JPS6240291A - Ultrahigh copy number streptomyces plasmid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a double-stranded deoxyribonucleic acid sequence of the 5cp2' minimal replicon, a recombinant DNA Streptomyces vector containing the sequence, and a Streptomyces vector transformed with the vector. Concerning Myces host cells and replicon probes.

-Technical background S CP 2 Hastreptomyces coelicolor (
Streptomyces coelicolor
), and 5CP2 rice is a mutant with enhanced pock formation associated with Streptomyces sex factor activity. These low copy number plasmids can be transferred to various Streptomyces species by genetic hybridization and protoplast alt transformation. Most of this plasmid DNA is not essential for replicon use or replication in recombinant DNA vectors.

The present invention extends our knowledge of 5cp2* to derive several small, self-transmissible plasmids and to identify fragments containing the basic replicon (containing information necessary for normal replication). , identify the DNA segment controlling plasmid DNA1, and identify the ``minimal replicon, which is the smallest self-replicating plasmid segment.''

SUMMARY OF THE INVENTION The present invention provides a minimal replicon of 5CP2'', 1 or more DNA segments that convey resistance to at least one antibiotic when introduced into and transformed into a susceptible, non-restrictive host cell, and E. coli ( We disclose various recombinant DNA cloning vectors containing a truncated Streptomyces replicon (hereinafter referred to as E. coli) replicon (when constructing a shuttle vector). ,
This replicon increases the copy number of the plasmid containing the truncated minimal replicon compared to the plasmid containing the 5ep2N' wild-type replicon. Furthermore, the present invention also includes transformants containing the above vector.

The present invention provides selectable, ultra-high copy number (greater than 1000 copies per cell) plasmids for use in Streptomyces and related host cells. To date, the development and use of recombinant DNA methods in the genus Streptomyces has been particularly difficult and slow due to the general lack of functional ultra-high copy number vectors. Low copy number vector (1 per chromosome
~5 copies), medium copy number vectors (~5 copies per chromosome)
50 copies), and high copy number vectors (50-1000 per chromosome) were previously available. Due to the appearance of ultra-high copy number plasmids, the production yield of proteins encoded by genes carried by these ultra-high copy number plasmids can be reduced by reducing the production yield of proteins encoded by genes carried by plasmids whose copy numbers are any of the above. This provides a means to dramatically increase expression levels compared to those from other sources. Ultra-high copy number plasmids are thus advantageous for expressing non-toxic gene products at high levels. Since most, if not all, genes express protein in proportion to the number of copies of the gene, cloning genes into ultra-high copy number vectors allows for high levels of accumulation of gene product. I can do it.

Deletion analysis of the vector of the invention containing a segment of 5ep21F is shown in the accompanying FIG. 1.

A restriction site map of the 5CPz rice derivative is obtained. 5cp2
The smallest replicon of * is ~1.4 kilobases (kb) of B
It was identified as a cl age 4, 3^ restriction fragment (this fragment specifies ultra-high level plasmid DNA with more than 1000 copies per chromosome).

As part of the study to identify the minimal replicon of SCP 2', additional cloning vectors were developed.

This replicon probe vector p HJ L 1
0 is useful for isolating replicons from various Streptomyces sp., while the shuttle vector PHJL2
25. p) IJL4QQ and P) IJL401 (these are relatively small 5CIs characterizing moderate plasmid DNA1 in Streptomyces) 2 *
'containing Replifun) is useful for shotgun cloning of genomic DNA.

The vectors of the invention are particularly useful because they are relatively small, versatile, and can transform and be selected in all Streptomyces cells that are sensitive to antibiotics. (The cell has been rendered resistant to the antibiotic and the excised replicon provides sufficient information to self-replicate in the cell).

Since more than 70% of naturally occurring antibiotics are produced by strains of the genus Streptomyces, the development of cloning systems and vectors applicable to this industrially important group is desirable. The present invention provides such vectors, thus making it possible to clone genes into Streptomyces populations, both for the production of new antibiotics and antibiotic derivatives, and for increasing the yield of known antibiotics. It is what makes it possible.

Terms used in this specification are defined as follows.

Ap or ApR - an ampicillin resistance phenotype or a gene that confers it.

Basic replicon - the smallest piece of DNA that replicates at the wild type copy number.

Km - kanamycin resistance phenotype or gene conferring this.

NmR or aph - a neomycin resistance phenotype or a gene conferring this.

2i″5DIIA@″−”−〇″”v :zKJ, :+
“C$*” produces a copy number effect that appears to be unrelated.

Recombinant DNA cloning vector - an autonomously replicating vehicle, including, but not limited to, plasmids and phages, to which one or more DNA segments can be further added, or to which DNA has been added.
Vehicle containing molecules.

Recombinant DNA expression vector - A recombinant DNA cloning vector into which a promoter has been introduced.

Replicon - A DNA sequence that controls and allows autonomous replication of a plasmid or other vector.

Restriction fragment - A linear DNA sequence produced by the action of one or more restriction endonuclease enzymes.

Susceptible host cells - DN encoding selectable resistance traits
Host cells that cannot grow without the A segment.

Transformation - The introduction of DNA into a recipient host cell that changes the genotype of the recipient host cell.

TsR or tsr-thiostrepton resistance phenotype or a gene conferring this.

[Brief explanation of drawings]

