MXPA01000184A

MXPA01000184A - Propionibacterium vector

Info

Publication number: MXPA01000184A
Application number: MXPA/A/2001/000184A
Authority: MX
Inventors: Pieter Hendrik Pouwels; Luijk Nicole Van; Johannes Petrus Maria Jore; Rudolf Gijsbertus Marie Luiten
Original assignee: Dsm Nv
Priority date: 1998-06-25
Filing date: 2001-01-08
Publication date: 2002-07-25

Abstract

An endogenous plasmid of Propionibacterium is described, isolated from Propionibacteria freudenreichii LMG 16545 (deposited as CBS 101022), and its sequence provided. This plasmid can be used to transform Propionibacteria to express homologous or heterologous proteins, in the production of recombinant proteins or products of enzymes, for example vitamin B12.

Description

VECTOR OF PROPIONIBACTERIÜM DESCRIPTION OF THE INVENTION This invention relates to an endogenous plasmid of Propionibacterium, with vectors derived therefrom and with the use of these vectors to express (heterologous) proteins in bacteria, especially Propionibacteria. In particular, the transformed bacteria can be used either for • 10 produce, by fermentation, vitamin B12 or in the production of cheese.

INTRODUCTION Propionibacteria are gram-positive bacteria capable of producing various compounds useful in various industrial processes. For example, it is known that several species • Propionibacterium produces vitamin B12 (cobalamin) in large-scale fermentation processes. Other species use in dairy applications such as cheese making where they contribute, and in many cases they are even primarily responsible for the specific flavor and texture of the cheese. Many species of Propionibacterium are considered safe for inclusion, as living organisms, in food and in animal feed products.

R? F: 126197 To be able to take full advantage of the biotechnological potential of Propionibacterium, efficient and flexible genetic engineering techniques are required. Such techniques are based on the availability of a suitable plasmid to express a protein from a heterologous gene in Propionibacterium EP-A-0400931 (Nippon Oil) refers to an endogenous plasmid (pTY-1) of Propionibacterium pentosaceum • (ATCC 4875) but does not describe its sequence or exemplify how can be used to express a heterologous gene. JP 08-56673 relates to plasmid pTY-1 to produce vitamin B12, but provides no evidence that the plasmid remains as a free replicating extrachromosomal element nor that the plasmid is stable within the transformed cells. The invention therefore seeks to provide vectors that are more efficient than those in the prior art and • that they may remain extrachromosomic or stable, or both. In particular, the invention attempts to provide a Efficient vector for the cloning or expression of Propionibacterium or foreign genomic fragments or genes within a host strain (of Propionibacterium). This can allow a detour of the specific restriction enzymes of the host and thus prevent the host treat the plasmid as a foreign polynucleotide.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, the present invention, in a first aspect, provides a polynucleotide comprising a sequence capable of selectively hybridizing to: (a) SEC. FROM IDENT. NO: 1 or the complement thereof; (b) a 3.6 kb plasmid sequence of Propionibacterium freudenreichii CBS 101022; (c) a 3.6 kb plasmid sequence of Propionibacterium freudenreichii CBS 101023; or (d) a sequence encoding a polypeptide of the invention such as (at least part of) the amino acid sequence of SEQ. FROM IDENT. NO: 2 or SEC. FROM IDENT. NO: 3, or the complement of it. The SEC. FROM IDENT. NO: 1 establishes 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. FROM IDENT. NO: 2. The second coding sequence runs from nucleotides 1181 to 1483 and the predicted amino acid sequence of this coding sequence is shown in SEQ. FROM IDENT. NO: 3 The inventors subjected to analysis a large collection of Propionibacterium isolates identified two strains harboring cryptic plasmids with a size of 3.6 kb. One of the strains is Propionibacterium freudenreichii LMG 16545 which was deposited in Centraalbureau voor Schimmelcultures (CBS), Oosterstraat 1, Postbus 273, NL-3740 AG Baarn, The Netherlands, with the name of Gist-brocades B.V. from ateringse eg 1, P.O. Box 1, 2600 MA Delft, The Netherlands on June 19, 1998 ^ aw under the terms of the Budapest Treaty and provided the access number CBS 101022. The other strain is Propionibacterium freudenreichii LMG 16546 which was also deposited by the same depositor on June 19, 1998 under the terms of the Budapest Treaty on CBS and provided with accession number 101023. 15 Through a complete characterization and analysis aided by nucleotide sequence computer ^^ of LMG 16545, the inventors have been able to identify insertion sites for foreign DNA fragments. These sites have allowed the construction of plasmids that are still capable of autonomous replication in Propionibacterium. Surprisingly, it has been found that an actinomycete erythromycin resistance gene Saccharopolyspora erythraea is efficiently expressed in Propionibacterium and can therefore be used as a selection marker for transformed cells.

Bifunctional vectors are also constructed, which can be stably maintained and selectable in both E. coli • as in Propionibacterium. This allows the use of E. coli for vector construction, as well as the functional expression of 5 homologous or heterologous genes in Propionibacterium. The construction of a vector using E. coli is comparatively easy and can be easily performed. The polynucleotide of the invention can be replicated either autonomously or extrachromosomally, for example in a bacterium such as a Propionibacterium. Therefore, another aspect of the invention provides a vector which comprises a polynucleotide of the invention. The invention also provides a process for By preparing a polypeptide, the process comprises culturing a host cell transformed or transfected with a vector of the invention under conditions to provide the expression • of the polypeptide. The invention further provides a polypeptide which comprises the sequence set forth in SEQ. FROM IDENT. NO: 2 or 3, or a sequence substantially homologous to that sequence, or a fragment of any sequence, or a protein encoded by a polynucleotide of the invention. DETAILED DESCRIPTION OF THE INVENTION POLYUCLEOTIDES A polynucleotide of the invention may be capable of selectively hybridizing with the sequence of SEQ. FROM IDENT. NO: 1, or a portion of the SEC. FROM IDENT. NO: 1, or the sequence complementary to this sequence or portion of the sequence. The polynucleotide of the invention may be capable of selectively hybridizing with the 3.6 kb plasmid sequence of P. freudenreichii, CBS 101022 or CBS 101023, or a portion of the sequence of any of the plasmids. Typically, a polynucleotide of the invention is a contiguous sequence of nucleotides which is capable of selectively hybridizing with the sequence of SEQ. FROM IDENT. NO: l or any 3.6 kb plasmid, or a portion of any of these sequences, or with the complement of these sequences or a portion of any of these sequences. A polynucleotide of the invention and the sequence of SEQ. FROM IDENT. NO: 1, or any of the 3.6 kb plasmids, or a sequence encoding a polypeptide, or a portion of these sequences, can hybridize at a significant level above the background. Background hybridization may occur, for example, due to other polynucleotides present in the preparation. The level of signal generated by the interaction between a polynucleotide of the invention and the sequence of SEQ. FROM IDENT. NO: 1 or any 3.6 kb plasmid or a portion of these sequences is typically at least 10 times, preferably at least 100 times as intense as the interactions between other polynucleotides and the coding sequence of SEC. FROM IDENT. NO: 1 or any of the 3.6 kb plasmids or a sequence encoding the polypeptide or a portion of these sequences. The intensity of interaction can be measured, for example, by radiolabeling the probe, for example with 32P. Selective hybridization is typically obtained using medium stringency conditions (eg 0.3M sodium chloride and 0.03M sodium citrate at about 50 ° C) to high stringency (the same conditions but approximately 60 ° C). The polynucleotides included in the invention can generally be at least 70%, preferably at least 80 or 90%, more preferably at least 95%, and optimally at least 98% homologous (to the sequences (a) to (d)) over a region of at least 20, preferably at least 30, for example at least 40, 60 or 100 or more contiguous nucleotides. Any combination of the aforementioned degrees of homology and minimum sizes can be used to define the polynucleotides of the invention, with the most astringent combinations being preferred (ie, greater homology in longer lengths). Thus, for example, a polynucleotide which is at least 80% or 90% homologous on 25, preferably on 30 nucleotides forms an embodiment of the invention, as well as a polynucleotide which is at least 90% or 95% homologous in 40 nucleotides. The portions referred to above may be the coding sequences of the SEC. FROM IDENT. NO: 1 or of any of the 3.6 kb plasmids. Other preferred portions of SEC. FROM IDENT. NO: 1 are the origin of replication, the promoter or regulatory sequences, or sequences capable of carrying out or assisting autonomous replication in a host cell, such as a Propionibacterium. It has been found that the portion of the plasmid from the SalI to AlwNl restriction site appears to be the region that is required for replication of the plasmid. Other parts of the plasmid have been deleted and still replication does not appear to have been adversely affected. Therefore in the sequence of the invention (b) and (c) may be the region delineated by the restriction sites Sali and AlwNl. This is about 1.7b in length. Alternatively, sequences (b) or (c) can be replaced by the sequence corresponding to nucleotides 1 to 1800 such as 100 to 1700, suitably 150 to 1,500, advantageously from 200 to 1,300 and optimally of 250 to 1200 of the SEC. FROM IDENT. NO: 1.

