AU630387B2 - A method of site-specific mutagenesis of dna and development of plasmid vectors - Google Patents

Description

i~ ;i S F Ref: 111298 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Spec ification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service: Degussa Aktiengesellschaft Rodenbacher Chaussee 4 D-6450 Hanau 1 FEDERAL REPUBLIC OF GERMANY Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: A Method of Site-Specific Mutagenesis of DNA and Development of Plasmid Vectors The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 88 224 AM/BT

ABSTRACT

The inventioi relates to a new method of sitespecific mutagenesis of DNA at the restriction cleavage sites using hydroxylamine and the construction of plasmid vectors with cloned resistance genes from C.xerosis, and also relates to plasmids mutated by the novel method and possessing unique restriction cleavage sites.

0 0 0 t 0 88 224 AM/BT A method of site-specific mutagenesis of DNA and development of plasmid vectors.

The invention relates to a method of site-specific mutagenesis of DNA and development of plasmid vectors.

Corynebacterium glutamicum, Brevibacterium flavum and related strains or mutants derived therefrom are known organisms for producing L-amino acids such as lysine and threonine by fermentation.

Plasmid vectors are an essential precondition for genetic improvement of strains. The construction of plasmid vectors for Corynebacterium or Brevibacterium is in general based on encoding plasmids which can be found in this group of bacteria. Examples thereof are plasmid pCG1 from Corynebacterium glutanicum ATCC31808 (US-PS 4,617,267), plasmid pAM330 from Brevibacterium lactofermentum ATCC 13869 (EP-A- 0 093611) and plasmid pHM1519 from Corynebacterium glutamicum ATCC 13058 (Miwa et al. 1984). The plasmid vectors also contain at least one DNA region which gives the host resistance to an antibiotic. Examples thereof are the kanamycin resistance gene of the transposon Tn5 (Santamaria et al. 1984), the kanamycin resistance gene of plasmid pUB110 from Staphylococcus aureus (EP-A- 0 093611), the hygromycin resistance gene from Streptomyces hygroscopicus (Santamaria et al. 1987) and the chloramphenicol resistance gene from Streptomyces acrimycini (Santamaria et al. 1987).

Plasmid vectors for Corynebacterium glutamicum and Brevibacterium can be used to clone genes for 0 6 biosynthesis of amino acids, to express the corresponding gene product or enzyme to a greater extent, and thus improve the evolution of amino acids. Examples are the improvement of the evolution of L-lycine by Corynebacterium glutamicum by cloning and over-expression of the phosphoenol pyruvate carboxylase gene of Corynebacterium glutamicum (British Patent application 8821319.4).

Plasmid vectors are made uo of a number of DNA regions. One DNA region enables the plasmid to replicate in the corresponding host organism. A second DNA region gives the cell resistance to an antibiotic and can be used as a genetic marker for selecting the plasmid-bearing cells of a population. A third DNA region gives resistance to another antibiotic and can be used as a genetic marker for insertion inactivation.

If suitable restriction cleavage sites are present in one of the two genetic markers, each one can be used for insertion activation whereas the other marker is used for selection. The plasmid vectors pBR322 (Bolivar et al. 1979) and pACYC177 (Chang et al. 1978) for E.coli are examples known to the skilled addressee.

The plasmid vectors have to be constructed from genes which give the host resistance to antibiotics.

Another precondition is the presence of unique restriction cleavage sites in the corresponding resistance genes of the plasmid vectors, in order to use them for insertion inactivation.

In order to use restriction cleavage sites in cloned antibiotic resistance genes for insertion inactivation, it is necessary to remove endogenous cleavage sites from the plasmid. This can be done e.g. by filling the cleavage site with nucleoside triphosphates or treatment with nuclease. These methods, however, are of very :3 limited use if the cleavage site is in a gene or another essential region, because this involves a change in the reading frame or a deletion of DNA. The introduction of point mutations is free from these disadvantages. On the other hand a method involving point mutations, such as mutagenesis with hydroxylamine (Birch et al. 1985) involves the risk of producing a number of mutations non-specifically distributed over an entire plasmid.

The invention aims at a method of site-specific mutagenesis of DNA in order e.g. to use the restriction cleavage sites in cloned antibiotic resistance genes for insertion inactivation, and construction of novel plasmid vectors.

The invention relates to a method of site-specific mutagenesis of DNA at the restriction cleavage sites, characterised in that the DNA is isolated and cleaved with a suitable restriction enzyme, the thus-treated DNA is mixed with a mutagenesis preparation usually containing 0.5 2 mol/l or preferably 1 mol/l hydroxylamine and is incubated for a sufficient time, usually 10 to 60 minutes and preferably 20 to 30 minutes at elevated temperature, preferably at 65 to 700C, and care is taken that the DNA double strand does not "melt", or only in the cleavage site region, after which the DNA is separated from the hydroxylamine-containing mutagenesis preparation, treated with a ligase, and a suitable microorganism is transformed with the thus-mutated DNA and the transformed microorganisms are isolated.

DNA is isolated from cells of microorganisms by generally known methods, and the same applies to cleavage at the restriction cleavage sites, treatment with ligase and the subsequent steps.

The mutagenesis preparation contains the v ~4 I r1\/ KXW/111298.do components described by Birch et al.

The invention also relates to the DNA treated by the method described, and comprising one or more inserted restriction cleavage sites.

A DNA is particularly suitable if, as a result of this mutagenesis, it contains one or more unique restriction cleavage sites in one or, if present, a number of resistance genes and/or the replicon.

These DNA portions coded for a resistance can also be derived from an inserted transposon, as is universally known.

It is preferred to use DNA in the form of plasmids, plasmid vectors or phage vectors, which for simplicity are called plasmids in the claims.

The invention also relates to the micoorganisms carrying the mutated DNA, more particularly strains of the genera Corynebacterium, Brevibacterium or mutants evolving derived amino acids.

The invention also relates to mutated forms of DNA produced by the method according to the invention.

Examples are the plasmids pCV34 (Fig. 6a) and pCV36 (Fig. 6b), which as compared with the starting plasmid have a mutated EcoRI cleavage site (pCV34) or a mutated EcoRI and a mutated pstI cleavage site (pCV36) and are therefore mutated forms of the pHM1519 replicon (Miwa et al. 1984).

These plasmids cannot be clehved by the corresponding restriction enzymes and are therefore preferable to the starting plasmids for construction of 0.30 plasmid vectors. Also, DNA fragments in pla mid molecules can be exchanged for fragments carrying mutations produced by the method described. A process of this kind is shown in example 5.4 in the construction of the plasmid vector pZ9 (Fig. The hosts for plasmids pCV34, pCV36 and pZ9 are strains of the genera Corynebacterium and Brevibacterium and, more particularly, aminoacid-evolving mutants derived therefrom. In the case of plasmid pZ9, E.coli is also a host. The plasmid-carrying strains of the genera Corynebacterium, Brevibacterium and Escherichia can be cultivated by conventional methods known to the skilled addressee.

The following strains have been deposited in the Deutsche Sammlung von Microorganismen (German Collection of Microorganisms): Corynebacterium glutamicum ATCC 13032/pCV34, no. DSM 5025, Corynebacterium glutamicum ATCC 13032/pCV36 no. DSM 5026 and Escherichia coli DH5 /pZ9, no. DSM 4938.

Another aspect of the invention is the use of antibiotic-resistant genes from Corynebacterium xerosis for constructing plasmid vectors which replicate in gram-negative and gram-positive bacteria and more particularly in aminoacid-evolving strains of the species Corynebacterium glutamicum, Brevibacterium flavum and related species.

