CN101724672B - 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 built 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) in 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 to carry out expressing to improve the method for lidamycin (LDM) output to positive regulating gene.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 anti-tumor activity of finding up to now, and is stronger more than 10000 times [2] than Zorubicin commonly used clinically.Through the new drug researches of more than ten years and exploitation, at present lidamycin (LDM) has entered the clinical II phase and has studied [3], is expected to develop into the new and effective antitumor drug with China's independent intellectual property right.

Lidamycin (LDM) belongs to Enediyne Antibiotic, is comprised of small molecules chromophoric group and apoprotein, and chromophoric group has ring-type enediyne structure, is that it has the active centre of extremely strong lethal effect to tumour cell.Enediyne Antibiotic 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 mechanism of action of Enediyne Antibiotic is unique, and the rear enediyne core of being combined with DNA is reset, and causes into the ring aromizing and forms a temporary benzenoid diradical compound, and unsettled intermediate product impels DNA single chain or double-strand break [4,5].Enediyne Antibiotic is because of its unique 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 lidamycin (LDM) is carrying out clinical study in China, 1997 neocarzinostatin poly (styrene-co-maleic acid) conjugate (trade(brand)name SMANCS) in Japan listing [6], be used for clinical therapy of tumor more than ten years in Japan, and calendar year 2001 Calicheamicin-CD33 antibody coupling matter (trade(brand)name Mylotarg) becomes first by the Monoclonal anti EGFR antibody leading chemotherapy [7] of FDA approval for oncotherapy.Lidamycin (LDM) can develop into new and effective antitumor drug due to its strong biological activity, also can be used as " bullet ", makes targeted drug miniaturization, high efficiency, and wide prospect has been showed in its research and application.Although 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 traditional breeding method is effective within the specific limits, superior strain directed, that build reasoningly lidamycin (LDM) need to be to the deep parsing of its biosynthesizing mechanism.

The lidamycin (LDM) biological synthesis gene cluster is cloned (GenBank accession number AY048670).The lidamycin (LDM) biological synthesis gene cluster is 75kb approximately, totally 56 genes.Infer that its chromophoric biosynthetic process is at first to synthesize 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 the research of its biosynthetic controlling mechanism be there is not yet report.

Streptomycete can produce a large amount of secondary metabolites, has important value in medicine and agriculture production, and approximately 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, activating transcription factor actII-ORF4[18 as special in 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 Cloning and sequencing of increasing microbiotic biological synthesis gene cluster, find to have a plurality of approach specificity regulatory genes in some larger 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 the biological synthesis gene cluster of these 3 Enediyne Antibiotics.

As everyone knows, strain improvement is the key that improves microbiotic output always.Traditional breeding method blindness is large, and workload is large, and efficient is low.Utilize the breeding of metabolic engineering method, design cell on the basis of metabolic analysis, and then use the instrument of modern molecular genetics to come engineered cells, can rationality, output improved significantly.The means of metabolic engineering breeding on the one hand can the reconstruction structure gene, can improve antibiotic output [35] by the 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 the expression of a plurality of important feature genes, finally affects antibiotic output.The method of the positive regulatory factor of overexpression is to import the regulatory gene of other copy in wild strain, can be take integrative plasmid as carrier, the regulatory gene of single copy is incorporated on genome, realize overexpression, can be also carrier by self-existent multiple copied plasmid, import the regulatory gene of a plurality of copies, can also add by the upstream of regulatory gene in expression plasmid the controlling element of promote expressing (as the strong promoter of constitutive expression etc.), 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 sgcR3, by improving gene copy number and utilizing the strong promoter of constitutive expression these three positive regulating genes to be carried out the method for expression, 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 lidamycin (LDM) biological synthesis gene cluster in GenBank.Albumen to these ORF coding carries out finding after homology analysis, and the albumen of sgcR1 coding and the known consistence of modulin StrR (GenBank accession number CAA07385) in length range are 44%, and similarity is 59% (as Fig. 1 a); Modulin (GenBank accession number ABP55137) consistence in length range of the albumen of sgcR2 coding and an AraC/XylS family is 50%, and similarity is 62% (as Fig. 1 b); The albumen of sgcR3 coding and the known consistence of modulin TylR (GenBank accession number AAF29380) in length range are 33%, and similarity is 47% (as Fig. 1 c).In addition, to C-1027, the neocarzinostatin that is all Enediyne Antibiotic, 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, there is the homologous gene of 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 Enediyne Antibiotic biosynthesizing.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 genes involved thereof, and sequence in the gene forms also closely similar (as Fig. 2), shows that the biosynthetic transcriptional control of this class microbiotic has general character.

