Plasmids from Corynebacterium glutamicum and use thereof

Details Plasmids from Corynebacterium glutamicum and use thereof Plasmids from Corynebacterium glutamicum and use thereof

2000-11-03

2004-08-17

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Vector, per se

C435S183000, C435S106000, C536S023100, C536S023200, C536S023700, C536S024100

Reexamination Certificate

active

06777229

ABSTRACT:

This application claims priority from German Application No. DE 199 53 206.0, filed on Nov. 5, 1999, the subject matter of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides the novel plasmids pTET3 and pCRY4 and the use thereof for the production of vector plasmids.
2. Background Information
Naturally occurring plasmids and plasmid vectors produced therefrom are vital to the improvement of the production characteristics of coryneform bacteria. Constructing plasmid vectors for this group of industrially significant bacteria is substantially based on cryptic plasmids, which are provided with suitable selection markers capable of functioning in Corynebacteria or Brevibacteria (U.S. Pat. No. 5,158,891 and U.S. Pat. No. 4,500,640). These plasmid vectors may be used for cloning and amplifying genes which are involved in the production of L-amino acids, vitamins or nucleotides. Expression of these particular genes may have a positive influence on the production of the desired substances. Thus, for example, cloning a DNA fragment which encodes a protein for a lysine exporter resulted in an improvement in the fermentative production of L-lysine with
Corynebacterium glutamicum
strain MH20-22B (DE-A 19548222).
In contrast with the known and equally industrially significant bacterium
Escherichia coli
, only a limited number of natural plasmids and suitable selection markers are available for developing cloning and expression vectors for Corynebacteria and Brevibacteria. Many plasmids known by different names prove to be identical on more detailed analysis of their genetic organisation. These plasmid isolates have thus been classed in two groups (Sonnen et al., Gene 107, 69-74 (1991)).
The pBL1 group includes the plasmids pAM286 from
Corynebacterium glutamicum
AJ11560 (EP-A 0093611), pAM330 from
Brevibacterium lactofermentum
ATCC13869 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)), pX18 from
Brevibacterium lactofermentum
ATCC21086 (Yeh et al, Gene 47, 301-308 (1986)) and pBL1 from
Brevibacterium lactofermentum
ATCC2179B (Santamaria et al., Journal of General Microbiology 130, 2237-2246 (1984)).
The pHM1519 group comprises plasmids pCG1 from
Corynebacterium glutamicum
ATCC31808 (U.S. Pat. No. 4,617,267), pHM1519 from
Corynebacterium glutamicum
ATCC13058 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)), pSR1 from
Corynebacterium glutamicum
ATCC19223 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) and pRN3.1 from
Corynebacterium glutamicum
ATCC39269 (U.S. Pat. No. 4,559,308).
In addition to members of these two groups of plasmids, the cryptic plasmids pCG2 from
Corynebacterium glutamicum
ATCC31832 (U.S. Pat. No. 4,489,160) and pAG3 from
Corynebacterium melassecola
22220 (U.S. Pat. No. 5,158,891) have also been isolated.
The only selection systems which have hitherto been available are two antibiotic resistance markers which were identified on the streptomycin/spectinomycin resistance plasmid pCG4 from
Corynebacterium glutamicum
ATCC31830 (U.S. Pat. No. 4,489,160) and on the tetracycline resistance plasmid pAG1 from
Corynebacterium melassecola
22243 (U.S. Pat. No. 5,158,891). Plasmid pCG4 also bears the sulI gene which imparts sulfamethoxazole resistance, the sequence of which gene was determined by Nesvera et al. (FEMS Microbiology Letters 169, 391-395 (1998)).
If strains which produce amino acids, vitamins or nucleotides are to be rapidly investigated and improved, it is important to have plasmid vectors which are mutually compatible and are sufficiently stable.
It is known from the prior art that pHM1519 plasmid derivatives and pEL1 plasmid derivatives may coexist. It is furthermore known that the plasmids pGA1 and pGA2 described in U.S. Pat. No. 5,175,108 are compatible. Plasmid vectors having high, moderate or low copy numbers so that expression of the gene under consideration may be graduated are also of significance. Most known plasmids have a high copy number. Only the plasmid pGA2 described in U.S. Pat. No. 5,175,108 is known to have a low copy number.