A resistance plasmid containing about 50 kilobase pairs (kb) and present in the strain of Corynebacterium xerosis M82B isolated by Kono et al. (1983) was 0 isolated by modified lysis after Birnboim and Doly (1982) and called pCXM82B, After'digestion of the plasmid DNA with restriction enzymes, the plasmid could be characterised by comparison of the DNA fragments o, obtained by single and double digestion. After cloning mnmmm-m of overlapping DNA fragments, the restriction maps shown in Figs. la and lb were obtained. Using the E.coli vector pUC19 known to the skilled addressee, DNA fragments were isolated from plasmid pCXM82B and carried genes which give the host resistance to the antibiotics chloramphenicol, kanamycin and erythromycin. The chloramphenicol resistance gene lies on a 5-kb long BglI! DNA fragment (Fig. 2a). The kanamycin resistance gene lies on a 1.7-kb SalI DNA fragment (Fig. 2b). The erythromycin resistance gene lies on an 8.5-kb SalI DNA fragment (Fig. 2c). A tetracycline resistance gene, which expresses a resistance not in E.coli but in C.glutamicum, was identifiable after fusion of a pUC19 plasmid containing the gene (Fig. 2d) with a Corynebacterium replicon and transformation into C. glutamicum. The strain Corynebacterium glutamicum ATCC 13032 with the pendulum vector p2Hi4s (Tc has been deposited at the German Collection of Strains of Microorganisms, no. DSM 5396.

The aforementioned DNA fragment having the correspoding resistance genes can be isolated by any skilled addressee from the strain of Corynebacterium xerosis M82B deposited at the German Collection of Strains of Microorganisms, no. DSM 5021. The DNA sequence of DNA fragments giving resistance to chloramphenicol has been defined as a feature of the invention. The oo0 analysis of the coding regions is ,iven in Fig. 3b and 0 co the DNA sequence in Fig. 4. The structural gene comprises 1173 base pairs with an ATG codon at the beginning and two TGA codons at the end of the structural gene.

Sg0. According to another feature of the invention, o starting from the previously-described resistance genes from Corynebactei"ium xerosis, plasmids were constructed "5"s which replicate in amino acid-evolving strains of the So,. species Corynebacterium glutamicum and Brevibacterium 7 I- flavum or related species. For example, starting from the previously-described plasmid pCV36, the chloramphenicol resistance gene was used to construct the plasmid vector pCVX4 shown in Fig. 7, which carries the chloramphenicol resistance gene and also the kanamycin resistance gene of the trasposon Plasmid pCVX4 has a length of 6.5 kb and is of use for insertion inactivation by the unique restriction cleavage sites EcoRi, PstI and MluI in the chloramphenicol resistance gene. Also, the plasmid vector pCVX10 (Fig.

8a) was constructed and has a length of 7 kb and carries the Kanamycin resistance gene of Corynebacterium xerosis with the unique restriction cleavage sites XhoI and Clal and the previously-described chloramphenicol resistance gene of Corynebacterium xerosis. Finally the plasmid vector pCVX15 shown in Fig. 8b was constructed; it has a length of 13.8 kb and carries the erythromycin resistance gene and the previouslydescribed chloramphenicol resistance gene of Corynebacterium xerosis.

The following strains have been deposited at the German Collection of Strains of Microorganisms: Corynebacterium glutamicum ATCC 13032/pCVXIno. DSM 5022 Corynebacterium glutamicum ATCC 13032/pCVXi!no. DSM 5023, Corynebacterium glutamicum ATCC 13032/pCVX4 no. DSM 5024.

u Another feature of the invention is the advantageous expression vector pZ8-1 shown in Fig. Plasmid pZ8-1 has a length of 7.0 kb and carries the mutated form of the pHM1519 replicon produced by the aforementioned novel method of hydroxylamine mutagenesis, and introduced into the plasmid during the on a construction, by replacement of a DNA fragment. More particularly, plasmid pZ8-1 carries the tac-promotor 0".7 (De Boer et al, 1983) followed by a DNA fragment with S9,, 35 multiple cloning sites and finally by the TIT2 0 V I BU J terminator of the rrnB-gene of Escherichia coli (Brosius et al. 1981). The T1T2 terminator was used to ensure stable replication of plasmid pZ8-1 in C.glutamicum. Escherichia coli DH5/pZ8-1 has been deposited in the German Collection of Strains of Microorganisms, no. DSM 4939. The suitability of plasmid pZ8-1 as an expression vector was demonstrated by insertion of the phosphenol pyruvate carboxylase gene of Corynebacterium glutamicum ATCC 13032 (British patent application 8821319.4) into the multiple cloning site. The resulting plasmid pDM7 (Fig. 11) has a length of 10.4 kb and produces an approximately increase in expression of the enzyme phosphoenol pyruvate carboxylase in Corynebacterium glutamicum.

Plasmid PDM7 can be constructed by the skilled addressee by known methods using the deposited plasmids pZ8-1 (DSM 4939) and pDM6 (DSM 4242), which contains the ppc gene (Fig. 12).

The invention also relates to microorganisms of the genera Escherichia, Corynebacterium and Brevibacterium, more particularly the amino acidevolving mutants, which contain the inserted DNA or plasmids mutated according to the invention and resistance genes of C. xerosis M82B.

Examples 1. Isolation and characterisation of a resistance plasmid from Corynebacterium xerosis.

Strains of C.xerosis carrying a resistance plasmid were first described in 1983 (Kono et al). The authors examined clinical isolates and discovered large plasmids (>40 kb) having a wide resistance spectrum in some strains of C.xerosis, The R t plasmid of strain M82 B (pCXM82B), which carries resistances against erythromycin, chloramphenicol, kanamycin and tetracyline has been specified in detail in the invention.

1.1 Modified lysis for obtaining plasmid DNA.

The method of lysis after Birnboim and Doly (1982) with the modifications for C.glutamicum (Thierbach et al. 1988) is of only limited applicability to C.xerosis. For this reason the following pretreatment, based on acetone treatment after Heath et al. (1986), has been developed for lysis of C.xerosis after Birnboim and Doly.

millilitres (ml) of an overnight culture of C.xerosis in LBG (Luria Broth with 2g/1 glucose Maniatis et al. 1982) with added antibiotic were mixed with 200 ml of LBG with antibiotic and 1C% glycine and grown for 22 hours at 300C in an agitator (120 rpm). The cells were harvested by centrifuging (10 minutes at 6000 rpm) in the Beckman J2-21 centrifuge (Rotor JA14) and were taken up in 1 ml TES (50mM Tris, 5mM EDTA, NaCl, pH After 40 ml acetone had been added, the cells were incubated in ice for six minutes and mixed by agitation at 30-second intervals.

The cells were then converted into pellets and washed twice with 40 ml TES. After being centrifuged again, the pellet was taken up in ml F1 buffer (410 mM saccharose, 10 mM MgCl, MMC medium Katsumata et al. 1984) with 20 mg/ml lysozyme and was repeatedly pressed through a ml plastic injector (Fresenius). The resulting homogenisate was incubated with agitation (100 rpm) at 37 0 C for 5 hours and then centrifuged.

The subsequent steps in the process correspond to 1 lysis after Birnboim and Doly (1982) without treatment with lysozyme. The plasmid DNA, which was precipitated with 96% ethanol, was then taken up in 150 pl TE and the concentration of DNA at 280 nm was determined in the photometer. In this manner about 20 pg of pCXM82b DNA was obtained from a 20-ml stationary culture of C.xerosis.