The present invention is studied the adjusting function of sgcR1, sgcR2 and sgcR3 in gene cluster.At first, respectively regulatory gene in gene cluster is carried out gene disruption by the homology double exchange, analyze it to the biosynthetic impact of lidamycin (LDM), find after sgcR1R2 (sgcR1 and sgcR2), sgcR2 and 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, in the blocking-up bacterial strain, the expression of these structure genes all obviously reduces (as Fig. 9), 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 respectively wild strain 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 impact of lidamycin (LDM), 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, result shows, in sgcR3 blocking-up strain, the expression level of sgcR1 and sgcR2 all significantly reduces, and the expression uninfluenced (as Fig. 8) of sgcR3 in 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, further illustrating SgcR3 is by regulating and controlling the expression of transcribing sgcR1 and sgcR2 (as Figure 10) of sgcR1R2.

The present invention conducts in-depth research the regulation relationship between them again after 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 fermented liquid always.Conventional strain improvement method adopts " random mutation+directed screening ", to be based upon on the basis that the secondary metabolites generation structure is processed as "black box", if the bacterial classification of the uncomprehending meta-bolites of gene of the enzyme that seed selection pathways metabolism and participation are synthesized, it is effective adopting this method.But it is large that the shortcoming of this method is 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 the amplification of approach fragment or rate-limiting step enzyme gene) of structure gene, improves the speed of product cross-film output etc.On the other hand, from the viewpoint of regulating, metabolic engineering can start with to improve the throughput of secondary metabolite from many aspects, can increase antibiotic growing amount by the copy number that increases positive regulator gene, also can remove those negative regulator (repressor), so that this approach realizes constitutive expression.In addition, regulatory gene is become derivable, 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 come the overexpression positive regulating gene with the method for strong promoter, thereby increasing 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 the 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, the sgcR2 that the present invention is used and sgcR3 gene coded sequence are known array [8], according to the design of the known array in GenBank primer (seeing the primer table), take lidamycin (LDM) wild mushroom genomic dna as 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 builds 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 copies and does not rely on chromosomal copying, can exist with the form of multiple copied, for example, plasmid pKC1139.In addition, carrier can be also a kind of like this carrier, and after introducing host cell, it is incorporated in genome and together with the karyomit(e) of integrating and copies, 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 copies, the replication orgin of pSG5, pSGL1 in streptomycete.

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

Target gene to be expressed and selected prokaryotic host cell are depended in the selection of suitable expression vector.Be applicable to expression vector of the present invention and include 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 namely identified by host cell for the expression of nucleotide sequence.Contain the transcriptional control sequence that mediates expression of polypeptides in 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 the born of the same parents of coding and host cell homology or allos or in born of the same parents obtains the gene of polypeptide.include but not limited to the promotor that obtains: aphD (aminoglycoside phosphotransferase gene) for produce available promotor that in bacterium, guiding recombinant expression plasmid of the present invention is transcribed at lidamycin (LDM) from following gene, 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 shallow Streptomyces glaucoviolaceus) etc. and sudden change.

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

The high copy number plasmid pKC1139 that adopts self-replicating in streptomycete in the present invention is as carrier, and the promotor of the ermE*p of constitutive expression and gene self is as promotor.Import respectively some genes or a plurality of gene in positive regulating gene sgcR1, sgcR2 and sgcR3 in wild strain, coordinate 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 after 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 respectively the output 206% and 35.7% (as Figure 11 b, 11c) of lidamycin (LDM) than wild strain.