The proteins (SEQ ID NO: 2 and 3) encoded by ORF1 and 0RF2 respectively are considered to help, • both, for the plasmid to replicate. The plasmid is replicated by the known replication method of a rotating circle in which the original double-stranded DNA plasmid is cut by any of the proteins which aid in the production of a copy of the outer chain using the inner chain as a template. The copy of the outer ring is removed and the ends are joined. The host then replicates a • 10 new inner ring using the outer ring generated as a template. The coding sequences of the invention can be modified by nucleotide substitutions, for example 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotides of the sequences (a) to (d) alternatively or additionally may be modified by one or more insertions or deletions or by an extension on either or both ends, or any of them. Degenerate substitutions can be made, or substitutions can be made which result in a A conservative substitution of amino acids when the modified sequence is translated, for example as discussed below in relation to the polypeptides of the invention. The polynucleotides of the invention may comprise DNA or RNA. They can also be polynucleotides which include within the same synthetic or modified nucleotides. Many different types of modifications to polynucleotides are known in the art. These include methyl phosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3 'or 5' ends, or both, of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein can be modified by any method available in the art. Such modifications can be carried out in order to improve the activity or duration in vivo. The polynucleotides of the invention can be used as a primer, for example a primer for PCR (polymerase chain reaction), a primer for an alternative amplification reaction, a probe for example labeled with a developing tag by conventional means using radioactive or non-radioactive tags, or the polynucleotides can be incorporated or cloned into vectors. Such primers, probes and other fragments will have at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length. Typically they will be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. The probes and fragments may be larger than 150 nucleotides, for example up to 200, 300, 500, 1,000 or 1,500 nucleotides in length, or even up to some nucleotides, such as 5 or 10 short nucleotides of any of the sequences (a ) to (d). Polynucleotides such as a DNA polynucleotide and the primers according to the invention can be produced recombinantly, synthetically or by any means available to those skilled in the art. They can also be cloned by standard techniques. Polynucleotides are typically provided in isolated or purified form. In general, the primers will be produced by synthetic means, which involve a gradual elaboration of the desired nucleic acid sequence by one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Larger polynucleotides will generally be produced using a recombinant medium, for example using PCR cloning techniques. This will involve making a pair of primers (eg, about 15-30 nucleotides) to the SEC region. FROM IDENT. NO: 1 of any of the 3.6 kb plasmids which one wishes to clone, starting with the primers in contact with DNA obtained from a Propionibacterium, performing a polymerase chain reaction under conditions which carry out the amplification of the desired region , isolating the amplified fragment (for example, by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Such techniques can be used to obtain all or part of the SEC. FROM IDENT. NO: 1 of any of the 3.6 kb plasmids. The techniques mentioned herein are well known in the art10. The polynucleotides which are not 100% homologous to SEC. FROM IDENT. NO: l or any of the 3.6 kb plasmids but which are within the scope of the invention can be obtained in many ways. The homologous polynucleotides of SEQ. FROM IDENT. NO: 1 of any of the 3.6 kb plasmids can be obtained, for example, by screening genomic DNA libraries made from a range of Propionibacteria, such as P. freundenreichii, P. jensenii, P. thoenii, P. acidipropionici , or other strains of bacteria of the class Actinomycetes, or other gram-positive bacteria, or those that are rich in G: C. All these organisms are suitable sources of homologous or heterologous genes, promoters, extenders or host cells for use in the invention. Such homologs and fragments thereof will generally be able to selectively hybridize to the coding sequence of the SEC. FROM IDENT. NO: 1 or its complement of any of the 3.6 kb plasmids. Such sequences can be obtained by probing libraries of Propionibacterium genomic DNA with probes comprising all or part of the coding sequence of SEQ. FROM IDENT. NO: 1 or any 3.6 kb plasmid under medium to high stringency conditions (eg 0.03M sodium chloride and 0.3M sodium citrate from about 50 ° C to about 60 ° C. Homologs can also be obtained using PCR degenerate which will use primers designed to direct conserved sequences within the homologs The conserved sequences can be predicted from the alignment of SEQ ID NO: 1 or other sequence of any of the 3.6 kb plasmids with their homologs. primers will contain one or more degenerate positions and will be used in less stringent conditions than those used for the cloning of sequences with unique sequence primers against known sequences., such polynucleotides can be obtained by site-directed mutagenesis of the SEC. FROM IDENT. NO: 1 or of any of the 3.6 kb plasmids, or their homologs. This may be useful when, for example, changes from a silent codon (which is not expressed) to sequences are required to optimize the codon preferences for a particular host cell in which the polynucleotide sequences are to be expressed. Other changes in the sequence may be desired in order to introduce restriction enzyme recognition sites or to alter the property or function of the polypeptides encoded by the polynucleotides. Methods for measuring polynucleotide homology are well known in the art. For example, the U GCG package provides the BESTFIT program which can be used to calculate homology, for example used in its intrinsic setting7. For amino acid homology with respect to the polypeptides of the invention which will be discussed later, one can use the BLAST program (Basic Local Alignment Search Tool1), which produces alignments of amino acid sequences (and of nucleotide sequences if necessary) to determine similarity in the sequence. Therefore BLAST can be used to determine exact matches or to identify homologs, and is particularly useful for those matches which do not contain separations. The BLAST technique uses the algorithm based on the high qualification segment pair (HSP). The invention includes double-stranded polynucleotides comprising a polynucleotide sequence of the invention and its complement. The polynucleotides (e.g., the probes or primers) of the invention may have a developer tag. Suitable labels include radioisotopes such as 32P or 35S, enzymatic labels or other protein labels such as biotin. The techniques for detecting these labels are known per se. Polynucleotides (labeled or untagged) can be used in nucleic acid-based assays to detect or sequence another polynucleotide of the invention in a sample. The polynucleotides of the invention include variants of the sequence of SEQ. FROM IDENT. NO: 1 of any of the 3.6 kb plasmids which are able to replicate autonomously or remain extrachromosomally in a host cell. Such variants may be stable in a bacterium such as a Propionibacterium. Generally, the polynucleotide will comprise the origin of replication or the coding region or regions, or both, of SEC. FROM IDENT. NO: 1 of any of the 3.6 kb plasmids, or homologs of these sequences. A polynucleotide of the invention which is stable in a host cell such as Propionibacterium or E. coli is one in which it is not lost from the host in the following five generations, for example in fifteen generations, preferably thirty generations. Generally, such a polynucleotide will be inherited by both daughter cells in each generation.

The polynucleotide may comprise a promoter or an origin of replication (e.g., toward the 5 'end of any of the sequences encoding a replication protein). The polynucleotide of the invention can be transformed or transfected into a bacterium such as a Propionibacterium or E. coli, for example by an appropriate method11. It may be present in a bacterium at a copy number of from 5 to 500, such as 10 to 100. The polynucleotide may be capable of autonomous replication in a bacterium other than Propionibacterium. Such a bacterium may be E. coli or a gram-positive bacteria or one rich in G: C or one of the Actinomycetes class. Such a polynucleotide will generally comprise sequences which allow the polynucleotide to replicate autonomously in that bacterium. Such sequences can be derived from plasmids which are capable of replicating in that bacterium. A polynucleotide of the invention may be one which has been produced by replication in a Propionibacterium. Alternatively, it could have been produced by replication in other bacteria, such as E. coli. The polynucleotide may be able to avoid Propionibacterium host restriction systems.

Vector A second aspect of the invention relates to a vector comprising a polynucleotide of the first aspect. The vector may be capable of replication in a host cell, such as a bacterium, e.g. Actinomycetes, e.g. Propionibacterium or E. coli. The vector can be a linear polynucleotide or, more usually, a circular polynucleotide. The vector can be a hybrid of the polynucleotide of the invention and another vector. The other vector can be an E. coli vector such as pBR322 or a vector of the pUC Rl, ColD or RSF1010 family or a vector derived therefrom. The polynucleotide or vector of the invention can be a plasmid. Such a plasmid may have a restriction map equal or substantially similar to the restriction maps shown in Figures 1, 2a or 2b. The polynucleotide or vector can have a size of 1 kb to 20 kb, for example 2 to 10 kb, and optimally 3 to 7 kb. The polynucleotide or vector may comprise multiple functional cloning sites. Such cloning sites generally comprise the restriction enzyme recognition sequences. The polynucleotide or vector may comprise the sequence shown in SEQ. FROM IDENT. NO: l or contain restriction recognition sites for EcoRI, Sacl, AlwNl, Bsml, BsaBI, Bell, ApaJ, HipIII, SalI, Hpal, PstI, Sphl, SamHI, Acc651, EcoRV and BglII, or both. The polynucleotide or vector may therefore comprise one, more than one or all of these restriction enzyme sites, generally in the order shown in the figures. Preferably, when present in a bacterium such as a Propinibacterium or E. coli, the polynucleotide or vector of the invention is not integrated into the chromosome of the bacterium. Generally, the polynucleotide or vector is not integrated in the following five generations, preferably 20 or 30 generations. The polynucleotide or vector can be a self-replicating plasmid that can remain extrachromosomally within a host cell, a plasmid which is derived from an endogenous plasmid of Propiopijbacterium, and when it comprises a heterologous gene (for the host) it is capable of express that gene inside the host cell. The term "derivative of" means that the plasmid that replicates autonomously includes a sequence equal to that of the polynucleotide of the invention. The vector of the invention may comprise a selectable marker. The selectable marker can be one which requires resistance to antibiotics, such as genes that provide resistance to ampicillin, kanamycin or tetracycline. The selectable marker can be an erythromycin resistance gene. The erythromycin resistance gene can be from Actinomycetes, such as Saccharopolyspora erythraea, for example Saccharopolyspora erythraea NRRL2338. Other selectable markers which may be present in the vector include genes conferring resistance to chloramphenicol, thiostrepton, viomycin, neomycin, apramycin, hygromycin, bleomycin or streptomycin. The vector can be an expression vector and thus can comprise a heterologous gene (which does not occur naturally in the host cell, e.g. Propionibacteria) or an endogenous gene or host cell homolog, e.g. Propionibacteria. In the expression vector, the gene to be expressed is usually operably linked to a control sequence which is capable of providing expression of the gene in a host cell. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship that allows them to function in the manner proposed. A controlled sequence "operably linked" to a coding sequence binds such that expression of the coding sequence is obtained under conditions compatible with the control sequences. The heterologous or endogenous gene can be inserted between nucleotides 1 and 200 or between nucleotides 1500 to 3555 of the SEC. FROM IDENT. NO: l or an equivalent position in a homologous polynucleotide. Such genes can comprise homologous or endogenous genes for example for elongation factors, promoter regulatory sequences or elements and replication proteins. Other genes (which may be heterologous to the host) include those that code for or that aid in the production of nutritional factors, immunomodulators, hormones, proteins and enzymes (eg proteases, amylases, peptidases, lipases), texturizing agents, flavoring substances (for example diacetylacetone), gene pooling agents, antimicrobial agents (for example nisin), substances for use in food products (for example in sauces, cheese), metabolic enzymes, vitamins (for example B12), uroporphyrinogen (III) methyltransferase (UO) III MT), cobA, antigens and (for example for vaccines) therapeutic agents. As will be seen, guests can produce a wide variety of substances, and not only polypeptides, which can be the desired product or can be used to produce the desired product. Heterologous genes may have a therapeutic effect in humans or animals. Such a gene can comprise an antigen, for example from a pathogenic organism. The host, such as Propionibacterium, comprises a polynucleotide with such a heterologous gene and can be used as such or in a vaccine, and can provide protection against pathogens. The heterologous antigen can be a whole protein or a part of a protein that contains an epitope. The antigen can be a bacterium, a virus, a yeast or a fungus.