1.2 Restriction mapping of plasmid pCXM82B.

Plasmid pCXM82B isolated from C.xerosis was mapped by using restriction enzymes which relatively seldom cut this plasmid, which is about kilobases (kb) large. The enzymes mentioned hereinafter (Table la) have between one and four recognition sites on the plasmid, Table la Enzymes used for rough mapping of pCXM82B.

Restriction enzyme Number of cleavage sites Clal 3 EcoRV 3 HpaI 1 Ns-I 4 All restriction digestion operations were performed as instructed by the manufacturer. The position of the cleavage sites relative to one another could be determined by double digestion.

Exact mapping was obtained by cloning of overlapping restriction fragments of the olasmid, which were first cloned with more frequently cutting enzymes (Table ib) in the E,colf plasmid vector pUC19 (Norrander et al.1983), then mapped and later combined in a circular map of the plasmid. In the process the o 0 rough mapping yielded the starting points for obtaining the exact map (Fig. The strain Cornebacterium xerosis M32B with the R plasmid pCXM82B has been deposited, no. DSM 5021, at the Gernan Collection of Strains of Microorganisms.

Table lb Enzymes used for sub-cloning and fine-mapping of pCXM82B Restriction enzyme BamHI BglII EcoRI HindIII KpnI PstI SalI XbaI Number of cleavage sites 7 8 8 13 13 All the length measurements were made by gel electrophoresis in agarose gels and comparison with standard lengths (DNA of bacteriophage digested with EcoRI and HindIII or PStI).

2. Cloning of antibiotic resistance genes from C.xerosis in E,coli, The overlapping sub-clones in E.coli plasmid pUC19 (see 1.2) were used for isolating DNA fragments with antibiotic-resistance properties, 2.1 Cloning of the chloramphenicol-resistance genes.

DNA of plasmid pCXM82B was isol;ted from C.xerosis by the method described in 1.1 and cleaved with the restriction enzyme OglII. Plasmid DNA of vector pUC19 obtained from E,coli by known methods (Maniatis et al. 1982) was digested with BamHI and treated with alkaline phosphatase. The two plasmids were mixed and the mixture was treated with T4-DNA ligase. This mixture was then used to transform E.coli JM83 (Messing 1979). A plasmid isolated from the transformants gave resistance to the antibiotic chloramphenicol (25 ,g/ml on agar plates of antibiotic medium No. 3 Messrs.

Oxoid). This plasmid, called pBg12, is new and consists of the pUC19 vector with a 5-kb long insert of pCXM82B DNA (Fig. 2a). The plasmid pBg12 was treated with the enzymes BamHI and PstI, Digestion with PstI was partially carried out.

The plasmid pSVB21 isolated from E.coli JM83 (Arnold and Puhler, 1988) was likewise digested with BamHI and PstI. The two were then mixed and the mixture treated with T4-DNA-ligase. After transformation of E.coli JM83, the plasmid pCXi0 was isolated. This plasmid is new and carries a 1.9-kb large BamHI-PstI sub-fragment of the insert of pBg12 in the vector pSVB21 (Fig, 2a).

2,2 Cloning of the kanamycin resistance genes, Plasmid DNA from pCXM82B was digested with the enzyme EcoRV and mixed with pUC19 DNA cleaved with Smal and treated with alkaline phosphatase. The mixture was incubated with T4-DNA ligase and transformed into E.coli JM83. Plasmid pEVK1 was isolated from a kanamycin-resistant transformant and gives resistance to 25 ug/ml kanamycin and 10 rg/ml neomycin (antibiotic medium No, 3) to the strain JM83. pEVK1 is new and consists of the plasmid pUC19 with a 2.7-kb long DNA fragment of pCXM82B (Fig. 2b).

2.3 Cloning of the erythromycin resistance gene.

Plasmid DNA from pCXM82B isolated by the method described in 1.1 was cleaved with the enzyme SalI and mixed with plasmid pUC19, which likewise had been cleaved with SalI and treated with alkaline phosphatase. The mixture was treated with T4-DNA ligase and transformed into E.coli JM83. A transformant was isolated and was resistant to 120 jg/ml erythromycin on Antibiotic Medium No. 3 and, after this primary selection, had a resistance of more than 2 mg/ml, This plasmid (pSalE2) is new and consists of an 8.5-kb long insert in the vector pUC19 (Fig. 2c). The localised erythromycin resistance on this DNA fragment can be induced by small quantitites (10 pg/ml) of erythromycin.

2 4 Expression of the resistance genes in E.coli.

The DNA fragments of pCXM82B giving resistance to antibiotics and cloned in the E.coli vector pUC19 (see were tested for their minimum inhibiting concentration (Table To this end, the clones present in E.coli strain JM83 in LBG liquid medium were inoculated from an antibiotic-containing preculture with a concentration of about 10 6 cells per millilitre, exposed to various concentrations of the corresponding antibiotics and incubated at 37C0 for about 16 hours, 3. DNA sequence analysis of the choramphenicol resistance gene.

The nucleotide sequence of the 1.9-kb long BamHI- I4 \H PstI DNA fragment was completely determined by the method of Maxam and Gilbert (1977) with the modifications of Arnold and Puhler (1988) and by the method of Sanger et al. (1978). Subcloning was brought about in the E.coli sequencing vectors pSVB21, pSVB25 and pSVB27 (Arnold and Puhler 1988). The sequencing strategy is shown in Fig. 3a. The DNA fragment carries restriction cleavage sites for the enzymes BamHI, EcoRI, NruI, PstI, SacI and SmaI, which were also used to produce the sub-clones.

The sequence of the two DNA strands was analysed using the sequence-analysis program package ANALYSEQ (Staden 1986). The coding-region analysis (Fig, 3b) gives a region coding for protein between positions 520 and 1720. In this region there is a long open reading frame in the nucleotide sequence of the 1,9-kb DNA fragment (Fig. It begins at position 545 with the starting codon ATG and ends at the position 1717 with two successive stop codons (TGA). Six nucleotides upstream of the starting codon there is ribosome binding site (GGAG). The molecular weight of the protein is 39.3 Mdals. It has no sequence homology with the known chloramphenicolacetyl transterase genes of other organisms (Stand EMBL DATA Liorary Release

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Table 2. Minimum inhibiting concentrations (MIC) of the clone resistances Strain Plasmid Kanamycin Ch Ioramphen icol Erythromycin C.xerosis M82B pCXM82B >1000 >200 >2000 E, coli JM83 -20 5 300 E. coi JH83 PUC19 20 5 300 E. coi JM83 pEYK'I )1000 n.t. n.t.

coli JH83 pBgl2 n.t, >100 n~t, E. coi JM83 pSA1E2 rnt. n.t. )2000 All values are given in~ug/ml.

4. Construction of plasmid vectors (pCV3O, pCV33) for Corynebacterium and Brevibacterium and production of mutated forms of replicon pHM15l9 and the kanamycin resistance gene.

4.1 Construction of vector plasmids for Corynebacterium and Brevibacterium, The construction of the vectors is shown in Fig.

The pendulum vector pECS300 (Fig. 13) described in British Patent Application 8821319,4

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00 0 15 was isolated from E.coli JM83 and transformed as per C.glutamicum ATCC 13032 (Theirbach et al 1988). Plasmid pHM1519 (Miwa et al. 1984) was partially restricted with HindIII and ligated with completely digested HindIII pECS300. The ligation mixture was transformed as per C.glutamicum, and plasmid DNA was isolated from 24 of the transformants and cleaved with HindIII. In the shortest of these plasmids, the HindIII fragment carrying the kanamycin resistance was cloned in one of the HindIII cleavage sites of the encoding replicon pHM519, The 4.5-kb large plasmid was then cleaved with SmaI Sall and an about long Smal-Sall piece from the E.coli vector pUC19 (Norrander et al. 1983) was inserted. The resulting plasmid pCV33 is 4.5 kb long and carries single cleavage sites for the enzymes Smal, BamHI, XbaI and Sail, 4.2 In vitro mutagenesis of plasmids from Corynebacterium glutamicum In order to use restriction cleavage sites in cloned antibiotic resistance genes for insertion inactivation, it may first be necessary to remove endogenous cleavage ,ites from the plasmid. This can be done e.g. by filling the cleavage site with nucleoside triphosphates or by treatment with nuclease.