Description of drawings

Fig. 1: SgcR1, SgcR2 and SgcR3 sequence analysis are analyzed, and 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, Ca1R7, 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 schematic 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 schematic diagram of crossing expression plasmid of sgcR1, sgcR2 and sgcR3, and Fig. 5 b has added sgcR1, the sgcR2 of strong promoter ermE*p and the expression plasmid structure schematic 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: the gel blocking method is analyzed the combination of SgcR3 albumen and sgcR1 and sgcR2 promoter region.Swimming lane 1, sgcR1 and sgcR2 promoter fragment; Swimming lane 2-6, 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 lidamycin (LDM) wild mushroom 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 is cloned in commercial carrier pGEM-T (U.S. Promage company) check order (Shanghai living work company), and is in full accord during result shows resulting fragment nucleotide sequence and reports.The gene coded sequence of sgcR1, sgcR2 and sgcR3 is illustrated respectively in SEQ IDNo.1, in SEQ ID No.2 and SEQ ID No.3.

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 respectively two DNA fragmentations (being called forearm and postbrachium) of goal gene upstream and downstream, 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 multiple clone site for suicide plasmid pOJ260 or the temperature sensitive type matter pKC1139 of conjugal transfer, be converted in 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, after restructuring, the thiostrepton resistant gene is replaced the gene between two arm homologous regions in genome, obtain to occur the zygote of double exchange with the antibiotics resistance screening, it is the goal gene mutant strain, gene function is blocked, the blocking-up bacterial strain is called after styreptomyces globispotus strain R2KO after Southern blot checking, R1R2KO and R3KO.

Structure take sgcR3 blocking-up strain styreptomyces globispotus strain R3KO as three blocking-up strains of example explanation:

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～78,205bp and 78,713bp～80, and 151bp is positioned at the both sides (primer sequence is seen the primer table) of sgcR3 gene.Take the total DNA of wild strain as template, the Pfu archaeal dna polymerase carries out the PCR reaction, introduces EcoRI and BglII two sections of WAB, and WCD introduces BglII and HindIII in 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 is connected with pGEM-T respectively, connects product and is converted into respectively E.coli TG1 competent cell.The recombinant plasmid that obtains carries out enzyme and cuts checking, and correct rear to Insert Fragment order-checking in recombinant plasmid, the sequence of reporting in sequencing result and GenBank is in full accord.

Plasmid pUC18 reclaims large fragment after 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 with the A Pu mycin resistant gene, without the streptomycete replicon, can import in streptomycete by the mode of conjugal transfer, could exist in the streptomycete host after only having plasmid and genome generation homologous recombination.POJ260 reclaims large fragment after 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 first is transformed to E.coli ET12567/pUZ8002, then imports in streptomycete by conjugal transfer (method of conjugal transfer sees streptomycete operational manual [37] for details).Utilize antibiotics resistance screening zygote, single cross may occur 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, can not grow in the flat board that contains the A Pu mycin, double exchange may occur.

Extract the correct zygote genomic dna of resistance checking that obtains, use respectively different endonuclease digestions, 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 4,176bp band (Fig. 3 b swimming lane 1), and mutant strain should be hybridized and obtained 4,668bp hybridization 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 of the dual-gene blocking-up strain of sgcR2 R1R2KO are basically identical.

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

Table 1: bacterial strain and plasmid list

Table 2: primer list

Each bacterial strain is cultivated 7-8 days in the inclined-plane after, dig down the approximately inclined-plane that carries disease germs of 2 * 1cm size with inoculating needle, be seeded in fermention medium, 28 ℃ of shaking culture 48 hours.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 substratum.The detection substratum that adds 15mL to carry disease germs in flat board is placed the Oxford cup on substratum after solidifying 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.

Result shows, styreptomyces globispotus strain R2KO, R1R2KO and R3KO fermented liquid do not produce lidamycin (LDM) (as Fig. 4) without bacteriostatic activity.

