LDM) in preparation and produces the purposes of recombinant vectors that bacterial strain is produced the output of lidamycin (LDM).

7. the purposes of claim 6, recombinant vectors wherein is to be operably connected to be selected from sgcR1 one or more gene in sgcR2 and the sgcR3 gene in carrier pKC1139.

8. the purposes of claim 6, recombinant vectors wherein are the plasmid pKCER1R2 of sgcR1 and sgcR2 gene of being operably connected in pKC1139.

9. the purposes of claim 6, recombinant vectors wherein are the plasmid pKCR3 of sgcR3 gene of being operably connected in pKC1139.

10. it is that the lidamycin (LDM) of culture presevation CGMCCNo.0135 is produced bacterial strain that the purposes of claim 6, lidamycin (LDM) are wherein produced bacterial strain.

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