Propionibacteruim vector

Details Propionibacteruim vector Propionibacteruim vector

2001-06-25

2004-12-14

Prouty, Rebecca A. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S320100, C435S252300, C435S252500, C536S023100, C536S026400

Reexamination Certificate

active

06830901

ABSTRACT:

This invention relates an erogenous plasmid of
Propionibacterium
, derived from it and the use of these vectors to express (heterologous) proteins in bacteria, especially Propionibacteria. In particular transformed bacteria can be used either to produce, by fermentation, vitamin B
12
or in cheese making.
INTRODUCTION
Propionibacterium
Gram-positive bacteria capable of producing various useful compounds in a variety of industrial processes. For example several
Propionibacterium
species are known to produce vitamin B
12
(cobalamin) in large scale fermentation processes. Other species are used in dairy applications such as cheese where they contribute, and in many cases even are mainly responsible, for the specific flavour and texture of the cheese. Many
Propionibacterium
species are considered safe for inclusion, as live organisms, into food and animal feed.
To be able to fully exploit the biotechnological potential of
Propionibacterium
, efficient and flexible genetic engineering techniques are required. Such techniques rely on the availability of a suitable plasmid to express a protein from a heterologous gene in
Propionibacterium.
EP-A0400931 (Nippon Oil) refers to an endogenous plasmid (pTY-1) from
Propionibacterium pentosaceum
(ATCC 4875) but does not describe its sequence or exemplify how it may be used to express a heterologous gene.
JP 08-56673 refers to the plasmid pTY-1 for producing vitamin B
12
but does not provide any evidence that the plasmid remains as a freely replicating extrachromosomal element nor that the plasmid is stable inside the transformed cells.
The invention therefore seeks to provide vectors that are more efficient than those in the prior art, and can remain extrachromosomal and/or are stable. In particular the invention aims to provide an efficient vector for the cloning or expression of
Propionibacterium
or foreign genomic fragments or genes into a (
line
821
) host strain. This may enable host specific restriction enzymes to be circumvented and thereby avoid the host treating the plasmid as a foreign polynucleotide.
SUMMARY OF THE INVENTION
Accordingly, the present invention in a first aspect provides a polynucleotide comprising a sequence capable of hybridising selectively to
(a) SEQ ID NO: 1 or the complement thereof
(b) a sequence from the 3.6 kb plasmid of
Propionibacterium freudenrsichii
CBS 101022;
(c) a sequence from the 3.6 kb plasmid of
Propionibacterium freudenreichii
CBS 101023; or
(d) a sequence that encodes a polypeptide of the invention, such as (at least part of) the amino acid sequence of SEQ ID NO: 2 or SEQ ID No: 3, or the complement thereof.
SEQ ID NO: 1 sets out the DNA sequence of the endogenous plasmid of
Propionibacterium
LMG 16545 which the inventors have discovered. The first coding sequence runs from nucleotide 273 to nucleotide 1184 and the predicted amino acid sequence of this coding sequence is shown in SEQ ID NO. 2. The second coding sequence runs from nucleotides 1181 to 1493 and the predicted amino acid sequence of this coding sequence is shown in SEQ ID No: 3.
The inventors screened a large collection of
Propionibacterium
isolates and identified two strains both harboring cryptic plasmids with a size of 3.6 kb. One of the strains is
Propionibacterium freudenreichii
LMG 16545 which was deposited at Centraalbureau voor Schimmolcultures (CBS), Oosterstraat 1, Postbus 273, NL-3740 AG Baarn Netherlands, in the name of Gist-brocades B. V. of Wateringseweg 1, P.O. Box 1, 2600 MA Delft, The Netherlands, on 19 Jun. 1998 under the terms of the Budapest Treaty and was given accession number CBS 101022. The other strain is
Propionibacterium freudenreichii
LMG 16546 which was also deposited by the same depositor 19 Jun. 1998 under the terms of the Budapest Treaty at CBS and was given accession number CBS 101023.
Through full characterization and computer assisted analysis of the nucleotide sequence of LMG 16545 the inventors have been able to identify insertion sites for foreign DNA fragments. These sites have allowed construction of plasmids that are still capable of autonomous replication in
Propionibacterium.
Surprisingly it was found that an erythromycin resistance gene from the actinomycete
Saccharopolyspora erythraea
is efficiently expressed in
Propionibacterium
and thus can be used as a selection marker for transformed cells.
Also constructed are bifunctional vectors, stably maintainable and selectable in both
E. coli
and
Propionibacterium
. This allows the use of
E. coli
for vector construction, as well as functional expression of homologous or heterologous genes in
Propionibacterium
. Vector construction using
E. coli
is comparatively easy and can be done quickly.
The polynucleotide of the invention may be autonomously replicating or extrachromosomal, for example in a bacterium such as a
Propionibacterium
.
Hence another aspect the invention provides a vector which comprises a polynucleotide of the invention.
The invention also provides a process for the preparation of a polypeptide, the process comprising cultivating a host cell transformed or transfected with a vector of the invention under conditions to provide for expression of the polypeptide.
The invention additionally provides a polypeptide which comprises the sequence set far out in SEQ ID NO: 2 or 3 or a sequence substantially homologous to that sequence, or a fragment of either sequence, or a protein encoded by a polynucleotide of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Polynucleotides
A polynucleotide of the invention may be capable of hybridising selectively with the sequence of SEQ ID NO: 1, or a portion of SEQ ID No: 1, or to the sequence complementary to that sequence or portion of the sequence. The polynucleotide of the invention may be capable of hybridising selectively to the sequence of the 3.6 kb plasmid of
P. freudenreichii
CBS 101022 or CBS 101023, or to a portion of the sequence of either plasmid. Typically, a polynucleotide of the invention is a contiguous sequence of nucleotides which is capable of selectively hybridizing to the sequence of SEQ ID. No: 1 or of either 3.6 kb plasmid, or portion of any of these sequences, or to the complement of these sequences or portion of any of these sequences.
A polynucleotide of the invention and the sequence of SEQ ID NO: 1, or either of the 3.6 kb plasmids, or a sequence encoding a polypeptide, or a portion of these sequences, can hybridize at a level significantly above background. Background hybridization may occur, for example, because of other polynucleotides present in the preparation. The signal level generated by the interaction between a polynucleotide of the invention and the sequence of SEQ ID NO: 1 or of either 3.6 kb plasmid, or portion of these sequences, is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1 or of either 3.6 kb plasmid, or a sequence encoding the polypeptide, or portion of these sequences. The intensity of interaction may be measured for example, by radiolabelling the probe, e.g. with
32
P. Selective hybridisation is typically achieved using conditions of medium stringency (for example 0.3M sodium chloride and 0.03M sodium citrate at about 50° C.) to high stringency (same conditions but at about 60° C.). Polynucleotides included in the invention can be generally at least 70%, preferably at least 80 or 90%, more preferably at least 95%, and optimally at least 98% homologous (to the sequence (a) to (d)) over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
Any combination of the above mentioned degrees of homology and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 80% or 90% homologous over 25, preferably over 30 nucleotides forms one embodiment of the invention, as does a polynuc

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