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

The design primer, take the total DNA of lidamycin (LDM) wild mushroom as template, the PCR method amplification comprises the upstream promoter zone of sgcR1, sgcR2 and sgcR3 gene nucleotide series and gene self and at fragment two ends introducing restriction enzyme site, primer sees Table 2.At first the fragment that obtains that increases is cloned in the pGEM-T carrier, after determining correctly through checking order, is cloned into the multiple clone site of pKC1139, obtains to be used for plasmid pKCR1, pKCR1R2, the pKCR3 of expression, and plasmid construction figure sees Fig. 5 a.The sgcR1 that will increase again simultaneously,, sgcR2 and sgcR1R2 fragment and strong promoter ermE ^*P (450bp[37]) fragment jointly is cloned into the multiple clone site of pKC1139, and is placed in strong promoter ermE ^*The p downstream obtains 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 rear (seeing Fig. 5 c) by enzyme, carry out fermenting experiment and bacteriostatic activity and detect.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 thus sgcR1, sgcR2 and sgcR3 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 in styreptomyces globispotus strain R1R2KO by conjugal transfer, plasmid pKCER1, pKCR1R2, pKCER1R2 are imported in styreptomyces globispotus strain R3KO by conjugal transfer, verify and after enzyme cuts checking, carry out fermenting experiment and bacteriostatic activity and detect by PCR.Result shows, pKCR3 imports in R1R2KO, the generation that can not recover lidamycin (LDM), and the importing of pKCER1, pKCR1R2 and pKCER1R2 has recovered the generation of lidamycin (LDM) in 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 Cascade Regulation.

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.Result shows, sgcR1, sgcR2 obviously reduce than wild strain in R3KO bacterial strain transcription level, and sgcR3 is at R1R2KO bacterial strain transcription level and wild strain suitable (as Fig. 8), verified the result of cross complementary 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, result shows, in the blocking-up bacterial strain, the expression of these structure genes all obviously reduces (as Fig. 9), 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 target sequence of SgcR3 albumen

Take the genomic dna of lidamycin (LDM) wild strain as template, the encoding sequence of amplification sgcR3 is cloned into plasmid pET-16b, and enzyme transforms e. coli bl21 (DE3) after cutting checking.Recombinant bacterial strain is 37 ℃ of overnight incubation in the LB substratum, and 1% culture is inoculated in 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, uses the His tag purification column purifying of GE company after Expression in Escherichia coli, dialysis, desalination, cold dried rear in-80 ℃ of preservations.

Design primer amplification sgcR1R2 upstream promoter district approximately 450bp fragment as probe, carries out gel retardation assasy with the test kit of PIERCE company.Result probe migration band (as Figure 10) occurs after showing electrophoresis, shows that SgcR3 albumen can be at the promoter region of external specific binding in sgcR1R2.

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, can control the expression of sgcR1 and sgcR2, SgcR3 albumen regulates and controls the expression of sgcR1 and sgcR2 by the promoter region in conjunction with 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, import positive regulatory factor sgcR1 and sgcR2, called after styreptomyces globispotus strain ER1R2C in wild strain.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 centrifugal (3,000rpm, 5min) is got supernatant, 4 ℃ of 100% ammonium sulfate saturation ratio precipitation, transfers to pH4.0 with 0.1N HCl, hold 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 analyzes with Sweden Kromasil C-18 reversed-phase column (5 μ m, 150 * 4.6mm, Bohus, SE), and moving phase is 20mM potassiumphosphate (pH6.86)/acetonitrile (50:50, v/v), and flow velocity is that 1.0ml/min detects at UV absorption value 350nm.

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

Reference

1Hu JL, Xue YC, the people such as Xie MY, a kind of new macromole Antitumor antibiotic C-1027 .I finds, produces classification, fermentation and biologic activity (A new macromolecular antitumor antibiotic, C-1027.I.Discovery, the taxonomy of producingorganism of bacterium, fermentation and biological activity), microbiotic magazine (J Antibiot) Tokyo, 1988,41 (11): 1575-1579

2Zhen YS, Ming XY, the people such as Yu B, a kind of new macromole Antitumor antibiotic C-1027 .III anti-tumor biological is learned active (A newmacromolecular antitumor antibiotic, C-1027.III.Antitumor activity), microbiotic magazine (J Antibiot) Tokyo, 1989,42 (8): 1294-1298