Host cells and expression The host cell forms the third aspect of the invention and comprises a polynucleotide or vector of the first or second aspects. The host cell can be a bacterium, for example of the Actipoipycetes class. The bacteria can be a Propionibacterium or E. coli. The Propionibacterium can be P. freudenreichii, P. jensenii, P. thoenii or P. acidipropionici. In a fourth aspect, the invention provides a process for producing a host cell of the third aspect, the process comprises transforming or transfecting a host cell with a polynucleotide or vector of the first or second aspects, for example with known transformation techniques11. In a fifth aspect, the invention provides a process for the preparation of a polypeptide encoded by the polynucleotide or vector of the invention present in the host cell of the invention comprising placing or culturing the host cell under conditions where the expression of the polypeptide. This aspect of the invention therefore provides a process for the preparation of a polypeptide encoded by a given gene, which process comprises culturing a host cell transformed or transfected with an expression vector comprising the gene, under conditions to provide the expression of such a polypeptide, and optionally recovering the expressed polypeptide. The host cell can be of the Actinomycetes class, or a gram-positive bacteria such as Propionibacterium or E. coli. Promoters, translation initiators, translation terminators, elongation factor genes, ribosomal RNA, antibiotic resistance genes, synthetic promoters (for example those designed based on consensus sequences) or other expression regulation signals present in the The polynucleotide or vector can be those which are compatible with the expression in the host cell. Such promoters include the promoters of the endogenous genes of the host cell. The culture conditions can be aerobic or anaerobic conditions, based on the host. For a fermentation process, the host cell should be placed in an anaerobic environment and then possibly under aerobic conditions. The produced compound, such as an expressed polypeptide, can then be recovered for example from the host cell or a fermentation medium. The expressed polypeptide can be secreted from the host cell. Alternatively, the polypeptide may not be secreted from the host cell. In such a case, the polypeptide can be expressed on the surface of the host cell. This may be desirable, for example, if the polypeptide comprises an antigen for which an immune response is desired in the human or animal. A homologous gene that may be present in the vector of the invention may be cobA. A host cell comprising this vector may therefore be capable of producing a compound such as vitamin B12 from a substrate or the compound may be the product of an enzyme. The invention specifically provides a process for the preparation of vitamin B12 which comprises culturing or fermenting such a host cell under conditions in which the UP (III) MT gene is expressed. The expressed enzyme can be contacted with a suitable substrate under conditions in which the substrate is converted to vitamin B12. This may result in increased production of vitamin B J1,2 'Therapeutics As described above, the polynucleotide of the invention may comprise a heterologous gene which is a therapeutic gene. Therefore, the invention includes a host cell comprising a vector of the invention which comprises a therapeutic gene for use in a method of treating the human or animal body by therapy. Such a host cell can be Propionibacterium. The host cell may be alive or dead. The host cell can be formulated for clinical administration by mixing it with a pharmaceutically acceptable carrier or diluents. For example, it can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, oral or transdermal administration. The host cell can be mixed with any vehicle which is pharmaceutically acceptable and appropriate for the desired route of administration. The pharmaceutically acceptable carrier or diluent for injection may be, for example, a sterile or isotonic solution such as water for injection or physiological saline. The dose of the host cells can be adjusted according to various parameters, especially according to the type of host cells used, the age, weight and condition of the patient to be treated.; the administration mode used; the condition to be treated and the clinical regimen required. As a guide, the number of host cells administered, for example by oral administration, is from 107 to 1011 host cells per dose for a 70 kg adult human. The routes of administration and dosages described are only as a guide since the skilled practitioner will be able to easily determine the optimal route of administration and dosage for any particular patient and condition.

Polypeptides A sixth aspect of the invention provides a polypeptide of the invention comprising one of the amino acid sequences set forth in SEQ. FROM IDENT. NO: 2 or 3, or a substantially homologous sequence, or a fragment of any of these sequences. The polypeptide may be one encoded by a polynucleotide of the first aspect. In general, the naturally occurring amino acid sequences shown in SEQ. FROM IDENT. NO: 2 or 3 are preferred. However, the polypeptides of the invention include homologs of the natural sequences and fragments of the natural sequences and their homologs which have the activity of the naturally occurring polypeptides. One of such activity may be the replication effect of the polynucleotide of the invention. In particular, a polynucleotide of the invention may comprise: (a) the protein of SEQ. FROM IDENT. NO: 2 or 3; or (b) a homologue thereof of Actinomycetes such as Propionibacterium freudenreichii or other strains of Propionibacterium; or (c) a protein at least 70% homologous to clauses (a) or (b). A homolog can naturally occur in a Propionibacterium and can function in a manner substantially similar to a polypeptide of SEQ. FROM IDENT.

NO: 2 or 3. Such homolog can be presented in Actiiaoinycetes or Gram-positive bacteria. A protein with at least 70% homology with SEC proteins. FROM IDENT. NO: 2 or 3 or a homologue thereof will preferably be at least 80 or 90%, and more preferably at least 95%, 97% or 99% homologous thereto over a region of at least 20, preferably at least 30, for example at least 40, 60 or 100 or more contiguous amino acids. Methods for measuring protein homology are well known in the art and will be understood by those ordinarily skilled in the art in the present context, that homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology"). "). The sequences of the proteins of SEC. FROM IDENT. NO: 2 and 3 and homologs can therefore be modified to provide other polypeptides within the invention. Amino acid substitutions can be made, for example 1, 2 or 3 up to 10, 20 or 30 substitutions. The modified polypeptide will generally retain its natural activity. Conservative substitutions can be made, for • 10 example according to the following table. The amino acids in the same block in the second column and preferably in the same line in the third column can be substituted for each other: • The polypeptides of the invention also include fragments of the full length polypeptides mentioned above and variants thereof, which include fragments of the sequences set forth in SEQ. FROM IDENT. NO: 2 or 3. Such fragments can retain their natural activity of full-length polypeptides. Suitable fragments will be at least about 5, for example 10, 12, 15 or 20 amino acids in size. The polypeptide fragments of SEC. FROM IDENT. NO: 2 and 3 and homologs thereof may contain one or more (for example 2, 3, 5 or 10) substitutions, deletions or insertions, including conserved substitutions. The polypeptides of the invention may be in substantially isolated form. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, for example 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention. A polypeptide of the invention may be labeled with a developer tag. The revealing label can be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, for example 125 I, 35 S, enzymes, antibodies, polynucleotides and linkers such as biotin.

Industrial Applications As will be apparent from the discussion, the host cells of the third aspect can be used to produce not only the recombinant proteins, but also other compounds of interest, which include non-protein chemicals such as inorganic chemicals, in particular vitamins. A seventh aspect of the present invention therefore relates to a process for the production of a compound, a process comprising culturing or fermenting host cells of the third aspect under conditions whereby the desired compound is produced. Although this compound can be a polypeptide, for example a polypeptide of the sixth aspect, it can also be one of the compounds mentioned in the previous discussion regarding the genes to be expressed. Clearly, the inorganic compounds will not be expressed by a gene but will be produced by an enzyme, or the polypeptide or the enzyme can help the host cell in the production of the desired compound. These compounds can be produced within the cell and can be subsequently isolated, for example after lysis of the host cell, or they can pass through the wall of the host cell to the surrounding medium, which can be a fermentation medium, for example an aqueous solution. In this manner, the host cells can be cultured in an aqueous medium comprising cells and nutrients for the cells, for example assimilable sources of carbon or nitrogen, or both. The polypeptides produced in this way can have therapeutic uses. They can be drugs or other pharmacologically active compounds, or they can be antigenic or immunogenic, in which case they can find use in vaccines. The invention additionally encompasses compounds produced by this process, whether or not of a recombinant polypeptide. The compounds contemplated specifically are vitamins such as vitamin B12 (cobalamin). In some cases, the compound does not need to be isolated from the fermentation medium or from the host cells. The host cells themselves can be used in particular applications, for example in, or in the manufacture of food products such as sauces, or cheese making, or the host cells can be included, for example from animal feeds, for example when the host cells contain a compound that is to be ingested by the animal in question. The invention therefore extends to the use of these compounds or host cells, the production of food products such as cheeses and sauces. The invention also contemplates food products or animal feeds made up of host cells or a compound produced by the invention.