FIG. 1 illustrates a deletion derivative of the 5cpz portion of plasmid pHJL192. The top line is
EcoRI and Hindu [5cp2 opened at the site
” is shown.The restriction site in parentheses is that 1
It means that there is more than just one. Segments can be identified by aligning the black horizontal bar with the plastic limited map named at the left end of the bar. FIG. 2 is a restriction site map of plasmid PHJL192. FIG. 3 illustrates the construction of replicon probes. Dotted bars indicate segments from 5cpz rice, blank bars indicate segments containing the aph gene for neomycin resistance, and hatched bars indicate segments containing the tsr gene for thiostrepton resistance. Plasmid pKC7 is an E. coli plasmid. Arrows indicate the direction and approximate position of antibiotic resistance gene expression. FIG. 4 is a restriction site and functional map of plasmid pHJL224. FIG. 5 is a restriction site and functional map of plasmid pHJL225. FIG. 6 is a restriction site map of plasmids pHJL120 and 121. FIG. 7 is a restriction site and functional map of plasmid pI-IJL125. FIG. 8 is a restriction site and functional map of plasmid pHJL236. FIG. 9 is a restriction site and functional map of plasmid pHJL241. Figure 10 illustrates the construction of plasmid pi (JL302). DNA of various origins is identified with the subsequent explanation in Figure 3, except that pUc19 is an E.coli plasmid. An arc with a bar at is −
5, shows a deletion in Lividans TK23 that renders the plasmid incapable of replication. FIG. 11 illustrates the method for sequencing the 5cp2' minimal replicon. FIG. 12 is a restriction site and functional map of plasmid pHJL400. The present invention provides a minimal replicon DNA sequence for 5CP2 rice that confers a pleiotropic copy number phenotype to plasmids transformed into various strains of the genus Streptomyces. Showing only one strand of the DNA sequence for simplicity, this novel 5CP2 American minimal replicon has the sequence: CACCTGCCGG AAGGCCC''ITA
ACCGGC:'IGG'1CGGTGCACGA
GGCTCTCGCCCGCCTGGg [in the sequence, A is deoxyadenyl, C is deoxycytidyl, G is deoxyguanyl, and T is thymidyl]. The present invention further provides novel recombinant DNA Streptomyces vectors comprising: (a) a DNA sequence that confers a pleiotropic, expressible copy number phenotype and has at least a 5cp2" minimal replicon sequence; and Tb) a susceptible and non-susceptible The present invention also encompasses vectors comprising one or more DNA segments that, when introduced and transformed into a restricted host cell, convey resistance to at least one antibiotic. and transformants using the above vectors. The vector of the present invention is constructed by enzymatic digestion of plasmid pHJL192. In this specification, plasmid pHJ
L192 is also referred to as pJL192. By various digestions taught herein, ~1.4 kb Bcl I
- Identifying and isolating the Sau3A minimal replicon fragment. This fragment was then ligated with a Streptomyces antibiotic resistance-conferring gene and a restriction fragment of an E. coli plasmid that contains a functional replicon and confers antibiotic resistance. Self-replicating bifunctional vectors can be prepared that are selective in both genera. Plasmid PHJL192 is 12C600R in E. coli
k-Mk-/PJL192 (This strain is from Northern Regional Research Laboratory (N
other Regional Research
Laboratory. Peoria, 1llinois $15Q4)
It can be isolated by conventional methods from the following: This strain has received accession number NRI as the preferred source and storage recipient for plasmids.
Anyone can use LLB-15040. The restriction site map of plasmid pHJL192 is shown in attached FIG. 2. For the purpose of this application, the attached second
The figure and all of the figures that follow are not drawn to scale. Plasmid pHJL192 is ~5.9kb ECOR
1-5al l5CP2 rice replicon fragment. This fragment is a normal reproduction
tl. Contains all the information necessary for Plasmid pH
Deletion derivatives of JL192 provided the opportunity to obtain a functional map of 5ep2* and to identify sequences that are essential for plasmid replication. Partial digestion of PHJL1920 by Kpn II yielded a series of linear products, and 4 DNA ligase yielded a circular molecule that transformed E. coli with 12C60C600Rk- to ampicillin resistance. Restriction mapping yielded plasmids pHJL200~ 205 was identified. Plasmid P"JL2ot is a Streptomyces griseophsuchus
eofuscus) C581 (C581) was transformed to be neomycin resistant, and this transformed host was
C58 substantially containing plasmid pHJL192
More than 1 plasmid DNA was shown. It is believed that the regulatory sequences controlling 5CP21 copy number were substantially mutated by truncating the recon fragment to the plasmid p)IJL2Ql. Plasmid pHJL200-205 is described in co-pending U.S. patent application &639,566 [Hershberger and Lars 77].
on), filed August 10, 1984]. Additionally, plasmid pHJL125, p I-I, is a useful and convenient plasmid for use in the present invention.
JL 196 and pHJLIQ7 are co-pending U.S. Patent Applications A478,133 () - Schuberger and Larson (supra), issued March 23, 1983], and the Journal of Bacteriology (J. Ba
cteriol, Larson and Bershberger (
157, p. 314 (1984)]. These articles and references are included for reference only. ~2.9 common to both pHJL201 and 202
or 3.0 kb Kpn I 7 fragment to Kp
By subcloning into pHJL5 which had been digested by n-engineering. Plasmid PHJL206 was obtained. Plasmid pHJL
5 arose as a spontaneous deletion in pHJLIQ2, but PHJ
In contrast to L192, it does not replicate in Streptomyces midges. Plasmid pHJL206 is pHJL
Replicates in C581 with plasmid yields similar to 201. The next step in replicon identification used a replicon probe vector that carries a Streptomyces resistance marker but cannot replicate in Streptomyces midline. The construction of this replicon probe vector is illustrated in the accompanying FIG. 3. Plasmid pKC7 (ATCC370
The ~5.2 kb Sal I7 fragment of 84) still contains the kanamycin resistance gene of Tn5 and the replicon and ampicillin resistance genes of plasmid pBR322. Partial Sal digestion of plasmid pHJL197 resulted in a ~3.6 kb fragment containing the neomycin resistance (aph) and thiostrepton resistance (1Sr) genes for Streptomyces. This purified fragment was converted into T4 DN
A. It was ligated with ligase to obtain pHJLg. This replicon probe vector pHJLQ was ligated with several species of Streptomyces, and C58
1 was transformed into thiostrepton and neomycin resistant. Because the Tn5 kanamycin gene was found to be unstable in pHJLg. ~1.3 kb Eco Rl containing the Tn5 gene
- pHJLIQ was prepared by deleting the Shimitake fragment. T4D allows T4 DNA ligase to circularize linear plasmids by blunt-end ligation.
The EcoR and Sal ends were filled with NAIJ gauze. pHJLIQ by rescue experiment
We tested the usefulness of S. The BamHI fragment of Coelicolor DNA is C5
Two types of T s R clones were generated in 81. Kpn efragment of S, coelicolor DNA is 0
One type of NmR1Tsλ clone was generated in 581. The BamH fragment of S. griseofuchus DNA generated two T s R clones in C581. Since neither this restriction fragment nor the self-ligated pHJL IQ transformed Oij'581 to antibiotic resistance,
! These experiments demonstrate the usefulness of pHJLIQ for precon.
It's inside O. The replicon of 5ep2 rice was further investigated by gun cloning. pHJL192~5
．． ~5.9 kb of p'HJL125 corresponding to the 9 Kb Eco R1-Sal I fragment
5au3A digestion of the alJ fragment produced a series of partial digestion products, and these fragments were purified with BamHI.
Digested pHJLlo was conjugated with T0n NA IJ glue. A thiostrepton-resistant transformant of 0581 gave plasmid pHJ L220-225. B
Plasmid p HJ L 22 with amHI restriction enzyme
~2.2 kb Ba from 4 and p HJ LP01
The m Hl 7 fragment was cut out. This~2,2k
The b fragment is the smallest self-replicating fragment in this series. Since these exact locations cannot be easily reproduced, plasmids pHJL224 and pH
JL225 was deposited at NRRL. These two strains, E. Cori Kl 2 C600Rk-Mk-/PH
JL 224 and E, 12C5QQRk-M for Cori
k-/pHJL225 has accession number NR as the preferred source and storage receptor for each plasmid.
Available to everyone under RLB-15988 and NRRL B-18052. Plasmid pHJL224
The restriction sites and functional maps of pHJL225 and pHJL225 are shown in the attached FIGS. 4 and 5. Plasmid p HJ L 225 provided a replicon for constructing a small and selectable plasmid of Streptomyces vera. pHJL225~2.2
The kb Ram HI restriction fragment and the ˜1,1 kb Bcl I fragment containing the thiostrepton resistance gene were ligated with T4 DNA ligase to yield the intermediate plasmid pHJL399. pHJL399N
de ■ Restriction enzyme Xte digestion. This linearized fragment was digested with Nde1 using PCU.