3Shao RG, Zhen YS, Enediyne Antitumor Antibiotics lidamycin (LDM): chemistry, biology and pharmacology (Enediyne anticancerantibiotic lidamycin:chemistry, biology and pharmacology), cancer therapy drug pharmaceutical chemistry (Anticancer AgentsMed Chem), 2008,8 (2): 123-131

4Dedon PC, Goldberg IH, the new chemical mechanism (Sequence-specificdouble-strand breakage of DNA by neocarzinostatin involves different chemical mechanisms within astaggered cleavage site) of neocarzinostatin sequence-specific DNA breakage two strands, journal of biological chemistry (J Biol Chem), 1990,265 (25): 14713-14716

5Smith AL and Nicolaou KC, Enediyne Antibiotic (The enediyne antibiotics), pharmaceutical chemistry magazine (J Med Chem), 1996,39 (11): 2103-2117

6Maeda, H., Edo, K., and Ishida, N.E. (1997), neocarzinostatin: a kind of past of cancer therapy drug, present and following (Neocarzinostatin:The Past, Present, and Future of an Anticancer Drug.Springer-Verlag, NY

7Bross PF, Beitz J, the people such as Chen G, approval summary: the WAY-CMA 676 (Approval summary:gemtuzumab ozogamicin in relapsed acute myeloid leukemia) in the myeloid leukemia recurrence, clinical tumor research (Clin Cancer Res), 2001,7 1490-1496

8Liu W, Christenson SD, the people such as Standage S, the biosynthesizing of Enediyne Antibiotic C-1027 (Biosynthesis of theenediyne antitumor antibiotic C-1027), science (Science), 2002,297 (5584): 1170-1173

9Lin S, Van Lanen SG, and Shen B, SgcC3 catalysis (S) in the enediyne antitumor antibiotics-beta-tyrosine-S-carrier proteins specificity chlorination (Regiospecific chlorination of (S)-beta-tyrosyl-S-carrier protein catalyzed by SgcC3 in thebiosynthesis of the enediyne antitumor antibiotic C-1027), U.S. chemical institute magazine (J Am Chem Soc), 2007,129 (41): 12432-12438

10Van Lanen SG, Lin S, the people such as Dorrestein PC, the substrate specificity of VITAMIN B4 enzyme SgcC1 in the biosynthesizing of enediyne Antitumor antibiotic C-1027 (Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesisof the enediyne antitumor antibiotic C-1027), journal of biological chemistry (J Biol Chem), 2006,281 (40): 29633-29640

11 Van Lanen SG, Lin S, and Shen B, the synthetic new branch (C-1027Biosynthesis of the enediyne antitumor antibiotic C-1027involves a new branching point inchorismate metabolism) that comprises the chorismate metabolism of enediyne Antitumor antibiotic C-1027, NAS's journal (Proc NatlAcad Sci USA), 2008,105 (2): 494-499

12Van Lanen SG, Dorrestein PC, Christenson SD etc., the biosynthesizing of Antitumor antibiotic C-1027 beta-amino acid main body is by beta amino acid acetyl carrier proteins mediation (acyl-S-carrier Biosynthesis of the beta-amino acid moiety of theenediyne antitumor antibiotic C-1027featuring beta-amino acyl-S-carrier protein intermediates), U.S. chemical institute magazine (J Am Chem Soc), 2005,127 (33): 11594-11595

13Murrell JM, Liu W, and Shen B, SgcA1 in biochemical definite Enediyne Antitumor Antibiotics C-1027 biosynthesis pathway expresses with crossing the output (Biochemical characterization of the SgcA1alpha-D-glucopyranosyl-1-phosphate thymidylyltransferase from the enediyne antitumor antibioticC-1027biosynthetic pathway and overexpression of sgcA1 in Streptomyces globisporus to improveC-1027production) that sgcA1 has improved C-1027, natural products magazine (J Nat Prod), 2004, 67 (2): 206-213