In a particularly preferred embodiment of the present invention the host cells can be used in a cheese-making process, and in this way the invention further includes a process for making cheese wherein the microorganisms that are used are host cells of the cheese. invention. The host cells can be used instead of or in addition to other bacteria such as lactic acid bacteria. Propionic acid bacteria which are currently used in cheese making processes, for example with mesophilic cultures (type of Maasdam cheese) as well as in thermophilic cultures (Emmental). Both mesophilic and thermophilic organisms may be responsible for the addition of milk or cheese. In this way, the host cells of the invention can be used not only for cheese making but also for the production of other fermented dairy products (for example yoghurts). The propionic acid bacteria are used in cheese making due to their ability to convert lactate and carbohydrates to propionic acid, acetic acid and carbon dioxide. The host cells of the invention, especially if they are propionibacteria, can be used because they are less sensitive to nitrates and salt, which may allow the reduction or omission of bactofugation of the milk (usually used to reduce the concentrations of Clostridia). ).

The fermentation of the host cells can have one or two phases or stages. These may be, for example, a growth or production phase, or both, or an anaerobic and an aerobic phase or both. Preferably, there will be a growth or anaerobic phase or both, and in a suitable manner also (eg subsequently) a production or aerobic phase, or both. Both carbon and nitrogen sources, or both, can be complex sources or individual compounds. For carbon, it is preferred that it be glucose. For nitrogen, appropriate sources include yeast extract or ammonia or ammonium ions. Preferred features and characteristics of one aspect of the invention are suitable for other aspects mutatis mutandis.

Figures The invention is illustrated by the accompanying drawings in which: Figure 1 is a restriction map of a vector within the invention, p545 which is obtained from P. freudenreichii LMG 16545 (CBS 101022); and Figures 2a and 2b each contain two maps of two vectors, all of the four vectors are within the invention. The invention will now be described by way of example with reference to the following examples, which should not be considered as limiting.

EXAMPLE 1 Analysis of the strains of Prooionibac ter ium A collection of 75 non-pathogenic strains of Propionibacterium was analyzed to determine the presence of natural plasmids. Most of the strains are obtained from the BCCM / LMG culture collection (Ghent, Belgium), although some strains are obtained from ATCC (Rockville, Md., USA) and from DSM (Braunschweig, Germany). The analysis is performed using a small-scale plasmid isolation procedure. First bacteria are grown anaerobically in MRS2 medium for 48 hours at 30 ° C. The plasmids are then purified from the bacteria using a plasmid DNA isolation procedure originally developed for E. coli * with some modifications: cells from a 5 ml culture are washed in a solution of 25% sucrose, 50 mM Tris-HCl pH, are resuspended in 250 μl of TENS (25% sucrose + 50 mM NaCl + 50 mM Tris-HCl + 5 mM EDTA pH 8) containing 10 mg / ml lysozyme (Boehringer Mannheim), and incubated at 37CC for 20 minutes. -30 minutes. The bacterial cells are then lysed in 500 μl of 0.2 N NaOH / 1% SDS (incubation for 2-5 minutes on ice). After the addition of 400 μl of 3M NaAc, pH 4.8 (5 minutes on ice) and subsequent extraction with phenol / chloroform, the DNA is precipitated by the addition of isopropanol. The DNA is analyzed by electrophoresis in 1% agarose gels and visualized by ethidium bromide. While most of the strains are negative, that is, they do not show the presence of natural plasmids in this analysis, most of the strains that gave a positive result contain large plasmids (= 20 kb.). 6 strains Of these, P. jensenii LMG16453, P. acidipropionici ATCC4875, P. acidipropionici LMG16447 and an unspecified strain of Propionibacterium (LMG16550) contains a plasmid in the size range of 6-10 kb Two strains (P. freudenreichii LMG16545 and P. freudenreichii LMB16546) show an identical plasmid profile of the two plasmids One plasmid is large (size not determined) and the other is smaller, more abundantly present and has a size of 3.6 kb These 3.6 kb plasmids of LMG16545 and LMG16546 are chosen for further analysis.

EXAMPLE 2 Analysis of a natural plasmid of strains LMG16545 and LMG16546 Plasmids of 3.6 kb were isolated from both strains and were further purified by ultracentrifugation in a density gradient of CsCl-ethidium bromide11. Limited restriction maps were prepared for preparations and found to be identical11. The restriction map of the 3.6 kb plasmid is shown in Figure 1. T4 ligase and restriction enzymes are obtained from New England Biolabs or GIBCO BRL. The 3.6 kb plasmid of strain LMG16545 (hereinafter referred to as p545) is radioactively labeled and used in Southern blot hybridization experiments. Hybridization conditions were 0.2 x SSC, 65 ° C. They react equally well with both DNA extract of the plasmids LMG16545 and LMG16546, substantiating the close relationship of these strains, while a plasmid DNA extract of P. acidipropionici ATCC4875, harboring a 6.6 kb plasmid named pTYl or pRGOl8 did not react. The DNA sequence of plasmid p545 was determined with dideoxyribonucleotides labeled with fluorescent dye in an Applied Biosystems 373A automated sequencer and is included as SEQ. FROM IDENT. NO: l in the sequence list. Sequence analysis is performed on the plasmid DNA that has been linearized with EcoRI and inserted into pBluescript SKII + DNA digested with EcoRI (Stratagene, La Jolla, Ca., USA). The computer-assisted analysis of the sequence obtained in this manner using the BLAST1 investigation shows homologies with the proteins involved in the replication of plasmids of several GC-rich organisms (eg pAL5000 encoded by repA and repB of Mycobacterium fortuitum8,14 sample 28-30% identity and 34-38% similarity with the respective putative replication proteins of the p545 plasmid; pXZ10142 of Corynebacterium glutamicum [PIR Accession Number S32701] is another example of plasmids encoding replication proteins homologous to putative replication proteins p545). The results of the database comparisons with homologous sequences are detailed in Examples 7 and 8.

EXAMPLE 3 Construction of shuttle vectors of E. coli / Propionibacterium Plasmid pBR322 from E. coli is digested with EcoRI and Aval and the smallest fragment generated in this way (measured 1.4 kb and encompasses the gene conferring resistance to tetracycline) is replaced by a synthetic duplex DNA. The synthetic duplex DNA is designed so that it binds to the EcoRl and Aval ends to supply several unique restriction enzyme restriction sites: '- AATTCAAGCTTGTCGACGTTAACCTGCAGGCATGCGGATCCGGTACCGAT ATCAGATCT-3' (SEQ ID NO: 4) 3 '- GTTCGAACAGCTGCAATTGGACGTCCGTACGCCTAGGCCATGGCTATAGT CTAGAAGCC - 5' (SEQ ID NO: 5) In this manner, the following restriction enzyme recognition sites were provided: EcoRI (restored), ffindlll, SalI, Hpal, PstI, SphI, Ba KI, Acc651, EcoRV, BglII (Aval is not restored). The synthetic DNA is ligated to the large fragment and the ligation mixture is transferred back to E. coli (T4 ligase is used). A plasmid of the expected composition is obtained (pBR322? L). The multiple cloning site can be used to introduce a selection marker as well as the p545 DNA plasmid. As an example of the construction of the E. coli / Propionibacterium leader plasmid conferring resistance to erythromycin, it is carried out as described now.

A fragment of 1.7 kb Acc65I is inserted Saccharopolyspora erythraea NRLL2338 from an erythromycin biosynthesis group and containing the gene that confers resistance to erythromycin15'2 within pBR322? L linearized with Acc65I. The newly derived construct, called pBRES, is linearized with EcóRV and binds to p545 DNA that has been digested with BsaBI.

It is found that E. coli transformants harbor a vector with the correct insert, in both orientations. The resulting plasmid vectors are named pBRESP36Bl and pBRESP36B2 (figures 2a and 2b). Plasmid vector constructs with linearized p545 DNA are also obtained at another restriction site located outside the putative replication region, specifically AlwNl. For this construction, the vector pBRES should be provided with a suitable cloning site. An adapter consisting of two complementary oligonucleotides of the following composition is designed (SEQ ID NOS: 6 and 7): 'GTACCGGCCGCTGCGGCCAAGCTT 3' 5 'GATCAAGCTTGGCCGCAGCGGCCG 3' The annealing of these oligonucleotides creates a double-stranded DNA fragment with Acc65I and BglII cohesive ends, respectively, which also contains an internal Sfil restriction site that provides compatible ends with the plasmid p545 digested with AlwNl. This adapter is cloned in pBRES between the BglII site and the proximal Acc65I. The pBRES-Sfi vector obtained in this way is subsequently digested by Sfil and ligated to p545 digested with AlwNl. Transformation of E. coli provides transformants with the corrected vector as confirmed by restriction enzyme analysis. The obtained vector is called pBRESP36A (figure 2).