19 (a plasmid containing a polylinker with a unique restriction site, available commercially) (Pharmacia, Inc., 8QQCent
annual Dr., Piscataway,
NJ 08854)] and pHJ
A shuttle vector was constructed from L399. This ligation mixture was used to transform E. coli to ampicillin resistance and Streptomyces to thiostrepton resistance. Plasmids with the reverse orientation were isolated and designated PHJL400right and pHJL401. The restriction sites and functional map of plasmid pHJL400 are shown in the attached FIG. 12. Plasmid DNA levels in medium copy number vectors are high enough to isolate the amount of DNA necessary for characterization and further manipulation. For this reason,
medium copy number vector PBR322 (in E. coli;
15-30 copies per cell) and its derivatives are expected to be used more frequently than any other available vector. Medium copy number vector pH
J LP01, pl (JL400 and pi (JL40
1 is an equally useful vector. Furthermore, the broad host range of plasmids pH'JL400 and pHJL401 makes these vectors more widely available for shotgun cloning. At moderate gene copy numbers, most gene products do not accumulate to the level of repression, so only the most toxic genes are lost from a promiscuous genomic library. It will be appreciated that the present talloning vectors are convenient for expressing poorly transcribed or translated genes cloned into Streptomyces. 5C in p HJ L 206 and p HJ L 224
Comparison of the P2 rice segments identified an overlap of ~1,4 kb (see Figure 1). Kpn ■ and Ba
Kono~1.4kb from pHJL224 by mHI digestion
We cut out common fragments. This ~1.4kb fragment was converted into a 0.755kb Kpn of IJLIO.
J-BamHI 7 fragment replaced with pHJL2
I got 36. Thiostrepton-resistant transformants of S. lividans TK23 (TK23) containing pHJL236 contained 100 times more plasmid D compared to similar cultures containing p) IJL201 or 225.
□Produces 1000 times more plasmid DNA when compared to similar cultures containing NA and pHJLlo2 or 197. Plasmid pHJL236 is thus the first example of a Streptomyces plasmid containing a replicon specifying ultra-high levels of DNA. The 5ep2'' segment in pHJL236 was further analyzed to identify the smallest replicon. With T4 DNA ligase, ~1.4k of pIIJL236
b Kpn r-Bam HI 7 fragment, p
i-1.9 kb Kpn I-Ram I'I containing the neomycin resistance gene derived from IJL196
Connected to I fragment. This new plasmid pHJ
L241 transformed TK23 to neomycin resistance. BamHI- and Bcl■- with T4 DNA ligase
Digested pI-I JL 241 was transformed into ~1.1kb BclE containing the thiostrepton resistance gene.
The fragment was ligated to obtain P) IJL250. 15
Several copies of the 9hp Bcl I fragment pH
It existed in JL250. These additional Bcl I fragments were removed during Bcl I digestion of pi-IJL250. Cyclize with T4DNAIJ gauze and
pHJL251 containing only one copy of the 9bp Bcl I fragment was obtained. 5 in pHJL251
Only one 159b from the other end of the cp2 rice segment
pBcli fragment or ~0.32 kb
Attempts to delete either of the Pvu ■-Bam1 (I fragments did not result in a functional replicon. Therefore, the ~1.4 kb Bcl in PHJL251
The l-3au3A fragment contains the minimal replicon of 5CPz rice specifying ultra-high levels of plasmid DNA. A shuttle vector was constructed from pHJL251 and puc19. These plasmids were ligated at their unique Nde site using T4 DNA ligase.
Cori JMI O9 [: Various meshing (Messi
p)I J L302 was obtained by transforming the ng) strain (commercially available from BRL) to ampicillin resistance and TK23 to thiostrepton resistance. Plasmid p HJ L 302 is E. In coli, cloning of pUC19
and Streptomyces
Lividance (5trep [omyces 1ivid
ans) TK 23 is a shuttle vector that produces ultra-high levels of plasmid DNA. While all aspects of the invention are useful, several of the present recombinant DNA cloning vectors are preferred. A preferred vector is the plasmid PHJLIO. pHJL224. pHJL225. pHJL236, p
HJL241, p I-I J L 251, pHJ
L 302, P) IJL400 and p HJ L
401, and preferred transformants are S. lividans TK23/pHJL 241,
S, Lividans TK23/PHJL251, S, griseofuscus/pl
IJL 4QQ, S, griseophsuchus/PHJL40
1, S, griseophsuchus/pHJL302, S, lividans TK23/pHJL 302. S, 7 Rajie (
fradiae)/pHJL400. S-'7G1
-/P) IJL401, S, Flagey/pHJL 30
2. E, Cori 12C600Rk-Mk-/PHJL3
02, E, Cori Kl 2 CsoooRk-Mk-/p
I-IJlaoo, E, Cori ni 12 C600Rk-M
k-/pHJL401, S, Lividans TK23/PH
JL400 and S, Lividance TK23/PHJL
It is 401. The shuttle vectors p HJ L 236 and pHJL302 of the invention contain replicons that are functional in E. coli and Streptomyces medica, thus providing flexibility in host selection. Therefore, the cloned DNA sequences can be used for construction of new plasmids, physical analysis,
and E. coli for restriction site mapping and then into Streptomyces midge for strain improvement and functional analysis. This means that Streptomyces in E. coli is more positive than in Streptomyces in middle.
This is particularly advantageous because it allows for rapid and convenient manipulation and amplification of single molecules. for example,
with spectinomycin or chloramphenicol
By propagating this vector with E. It can be conveniently amplified in coli. this is
- This is not possible with the Streptomyces host system. Additionally, all plasmid vectors. Since it contains the resistance marker expressed in E. and Ko IJK12, selection of recombinants is easy. Therefore, large quantities of plasmid DNA can be conveniently isolated in a shorter period of time than would be required when performing a similar operation in Streptomyces. The recombinant DNA cloning vectors of the present invention are not limited to use in a single species or strain of Streptomyces. On the contrary, the present vector has a wide range of applications and can be used economically to produce antibiotics such as aminoglycosides, macrolides, β-lactams, polyethers and glycopeptides, especially in host cells belonging to the genus Streptomyces. An important group of non-restricted strains can be introduced and transformed into host cells. Such non-restrictive strains can be obtained using conventional methods well known in the art [Lomovskaya et al., Microbiological Reviews (Microbiological Reviews)].
crobiological Reviews),
44.206 (1980)) or from the group of Streptomyces [Cox and Baltz
, Journal of Bacteriology (J,
Bacteriol. ), 159.499 (1984)). Host cells of non-restricting strains lack restriction enzymes and therefore do not cut or degrade plasmid DNA during transformation. Host cells containing restriction enzymes that do not cut any of the restriction sites of the vector are also considered non-restricting in this application. Various replicon restriction fragments such as plasmids pHJL 192 , pUc19 and various antibiotic resistance-conferring DNA segments can be modified to facilitate ligation. For example, molecular linkers can be attached to replicon fragments as well as specific resistance-conferring DNA segments to provide specific sites for subsequent ligation. Additionally, restriction fragments containing origins of replication can be modified by adding, deleting or substituting certain nucleotides to create different restriction sites for DNA ligation and to change properties. Those skilled in the art are familiar with nucleotide chemistry and the genetic code and will know which DNA modifications are desirable and which nucleotides are internally exchangeable for a particular purpose. The recombinant DNA cloning vectors and transformants of the present invention fill the need for cloning carriers suitable for use in Streptomyces and related microorganisms and have wide utility value. Furthermore, the ability of one vector to confer antibiotic resistance also provides a functional means for selecting transformants. This is important because it is practically necessary to determine and select the specific cells that have acquired the vector DNA. DNA that cannot be inserted into this vector and subsequently screened for additional NA segments for which there is no functional test to determine its presence.
Transformants containing A can also be isolated by appropriate antibiotic selection. Such non-selectable DNA segments can be inserted at all sites other than those necessary for plasmid function, maintenance and replication. Such non-selectable DNA segments include, but are not limited to, all types of antibiotic modifying enzymes, antibiotic resistance, genes specifying antibiotic biosynthesis, and regulatory genes. The antibiotic resistance-conferring vector of the present invention comprises a linked DNA
It is also useful to ensure that the segment is stably maintained in the host cell over many generations. These genes or DNA fragments, which are covalently linked to fragments that confer antibiotic resistance and grow in either Streptomyces or E. coli, are transformed to levels of antibiotics that are toxic to non-transformed cells. Conversion. Maintain by exposing your body. Therefore, we do not lose this vector and, as a result, share