14Christenson SD, Liu W, the people such as Toney MD, a kind of novel 4-methylideneimidazole-5-one-containing tyrosine aminomutase in Enediyne Antitumor Antibiotics C-1027 biosynthetic pathway (A novel4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibioticC-1027biosynthesis), U.S. chemical institute magazine (J Am Chem Soc), 2003,125 (20): 6062-6063

15Christenson SD, Wu W, the people such as Spies MA, 4-methylideneimidazole-5-one-containing tyrosine aminomutase dynamic analysis in the Enediyne Antitumor Antibiotics biosynthetic pathway (Kinetic analysis of the4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibioticC-1027biosynthesis), biological chemistry (Biochemistry), 2003,42 (43): 12708-12718

16Christianson CV, Montavon TJ, the people such as Van Lanen SG, TYR-2 in Enediyne Antitumor Antibiotics C-1027 biosynthetic pathway, the structure of 3-aminomutase (The structure of L-tyrosine2,3-aminomutase from the C-1027enediyne antitumor antibiotic biosynthetic pathway), biological chemistry (Biochemistry), 2007,46 (24): 7205-7214

17Bibb MJ, the regulation and control of the secondary metabolism of streptomycete (Regulation of secondary metabolism in streptomycetes), microbiology progress (Curr Opin Microbiol), 2005,8 (2): 208-215

18Fernandez-Moreno MA, Caballero JL, the people such as Hopwood DA, the actiorubin gene cluster comprises regulation and control and microbiotic output gene, directly control (The act cluster contains regulatory and antibiotic export genes, directtargets for translational control by the bldA tRNA gene of Streptomyces), cell (Cell) by bldA tRNA, 1991,66 (4): 769-780

19Arias P, Fernandez-Moreno MA, and Malpartida F, the discovery (Characterization of the pathway-specific positive transcriptionalregulator for actinorhodin biosynthesis in Streptomyces coelicolor A3 (2) as a DNA-binding protein) of actiorubin biosynthetic pathway specificity positive regulator protein in Streptomyces coelicolor A3 (2), bacteriology magazine (J Bacteriol), 1999,181 (22): 6958-6968

20Retzlaff L and Distler J, the modulin StrR that Streptomyces griseus streptomycin is expressed has the DNA of a plurality of recognition sites in conjunction with positive regulator protein (The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is aDNA binding activator protein with multiple recognition sites), molecular microbiology (Mol Microbiol), 1995,18 (1): 151-162

21Tomono A, Tsai Y, the people such as Yamazaki H, Streptomyces griseus streptomycin biosynthetic pathway specific transcriptional incitant strR is subject to regulation and control (the Transcriptional control by A-factor of strR of the A factor, the pathway-specific transcriptionalactivator for streptomycin biosynthesis in Streptomyces griseus), bacteriology magazine (J Bacteriol), 2005,187 (16): 5595-5604

22Furuya K and Hutchinson CR, the DnrN albumen of Streptomyces peucetius, pseudoreaction albumen, a kind of DBP (The DnrN protein of Streptomyces peucetius that regulation and control erythromycin produces, a pseudo-response regulator, is aDNA-binding protein involved in the regulation of daunorubicin biosynthesis), bacteriology magazine (J Bacteriol), 1996,178 (21): 6310-6318

23Furuya K and Hutchinson CR, DrrC albumen, a kind of UvrA sample albumen, a kind of DBP (The DrrC protein of Streptomyces peucetius that is induced generation by daunomycin, a UvrA-like protein, is a DNA-binding proteinwhose gene is induced by daunorubicin), FEMS microbiology communication (FEMS Microbiol Lett), 1998,168 (2): 243-249

24Stutzman-Engwall KJ, Otten SL, and Hutchinson CR, the overexpression of daunomycin in the regulation and control of secondary metabolism of Streptomyces and Streptomyces peucetius (Regulation of secondary metabolism in Streptomyces spp.and overproductionof daunorubicin in Streptomyces peucetius), bacteriology magazine (J Bacteriol), 1992,174 (1): 144-154