These methods however are of very limited use if the cleavage site is in a gene or another essential region, since the result is a change in the reading frame or a deletion of DNA. On the other hand a method involving point mutations such as mutagenesis with hydroxylamine (~sL et al 1985) brings the danger of producing a number of mutations unspecifically distributed over an entire plasmid, Our modified hydroxylamine 4.2.1 mutagenesis is characterised mainly by high local specificity. This is achieved by linearising the plasmids with the corresponding enzyme and by lower temperatures than those described, so that the DNA double strand melts only near the cleavage site, if at all. The EcoRI cleavage site in 4.2.1 (pHM1519 Miwa et al. 1984) and also the PstI cleavage site in the kanamycin resistance gene both lie in essential regions and cannot be removed either by filling up or by treatment with Si nuclease. A point mutation (cytosine thymine) in the kanamycin resistance gene would however preserve both the reading frame and the amino acid sequence of the protein.

Mutagenesis of the EcoRI cleavage site in replicon from pCV33 and production of pCV34.

Plasmid pCV33 was isolated after Thierbach et al (1988) and cleaved with EcoRI enzyme. The mutagenesis preparation consists of about 2ug plasmid DNA in 60 rl TE buffer, 180,/l of 1.5 M hydroxylamine-HC1 in 25mM EDTA, 5/p1 0.25 EDTA and 13ul 1M Tris-HC1, pH 8.0. The preparation was mixed and incubated at 68 0 C for 30 minutes, After treatment with phenol and precipitation of the plasmid DNA with ethanol (Maniatis et al. 1982) the DNA pellet was taken up in 20//1 of TE and treated with T4-DNA ligase. After transformation of C.glutamicum ATCC 13032 with the plasmid DNA, it was possible to isolate transformants which were no longer cleaved by enzyme EcoRI. The plasmid pCV34 (Fig, 6a) is new. The strain Corynebacterium glutamicum ATCC 13032 with plasmid pCV34 was deposited at the German Collection of Strains of Microorganisms, no. DSM 5025.

a o0 a 3 o 25 0 30 o 1 I ~"Bi I 4*" t a 1

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I _~~rj 4.2.2 Mutagenesis of PstI cleavage site in the kanamycin resistance gene of pCV34 and production of plasmid pCV36.

In similar manner to that described in pCV34 plasmid DNA was linearised with enzyme PstI and subjected to hydroxylamine mutagenesis. The duration and temperature of the reaction were minutes and 68 0 C. After ligation with T4 DNA ligase and transformation into C,glutamicum it was possible to isolate the plasmid pCV36 (Fig, 6b), which is resistant to restriction by EcoRI and PstI. pCV36 is new and has the same amount of resistance to kanamycin as pCV33. C.glutamicum ATCC 13032/pCV36 was deposited at the German Collection of Strains of Microorganisms, no. DSM 5026, Construction of plasmids with two antibioticresistance genes and the possibility of insertion inactivation, 5,1 Production of plasmid pCVX4 using the chloramphenicol resistance gene from C,xerosis.

A 1.9-kb long BamHi-SalI fragment was isolated from the E,coli plasmid pCX10 which carries a 1.9-kb long insert of pCXM82B DNA (Fig. 2a), The Sall cleavage site does not come from the C,xerosis DNA fragment, but is in the immediate neighbourhood of the PstI cleavage site bounding the DNA fragment iui vector pSVB21. The isolated piece of DNA was mixed with the plasmid vector pCV36, restricted with BamHi and Sail and treated with alkaline phosphatase. This mixture was ligated and transformed into C.glutamicum. It was possible to isolate the plasmid pCVX2,1 from a

I

choramphenicol-resistant transformant (Fig. 7).

The new plasmid was then linearised with Sail and treated with the nuclease Bal31. After treatment with T4-DNA polymerase and ligation with T4 DNA ligase, C.glutamicum was transformed with the plasmid mixture and plasmid pCVX4 was isolated from a transformant. pCVX4 is new, 6.5 kb long and gives the cell resistance to kanamycin and chloramphenicol. It has lost the cleavage site for Sall and one of the PstI recognition sites and provides insertion inactivation possibilities owing to the enzymes EcoRI, PstI and M1ul in the Cm gene (Fig, The strain C.glutamicum ATCC 13032 carrying the plasmid PCVX4 was deposited at the German Collection of Strains of Microorganisms, No.

DSM 5022.

5,2 Production of Plasmids pCVX10, using the kanamycin resistance gene Cxerosis.

Plasmid PCVX2.1 (see 5.1) was isolated from Cglutamicum, digested with BamHI and BglII, treated with T4 DNA ligase and transformed into C.glutamicum. Plasmid pCVX2.1,BB was isolated from a kanamycin-sensitive and chloramphenicolresistant transformant (Fig, The plasmid has a deletion of 1.4 kb opposite pCVX2.1, which includes the entire kanamycin resistance gene of pCVX2.1nBB was linearised with Sail, treated with alkaline phosphatase and mixed with plasmid pCXM82B, which had also been cleaved with Sall. After ligation and transformation, the plasmid pCVX10 was isolated from a kanamycin- Sresistant transformant (Fig.' 8a). pCVXO is new, 7 kb long and carries resistances against kanamycin and chloramphenicol. In addition to 35 the Cm gene, the plasmid offers new insertion 4 inactivation possibilities via the enzymes Xhol and Clal in the kanamycin resistance gene. The strain C,glutamicum ATCC 13032/pCVX10 carrying the plasmid pCVX10 was deposited at the German Collection of Strains of Microorganisms, No. DSM 5023.

5.3, Production of plasmid pCVX15, using the erythromycin resistance gene from C.xerosis, Plasmid pCVX2,1 BB was cleaved with the restriction enzyme Sail and treated with alkaline phosphatase. pSalE2 (see 2.3) was restricted with Sall and the two plasnids were mixed together.

The mixture was treated with T4 DNA ligase and transformed into C.glutamicum Plasmid (Fig. 8b) was isolated from a transformant resistant to 10/jg/ml erythromycin, and carries the DNA fragment, which is 8.5 kb long and also gives Em resistance in E,coli, Plasmid pCVX15 is new and carries unique cleavage sites for enzymes BglII, BamHI and Xbal on the Erythromycinresistance bearing DNA fragment and for Ecot and PstI in the Cm gene. The strain C.glutamicum ATCC 13032/pCVX15 carrying the plasmid pCVX15 was deposited at the German Collection of Strains of Microorganisms, no. DSM 5024, 5.4 Production of E.cg!jC..glutamicum pendulum vector p2Hi4S and identification of the tetracyline resistance gene from _exero js in pCyq.lutjymip9 The Ecoli vector pUC19 was linearised with HindIII enzyme and ligated with plasmid pCXM82B, also cleaved with HindIII, A pUC19 derivative was isolated containing a 10.7-kb long DNA piece of pCXM82B. Plasmid p2Hi4 is new and carries the erythromycin resistance gene on the cloned 21 _i

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fragment.

p2Hi4 was cleaved with XbaI. The Corynebacterium vector pCV33 (see 4.1) was also digested with XbaI, and treated with alkaline phosphatase. After Si gation of both pl asmi ds by T4-DNA li gase and transformation into the strain DH5C it was possible to isolate the plasmid e2Hi4S (Fig, 14) which gives resistance in E.,coj against ampicillin, kanamycin and erythromycin, p2Hi4S is new and can replicate as a pendulum vector in E~oiand C...lutmiun After transformation of C. 1tmiu by p2Hi4S, kanamycin and erythromycin-resistant colonies were isolated, and were additionally resistant to tetracycline, It was shown by plasmid analysis and retransformation that the resistance to tetracycline is given by the C~xop~is DNA fragment, Replacement of mutatod DNA fragments in plasmid vectors, Construction of plasmid pZ9.