;' Transformants that have lost the combined DNA are unable to proliferate and are excluded from culture. This is especially important in large scale fermentations where maximum efficiency of product expression is desired. Thus, the vectors of the invention can stabilize and maintain any important DNA sequences, provided that the cloned DNA or expression product is not lethal to the host cell. The cloning vectors and transformants of the present invention are for cloning genes and improving the yield of various products currently produced in Streptomyces and related cells. Examples of such products include streptomycin, tyrosine, cephalosporins, actaplanin, avoparcin, naragin, monensin, abramycin, tobramycin, erythromycin, tetracycline, chloramphenicol, vancomycin and ticomycin. , but not limited to these. Furthermore, the present invention can be applied to, for example, human insulin, human proinsulin, glucagon, interferon, human growth hormone, avian growth hormone, bovine growth hormone, porcine growth hormone, interleukin
and commercially important proteins such as interleukins; cloning and characterizing DNA sequences encoding enzymatic functions in metabolic pathways that lead to commercially important methods and compounds; or regulatory elements that improve the expression of genes; , and also provides selectable vectors useful for reconstruction. Such desired DNA sequences include, for example, streptomycin, cephalosporins, tyrosine, actaplanin, avoparcin, naragin, monensin, abramycin, tobramycin, tetracycline, chloramphenicol, erythromycin, ticomycin and derivatives such as vancomycin derivatives. including, but not limited to, DNA encoding enzymes that catalyze the synthesis of antibiotics, or that mediate and increase the biosynthesis of antibiotics or other Q-products. The ability to insert, stabilize, and shuttle E. coli into Streptomyces and E. coli facilitates recombinant genetic engineering to increase the utility and yield of Streptomyces-produced antibiotics. . moreover. ~1,4kb synapse of plasmid PHJL251
Because the u3A replicon fragment produces an ultra-high copy number plasmid, nearly all poorly transcribed or translated DNA sequences can be easily cloned into this vector and shuttled between Streptomyces and E. coli. ) can be done. The genus Streptomyces has an extremely high proportion (6
9-73%) of guanine plus cytosine (G-1-
C) has been reported. It is also known to exhibit a codon usage pattern for protein expression in which the ratio of G+C is relatively low at the first position of the codon and maximum at the third position. The 5ep2* minimal replicon nucleotide sequence of the invention is set out in claim (1). Complete sequence analysis revealed numerous open reading frames of varying lengths of amino acids. Close examination of this sequence reveals two potential initiation signals, both read in the same frame. These ATG codons are present at nucleotide positions 1068 and 1077 numbered in claim (1). When the ATC codon at position 1068 is used, G-1 at the third and first nucleotide positions
The percentages of -C are 91% and 68%, respectively. Therefore, the single precon sequence is consistent with literature for Streptomyces proteins. If one skilled in the art analyzes this minimal replicon sequence,
) Further interesting features of the present sequence include the possibility of a Z-DNA sequence with the potential for a main-wound helical configuration.
It will be appreciated that functional, direct repeat, and reverse complementary sequences are included. Escherichia coli K provides a starting material for constructing many of the vectors of the present invention.
12C600”k-Mk-/pJL 192 (NRRL
B-15040) can be cultured in a number of ways using any of several different media. Preferred carbohydrate sources for the culture medium include, for example, glucose and glycerin, and nitrogen sources include, for example, ammonium salts, amino acid mixtures and peptones. Nutrient inorganic salts are also added, including the usual salt layers that can produce ions such as magnesium, sodium, potassium, ammonium, calcium, phosphate, chlorine, sulfate, etc. It also adds essential trace elements, as they are also necessary for the growth and growth of microorganisms in the pond. Such trace elements are usually supplemented as impurities associated with the addition of other constituents of the medium. E. coli 12C600Rk-Mk-/pJL192 can be grown under aerobic culture conditions over a relatively wide pH range of about 6.5-7.5 and at a temperature range of about 25-42°C. However, the largest amount of plasmid pHJ
In order to produce L192, it is desirable to start with a culture medium at a pH of about 7.2 and maintain the culture temperature at about 37°C. E. coli cells are cultured under the conditions described above to accumulate cells from which plasmid pHJL192 is isolated by methods well known in the art. The invention disclosed in this specification will be explained in more detail by giving examples below. A number of methods used in constructing the invention are listed below. A description of the method is given in the appropriate examples. Example I E, Cori 12 C600Rk-Mk-/
Culture of pJL192 and isolation of plasmid pHJL192 E. coli K12 C600Rk-M k-/
A single bacterial colony of PJL192 (NRRL i; -15040) was cultured with 10 g of Bacto, tryptone, and 10 g of ampicillin per aqueous solution 11 with 25 μg/mt of ampicillin per normal microbiological procedure.
5 g yeast extract and NaC/(P)I 7.5)
LB medium containing 10 g was inoculated. This culture was incubated overnight at 37°C. The next morning, 1mM Mg
SO4, 0.2% glucose, 0.3-0.4% C
AA (casamino acid, Difco), 2
μg/mt of Bl [thiamine-IIC/, sigma (Si
gma)) and 10-M9 medium supplemented with additives [
Miller, Experiments in Molecular Genetics (E
experiments in molecular
Genetics), Cold Spring Harbor
・Laboratories (Co1d Spring Har
bor Laboratories, Cold Spring Harbor
Or), New York (1979)] was inoculated with 0.1 rnl of this late culture. This culture was incubated overnight at 37°C with vigorous shaking, and the next morning, the overnight culture sample was diluted 1/10 to 1150, inoculated into 500 m/M9 medium supplemented as above, and shaken vigorously. The cells were incubated at 37° C. for 2.5 to 3 hours. The turbidity of this culture, measured with a blue filter, was approximately 300-400 Klett units. This culture was added to chloramphenicol (150
~175 μg/mz) and continued incubation overnight with vigorous shaking. The bacterial cells were collected by centrifugation at 7500 rPm for 5 min at 4°C and then incubated with a SV of 200-[0.15M NaCl].
l, 0.1 M NaEDTA (p) 18.0)]
Washed twice. This pellet was suspended in a TS solution [25% sucrose, 50 mM Tris (pH 8)) to a wet weight of 10 m//g and placed on water. To this suspension was added 2-/g wet weight of lysozyme [50mM Tris-H
5 m9/rn in Cl (pH 7,8)! ] and
It was left to cool on ice for 5 minutes. Then 4-/g wet weight of 0.25M EDTA (pH 8.0) was added and cooled for an additional 5 minutes. xerrt/g wet lytic solution [0.4% deoxycholate, 1% Breezy 58.50mM 1-lith and 0
．． 0625M EDTA (PH8)]. This mixture was incubated at 37°C for 15-30 minutes. Sorvall for 15-30 minutes at 4°C.
) 5534 21,000 rPm in rotor
The DNA was collected by centrifugation. Remove the supernatant and dilute with 0.1 volume cr) 3 M NaOAc (pH 8) and 0.
．． 64 volumes of Inpropatool was added to this supernatant. The DNA was centrifuged at 10.00 Orpm for 10 min at 4°C and the pellet was resuspended in 0.1 volume of -rE[lQmM Tris, 1mM EDTA (pH 8). This plasmid DNA was purified by centrifugation equilibrated in a cesium chloride density gradient containing propidium diiodide according to known methods. Example 2 Intermediate plasmid pHJL120 and pH
JL121 construction DNA (NRRL t5041), 1 μl water, 2
μl of the ORI (containing 20 BRL units) restriction enzymes and 17 μj of the EcoRJ reaction mixture were incubated at 37° C. for 2.5 hours. The reaction was terminated by incubating at 60°C for 10 minutes. In all of the following examples, restriction enzyme digestion conditions were used as recommended by the commercial supplier. Vector fragments used for ligation were routinely digested with calf intestinal alkaline phosphatase (C1ap) to remove terminal phosphates and prevent self-ligation. Reactions with T4ONAIJ gauze were performed under standard reaction conditions to generate recombinant DNA molecules. [Dugaiczyk et al.
), Journal of Molecular Biology (J, Mol, Biol, ). 96:171 (1975)). Limit DN A c
i, analyzed by agarose gel electrophoresis or polyacrylamide gel electrophoresis [Fishman and Bershberger