EXAMPLE 4 Transformation of Propionibacterium with E. coli / Propionibacterium vectors The transformation of Propionibacterium freudenreichii strain ATCC6207 with pBRESP36Bl will be described. Bacterial cells are cultured in SLB (sodium lactate broth17 at 30 ° C to a stationary growth phase, and subsequently diluted 1:50 in fresh SLB.) After incubation at 30 ° C for approximately 20 hours, cells are harvested. (now in exponential growth phase) and washed extensively in cold 0.5M sucrose Subsequently the cells are washed once in the electroporation buffer, which consists of 0.5 M sucrose, buffered with 1 mM potassium acetate, pH 5.5, and they are finally resuspended in this electroporation buffer in approximately 1/100 of the original culture volume.The cells are kept on ice throughout the procedure.For electroporation (BIORAD apparatus), 80-100 μl of cell suspension is mixed with ± 1 μg of DNA (or smaller amounts), in a chilled 1 or 2 mm electroporation cuvette, and electric pulses are supplied. It is found that the optimal pulse conditions are 25 ^ / cm at 200 O of resistance and a capacitance of 25 μF. However, minor and major voltages (also at 100 O) also provide transformants. Immediately after the pulse, 900 μl of cold SLB containing 0.5 M sucrose is added to the suspension of pulsed cells and these are subsequently incubated for 2.5 to 3 hours at 30 CC before plating appropriate dilutions on SLB / agar plates. containing 0.5 M sucrose and 10 μg / ml erythromycin. After an incubation period of 5 to 7 days at 30 ° C under anaerobic conditions, the transformants are detected. DNA isolated from E. coli DH5a (Promega) provides a transformation efficiency of 20-30 transformants per i-μg of DNA. An efficiency greater than 10-200 fold is obtained when DNA is isolated from E. coli JM110 (strain darrf, dcrrf). The transformation of E. coli is carried out according to the BIORAD instructions.

The transformants contain the authentic, indistinguishable vectors of the original plasmid DNA used for transformation of ATCC6207. This is shown by the restriction enzyme analysis of the plasmid DNA isolated from the 5 transformants by the isolation procedure of small plasmid DNA mentioned above. The vectors are present exclusively as replicating plasmids autonomously. Hybridization with ^ j. Southern13 transfer (with total DNA isolates shows that The chromosomal DNA does not hybridize with the vector DNA used as a probe, indicating that no chromosomal integration of the plasmid DNA has occurred. The transformation is also successful with vectors pBRESP36B2 and pBRESP36A, indicating that the functionality of the The vector is independent of the orientation of p545 or the cloning site used. In addition, in this case the authenticity of the vectors was confirmed. • In addition, the transformation of P. freudenreichii strain ATCC6207 with DNA isolated from a transformant of Propionibacterium results in an increase of 105-106 fold in the efficiency of transformation compared to that obtained with DNA isolated from E. coli DH5a. The transformation of another strain of P. freudenreichii, LMG16545 (the same strain from which the plasmid p545 was obtained), results in a transformation efficiency comparable to that of the ATCC strain. • The results of the transformations, and the effect on the production of vitamin B12, are shown in the following Table. Eight of the 10 transformants provided 50% more vitamin B12 content compared to the control strain. • 10 fifteen twenty EXAMPLE 5 Construction of the plasmid vector containing the coiA gene The construction and application of a plasmid vector to increase the synthesis level of vitamin B12 (cobalamin) in P. freudenreichii strain ATCC6207 will be described. The promoter region of the gene that confers resistance to erythromycin in Saccharopolyspora erythraea2'2 is generated by PCR using the following primers (SEQ ID NOS: 8 and 9): direct primer: (51 - 3 ') AAACTGCAGCTGCTGGCTTGCGCCCGATGCTAGTC reverse primer: (5 '- 3') AAACTGCAGCAGCTGGGCAGGCCGCTGGACGGCCTGCCCTCGAGCTCGT CTAGAATGTGCTGCCGATCCTGGTTGC The PCR fragment generated in this way contains an AlwN1 site at the 5 'end followed by an authentic promoter region and the first 19 amino acids of the coding region of the erythromycin resistance gene, for • ensure proper transcription and initiation of translation. At the 3 'end, Xbal and Xhol sites are provided (for insertion of the cobA gene at a later stage), a terminator sequence as presented at the 3' end of the erythromycin resistance gene, and at an AlwNl site. The PCR product is digested with AlwNl and ligated to pBRESP36B2, partially digested with AlwNl. Of the two sites • 10 AlwNl present in pBRESP36B2, only one present in the specific part of p545 of the vector will accommodate the fragment of E. coli transformants that are obtained harboring the expected construct, called pBRES36pEt. This vector is used for additional constructions as described in the following. The coding sequence of cobA, the gene coding for uroporphyrinogen III methyltransferase, is generated by PCR from Propionibacterium freudenreichii strain ATCC6207, using the following primers (SEQ ID NOs. 11): direct: (5 '-3') CTAGTCTAGACACCGATGAGGAAACCCGATGA inverse: (5 '-3') CCCAAGCTTCTCGAGTCAGTGGTCGCTGGGCGCGCG 25 The cobA gene amplified in this way presents an Xbal site in the N-terminal coding region and HipdlII O and Xhol sites in the terminal coding region C. The functionality of this cobA gene is confirmed by cloning of the PCR product as a Xbal-HindIII fragment in pUC18, and the subsequent transformation of E. coli strain JM109. Transformants with a functional cobA gene show bright red fluorescence when illuminated with UV light.

^^ Plasmid DNA isolated from such a transformant is digested with Xbal and Xhol, is linked to pBRESP36B2. DNA digested in the same way is used to transform E. coli DNA from several transformants that are analyzed by restriction enzyme digestion and gel electrophoresis. It is found that the transformants present the correct insert in the vector of expression. This new vector is called pBRES36COB. This vector is subsequently transferred to P. freudenreichii ATCC6207 following the protocol described above. Ten of the obtained transformants were analyzed and found to harbor the pBRES36COB vector, which is again indistinguishable from the original vector used for transformation, as shown by analysis of the restriction enzyme digests. In these ten transformants, the level of vitamin B12 synthesis was determined as follows. Frozen cultures of transformants of Propionibacterium 1 to 10, as well as control strains containing only the plasmid vector pBRESP36B2, were inoculated into 100 ml flasks containing 50 ml of medium BHI (brain heart infusion) (Difco) and incubated during 72 h at 28 ° C without agitation. From this preculture, 4 ml were transcribed to 200 ml of production medium consisting of Difco yeast extract 15 g / 1, Nalactate g / 1, KH2P04 0.5 g / 1, MnS04 0.01 g / 1 and CoCl2 0.005 g / 1 in a 500 ml shake flask and incubated at 28 ° C for 56 h without shaking, followed by 48 h in a shaker New Brunswick revolving at 200 rpm. The titers of vitamin B12 were measured using a known method of CLAP5. Nine of the 10 transformants showed approximately 25% higher production of vitamin B12 compared to the control strain.

EXAMPLE 6 Stability of plasmids The totality of the three conducting vectors pBRESP36A, pBRESP36B1 and pBRESP36B2 were stably maintained for 30 generations of culture of the respective transformants: no loss of erythromycin resistance was observed as determined by the viability beads on selective agar plates (containing erythromycin) and non-selective The structural stability of the plasmid in the transformant population after 30 generations is established * f by isolation of the plasmid DNA and characterization by restriction enzyme mapping as described above: only restriction fragments similar to those of the authentic plasmid were observed.

EXAMPLE 7 • 10 Analysis of sequence sequence homology for the predicted polypeptide encoded by the first open reading frame (SEQ ID NO: 2) MDSFETLFPES LPRKPLASAEKSGAYRHVTRQRALELPYIEANPLVMQSLV 15 ITDRDASDADWAADLAGLPSPSYVSMNRVTTTGHIVYALKNPVCLTDAARR RPINLLARVEQGLCDVLGGDASYGHRITKNPLSTAHATL GPADALYELRA LAHTLDEIHALPEAGNPRRNVTRSTVGRNVTLFDTTRM AYRAVRHSWGG PVAEWEHTVFEHIHLLNETI IAD The previous sequence of 227 amino acids (ORF1) is aligned and compared with several other protein sequences (objective NBRF-PIR, version PIR R52.0 March 1997, section 45, KTUP: 2). With a protein of Mycobacteriu fortuitum pAL 5,000 (JS0052), a coincidence of 37.1% was found over 194 amino acids (INIT 167,292). With a protein of Corynebacterium glutamicum (S32701) is a coincidence of 32.0% over 125 amino acids (INIT 125, 116). A match of 29.9% over 221 amino acids (INIT 86, 259) is found with the ColE2 protein of E. coli (S04455). Precisely the same coincidence is found on the same number of amino acids for the ColE3 protein, also of E. coli (S04456).

EXAMPLE 8 Analysis of sequence sequence homology for the predicted polypeptide encoded by the second reading frame (SEQ ID NO: 3) MTTRERLPRN GYSIAAAAKK LGVSESTVKR TSEPREEFV ARVAARHARI RELRSEGQSM RAIAAEVGVS VGTVHYALNK NRTDA The second protein (ORF2) is also aligned and compared to another protein, using the same parameters and software as those described for Example 7. However, this sequence only has a length of 85 amino acids. The ORF2 sequence is compared with a Mycobacterium fortui tum protein (S32702) and a 53.3% match over 75 amino acids is found (INIT 207, 207).