Plasmid pZ1, which is described in German patent application 3737729.9, was isolated from E.coli by known methods (Mciniatis et al. 1982). After 1 inearisation with the restriction endonuclease PstI, the plazamid was shortened by about 1 kilobase using the nuclease Bal11. After treat~nont with T4 DNA ligjase and transformation of Ecoli OHS, (Hanahan 1985), it was pos~sible to isolate a plasmid called p2-1, After digestion of plasmid pCV34 with Hinoll and PstI, the 2,4-kb HincII-DNA fragment was isolated by electroelution. The resulting fragment was mixed with plasmid p22-i treated with Hincl and 'V 30 alkaline phosphatase, and the mixture was treated with TA-DNA ligase, After transformation of E.coli OH5, plasmid pZ3-4 was isolated from a kanamycin-resistant transformant, and could no longer be cleaved by the restriction endonuclease EcoRI.

Plasmid pCVX2.1, which carries the chiorarrphenicol resistance gene from C.xerosis (see was treated with B imHI and Sall. The 1 .9--kn long DNA fragment was then shortened by about 0,1 kb by treatment with Ba13l. After, treatment with T4-DNA polymerase, the aforementioned DNA fragment was inserted in the ScaI cleavage site of pZ3-4, producing the vector called pZ9 (Fig. E,coli was deposited at the German Collection of microorganisms, No, DsM49,' 8 Plasmid PZ9 is now and carries unique restriction cleavage sites for Xhol and Clal in the kanamycin renistance gene and Pstl, Wl and EcoRI in the chlorarnphenicol resistance gene, which e'ani be ured for insertion inactivation, 5.6 Expression of the genes for resistance to kanamycin, chloramphenicol erythromycin and tetracycline, from rt into Cltmc2 The previoualy-constructed plasmid vectors were tested~ after transformation (Thiorbach et al.

1988) for their minimum inhibition concentrations in C~lutIc~ATCC 13032 (Table 3).

Table 3 Minimum inhibiting concenitrations (MIC) of the cloned resistances, The minimum inhibiting concentration was determined in LBG medium as in 2.4. Cx stands for C e rosis M828 and Cq stands for ~.jtm~mATCC 13032. The kanamycin resistance of plasmid pCV34 and pCVX4 (*)comes from Tn5 and the resistance of plasmid pZ9 comes from Tn990.q All values are given in pg/ml.

6. Construction of expression vectors for Corynebacterium and Brevibactorium, using the mutated pHM1519 replicon.

6.1 Construction of the expression plasmid PZS-i1 with the tac-promotor and the rrnBO-T1T1 terminator, The expression vector pKK223-3 (Broslus 1964) was~ 4 4 4 4 4 4 obtained from Messrs. Pharmacia and treated with enzymes Scal and BamHI. Treatment with BamHI was in the form of partial digestion. The resulting 1.1-kb long Scal BamHI fragment, which carries the tac promotor, a DNA sequence with recognition sites for the enzymes PstI, Sall, BamHI and EcoRI and the T1T2 terminator of the rnB gene, was isolated by electroelution and treated with T4-DNA polymerase. The aforementioned fragment was mixed with plasmid pZ3-4 linearised with Scal, treated with T4-DNA ligase and E.coli DH5 transformed with the ligation mixture. Plasmid PZ8-1 was isolated from a kanamycin-resistant transformant, as shown in Fig. 10, E.coli DH5/pZ8-1 was deposited at the German Collection of Strains of Microorganisms, No, DSM 4939, After transformation of C.glutamicum ATCC 13032, the replication capacity of pZ8-1 could be demonstrated, Plasmid PZ8multiplies under non-selective culture conditions in stable manner in Corynebacterium glutamicum for at least 70 generations.

6,2 Over-expression of the PEP-carboxylase-gene (ppc) of C.glutamicum, using the vector p28-1.

Plasmid pDM2, which is described in British patent application 8821319.4, was treated with Sal ard Smal and mixed with the vector pZ8-1, which haa been linearised with Sall. The DNA mixture was treated with T4 DNA ligase and the ligation mixture was used for transformation of E.coli XH1I (Mountain et al, 1984). The plasmid pDM7 shown in Fig, 11 was isolated from a kanamycin-resistance and succinate-prototrophic transformant.

C.glutamicum ATCC 13032 was transformed with plasmid pDM7. After cultivation in MMYE medium (Katsumata et al. 1984), the specific PEPo I 0 0 0 4r o 04

I_

carboxylase content in the transformant ATCC 13032/pDM7 and in the control strain ATCC 13032/pZ8-1 was determined as described in British Patent Application 8821319.4.

The specific content of PEP carboxylase was 0.16 U/mg protein in strain ATCC 13032/pZ8-1 and 3.00 U/mg protein in strain ATCC 13032/pDM7.

References: Arnold and Paxhler (1988) Gene IQ, 171ff Si-rch et al. (1985) J. Gen. Microbial. In, 1299ff Birnbotmand Daly (1982) Nucl. Acids Res, 2, 1513ff Bolivar at al. (1979) Life Sciences 2j, 807ff Brosius at al. (1981) J. Mol. Biol. JAI, 107ff Brosius (1984) Gene 22., 161ff Chang et al. (1978) J. Bact. 12_4, 1141ff Do Boer at al. (1983) Proc. Natl. Acad, Sci. USA 21, 21ff Hanahan (1985) in Clover DNA cloning Vol.), IRL Press Heath at al. (1986) App. Environ. Hicrobial. 5, 1138ff Katsumata at al, (1984) Bactriol. X, 306ff Xono et al., Antimcrob. Agents Chemother. 2 1 506ff Xaniatis et (1982) Molecular cloring, Cold Spring Harbor Lab.

Maxan and Gilbert (1980) Methods Enzymol. i, 499ff Messing (1979) Recomb. DNA Tochn, Bull., NIH Publ. a-2.2, 2, 43ff KiwA et al. (1984) Agric. Biol., Chem, 48, 2901ff Horinaga at al, (1987) JBiotech. 1, 305ff Mountatn et al., (1984) Mol. Con. Genet. JU, 82ff Norrander at al,'(1983) Gene 2S, 101ff Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463ff Santamari, (1984) J. Gon. Microbiol. 130, 2237ff Santamaria (1987) Gene 56, .199ff Staden (1986) Nuci. Acids Rles. 14, 217ff Thierbach et al1. (1988) App). Microbiol. flotechnol. 29, 356ff A microorgani sml, .yjQryn Iexi-wn Y..Q~i~s CXM82B, was deposited with the Deutsche Sammiung von Mikroorganismen, Grisebachstra~e 8, 3400 Gbttingen, West Germany onl 19 November 1988 and accorded number OSM 5021.

A microorganism, Qnnj~rjvf gliitaicun CG/pCV34, was deposited with the Deutsche Samifung von Mikroorgani smen, Gri sebachstra3e 8, 3400 Gttingen, West Germany on 19 November 1988 and accorded number DSIA 5025.