shberger), Journal of Bacteriology (J, Bacteriol, ), 15
5:459 (1983)) Fragment sizes were calculated from measurements taken with a digital tablet [
Elder et al., Analytical Biochemistry
chem, ), 128:223 (1983)). Typically, the reactions were analyzed by agarose gel electrophoresis (AGE) to confirm completion of restriction. 0.1 times the capacity 3
DNA was precipitated by adding M Na0Ac (pH 8,0) followed by 2 volumes of 100% ethanol. This precipitation can be carried out either overnight at -20°C or at -70°C (on dry ice) for at least 15 minutes. This DNA precipitate was washed once with cold 70% ethanol, dried, and 50 pi T E [l Q mM)
Squirrel, 1 mM EDTA (pH 8,0)]. US: Restriction enzymes and instructions can be obtained from the following sources: New England Biolabs.
land BioLabs, Inc.
32 Tozer Road. Beverly, Massachusetts Q.
1915), Boehringer-Mannheim Biochemicals
Biochemicals. 7941 Castleway Drive, Ind.
ianapolis. Summer ndiana 46250), Bethesda
Research Laboratories, Inc. (Bethesda Re)
5earch Laboratories Inc.
. 8717 CyrovemonL C1rcle,
Gaitherburg. Maryland 20760). B. EcoRI digestion of plasmid PBR325 The desired digestion was carried out essentially according to the teachings of Example 2, except that plasmid pBR325 was used in place of plasmid 5CPz rice. The obtained DNA will be used later 4
Stored at °C. from Example A) approximately 40μz, EcoRI digested plasmid pBR325 (from Example B) 10μ/, 0.1
M MgCl2 10μ1% 0.1 M(NH4)28
0410μl, 2mM ATpioμl. 1 μl of T4 DNA ligase, and ligation
. Mixture [50mM Trix-HCl (pH 7,5),
10mM β-mercaptoethanol, 1mM ED'r
A, [50ug/, cDBsA]20. u
/ 4'C? The reaction mixture was incubated for 18 hours (AGE) to confirm proper ligation. This suspended DNA consisted of the desired ~35.8 kb plasmid P) IJL120 and pHJL121. . Since the EcoRI fragment of plasmid pBR325 can be oriented in both directions, recombinant plasmids with two different orientations were obtained. After isolation as described in Example 1, plasmids pHJL120 and PHJ
A restriction site map for each L121 was created and shown in the attached Figure 6. Example 3 Transformation of E. coli Kl 2 C, a widely available strain deposited with the ATCC under accession number ATCC 33525.
A fresh overnight culture of 600Rk-Mk- (E. coli) was added to fresh L-gloss [Miller et al.
(1972)) and were subcultured 1:10 and grown for 1 hour at 37°C. A total of 660 cells were collected,
Wash with 100 mM NaC/2.5 m/15
Suspended in 0 mM Ca Cl 22.5- and incubated for 20 minutes at room temperature. The cells were collected by centrifugation, suspended in 0.5 d of 150 mM Ca Cl 2 /10% glycerol, chilled on ice for 3-5 minutes, and frozen. This cell suspension was stored in liquid nitrogen until use. This preservation and storage is due to the covalently closed circular D
There was no adverse effect on the frequency or viability of transformation by NA. B. Transformation The competent cells were thawed in a water bath and the o6°5-DNA (12,5 m/sample of Example 2c and 37
．． 5 pl)0. I X SSC (0,015M)N
aCl, 0.0015M sodium citrate, pH
7)] to cells at a ratio of 0.1-. The transformation mixture was chilled on ice for 20 minutes, heat-shocked at 42°C for 1 minute, and chilled on ice for 10 minutes. This sample was then diluted with L-broth o,85- and incubated at 37°C.
．． Incubate for 5 hours, ampicillin (50 μg/
y) was spread on Noshiichi agar and incubated at 37°C for 18 hours. The correct phenotype obtained (ApR, TcR
) colonies, essentially Nickhart et al. [Ec
khardt et alo, P5 Sumi F (Pla
Plasmids were screened for size according to the in-well lysis method described by J. Smid, 1:584 (1978). These colonies obtained were injected into the desired E. coli with 12C600Rt-Mk-/pHJ.
L 120 and E, Cori K12 C600Rk
-MB, -/PHJL121 transformants. The ampicillin-resistant and tetracycline-resistant colonies were isolated and cultured according to known methods,
This was then used to purify a covalently closed circular DNA, which was then identified by restriction enzyme and AGE analysis of the constituent plasmids using conventional methods. Next, using this identified transformant, E, Ko IJ K 12C
Plasmids pHJL120 and PHJL were prepared as described in Example 1, except that a strain containing the desired plasmid was used instead of 600Rk-M+c-/pJLt92.
121 was prepared and isolated. Example 4 Intermediate plasmid pl-IJL 125 F
) Construction and isolation of the ~10.2 kb Sal fragment The procedure was essentially the same, except that the reaction was stopped before the digestion was complete, and plasmid pHJL121 and the Sal fragment were used in place of plasmid 5ep2* and KanRI. The desired digestion was carried out as described in Example 2. The resulting Sal restriction fragments were not separated by preparative AGH but precipitated by standard ethanol precipitation. Dissolve this restriction fragment in TE buffer and immediately
□I then performed ligation. Self-ligation of isolated DNA
I let it happen. The obtained DNA is the desired plasmid pHJL1
25 and 12 other plasmids (this plasmid was later isolated and the Sag I of pHJL121
(shown to further contain restriction fragments). Isolated by conventional methods and derived from plasmid PBR325 ■ Origin of replication and plasmid 5ep2
Plasmid pHJL125 containing the ~5.9 kb origin of replication-containing EcoRI-SalI fragment of rice was transformed into T
E buffer and stored at 4°C for later use. The restriction site map of plasmid pI (JL125 is shown in the attached Figure 7. The restriction site map was created using transformed E.
The experiment was carried out using plasmid DNA derived from E. coli 12 C60C6001tk-. Example 5 Plasmids pHJI-196 and pHJL
The construction plasmid pHJL125 of 197 was completely digested with the restriction enzymes SHRI and SAL. The ~5.9 kb EcoRne Sal fragment containing the 5cp2* replicon was gel purified as described in Example 6B (all partial digestions were performed using the method described in Example 6B) and ~7,5k of plasmid pHJL192
b The fragment was partially ligated into the fragment. This ligated material can be used to generate competent E.
, transform coli cells and transform the cells with plasmid PI
-IJL196 was isolated. Transformants were fixed by ampicillin resistance phenotype and restriction enzyme analysis of plasmid DNA. Using the obtained cells, each plasmid DNA was isolated substantially according to the method of Example 1. The ~1.1 kb Bcl efragment from plasmid plJ 702 (ATCC3g155) contains the tsr gene that specifies resistance to thiostrepton in Streptomyces. BamHI
This ˜1,1 kb Bcl fragment was ligated into pHJL196, which had been isolated as a full-length linear molecule by partial digestion with AGE and subsequent AGE separation. Ligation of the BamHI fragment to the Bcl fragment results in a bond that is no longer recognized by either enzyme. In the figure, this connection is represented by the letters "B a m HI / B Sho Earthwork". This plasmid DNA was used to generate competent E. The plasmid pHJL1 obtained by transforming coli cells
Transformants containing 97 DNA were identified by restriction enzyme analysis of plasmid DNA. Plasmid pHJL1
The restriction site and function map of 97 is shown in the attached Figure 3. Example 6 Construction of Replicon Probe p) IJLIO Approximately 70μ/(20μg) of plasmid PHJL197 was digested under partial digestion conditions with 7μ1 (70 units) of the restriction enzyme containing neomycin resistance and thiostrepton resistance conferring genes. A ~3.5 kb fragment was obtained. This ~3.6 kb Sal e fragment was purified by AGE. Complete fl of plasmid pKC7 (ATCC37084)
SJS digestion and C1ap treatment yielded a ~5.25 kb fragment containing the kanamycin resistance-conferring gene of 凰5, the replicon of plasmid pBR322, and the ampicillin resistance-conferring gene. This~5.
The 25 kb Sal E fragment was ligated under standard conditions to the ~3,5 kb Σ Lee fragment and used to transform competent E. coli cells. Isolates containing pHJL8 were obtained by selecting ApR and KmR transformants. The restriction site and functional map of plasmid pl-IJL$ is shown in the accompanying FIG. 3. Plasmid pHJL $100μ! (24 μg) was digested with 3 μl (10 units) of restriction enzyme under partial digestion conditions. The restriction enzyme reaction was inactivated by increasing the temperature to 70°C for 10 minutes. Next, to King Shi■
Restriction enzyme ~10μ1. (10 units) and incubation continued until EcoR digestion was complete. After ethanol precipitation, 100 μl of buffer (82064
μ/, 11n9/, IBSA I Oμl, 10X)
Lys acetate buffer 10μ/, 8 units/μ/T4 polymerase 4tit and lQmM each dNTP 3μ
! ) The DNA was resuspended for 30 minutes at 37°C. The reaction was terminated by raising the temperature to 70°C for 10 minutes, reducing the "loaded" DNA to 0.8