EXAMPLE 9 < (H Functional analysis of plasmid p545 In order to further improve the vector system, the essential plasmid functions for replication and stability were delineated more precisely by monitoring large regions of the original p545 plasmid. ^. The result, a smaller cloning vector, will allow the use of the vector system based on p545 to clone larger fragments of DNA. For this purpose, the vector pBRESP36A is digested (figure 2) with SstII and Bell, resulting in fragments of 1. 7 kb and 6.5 kb. The fragment of 1.7 kb, in fact, the fragment of 1.6 kb of AlwNI-BcII from plasmid p545, is replaced by a Synthetic duplex DNA, constituted by SEC. FROM IDENT. NO: 12 and SEC. FROM IDENT. NO: 13 with compatible ends with Sctll and Bell and many unique restriction enzyme recognition sites. 20 SEC. FROM IDENT. NO: 12 5 'GGAGATCTAGATCGATATCTCGAG 3' SEC. FROM IDENT. NO: 13 5 'GATCCTCGAGATATCGATCTAGATCTCCGC 3" The following restriction enzyme recognition sites were provided in this manner: SstII (restored), Bglll, Xbal, Clal, EcoRV, Xhol, (Bell is not restored.) The ligation mixture is transferred E.coli and the transformants containing a vector of the expected composition are selected. The vector is called pBRESA? S-B. The subsequent successful transformation of P. freudenreichii strain ATCC6207 with this vector indicates that the 1.6 kb region between AlwNl and B ll in p545 is not essential for plasmid replication. ^ * An additional deletion is made by removal of ^ 10 the 240 bp corresponding to the region between Sali and BcII in plasmid p545. This is obtained by digestion of pBRESA? S-B with Sall -Sstl and SstI -Xhol respectively, and isolation of the 1.3 kb fragment subjected to Sall-Sstl and the 6.6 kb fragment of SstI and Xhol. The fragments are linked and the mixture of ligation is transferred to P. freudenreichii ATCC6207, which provides numerous transformants. The newly derived construct, called pBRESA? S-S, is isolated and its structure confirmed by restriction enzyme mapping. Therefore, all the essential information for The replication of plasmid p545 is located in a 1.7 kb fragment delimited by the SalI and AlwNI restriction sites and encompasses the predicted replication proteins encoded by 0RF1 and ORF2, and can be deleted 1.8 kb without evidently altering the replication stability of the plasmid 25 EXAMPLE 10 Expression of a chloramphenicol resistance gene from Coryn eb cterium 5 A chloramphenicol resistance gene (cml) of Corynebacterium16 has been identified as coding for an export protein of chloramphenicol. This gene is inserted into the driving vector of Propionibacterium - E. coli pBRESP36B2. This vector is digested with BglII and HindIII, and with BglII and Hpal, respectively. The fragments of 2.9 kb BgrlII - Hindlll and 5.2 kb BglII - Hpal are isolated. The fragment containing the cml gene, which includes its own promoter, is obtained by digestion with PvuII and HindlII and the 3.3 kb fragment containing the gene is isolated. The two specific vector fragments and the cml fragment are linked: the extremes PvuII and Hpal become blunt, therefore • inserting the cml gene as well as restoring the ermE gene of the original vector. The ligation mixture is transferred to E. coli and a transformant is selected, in which the vector contains the correct cml insert. The vector is called pBRESBCM. Transformation of P. freudenreichii ATCC6207 with this vector, and selection on plates containing 10 μg / ml erythromycin, or 5 μg / ml chloramphenicol, provides colonies resistant to erythromycin and chloramphenicol, respectively, indicating that, in addition to the erythromycin resistance gene (shown above in the conductive vectors of Propionibacterium - E. coli), also the chloramphenicol resistance gene is expressed in Propionibacterium. Transformants can be cultured in liquid medium containing up to 20 μg / ml chloramphenicol.EXAMPLE 11 Expression of the gene for lipase (qehA) of P. acnes To illustrate the cloning and efficient expression of an extracellular protein using the current vector system, a gene for lipase gehA from P. acnes is used19. The vector pUL6001, which harbors gehA in an XhoI fragment, is digested with XhoI and the 2.75 kb fragment containing the gene is isolated. The pBRESA? S-B vector (from Example 9) is linearized with XhoI and the ends are dephosphorylated using bovine intestine phosphatase to avoid updating. The linearized vector and the fragment containing gehA are ligated and the ligation mixture is transferred to E. coli. The transformants are analyzed by restriction enzyme analysis to determine the presence of the correct recombinant plasmid, called pBRESALIP. This plasmid is subsequently transferred to P. freudenreichii strain ATCC6207.

The transformants are analyzed to determine the expression of the lipase gene, using agar plates containing fJP tributyrin as the indicator for increased expression of lipase. Transformants of P. freudenreichii harboring 5 pBRESALIP show significantly increased halo sizes in this assay, compared to untransformed strains or strains transformed with the parental vector.