25Gallo MA, Ward J, and Hutchinson CR, in Streptomyces peucetius, the outer locus of daunomycin gene cluster suppresses the phase shift mutation (The dnrMg ene in Streptomyces peucetius contains a naturally occurring frameshiftmutation that is suppressed by another locus outside of the daunorubicin-production gene cluster) of dnrM, microbiology (Microbiology), 1996,142 (Pt2) 269-275

26Otten SL, Olano C, and Hutchinson CR, dnrO genes encoding DBP is by the generation (The dnrO gene encodes a DNA-bindingprotein that regulates daunorubicin production in Streptomyces peucetius by controlling expression ofthe dnrN pseudo response regulator gene) of the gene regulating daunomycin of control dnrN pseudoreaction albumen in Streptomyces peucetius, microbiology (Microbiology), 2000, 146 (Pt6) 1457-1468

27Otten SL, Ferguson J, and Hutchinson CR, the generation (Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus) of dnrR2 locus regulation and control daunomycin in Streptomyces peucetius, bacteriology magazine (J Bacteriol), 1995,177 (5): 1216-1224

28Bate N, Stratigopoulos G, and

E, the not same-action (Differential roles of two SARP-encoding regulatory genes during tylosin biosynthesis) of two kinds of SARP coding regulatory genes in the tylosin biosynthesizing, molecular microbiology (Mol Microbiol), 2002,43 (2): 449-458

29Bate N, Bignell DR, and Cundliffe E, the relevant SARP-helper of tylosin biosynthesizing is active, and (Regulation of tylosinbiosynthesis involving ' SARP-helper ' activity), molecular microbiology (Mol Microbiol), 2006,62 (1): 148-156

30Bate N, Butler AR, the people such as Gandecha AR, a plurality of regulatory genes of tylosin biological synthesis gene cluster in Streptomyces fradiae (Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae), chemicobiology (Chem Biol), 1999,6 (9): 617-624

31Bignell DR, Bate N, and Cundliffe E, the regulation and control that tylosin produces: with the effect (Regulation of tylosin production:role of a TylP-interactive ligand) of the interactional part of TylP, molecular microbiology (Mol Microbiol), 2007,63 (3): 838-847

32Stratigopoulos G and Cundliffe E, tylosin biological synthesis gene cluster expression analysis: the crucial regulating and controlling effect of tylQ product (Expression analysis of the tylosin-biosynthetic gene cluster:pivotal regulatory role of the tylQproduct), chemicobiology (Chem Biol) 2002,9 (1): 71-78

33Stratigopoulos G, Bate N, and Cundliffe E, tylosin is biosynthetic just to be regulated and controled: the keying action of TylR (Positivecontrol of tylosin biosynthesis:pivotal role of TylR), molecular microbiology (Mol Microbiol), 2004,54 (5): 1326-1334

34Kuscer E, Coates N, the people such as Challis I, in Streptomyces hygroscopicus, rapH and rapG are to the biosynthetic positive regulating and controlling effect of rapamycin (Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyceshygroscopicus), bacteriology magazine (J Bacteriol) 2007,189 (13): 4756-4763

35 star units, Pan Junhua, the people such as Mei tiny stream once, the strategy of secondary metabolite strain improvement and means, Wuxi Light Industry Univ.'s journal, 2002,21 (4): 428-433

36Sambrook J, Russell DW, molecular cloning: an experimental implementation handbook, the third edition (Molecular Cloning:a LaboratoryManual, 3rd edn), the cold spring port, New York: cold spring harbor laboratory (Cold Spring Harbor, NY:Cold Spring HarborLaboratory) 2001

37Kieser T, Bibb MJ, the people such as Buttner MJ, streptomycete genetic manipulation handbook (Practical Streptomyces Genetics) Nowe is controlled (Norwich), The John Innes Foundation; 2000

38Zhao CY, WangYF, the people such as LR, microbial method is measured tiring of lidamycin (LDM), Chinese microbiotic magazine the 30th the 9th phase of volume 535-537 September in 2005