It was run on an agarose gel for X preparation. The location of the separated fragments in the gel was confirmed by staining with ethidium bromide and making the fluorescent bands visible under ultraviolet light. Make a cut near the desired ~7,5 kb band and DE
AE-Cellulose (Whatman)
DE-813 paper was inserted into this cut. The DNA was electrophoresed until it was completely bound to the paper. Take out this paper and add 100mM of KCI.
6 L at a time in l-lis-1-IC/ (pi-I8)
The mixture was washed, shaken vigorously and diffused into Sogo's solution [IM of NaC/and 10 mM Tris-11Cj (pHs) J5]. The paper was filtered off through siliconized Pyrex wool, and the DNA was either precipitated directly with 2 volumes of ethanol or diluted with 1 volume of water and then precipitated with ethanol. Both of these precipitation methods
It can be carried out at -70°C (1 hour on dry ice), or alternatively -20°C (-overnight). this DNA
was collected by centrifugation, and the precipitate was added to TE20 at a concentration of 8 ng/μj.
The suspension was resuspended to 0 μl. Approximately 100 μl of this DNA was added to 3M Na0AclQμ
j and 220 μl of ethanol and stored at −20° C. overnight. The DNA was reprecipitated, dried and treated with 25μ/0.66mM ATP, 1μ/(1μM ligase)
unit/μj, Boehringa 11 Mannheim), 14 μl of water
5X Kinase-Ligase Buffer (250mM) containing
Risto IC1 (pH 7,8), 5QmM MgC/2
．． 25mM+7)DTT. and 25% glycerin] overnight. Approximately 25 μl of this ligation reaction overnight was used to transform competent f, (E. coli cells). The transformants were selected and pl (JLIQ) was identified by restriction enzyme analysis. 12 c5QQILk-Mk-/
p [I J L 224 and E, Cori 12
Cultivation of C60C600Rk-/PHJL225 and Isolation of Plasmid DNA Desired cultivation and subsequent plasmid p HJL
The isolation of pHJL224 and pHJL225 was performed substantially as described in Example 1. E, col JK12C600
Rk-Mk-/pi (JL224 and E, 12
The c6ooRk-Mh-/pHJL 225 strain is commonly available under accession numbers NRRL B-15988 and B-18052, respectively, as a preferred source of plasmids and storage recipients. The restriction site and functional map of plasmid PIIJL224 is shown in the attached FIG. 4, and that of plasmid pHJL225 is shown in FIG. 5. Example 8 Construction of ultra-high copy number shuttle vector Plasmid pl-IJL224 Approximately 35 μl (17 μl
g) under standard conditions, Kpn I and B am t
Digested with II restriction enzyme. The ˜1,4 kb Kpn knee Bam11I replicon fragment was isolated by AGE and recovered as described in Example 6B. Similarly, plasmid pHJLIQ was digested with Kpn 1 and BamI-II, and 0.755kpmI-namH contained in the neomycin resistance gene was digested. I fragment was removed. The ~1.4 kb replicon fragment was ligated into kpnI-BamHI digested pHJLIQ, and this Lyde......P...
・+1i=i*... [Using the john mixture,
Transformants as described in Example 10 were identified by their thiostrepton resistance phenotype and by restriction enzyme analysis of their plasmid DNA. Plasmid pHJL236 was isolated using the obtained cells. This plasmid DNA was introduced into competent E. coli cells for transformation, and transformants were identified by their ampicillin resistance phenotype. This transformant can then be used to isolate plasmid DNA. Plasmid pHJL2
36 produces transformants with ultra-high levels of plasmid DNA in S. lividans TK23, but S.
No stable transformants are produced in Griseofsuchus. The restriction site and functional map of plasmid PHJL236 is shown in the accompanying Figure 8. Example 9 Transformation of Streptomyces griseofuchus A. Growth of protoplast steel culture typically 0.4%
S. griseofuchus (this strain was collected from the American Type Culture Collection) under immersion conditions for 20 hours at 30°C in TSB' supplemented with glycine.
an Type Cul cure Collection
n, Rockville, Maryland 2
A propagation inoculum was prepared by propagating the P. cerevisiae, deposited in the University of California, USA, which is part of the Permanent Archives Collection and is generally available under accession number ATCC 23916]. The method of converting S. griseofuchus into protoplasts is time-consuming;
This is usually done as follows. YMX agar (0,3
S. griseofuchus is inoculated in a line on plates containing yeast extract, 0.3% malt extract, 0.2 dextrose and 2% agar). Approximately 48 hours later, a single bacterial colony is inoculated with TSB10rnlK, homogenized and incubated overnight at 30°C. 4rnl of this overnight culture is then homogenized, added with TSBloo supplemented with 0.4% glycine, and incubated overnight at 30"C. The next afternoon, repeat this operation with a fresh overnight culture. The next morning, add 50% to this culture.
(v/v) Add 50 rnl of glycerin, sample 14 m/
Freeze at -20°C. The frozen cells can be stored for 6 months and used for transformation. Thaw the frozen cells by placing the test tube in water in a beaker at room temperature. The cells were placed on the bench top.
p) Collect in a centrifuge tube and wash 3 times in 10.3% sucrose. P medium supplemented with 1 my/mt Norizozyme [Hopwoo and Wright
d and Wright-), Journal of
Molecular and General Genetics (J
lMolecular and General Ge
netics), 162:307 (1978)) 1
Resuspend this cell pellet in 0 rnl and incubate at 30 °C for 2
Incubate for an hour. The protoplasts are pelleted by centrifugation and the pellet is washed three times in 10 ml of P medium (pipette the pellet into the solution and portex mix for each wash). This final pellet is resuspended in P medium 2- for subsequent transformation. Rice tributicase soy broth was obtained from Difco Laboratories, Det.
roit. Michigan) or Baltimore Biological Laboratories (Baltimore Biol.
logicalLaboratories, P, 0. B
ox243. Cockeysvi1e, Maryland 2
1031). B. Approximately 10 μl of plasmid DNA in transformation buffer
and S. griseofuchus protoplasts, approximately 150
The μl were mixed slightly in a test tube. In this mixture,
Approximately 101 pi of 5o% PEG1000 r polyethylene glycol, Sigma) in P medium was added and mixed using a pipette. After waiting 1-2 minutes, P
Medium was added to bring the volume to 11 nl. The transformed cells were plated in R2 medium and incubated overnight at 30°C. To this regenerated protoplast, 63 ml of R2 containing 400 μg/m/m of thiostrepton was layered and 3
It was incubated for 4 days at 0°C. The resulting thiostrepton-resistant colonies of S. griseofsuchus/pHJL224 were isolated and cultured according to known methods and then identified according to conventional methods and the teachings of Example 9C. This transformed culture was used for the subsequent preparation and isolation of plasmid DNA. C. S. Analysis of Griseofusuchus transformants The resulting transformants were cultured on YMX agar supplemented with thiostrepton (40 μg/−) to obtain single colonies. This colony was transferred to T containing thiostrepton (40ag/lri).
Inoculated into SB medium trap i0rnl. The culture was homogenized and grown overnight at 30°C in 4 rotations. This culture was homogenized and TS B 200 m/
Add 0.4% glycine and thiostrebutone (40μ
The cells, which were grown for 1 to 2 days at 30°C, were incubated with Sohal (5orvall) GSA o
- 15 min, 1 o, ooo rpm). The cells were incubated with 25% sucrose and 5 rq/mt
TEloo- containing lysozyme was resuspended. After incubation for 1 hour at 37°C, 0.25M (D
NaEDTA (p)(s,o) 50- was added. Sample 25- was transferred to a sawpal tube containing 20% w/v SDS (sodium dodecyl sulfate) and incubated at room temperature for 2
Cells were lysed by gentle mixing for 0-30 minutes. After adding 5M NaC/f3-, the lysate was mixed at room temperature for 30 min and then placed on ice for 1.5 h. Remove the cell debris in a sawpal rotor, ts, oo
It was removed by centrifugation at ORPM for 20 minutes. Collect the supernatant and DN with 0.64 times volume of Inproper Tools.
A was precipitated and centrifuged at 10.00 Orpm for 20 minutes. The supernatant was decanted and the DNA pellet was air-dried and then resuspended in TE, $1 street solution 1o- (the pellet was purified by C3Cj equilibrium gradient as described in Example 1). Example 10S, transformation of lJvidans S, Streptomyces lividans TK instead of Griseofsuchus
The desired constructions were individually made, screened, and recovered essentially as described in Example 9, except that No. 23 was used. S
, Lividans is a well-known strain from ancient times, and has been developed by the Northern Regional Research Laboratory (Nor).
thern Regional Re5earcb
Laboratory. Peoria, l1linois 515 Q 4
), and is publicly available under receipt number NRRLB-15826. Furthermore, the medium for converting and proliferating S. lividans into protoplasts, and the preparation and transformation method for protoplasts are as described in Example 2 of International Publication AWO 79101169 (International Patent Application PCT/BG79100095). This identified transformant is then transformed into plasmid PIIJL23, which is subsequently carried out according to known methods substantially as described in Example 9C.