REFERENCES 10 1. Altschul et al. , J. Mol. Biol. 215-403-410 (1990) 2. Bibb et al. , Gene 38, 215 (1985) 3. Bibb et al. , Mol. Microbiol. 14 (3), 533 (1984). 15 4. Birnboim and Doly, Nucleic Acids Res. 7, 1513 (1979) 5. Blanche, Anal. Biochem. 189, 24 (1990) 6. DeMan et al, J. Appl. Bacteriol. 36, 130 (1960) 7. Deveraux et al, Nucleic Acids Research. 12: 387-209 395 (1984) 8. Labidi et al, Plasmid 27, 130 (1992) 9. Rehberger and Glatz, Appl. Environ. Microbiol. 59, 83 (1990) 10. Rossi et al, Res. Microbiol. 147, 133 (1996) 11. Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press (1989) 12. Sattler et al, J. Bact. 177, 1564 (1995) 13. Southern, J. Mol. Biol. 98, 503 (1975) 5 14. Stolt and Stoker, Microbiol. 142, 2795 (1996) 15. Thompson et al, Gene 20, 51 (1982) 16. Uchijama and eisblum Gene 38, 103 (1985) 17. de Vries et al, J. Gen. Microbiol. 71, 515 ^ (1972) 9 10 18. Tauch et al, Plasmid 40 (2): 126 (1998) 19. Miskin et al, Microbiol 143: 1745 (1997) SUMMARY OF THE SEQUENCES 1. DNA sequence of plasmid LMG 16545 (CBS 101022), 3.6 kb ^ 2. Amino acids of the ORF1 protein (303 residues, bases 273-1184). 3. Amino acids of the ORF2 protein (85 20 residues, bases 1181-1438). 4-13. Primers / DNA oligonucleotides.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. - 1 LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT (A) NAME: Gist-brocades B.V. (B) STREET: Wateringseweg 1 (C) CITY: Delft (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 2611 XT (ii) TITLE OF THE INVENTION: Propionibacterium Vector (iii) SEQUENCE NUMBER: 13 (iv) READABLE COMPUTER FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: patentln Relay # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEC. FROM IDENT. NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: - 2 - (A) LENGTH: 3555 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: (genomic DNA) (iii) HYPOTHETICAL: NO (iii) ANTICIPATION: NO (vi) ORIGINAL SOURCE (A) ORGANISM: Propionibacterium freudenreichii (C) INDIVIDUAL ISOLATED: CBS101022 LMG16545 (ix) CHARACTERISTIC (A) NAME / KEY: CDS (B) LOCATION: 273..1184 (C) OTHER INFORMATION: / gene = "ORF2" (ix) FEATURE: (A) NAME / KEY: CDS (B) (LOCATION) 1181..1438 (D) OTHER INFORMATION: / gene = * ORF2 * (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1: GTCGACCCTG ACAGCCGGCG AGCAGTTCAG GCGAAGATCG CACAGCTGCG CGAGGAACTA 60-3 - GCCGCAATGC CCCAACACGC CCCAGCCATC CCTTGSAGCA GGTGGCAGC3 TCAGGGGAGT 120 CGGGGGATGT TTGGCAGGGG ATGTGGAAAG AGAGTTCGCT TTGCTCACAT GGCTCAACCO 180 GGTAACTAAC TGATATGGGG TCTTCGTCGC CCACTTTGAA CACGCCGAGG AATGGACCAC 240 ^ GCTGAACGTG ACTCGCATCC TTCACTGCAT GT TTC ATG GCT GAG ACG QAT TTG 253 Met Asp Ser Phe Glu Thr Leu 1 5 TTC CCT GAG AGC TGC CTG CCA CGC AAG CCß CTG GCG TCA GCC GAG AAG Phe Pro Glu Ser Trp Leu Pro 'Arg Lys Pro Leu Wing Ser Wing Glu Lya 10 15 20 0 TCT GGG GCG TAC CGG CAC GTG ACT CGG CAG AGG GCG CTG GAG CTG CCT 3B9 Ser Gly Ala Tyr Arg His Val Thr Arg Gln Arg Ala Leu Glu Leu Pro 25 30 35 TAC ATC GAA GCT AAC CCG TTG GTC ATG CAG TCC TTG GTC ATC ACC GAT 437 Tyr He Glu Wing Asn Pro Leu Val Mee Glp Ser Leu Val le Thr Aßp 40 45 SO 55 COA GAT GCT TCG GAT OCT GAC TGG GCC GAC GAC CTC GCT GGG CTG CCT 5 485 Arg Asp Ala Ser Aßp Ala Asp Trp Ala Ala Asp Leu Wing Gly Leu Pro '60 65 70 TCA CCG TCC TAC GTG TCC ATG AAC CGT GTC ACG ACC ACC GGA CAC A? C 533 Ser Pro Ser Tyr Val Ser Met Asn Arg Val Thr Thr Thr Gly His He 75 90 85 GTC TAT GCC TTG AAG AAC CCT GTG TGT CTG ACC GAT GCC GCG CGG CGA 0581 Val Tyr Ala Leu Lys Asn Pro Val CYSS Leu Thr Asp Ala Ala Arg Arg 90 95 100 CGG CCT ATC CCT CTG CTC GCC CGC GTC GAG CAG GGC CTA TGC GAC GTT 629 Arg Pro He Asn Leu Leu Ala Arg Val Glu Gln Gly Leu Cys Aap Val IOS 110 115 CTC GGC GGC GAT GCA TCC TAC GGG CAC CGG ATC ACA AAG AAC CCG CTC 677 5 Leu Gly Gly Asp Ala Ser Tyr Gly His Arg He Thr Lys Asn Pro Leu 120 125 130 135 AGC ACC GCC CAT GCG ACC CTC TGG GGC CCC GAC GAC GCG CTC TAC GAO 725 Ser Thr Wing His Wing Thr Leu Trp Gly Pro Wing Asp Wing Leu Tyr slu X40 145 150 - 4 - CTG CGC GCC CTC GCC CAC ACC CTC GAC GAG ATC ACC GCA CTG CCG GAG 773 Leu Arg Ala Leu Ala His Thr Leu Asp Glu lie His Ala Leu Pro Glu 155 160 165 GCA GGG AAC CCG CGT CGC AAC GTC ACC CGA TCA ACG GTC GGC CSSC AAC 821 Ala ßly Asn Pro Arg Arg Asn Val Thr Arg Ser Thr Val Gly Arg Asp 170 175 180 GTC ACC CTG TTC GAC ACC ACC CGC ATG TGG GCA TAC CGG GCC GTC CGG B69 Val Thr Leu Phe Aap Thr Thr Arg Mee Trp Wing Tyr Arg Wing Val Arg 185 190 195 CAC TCC TGG GGC GGC CCG GTC GCC GAA TGG GAO CAC ACC GTA TTC GAG 917 His Ser Trp Gly Gly Pro Val Ala ßlu Trp Glu His Thr Val Phe Glu 200 205 210 215 CAC ATC CAC CTA CTG AAC GAG ACG ATC ATC GCC GAC GAA TTC GCC ACÁ 965 His He His Leu Leu Aßn Glu Thx He He Wing Asp Glu Phe Ala Thr 220 225 230 GGC CCC CTC GGC TTG AAC GAA CTT AAG CAC TTA TCT CGA TCC ATT TCC 1013 Gly Pro Leu sly Leu Asn Glu Leu Lyss His Leu Ser AXG Ser He Ser 235 240 245 CGA TGG GTC TGG CSSC AAC TTC ACC CCC GAA ACC TTC CGC GCA CGC CAG 10S1 Arg Trp Val Trp Arg Asn Phe Thr Pro Glu Thr Phe Arg Ala Arg Gln 250 2S5 260 AAA GCG ATC AGC CTC CGT GGA OCA TCC AAA GGC GGC AAA GAA GGC CIQC 1109 Lys Ala He Ser Leu Arg Gly Ala Ser Lyss Gly Gly Lys Glu Gly Gly 265 270 275 CAC AAA GGC GGC ATT GCC AQT GGC GCA TCA CGG CGC GCC CAT ACC CGT 1157 His Lys Gly Gly lie Wing Ser Gly Wing Ser Arg Arg Wing His Thr Arg 280 285 290 295 CAA CAG TTC TTG GAG GGT CTC TCA TGACCACACG TGAACGTCTC CCCCGCAACG 1211 Gln Gln Phe Leu Glu Gly Leu Ser 300 GCTACAGCAT COCCGCTGCT QCGAAAAAGC TCGGTGTCTC CGAGTCCACC GTCAAGCGGT 1271 GGACTTCCGA GCCACGCQAG GAGTTCGTGG CCCGCGTTGC CGCACGCCAC GCGCGGATTC 1331 GTGAGCTCCG CTCGGAGGGT CAGAGCATGC GTGCGATTGC TGCCQAGGTC GGGGTTTCCG 1391-5 - TGGGCACCGT GCACTACGCG CTGAACAAGA ATCGAACTGA CGCATGACCG TAACGCC5CA 1451 CGATGAGCAT TTTCTTGATC GTGCACCGCT TGGCACTACG TTCGCGTGCG GTTGCACAGT 1H11 GCsCGCCACG TTCTTATCCT GCGGCCATTG TGGCTACAGC CAATGGGGGG CATCAGCAAC 1S71 GGACGTTGAA CCCGGTGGGC AAGTGTTACT CAGGGGGACA TGCCCAGTC7 GCGGCGCTCG 1631 GATTGACGGT ATGGCAGTCG TGCATGCGGC CCCACCGTCA AACTCATTCA GGTATCAGTG 1691 AGAACCCTCA TGGCACCCCC TCGTGACACG TTCTCGTTGC GATCAGCTGC TGTGCGTGCG 1751 GGCGTGAGCG TTTCTACGCT GCGGCGCAGG AAATCAGAGC TTGAGGCTGC CGGAGCGACG 1811 GTAGACCCGT CCGGTTGGGT GGTGCCACTG CGTGCACTCA AGGTCQTTTT TGGGGTGTCA 1871 GATGAGACCT COAATGCGCC CGGTCATGAC GCTGAGTTAG TGGCGCAGCT GCGCTCTGAG 1931 AACGAGTTTT TACGGCGTCA GGTCGAGCAG CAGGCQCGCA CGATCGAACG GCAGGCTGAß 1991 GCACACGCGG TßaTCTCAGC GCAGCTCACA CGGGTTGGCC AGCTTGAGaC CGGCGACGCA 2051 GCAGCACCGA CACTGGCACC CGTTGAAAGG CCGGCTCCGC GACGGCGGTG GTGGCAGCGT 2111 CGGTAGCGGT CAGGATCGCT CTGGCGTGAC GAGTGTGTCT GGCAGTGCGA ACAGTTGCTC 2171 GACCAGTGGC AGCAGAAGCG AGATCGCTGC GTGGTGCTGT TCCTCGGTCA GTTCGTCGAG 2231 GACTGGCGGG TCTTGCTGCa TCCAGCCGAT CGCCTCGGCG GCCAAGQTCA GTTCCAAGCT 2291 GTGCCAACGC ACACGCCCCT CGGCT3ACAG CTGAGTCTCG AACTGTGCAA CTGGACCGGC 2351 CGOAAGATGC ACGTTGCCGA GGTCGTGAGT GGCCAAGCGC ACGTCAAASA GTGCTGCTTC 2411 GTAGCCGCGC AGAAATGGCA GTGCTCGGTC GATTCGGATC GGCCTGCCCA GGTACATTCC 2471 GGGCCGCTTG ATGAACGCCT CCGCGTAGAA GCGCACCGTT CTCGGCCCGG CCTCGTGATC 2531 TGTCACTGTG CACGCTCCTC TCGATGGTTC TCGACGCTAC CGGAGACCAC CGACGTTCAT 2591-6 - GCCCAGCGCA GCGACCTGAA AßßACCAAGC CGAGTTAGCC GTGCTAACCG TATAGCTTGC 2651 TCCGTCGCCT CTGAGGGCAA CCACCTGCGC AGCAGGTGGG CGGCAGCCCG CGCGCAAGCG 2711 CCTACCGGGT TTGGGCACAG CCCATAAATC AACGC TCCG GTGTTGAAGC GATCGTGTGT 2771 CACGATTGCT ATGCTTGCTA CCCCTTCAGG GTTTTCGTAT ACACAAATCA AGTTTTTTCG 2831 TATACGCTAA TGCCATGAGT GAGCATCTAC TGCACGßCAA GCCCGTCACC AACßAGCAGA 2891 TTCAGGCATG GGCAGACGAG GCCGAGGCCG GATACGACCT GCCCAAACTC CCCAAGCCAC 2951 GGCGCGGACG CCCGCCCGTA GGAGACGGTC CGGGCACCGT CGTACCCGTQ CGTCTCGACG 3011 CGGCCACCGT TGCCGCTCTC ACAGAACGAß CAACAGCCGA GGGCATCACG AACCGTTCAG 3071 ACGCGATCCG AGCCGCAGTC CACGAGTGGA CACGGGTTGC CTGACCTCCA CGACTCAGCA 3131 CßCAAGCACT ACCAACGAGA CCGGCTCGAC GACACGGCCG TGCTCTACGC GGCCACCCAC 3191 GTTCTCAACT CCCGGCCACT CGACOACGAA GACGACCCGC GCC3CTGGCT CATGATCGGA 3251 ACCGACCCAG CAGGCCGCCT ACTCGAACTC GTCGCACTGA TCTACGACGA CGGCTACGAA 3311 CTGATCATCC ACGCAATGAA AGCCCGCACC CAATACCTCG ACCAGCTCTA ACCAAGAAAG 3371 GAACCTGATG AGCGACCAGC TAGACAGCGA CCGCAACTAC GACCCGATGA TCTTCGACGT 3431 GATGCGCGAG ACCGCSAACC GCGTCGTCGC CACGTACGTT GCATGGßAAG ATGAAGCCGC 3491 TGATCCCCGC GAGGCTGCGC ACTGaCAGGC CGAGCGATTC CGCACCCGGC ACGAGGTGCG 3SS1 CGCC 3S55 (2) INFORMATION FOR SEQ. FROM IDENT. NO: 2: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 303 amino acids - 7 - (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 Met Asp Ser Phe Glu Thr Leu Phe Pro Glu be Trp Leu Pro Arg Lys 1 5 10 15 Pro Leu Wing Ser Wing Glu Lys Ser Gly Wing Tyr Arg His Val Thr Arg 20 25 30 Gln Arg Wing Leu Glu Leu Pro Tyr He Glu Wing Asn Pro Leu Val Met 35 40 45 Gln Ser Leu Val He Thr Asp Arg Asp Wing Ser Asp Wing Asp Trp Wing 50 55 60 Wing Asp Leu Wing Gly Leu Pro 3er Pro Ser Tyr Val Being Met Asn Arg 65 70 75 80 Val Thr Tbr Thr Gly His He Val Tyr Ala Leu Lys Asn Pro Val Cys 85 90 95 Leu Thr Asp Ala Ala Arg Arg Pro He Asn Leu Leu Ala Arg Val 100, OS 110 Glu Gln Gly Leu Cyß Asp Val Leu Gly Gly Asp Ala Ser Tyr Gly Hls 115 120 125 Arg He Thr Lys Asn Pro Leu Ser Thr Wing His Wing Thr Leu Trp Gly 130 135 140 v? ° AI * ASP Ai L, eu Ty1 G ^ u Leu ^ 9 A ^ -a Leu & amp; a 3 Tnr Leu AS 145 150 155 160 Glu He His Wing Leu Pro Glu Wing Gly Asn Pro Arg Arg Asn Val Thr 165 170 175 Arg Ser Thr Val Gly Arg Asn Val Thr Leu Phe Asp Thr Thr Arg Met 180 185 190 Trp Wing Tyr Arg Ala Val Arg His Ser Trp Gly Gly Pro Val Ala Glu 195 200 205 Trp Glu His Thr Val Phe Glu His He His Leu Leu Asn Glu Thr He 210 215 220 He Wing Asp Glu Phe Wing Thr Gly Pro Leu Gly Leu Asn Glu Leu Lyß 225 230 235 240 His Leu Ser Arg Ser He Be Arg Trp val Trp Arg Asn Phe Thr Pro 245 250 255 Glu Thr Phe Arg Wing Arg Gln Lya Wing He Ser Leu Arg Gly Wing Ser 260 265 270 - 8 - Lys Gly Gly Lys Glu Gly Gly His Lys Gly Gly lie Wing Ser Gly Wing 275 280 285 • Being Arg Arg Ala His Thr Arg Gln Gln Phe Leu Glu Gly Leu Ser 5 290 295 300 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 3: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 85 amino acids ^ 10 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3: Met Thr Thr Arg Glu Arg Leu Pro Arg Asn Gly Tyr Ser lie Ala Ala 1 5 10 15 # 20 Ala Ala Lys Lys Leu Gly Val Ser Glu Ser Thr Val Lys Arg Trp Thr 20 25 30 Ser Glu Pro Arg Glu Glu Phe Val Wing Arg Val Wing Wing Arg His Wing 35 40 45 25 Arg lie Arg Glu Leu Arg Ser Glu Gly Gln Ser Met Arg Ala lie Wing 50 55 60 - 9 -Ala Glu Val Gly Val Ser Val Gly Thr Val His Tyr Ala Leu Asn Lys 65 70 75 80 Asn Arg Thr Asp 85 Wing (2) INFORMATION OF THE SEC. FROM IDENT. NO: 4: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 4: AATTCAAGCT TGTCGACGTT AACCTGCAGG CATGCGGATC CGGTACCGAT ATCAGATCT 59 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 5: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear - 10 - (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5: • CCGAAGATCT GATATCGGTA CCGGATCCGC ATGCCTGACAG GTTAACGTCG ACAAGCTTG 59 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 6: (I) CHARACTERISTICS OF THE SEQUENCE: 10 (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO 6 : GTACCGGCCG CTGCGGCCAA GCTT 20 24 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 7: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs 25 (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear - 11 - (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7 GATCAAGCTT GGCCGCAGCG GCCG 24 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 8: (I) CHARACTERISTICS OF THE SEQUENCE: 10 (A) LENGTH: 35 base pairs # (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8: AAACTGCAGC TGCTGGCTTG CGCCCGATGC TAGTC • 20 35 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 9: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 76 base pairs 25 (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear - 12 - (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: AAACTGCAGC AGCTGGGCAG GCCGCTGGAC GGCCTGCCCT CGAGCTCGTG TAGAATGTGC 60 TGCCGATCCT GGTTGC 76 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 10: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: CTAGTCTAGA CACCGATGAG GAAACCCGAT GA 32 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 11: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs - 13 - (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear • (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11 CCCAAGCTTC TCGAGTCAGT GGTCGCTGGG CGCGCG 10 36 (2) INFORMATION OF THE SEC. FROM IDENT. NO: 12: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs 15 (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) 20 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12 GGAGATCTAGATCGATATCTCGAG 24 25 (2) INFORMATION FROM SEC. FROM IDENT. NO: 13: (I) CHARACTERISTICS OF THE SEQUENCE: - 14 - (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 13 GATCCTCGAGATATCGATCTAGATCTCCGC 305