The widely used plasmid vectors are composed of components originating from the species
C. glutamicum
and components from another species of bacteria, typically
E. coli
. This method introduces foreign DNA into the species
C. glutamicum
. Functional plasmid vectors with a graduated copy number which contain only endogenous DNA and thus meet the criteria of self cloning are not known in specialist circles.
SUMMARY OF THE INVENTION
Object of the Invention
It is an object of the invention to provide novel plasmids that are suitable for constructing plasmid vectors having improved characteristics for coryneform bacteria which produce amino acids, vitamins and nucleotides.
Description of the Invention
Amino acids, vitamins and nucleotides are used in animal nutrition, in the food industry, in the pharmaceuticals industry and in human medicine. These substances are produced with strains of coryneform bacteria. Production characteristics are improved by amplifying suitable genes by means of plasmid vectors. There is accordingly general interest in providing novel plasmid vectors having improved characteristics.
The present invention provides the mutually compatible plasmids pTET3 and pCRY4, isolated from the strain of
Corynebacterium glutamicum
deposited under DSM number 5816, wherein
1.1 plasmid pTET3 is characterised by a length of ~27.8 kbp and the restriction map shown in
FIG. 1
, and an antibiotic resistance region and
1.2 plasmid pCRY4 is characterised by a length of ~48 kbp and the restriction map shown in FIG.
2
.
The present invention also provides composite plasmids of pTET3 and pCRY4 capable of autonomous replication in coryneform bacteria, said plasmids containing
2.1 a part or the entire quantity of the nucleotide sequences
2.2 at least one DNA replication region derived from one of the plasmids pTET3 or pCRY4
2.3 optionally a gene fragment which is derived from a plasmid which can multiply in
E. coli, B. subtilis
or Streptomyces and
2.4 at least one region for expressing active substance resistance, preferably from the plasmid pTET3.
The present invention also provides novel composite plasmids that contain at least part of an active substance resistance region.
The novel plasmid pTET3, the restriction map of which is, shown in
FIG. 1
, has
1. a replication region comprising the nucleotide sequence shown in SEQ ID NO:1 and
2. an antibiotic resistance region consisting of a tetA gene imparting tetracycline resistance and an aadA gene imparting streptomycin and spectinomycin resistance, shown in SEQ ID NO:6.
The novel plasmid pCRY4, the restriction map of which is shown in
FIG. 2
, has a replication region comprising the nucleotide sequence shown in SEQ ID NO:4.
The present invention also provides the production of amino acids, vitamins and nucleotides using plasmid vectors (composite plasmids) which contain pTET3 and pCRY4 and optionally pGA1 or pGA2 nucleotide sequences.
Corynebacterium glutamicum
LP-6, which was deposited as DSM5816 in the context of EP-B 0 472 869, contains the novel plasmids pTET3 and pCRY4 in addition to the plasmids pGA1 and pGA2 described therein. The storage period for DSM5816 has been extended pursuant to rule 9.1 of the Budapest Treaty.
Plasmids pTET3 and pCRY4 are prepared by culturing strain LP-6 in a conventional medium, such as for example brain-heart bouillon or Luria-Bertani medium. The cells were harvested by centrifugation, treated with lysozyme and digested by the alkaline lysis method. The DNA is then purified by anion exchange chromatography on silica gel particles, precipitated with ethanol or isopropanol and then resuspended in H
2
O. Complete systems for isolating plasmid DNA are commercially available as “kits”. One example of such a kit is the “NucleoBond Plasmid Kit” from Clonetech Laboratories GmbH. The person skilled in the art will find detailed instructions relating to the use of this kit in th

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