A microorganisin, QQyn~bacterivm glvtifcvii CG/pCVX10, was deposi ted with the eutche anilun von Mikroorganismen, Grisebachstra3e 31,00 GOttingen, West Germany onl 19 November 1988 and accorded number DSM 5023.

A microorganism, ,Qqrynieba& t~rium glitaimi.cum CG/pCVX4, was deposited with the Deutsche Sarml~ung von Mikroorganismen, Grisebachstrie 8, 3400 GOttingen, West Germany on 19 November 1988 and accorded number DS1W 5022.

Amicroorism g-rynQ _aCte.riun qI~tamicum CG/pCVX15, was deposited with the Deutsche Sammiung von MiKroorganismen, Grisebachstra~e 8, 3400 Gttingen, West Germany on 19 November 198' and accorded number DSIM 5024, A microorganism, gjutamicvum CG/pCV36, was deposited with the Deutsche Sammlung von Mikroorganismen, Grisebachstraf3e 8, 3400 G~ttingen, West Germany on 19 Novemiber 1988 and accorded number DSM 5026.

A microorganism, ~Q~lSeI!1 U4l~L3CG/p2H4S, was do-posited with the Deutsche SammIung von Mikroorganismen, Grisebachstr(e 8, 3400 Gbttingen, West Germany on 13 June 1989 and accorded number DSW 5396.

A microorganism, Qjj DHalpha/pZ9, was deposited with thle Deutsche Sammiung von Mikroorganismen, Grisebachstr(e 8, 3400 G~ttingen, West Germany on 4 November 1988 and accorded number DSW 4939.

A mi croorgani sm, B.h..icil j..i DHS/pZ8-1 was depos ited wi th the Deutsche SammIung von Mikroorganismon, Grisebachstra~e 8, 3400 Gbttinlgen, West Germany on 4 November 1988 and ac;covded number DSM 4938.

291 32391W

Claims (14)

1. A method of site-specific mutagenesis of DNA at the restriction cleavage sites, characterised in that the DNA is isolated and cleaved with a suitable restriction enzyme, the thus-treated DNA is mixed with a hydroxylamine-containing mutagenesis preparation and incubated at elevated temperature, taking care that the DNA double strand does not "melt" or only in the region of the cleavage site, after which the DNA is separated from the hydroxylamine-containing mutagenesis preparation, treated with a ligase and a suitable microorganism transformed with the thus-mutated DNA and the transformed microorganisms are isolated.

2. DNA with one or more mutated restriction cleavage sites, produced by the method according to claim 1.

3. DNA according to claim 2 with one or more unique restriction cleavage sites in one or more resistance genes and/or the replicon.

4. DNA according to claims 2 or 3, characterised in that it is a plasmid. A plasmid according to claim 4, characterised in that the DNA portion or portions (resistance gene or genes) which code for one or more resistances are derived from a transposon. 6, A plasmid according to claim 5, characterised in that the DNA :portion or portions which code for one or more resistances are derived from Corynebacterium xerosis M82B. 'V r

7. Plasmid p2Hi4S according to claim 3, containing the fragment of C. xerosis M82B carrying the tetracycline and erythromycin resistance, characterised by the restriction map shown in Fig. 14 and deposited in C. glutamicum ATCC 13032, number DSM 5396.

8. A plasmid according to claim 6 wherein said DNA portion or portions which code for one or more resistances are derived from plasmid pCXM82B which contains resistances against erythromycin, chloramphenicol, kanamycin and tetracycline and is characterised by the restriction map given in Figs. la and lb.

9. A plasmid according to claim 8 wherein said plasmid pCXM82B is contained in C, xerosis M82B and deposited under No. DSM 5021, A plasmid or DNA according to any one of claims 2 to 7, characterised in they are replicated in strains of the species Corynebacterlum or Brevibacterium or amino acid-evolving mutants derived therefrom. 15 11. Plasmid pCV34, characterised by the restriction map given in Fig. 6a and deposited in C. giutamicum number DSM 5025.

12. Plasmid pCV36, characterised by the restriction map given in Fig, 6b and deposited in C, glutamicum, number DSM 5026,

13. Plasmid vector pCVX4, characterised by the restriction map given in Fig, 7 and deposited in C, glutamicum, number DSM 5022,

14. Plasmid vector pCVX10, characterised by the restriction map given in Fig, 8a and deposited in C, glutamicum, number DSM 5023, Plasmid vector pCVX15, characterised by the restriction map given in Fig. 8b, and deposited in C. glutamicum, number DSM 5024. 31 KXW/111298.doo r- 32

16. Use of plasmids according to any one of claims 2 to 7 and 9 to 14 for replacing one or more DNA segments containing at least one or more replication regions and/or genes, in multicomponent plasmids by corresponding DNA segments which differ from the aforementioned segments by the presence of one or more unique restriction cleavage sites.

17. Use of plasmids according to claim 15, characterised in that the replaced gene or genes give the cell resistance to one or more active principles. 18, Use of plasmids according to any one of claims 2 to 7 and 9 to 14, characterised in that one or more resistance genes and, if required, an expression signal are additionally inserted into the plasmids.

19. Plasmid pZ9 according to claim 17, characterised by the restriction map given in Fig. 9 and deposited in E, coli under the number DSM