39Bierman M, Logan R, the people such as O ' Brien K, the plasmid cloning vector (Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp) that is used for conjugal transfer DNA between intestinal bacteria and streptomycete, gene (Gene) 1992,116:43-49

Sequence table

＜110〉biotechnology research institute

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

<130>IDC08068

<160>43

<170>PatentIn?version3.2

<210>1

<211>1080

<212>DNA

<213>sgcR1

<400>1

<210>2

<211>855

<212>DNA

<213>sgcR2

<400>2

<210>3

<211>1188

<212>DNA

<213>sgcR3

<400>3

<210>4

<211>24

<212>DNA

＜213〉primer

<400>4

<210>5

<211>25

<212>DNA

＜213〉primer

<400>5

<210>6

<211>22

<212>DNA

＜213〉primer

<400>6

<210>7

<211>22

<212>DNA

＜213〉primer

<400>7

<210>8

<211>22

<212>DNA

＜213〉primer

<400>8

<210>9

<211>21

<212>DNA

＜213〉primer

<400>9

<210>10

<211>22

<212>DNA

＜213〉primer

<400>10

<210>11

<211>22

<212>DNA

＜213〉primer

<400>11

<210>12

<211>28

<212>DNA

＜213〉primer

<400>12

<210>13

<211>24

<212>DNA

＜213〉primer

<400>13

<210>14

<211>29

<212>DNA

＜213〉primer

<400>14

<210>15

<211>29

<212>DNA

＜213〉primer

<400>15

<210>16

<211>26

<212>DNA

＜213〉primer

<400>16

<210>17

<211>26

<212>DNA

＜213〉primer

<400>17

<210>18

<211>22

<212>DNA

＜213〉primer

<400>18

<210>19

<211>21

<212>DNA

＜213〉primer

<400>19

<210>20

<211>26

<212>DNA

＜213〉primer

<400>20

<210>21

<211>28

<212>DNA

＜213〉primer

<400>21

<210>22

<211>28

<212>DNA

＜213〉primer

<400>22

<210>23

<211>26

<212>DNA

＜213〉primer

<400>23

<210>24

<211>24

<212>DNA

＜213〉primer

<400>24

<210>25

<211>25

<212>DNA

＜213〉primer

<400>25

<210>26

<211>20

<212>DNA

＜213〉primer

<400>26

<210>27

<211>20

<212>DNA

＜213〉primer

<400>27

<210>28

<211>20

<212>DNA

＜213〉primer

<400>28

<210>29

<211>20

<212>DNA

＜213〉primer

<400>29

<210>30

<211>22

<212>DNA

＜213〉primer

<400>30

<210>31

<211>20

<212>DNA

＜213〉primer

<400>31

<210>32

<211>21

<212>DNA

＜213〉primer

<400>32

<210>33

<211>20

<212>DNA

＜213〉primer

<400>33

<210>34

<211>20

<212>DNA

＜213〉primer

<400>34

<210>35

<211>20

<212>DNA

＜213〉primer

<400>35

<210>36

<211>22

<212>DNA

＜213〉primer

<400>36

<210>37

<211>21

<212>DNA

＜213〉primer

<400>37

<210>38

<211>21

<212>DNA

＜213〉primer

<400>38

<210>39

<211>21

<212>DNA

＜213〉primer

<400>39

<210>40

<211>19

<212>DNA

＜213〉primer

<400>40

<210>41

<211>20

<212>DNA

＜213〉primer

<400>41

<210>42

<211>18

<212>DNA

＜213〉primer

<400>42

<210>43

<211>18

<212>DNA

＜213〉primer

<400>43

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, and the expression of one or more gene in sgcR2 and 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 sgcR3 gene is operably connected imports to realizes in 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 lidamycin (LDM) production strain that described expression improves.

6. be selected from sgcR1, the purposes of one or more gene in sgcR2 and sgcR3 gene in the recombinant vectors of the output of producing bacterial strain production lidamycin (LDM) for the preparation of the raising lidamycin (LDM).

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

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 CGMCC NO.0135 is produced bacterial strain that the purposes of claim 6, lidamycin (LDM) are wherein produced bacterial strain.

Images