used for the preparation and isolation of 6. Example 11 Construction of ultra-high copy number plasmid pHJL241 ~1.4 kb Kp of pHJL224 isolated in Example 7
nI+Bamf[I replicon fragment of ~1,9k of plasmid pHJL 196 of Example 5
Plasmid pHJL241 was constructed by ligating bKpn 1 to a partial BamHI fragment. Using this ligation material, plasmid pHJL236 DNA was ligated to PHJL236 DNA substantially as described in Example 10.
JL241), S, Lividance TK23
was transformed. Furthermore, in the R2 upper layer, 50Mg/
Transformants were selected using mt neomycin instead of thiostrepton. Plasmid pi-IJL2
The restriction site and functional map of 41 is shown in the accompanying Figure 9. Total protein of S, lividans TK23/PHJL 241 and S, lividans TK23/PHJL197 was measured using PAGE (polyacrylamide gel electrophoresis). S containing pHJL241, Lividans TK23 containing S, Lividans TK 23 /
Compared to p HJ L 1 g 7, it produces more aminoglycoside phosphotransferase of 31 to 33 kd (cow Rodalton), which is the root cause of neomycin resistance. Therefore, S. Lividans TK23/pHJL241
is a preferred host for the purification of aminoglycoside phosphotransferases. Example 12 Construction of intermediate plasmid PI-IJL250 S, plasmid pHJL23 derived from Lividans TK23
Approximately 6 iooμ/(40Mg) was added to Ba under standard conditions.
Digested with mHI and Bcl I restriction enzymes. This Ram Hl + Bcl I 7 fragment was transformed into a protein containing the engineered gene isolated in Example 5.
I was ligated to a ~1,1 kb Bcl I fragment.
LS, Lividance TK23
This ligation iyo. Yo□1,
ga. , 7ke, Nio1,1□, ;250 thiostrepton-resistant transformants were analyzed by restriction enzyme analysis. Example 13 Ultra-high copy number plasmid pHJ LG*(
II-et*L, r. ,7゜. ,,. 643, ~. 4
)251 construction plasmid PHJL250 is one from the left end of the replicon.
Since it contained several copies of the 59bPBC YuI fragment (see Figure 1), the pH of ~14μ/(4μg)
JL250 was digested with Bcli restriction enzyme. This Bcl
A solution of 1-digested DNA was diluted with TE13Qμl. Approximately 4 μl was taken out and ligated in 40 μl of reaction solution. This relatively low DNA concentration reduced ligation between the two DNA fragments by T4 DNA ligase and promoted circularization. S6 lividans TK23 was transformed with this ligation mixture and pHJL251 was transformed with thiostrepton.
Transformants containing . The restriction site and functional map of plasmid PHJL251 is shown in the accompanying Figure 10. Example 14 Medium copy number plasmid p I-I J L
Construction A of 400 and ptlJL401, Nde1 digestion of plasmid pUC19, approximately 1 μg of plasmid p (JC19 (Pharmacia))
cia, Inc.), 8QQ Centenni
alDr., Piscataway, NJ) was completely digested with Ndel restriction enzyme to obtain a linear vector fragment, which was treated with C1ap. B. Construction of intermediate plasmid pHJL399 Plasmid pHJL225 isolated in Example 7 approximately 35μ/(17,
5 μg) was completely digested with upper l-I-[1 restriction enzyme,
The desired ~2.2k containing 5cp2 replicon
b Bam Hl fragment was purified by AGE. The 1.1 kb BclI fragment isolated in Example 5 was ligated to this BamtIl fragment to construct plasmid pHJL399. By thiostrepton, pHJL399-containing 5.
IJ bidans TK23 transformants were selected. Restriction enzyme analysis of this transformant revealed that plasmid PHJL 399
Isolate DNA and create plasmids pHJL400 and 4
It was used to construct 01. A, Nde1 digestion of plasmid pUC19 and ligation of fragments Approximately 30 μ/(1 μg) of plasmid pHJL399 was added to Nd
Digested completely with eI restriction enzyme. Plasmid pHJL
Since 399 has only one ±ie1 site, only one type of linear fragment was generated. This Ndef fragment of pHJL399 was ligated to NdeI-digested pUc19 by T4 DNA ligase. D. E. Transformation of coli JM109 E, -y! J JMI 09 cells (Yanisch-Perron et al.
), Gene, 33: 103 (198
5)) to become competent and to substantially develop the knowledge of Maniatiseta+, Molecular Cloning, Experimental Procedures, Cold Spring Harbor Laboratory (Molecular Cl
oning, A LaboratoryMan
ual Co1d Spring )Iarbor
Laboratory), p. 252 (1982))
The above ligation mixture was transformed using the calcium chloride/-vidium chloride method as described by. The transformants were resistant to ampicillin and X-gal (X-ga
l) Identified by the production of blue colonies in the containing medium and confirmed by restriction digestion of plasmid DNA. Plasmids pI (JL400 and P)IJL401 differ only in the orientation of the Nde ■ restriction fragment of plasmid pHJL399. Plasmid pHJL
The restriction site and function map of 400 is shown in the accompanying Figure 12. Both plasmids are S, griseofuchus and S,
lividans to be resistant to thiostrepton. Example 15 Transformation of Streptomyces Frazie The procedure was substantially as described in Example 9 except that S. Frazie was used instead of S. griseofuchus □
゛S, Frazier/PHJL400 and S. Frazier/PHJL401, m construction is carried out individually,
They were sorted and collected. S. frasier is a well-known strain that has been deposited with, and forms part of, the American Type Culture Collection, and is publicly available under accession number ATCC 19609. . Additionally, TSB for protoplasting and propagating S. frazie. The medium was changed slightly to contain only 0.2% glycine. S. Frazier is shaped due to an endogenous restriction system.
2□ The frequency of pawning and exchange is extremely low. Therefore, 1μg of DNA
The number of transformants per - is pi-IJL4
00 and PI (JL401) transformed when the host is S. fraziae than when the host is S. griseofsuchus or S. +) vidans. However, plasmid DNA isolated from S. frasiae gives a high frequency of transformation when transformed into S. frasiae. Example 16 Construction of shuttle vector plIJL302 Plasmid pHJL2s1 constructed in Example 13 about 10
μ/(2 μg) was completely digested with Nde restriction enzyme. This linearized vector fragment was then ligated into similarly digested pUD19 (from Example 14A). This ligation mixture was used to transform E. coliJJM109 as described in Example 14D, and transformants were identified as taught in Example 14D and confirmed by restriction digestion of plasmid DNA. Plasmid PI
A schematic of the IJL 302 construction is shown in the accompanying Figure 10. 7miF pHJ L 302 DNA, strain S, Griseofsuchus, S. according to the above examples. lividans and S. frasier were transformed. This vector transforms each host to thiostrepton resistance. Furthermore, this plasmid DNA was shown to be stably retained in these transformants, indicating that S.
griseophsuchus and S. It was different from the plasmid PHJL236 in Frazier. Example 17 Sequencing of the Minimal Replicon Substantially the Maxam and Gilbert Protocol/L/
m and G11bert protocol,
Methods in Enzymology
inEnzymology), 65(1):
497 (1980)), 5ep2'l'
The minimal replicon was sequenced. The physical map of the 5CP2* minimal replicon is shown in the accompanying Figure 11. All plasmids herein that reproduce in Streptomyces contain the complete 1404 bP sequence. This physical map is used in sequencing methods to identify replicons. and restriction enzyme sites within the various DNA fragments used for sequencing. Black circles indicate the position of 5' end labeling, and arrows indicate direction and distance of sequencing. 4. Brief explanation of the drawings Figure 1 is a schematic diagram for explaining the deletion derivative of the 5CP2'' portion of plasmid pHJL192, Figure 2 is a schematic diagram showing the restriction site map of plasmid pHJL192, and Figure 3 is a schematic diagram showing the restriction site map of plasmid pHJL192. The figure is a process diagram of replicon probe construction, and Figures 4 and 5 are plasmid pHJL2, respectively.
FIG. 6 is a schematic diagram showing the restriction site and function map of plasmid PI-IJ and pHJ L225.
FIG. 7 is a schematic diagram showing restriction site maps of L120 and 121, and FIGS. 7, 8, and SS9 are respectively plasmid pHJL 125. pHJL 236 and pH
FIG. 10 is a schematic diagram showing the restriction site and functional map of JL241, FIG. 10 is a process chart for constructing plasmid ptIJL302, FIG. 11 is a schematic diagram for explaining the method for sequencing the 5ep2* minimal replicon, and FIG. Figure 12 is a schematic diagram showing the restriction site and functional map of the plasmid pHJL400 (D). ■■■ pHJL200 ■ pHJL201 ■■■■ pHJL202 pHJL203 ■ ■■■■■ pHJL204 ■ ■pHJL20s
■ ■■ pHJL206 ■
■■ pHJL224 ■ pHJL225 ■■■ pHJL236 ■ FIG, 2 9-8 kb 9.4 kb FIG, 6 FIG, 7