4938. 20, Plasmid pZ8-1 according to claim 17, characterised by the tac- promotor and the T1T2 terminator of the rrnB-gene of E, coli and the restriction map given in Fig, 10 and deposited in E, coli under the number DSM 4939, 21. Plasmid pDM7, charactorised by the restriction map given in Fig, 22, Microorganisms of the genera Escherichia, Corynebacterium or Brevibacterlum, which microorganisms contain DNA or a plasmid according to one or more of claims 2 to 7, 9 to 14 and 18 to 23, Mutant microorganisms of the genera Eschorichia, Corynebacterium or Brovibactorium, which microorganisms secrete amino S' acids and contain DNA or a plasmid according to one or more of claims 2 to 7, S9 to 14 and 18 to 24, A method of siteospocific mutagenesis of DNA at the restriction cleavage sites substantially as hereinbefore described with reference to any one of the Examples. 25 DNA with one or more mutated restriction cleavage sites substantially as hereinbefore described with reference to any one of the Examples, KXWI 2Ot ,d -I 33 26. DNA with one or more mutated restriction cleavage sites substantially as hereinbefore described with reference to the accompanying drawings. DATED this TWENTY-SIXTH day of AUGUST 1992 Degussa Aktiengesellschaft Patent Attorneys for the Applicant SPRUSON FERGUSON SI 4 KZ OWMI126 mmmmmmm C!RCULt9R RE5TRICTION' MA~P OF THE R PLASNJ-D PCXPI92B FROM C,XEROS)S. THE CIRCULAR M1AP OF pCXM828 WAS OEBTAINED B'Y FIN'E MAPPING OF OVERLAPPING CLONES, THE MIAP SHOWS TIHE REGIONS OF THE RESISTANCE GENF-S. RESISTANCE 'TO CHLORAJIPHE NICOL, X<rn- RESISTANCE TO KANAMYCiN AND Em%= RES~ITANCE TO ERYTHROFIYciN~ AND R~ESTRICTION CLEAVAGE SITES (SEE FIG, 2 FOR ABBREVIATrIONS), FIG, 1 FIG. l b 1 Fig. 2a Restriction maps of the DNA tragments giving resistance to chloramphenicol. Cmr Hp P E II I PBa B c 5 b S H pXo0 P E P~a I I II 0 0 0 00 o 0 o o Ul 0 000 4 P0*4 Fig. 2b Restriction map of the DNA fragment giving resistance to kanamycin. K mr o PCI H H Fig. 2c Restriction map of the DNA fragment giving rcsintance to erythromycin Emr .P KF .8 9 1 pS&1E2: 0 'ii Xb LSkb I So I- 4*44 44 04 44 44 #4 *o 4 9 4 4.94 4 44, 44 1 I 9 The restriction Cleavag sitx3tie aibbreviaated ain fllot.: aptarnrf, )gp:RpnI, CI:Cial, E:Lco, MB:SO1I an u Xb: naXb Up~pl, Kt~pn, NnNnil, P:PsX, S:all and Xb:XbaX. rig. 2d Restriction map of the DNA fragment giving resistance to tetracycline and erythromycin I EmP T Tcr I SR~b ~5;t F Scz S 1RIP M 4 (L7k) P~7 M744 i{U1~.7~hj FIG. 3ai SEQU)EN'CING STRATEGY( OF THE CHLOR/941PHENicioL RESISTANCE GENE Ps~l Said SmallcolitNrul 5m~J sad f3I-HN-ul Gain H I lobs [REITNCE TO CH LORAMPH EN 100 THE ARROWS GIVE THE RESPECTIVE SEQUJENCED REGIONi OF A SUB- CLONE, THE POSITION OPTHE RES!STANCE. GENE 15 SHOWN IN THE DIAGRAMtI FI G. 3b CODING RANGE ANALYiSIS OF 1"HE DNA FRAGM'EINT CARR'YING THE CHL0RFAMPM-ENICOL RESISTANCE GENE FRAME' FRAME FRAM'E POSTIONAL BASE PREFERENCE MIETHOD 0 So0 1000 1500 I I II I 1 THE PRINT outr OF PROGRAM PANALYSEa SHOWS THE CmR AND ITS STARTING OONr THE READING F R/~t'1 2 ABOVE HAS THE H~IGHEST COOING PR08AIaILITY OVER THE REGION S00- 1700, Fig.- 4 Sequence of the DNA fragment carrying the chioramrphenicol resistance gene-. The nucleotide sequence of the 1.9-kb PstI-Bamli DNA fragment with the derived amino acid sequence of the chlorainphenicol resistance gene. CTGCAGGACATGGTC~tAAGACC;TCAACAtCCAGCGCTAiAGACCATCACATGGGAGCCAA-GC.CGCAGA.AATGCCTGTGCAGCA CAAGTC 20 30 4oQ 50 60 70 80 C,%GxTTAIacAGGGTGCAAGTGTTTTCTGTGACCGCitCTGACCGTGCAAGACCAACCACGAGAAACCAGGGCGAGATCACG i00 110 120 130 140 150 160 170 180 C GC, ,G G -1 TC-1A MG G GC C CG A GG C CC T CA AItC CT G TC C G TG G G T G C A AT G.GC T C A A CV-C.%C CC C G -t 190 200 210 220 250 20250 260 270 CIAAAT CGCTC GGCG &;CGCGAA C CC C ALG-GAAATI. TTzt.AC G, lC.TATTC AA G C G -G G C GCAT CC AC C GCTTGATT C C GC C TT GAA GCC ATOC 280 290 300 3110 320 330 3140 350 360 tTAGAG=TGGGGCATGTCGAGT-CCCGJJ~CGCG-,GTC.GGGGU7GTCGG.L(A.CCCCCACGCCCTTGAACCACTAGT±A.CC-ACG c 370 380 390 1900 1110 24 20 430 440 450 CCA; CGAt:CTGTGTGGGCGTATGTCTGGCGTCCCCGGGCGCTGGCCG TGGTCACAf AGAAG.-AuCCAT-£i CTTGAITGCGACACCTCGGAGTA' &GO 470 480 (,90 500 510 520 530 510 Fig-. 4 (continued) He tProPheAlaLeuCys .alLeuA1 aI-euAlaValiheVallle c~lyThirS er~iuPhelle tLeuAl a~lyLeuLeuProAlalle CTCGATI-GGrrGCCCTcTGCGTGTTGCCCTAGCGG4-TcECTcATGGGCACT-fcAGAA.TTCATcGTCGcc;GATTGcTCCCCGCGAT >.550 560 570 580 590 600 610 G20 630 AlaThr-Glu-teutspValSerVal~lyTlhri'laG-IyLcu~LeuThirS crid aPheAlaValGlyie cVaP .alGlyAlaProValValAl a CGCGACCCC^ITGCGTCTCGGTGGCACTCGGGCCGTGClGTCCGCTTCACGTCGGTA't-'GTCTCGGCGCGCCAGCGT.A.C 640 650 b360 670 680 690 700 710 720 AklaPheAla-.rg~krglrpSerProArgLeuThriLeuI I1eValCysLLuLauVa lPheAaGl1ySerllisva1IleGlyAla~c rtThrPro GGCATTCGCTCGCCGTTGGTCACCGCGGCTCACKi TGATCGTTTCCTCCGTGTTCGCGGGAGCCACGTCTCGGAGCGATACACC 730 740 750 760 770 780 790 800 810 Vai~lheSerLeuLeuLeuleThirArgVa 1Leu~.crA1 aLcuAl aiksilGlyPhe LeuAlaValAl aLeuSecrTh rAlaThirThrLeu ,AGTCTTCTGTCTCCTGCTCATCACCGGGTGCrCCGCTCTCGCAAACGC'GG.TTCCTCGCCGTAGCAGTGACACGGCCACTGCCT 820 830 840 850 860 870 880 890 900 Val~roAlatPsnGlnLysGlyArgAlaLeuSerIlLeuLuStGlyTtrTlrIl efdamhrValVa l~lyValFroAlaGlyAlaLeu