Claims

- 56 - CLAIMS Having described the invention as above, the content of the following 5 claims is claimed as property:

1. A polynucleotide characterized in that it comprises a sequence capable of selectively hybridizing with: ^ (a) SEC. FROM IDENT. N0: 1 or the complement of the 9 10 same; (b) a 3.6 kb plasmid sequence of Propionibacterium freudenreichii CBS 101022; (c) a 3.6 kb plasmid sequence of Propionibacterium freudenreichii CBS 101023; or 15 (d) a sequence encoding a polypeptide which comprises a SEC. FROM IDENT. NO: 2 or 3, a sequence of amino acids substantially homologous thereto or a fragment of any of the sequences. 20

2. A pj-ip? R_lßot-tdo, characterized in that it is a plasmid that replicates autonomously that can remain extrachromosomal within a host cell, plasmid which is derived from an endogenous plasmid of Propionibacterium, and when it comprises a heterologous gene is able to express that gene 25 inside the host cell. - 57 -

3. The polynucleotide according to claim 1, characterized in that it replicates autonomously in a host cell.

4. The polynucleotide according to claim 3, characterized in that the host cell is a Propi on i bacterium.

5. The polynucleotide according to claim 4, characterized in that the Propionibacterium is Propionibacterium freudereichii.

6. The polynucleotide according to any of the preceding claims, characterized in that it is capable of selectively hybridizing to one or more sequences in SEQ. FROM IDENT. N0: 1 which is, or are, necessary for autonomous replication in a Propionibacterium.

7. The polynucleotide according to claim 1, characterized in that it comprises the 1.7 kb fragment of SEQ. FROM IDENT. N0: 1 delineated by Sali and AlwNl restriction sites or by nucleotides 1 through 1750 of the SEC. FROM IDENT. N0: 1 - 58 -

8. A vector, characterized in that it comprises a polynucleotide according to which one of the preceding claims.

9. A vector, according to claim 8, characterized in that it is a plasmid.

10. The vector according to claim 8 ^^ or 9, characterized in that it additionally comprises a selectable marker 10.

11. The vector according to any of claims 8 to 10, characterized in that it replicates autonomously in E. coli.

12. The vector according to any of claims 8 to 11, characterized in that it is a vector of • expression.

13. The vector according to claim 12, characterized in that it comprises an endogenous gene of a Propionibacterium or a heterologous gene operably linked to a control sequence which is capable of providing expression of a gene. 25 - 59 -

14. The vector according to claim 13, characterized in that the gene is the coJbA gene.

15. The vector according to claim 13, characterized in that the heterologous gene encodes a polypeptide which is therapeutic in a human or animal.

16. A polypeptide, characterized in that it comprises 10 the sequence SEC. FROM IDENT. NO: 2 or 3, or a sequence substantially homologous thereto, or a fragment of any of the sequences, or which is encoded by a polynucleotide according to claims 1 to 7.

17. A host cell, characterized in that it comprises a heterologous polynucleotide or a vector according to any one of claims 1 to 15, or which can be transformed or transfected with a vector according to any of claims 13 to 15. 0

18. The host cell according to claim 17, characterized in that it is a bacterium. - 60 -

19. The host cell according to claim 18, characterized in that it is a Propionibacterium or E. coli.

20. A process for producing a host cell according to any of claims 17 to 19, characterized in that it comprises transforming or transfecting a host cell with a polynucleotide or vector according to any of claims 1 to 15.

21. A process for the preparation of a polypeptide, or other compound, the process is characterized in that it comprises culturing or fermenting a host cell as defined according to any of claims 17 to 19 under conditions that allow the expression or production of the polypeptide or compound .

22. The process according to claim 21, characterized in that it is a fermentation process in which the host cell is cultured under aerobic or anaerobic conditions.

23. The process according to claim 21 or 22, characterized in that the -61-expressed polypeptide or the compound produced is recovered from the host cell.

24. The process according to claim 23, characterized in that the polypeptide is a protease, amylase, lipase or peptidase or the compound is vitamin B12.

25. The process according to any of claims 21 to 24, characterized in that the polypeptide is selected from the host cell.

26. The process according to claim 25, characterized in that the polypeptide is expressed on the surface of the host cell, or the polypeptide is an antigen or immunogen.

27. A polypeptide or compound, characterized in that it is prepared by a process according to any of claims 20 to 26.

28. A process for the production of vitamin B12, cobalamin, the process is characterized in that it comprises culturing a host cell according to any of - 6.2 - claims 17 to 19, under conditions in which the vitamin is produced and, if necessary, isolate the vitamin.

29. Vitamin B12 characterized in that it is produced by a process according to claim 28.

30. The polypeptide according to claim 27, characterized in that it is used in a method for treating the human or animal body by therapy.

31. A host cell according to any of claims 17 to 19, characterized in that it is used in a method for treating the human or animal body by therapy or for use in an animal feed.

32. The use of a host cell according to any of claims 17 to 19, or a polypeptide or compound according to claim 27 either to make cheese for use in cheese making.

33. The use of a host cell according to any of claims 17 to 19, or a polypeptide or compound according to claim 27, characterized in that it is used in the manufacture of a food product or in an animal feed. - 63 -

34. A food product characterized in that it comprises a polypeptide or compound according to claim 27 or a host cell according to any of claims 17 to 19.

35. A food product according to claim 34, characterized in that it is for human consumption, for example a cheese, sauce, or for an animal.

36. A process for making cheese or other fermented milk product, the process is characterized in that it comprises the use of a host cell according to any of claims 17 to 19.

37. The process according to claim 36, characterized in that the host cell is used instead of or in addition to lactic acid bacteria.

38. The process according to claim 36 or 37, characterized in that the host cell is a Propionibacterium cell.