Claims (13)

[Claims] (1) Double-stranded deoxyribonucleic acid sequence of the smallest replicon of SCP_2^*, where one strand is the sequence: [There is a gene sequence] [In the sequence, A is deoxyadenyl, C is deoxytidyl, and G is deoxyguanyl. , and T is thymidyl]. (2) a recombinant DNA Streptomyces vector, comprising: (a) a DNA sequence that confers a pleiotropic copy number phenotype and has at least the minimal replicon sequence of SCP_2^* described in paragraph (1); (b) a vector containing one or more DNA segments that, when introduced and transformed into a susceptible, non-restrictive host cell, convey resistance to at least one antibiotic. (3) The vector according to item (2), which has an extremely large pleiotropic copy number phenotype. (4) The vector according to item (3), which is a plasmid pHJL241, pHJL250 or pHJL251. (5) The second (2nd
) Vectors listed in section. (6) The vector according to item (5), which is a plasmid pHJL225, pHJL400 or pHJL401. (7) The vector according to paragraph (2), wherein (c) susceptible and non-restrictive E. coli one or more DNA segments that, when introduced and transformed into an E. coli host, convey resistance to at least one antibiotic; A vector further comprising one or more restriction fragments containing a cory replicon. (8)E. The vector according to item (7), wherein the fragment containing the coli replicon is a fragment of plasmid pBR322, pBR325, pBR328 or PUC19. (9) Shuttle plasmid pHJL236 or pHJ
The vector according to item (8), which is L302. (10) A sensitive and non-restrictive Streptomyces host cell transformed with the vector according to any one of paragraphs (2) to (9). (11) The host cell according to paragraph (10), which is Streptomyces lividans TK23 transformed with the vector according to any one of paragraphs (4) to (9). (12) The host cell according to paragraph (10), which is Streptomyces griseofsuchus transformed with the vector according to any one of paragraphs (6) to (9). (13) Replicon probe which is plasmid pHJL10.