CGTGCCAGCGAA-tCCAGAACCGGCCTCACTGTCGiTCC-GCTTCCGCACGCGCATCCAACCCTCGTGGCCGTGCCCGCCCGGGGCAGT 910 920 930 910 950 960 970 980 990 LeuGlyThirAlaLeuGlyTrpArgThirThErPheTrpAlallCjlalleLeuCys 11cProAlaAlaValGIyValIl eAtgGlyValThr GCTCCCGCCG;TGCGGCTGGCCXACGAXCGTTGCCGTCCCTCCTCTCTAtrTCCCGCGGCCGTTCGAGTCATTCGTGGCGTCAC 1000 1010 1020 1030 10180 1050 1060 1070 1080 AsnA!.nalGlyArgSerGluThirSetAl~aThrSerProAtgl-CutgValGluLt1S rGlnLeuAlaTlhrProtrgLeulleLeuAla CAACAATGTTGGTCGCAGCaGAGTAGCGCG,XGGTCANCCAACGCTCCGTGTCGaGCCXCAGTGGCGACGCCGGGGTCATCCTGGC 1090 1100 111 1120 1130 lli6O 1150 1160 1170 Fig. 4 (continued) i1-ecAz^,aLeuGlyAlaLeu11eAs-nGlyGlyThirPheAlaAlaPh&eThrPheLct Ala Pro IleVallhrG; luThrAlaGlyLeuAlaGlu CATGGCACTCGGAtGCGCTGATCA.ACGGAGGGACGTTGCGCM'TCACCTTCCTGCACCCATCGTGACGAGACGCGGGCTTGG CCGA 1180 1190 1200 1210 1220 1230 1240 1250 1260 AlaTrpValSerVal~tlaLeuVal~e t~heGlyIleClySerPheLeuGlyVa1ThirIleAlaGlyArgLeuSerAspGlnArgProGly AGCGTGGGTGTCCGTCGCGGTGGTGATGTTCGGCtTCGATCGTTCTTGGCGTCGATCCAGGAGACTATCAGATCAACGAGCCTGC 1270 1280 1290 1300 1310 1320 1330 1340 135e LcxValLeulaValGlyGlyProLeuLcuLeuThrGlyTrpIleValLcuAlaxValVal-ilaSerllisProValAlaLeuIle'JalLeu CCTCGTGGTCGCAGTCGGCGGACCGCTATTCTGICAGGGTGGATCGTGTTCGCAGTGGTCGCA TGTCATCCGGTTGG TTATCQTCCT 1360 1370 1380 1390 14,00 1410 1420 1430 1440 ValLeuValGInGlyPheLeuS erPheGlyValGly~erThrLeuI leThrArgValLeuTyrAlatxlaS er~lyAl a~roThrM e tGly CGTCCTCGTTCtmGGGATTCCTGTCGTTCGGCGTCGGCACTCTCTGTC'CGCCGTGGTGTATGCAGCTGGGTGCGCCAACGT-GG 1450 1460 1470 1480 11490 1500 1510 1520 1530 GlySerTyrAlaThrAl a~tla Lau~tsnll cC 1yA18A1aAlaGlyProV ilLeuGlyAl aLe uC~yLeuA1 aThrG lyLeuiGlyLeuLeu C- T7-CGTACGCAACGGA.GCATTGAATATCGGAGCTGCAGCGGGGGCCCGTGCTTGGTGCGCTCGGGGTCGCGACCGGGCTGGGGCTGCT j-540 1550 1560 1570 1580 1590 1600 1610 1620 -A Fig-. 4 (continued) AlaProValTrpValAlaS erValLeuThrAlal leAl aLeu;,a illlzdlertLeuLeuTh r,,r Ar gAlaLeuThtrLys'rhrAl aAlaGlu CGCGCCGGTTTGGGTCGGTTCGGTGCTG'CGCG TCGGCTTCGTCAkTCAITGCTTCTGACCAGACG CGCGCTTAGGAAkGACCG CGGCGCX% 1630 1640 1650 1660 1670 1680 1G90 1700 1710 AlaAsn GGCCAATTG2J ,GtCCCATCCGAACGGC-iTTCCATCCTCGTGCCCGTCTCCG-TTGCTCGGCTATTGTCCAQACGGCTATCCGGCC 1730 1740 1750 i760 1770 1780 1790 1800 A.CGITCG CC GCAAGATGTTEATGGTTCrCCCG7CACT C(CAAGC, CQ -,GCCGcTCCGGGAAGAcGGTG A GTGG GAGc A G 1810 1820 1830 I -1;f 1850 1860 1870 1880 1890 GAti.-CGCTC CAG CAI CGCACCG GNTCG 1900 1910 The numbering of the bases is underneath the nucleotide sequence. The amino acid sequence of the Cm gene is given in the 3-letter code. The ribosome binding site is marked by At the end of the coding region tbh- e are 2 successive stop codons FIG. CONSTRUCTION OF VECTOR5 FOR C0R'INEAC TERILUM AND BREVIBACTERIUM Eco RI, I ,Nco! 200Eca RI I NcofFOO 7LO0,.Se.*4 I tn- t T B 9 /111 1500 PARTIAL HindliII LIGATION 14n 1 K L 1: L T T LIGATION L) -p 1I Sm a I*S aI I gco RI1 3900, Sph) tho) &ILI '00 /4Sobp Bcal 1100 NIi~'c'~issa 4 2300 f THE ABBREVIATIONS STAlND FOR THE FoLLoIN'G amps REISTANrCE ,To AIIPICILLIN, kan RESISTA~NCE TO RANAMN'ciN, ori (8c) t:ORIGIN OF REPLICATION FOR E. COIL FIG. 6a RESTRICTION MAP OF PLASTIID PpCV34 tto 1, 200 FIG. 6b RESTRICTIOPI MA~P OF PLAM)ID pCV36 0 0 0 0000 00 %1 000000 0 1 ABBREVIATION, kant K /ANAIIYCIN IRESISrANCE GENE, FIG. 7 CONSTRUCTION OF THE PLASrIID VECTOR pCVXL 4 smal, 4905BBE, Sphl B 925 Hin dil IlnI1t400 ScI 30 p058n ilCVX/ 4630bp c m Bell, 1220 485'8 11 6550 bp Psfhi 505 -Seao1, 280 orr(E) ma, 15 75 tik5,anI 1 f cm EoR2~ 246, SI1 c4,87C3B5~ Sphl tot R1,216 24GS, $ph Sal G 1 I C385 sO MN 47C 2 )4 1 S.l 46r frull 3l00 9 A5~~bRElOTlI' F01 3470,iLiN B~nlAamYC4 1 Stcm R~l2160C TOCLRMPEIO, r(c rqnv SE~la~ HI R Sal sao al LI1iIO A FIG. 8a RESTRICTION' MIAP OF PLAStMD pCVXIO 6650 5050, in dill EoR S 2h, ns FIG. Sb RESTRICTION MIAP OF PLASMIO Sail I IC~Ps 750 )<r I Ir ly,61450 A8BR6VI/VrIONi kan aKAPIA(YCIr4, cmn'L CHLORAMtPHLNICOL, er t RESISTANjCE TO ER~tTHROM YCI N. TH5 SOLOL? M1ARKED R8610N CORRESPONDS rO THE RESPECTIVE DNA FRAGMENT ONt WHICH THE RESISTANCE GENF. WAS LOCALISIZI, MA~P OF PLASJIO p79 7280, SinaI 7200,' 5Ca1 715 0, CCR 6270,lC n Fc GI 90,Sral h1,io 6160, Psh tk ll,11 Hind i.i 2370 h9ill. 30.90 Hird III $3220 411i0 Sphl fl'rc 1I, 3? A88REVIATIONS Ori (5a) ORIC1H OF REPICA TI0' F0R E. coji. cmr CHLORAIPHEN)COL, kan aRESISTANiCE TO KAPNAWY~CIN. FlIG. 9 RESTRICTION MA 1 P OF THE vXPRESSIOPI PLA5t1ID pZS'8-) 5300 Scat 5G10, Hincll, FiBBREVIATIONH: ori (Ec) t Or-loin of roplaliwi for E. coi, kan t, aramvcin resisfbnce. Pi'ac. foo promotor, rnn- TITZ *fr ierniator T I T or ihc3 rrrS gone~ oPE. col; FIG. PESTRICTION RESRICiO~ AP OF PLASJID pDNh7 ABBREVIATION: ori (Eca) eORIGIN OF RjtPLICFA71ON' FOR E. colf kan 100MIN~ PESISTNCt, PW D AriC PR611OrOR, 'rm- Tn TEPMN~rATaIP TIT OP THEt rrm8 6CrfE OF C. toli. 0r,-% Km p ECS "700 7 9kb m p /0FIG. 12 Sina i p oliga iian 7 9) b )c e flF.TRICT ION MAP Or PLAStIID p DIG F IG. 13 KpnIKhul FIGa 14 Ro~~~trictiHn 4fdt 0C 1i-'ju vo~to pCV3 (748iO bp ~h m mNt i'r Sa~r ll Psi1 (5OViO arrv tho I tuc t~ yifo fu tioy~,ad~ Hi I~~I HU1 12010 b) b~ie I~iu to ampci3.li (amp. kanmycin kan) n Imthoyi 0. 0 anKoi ic t pnmci ohce)n Psnf rythomci nIion ni th~ c'Biooo aibnO jnMwhc oxcy fO~l rh ep.cT ouoiun on .et aori4 io) al)o iILUlm (ivC) rom kd veto bo 148.Th xo-i i;maovi