NZ229125A

NZ229125A - Cloning vectors of the pfx family including a sequence from plasmid pdi 25 of l. lactis 5136

Info

Publication number: NZ229125A
Application number: NZ22912589A
Authority: NZ
Inventors: Pak-Lam Yu; Lindsay Ernest Pearce; Fengfeng Xu
Original assignee: New Zealand Dairy Res Inst; Univ Massey
Priority date: 1989-05-15
Filing date: 1989-05-15
Publication date: 1992-03-26

Description

<div class="application article clearfix" id="description"> * 0 0^1 C ? 25 S&S-o.** ia^gW° t'r receiver NEW ZEALAND PATENTS ACT, 1953 No: Date: COMPLETE SPECIFICATION IMPROVED RECOMBINANT DNA METHOD •> /[/We THE NEW ZEALAND DAIRY RESEARCH INSTITUTE, a body corporate incorporated under the Charitable Trusts Act 1957, of Dairy Farm Road, Fitzherbert West, Palmerston North, New Zealand and MASSEY UNIVERSITY, a body corporate established under the Massey University Act 1963, of Private Bag, Palmerston North, New Zealand hereby declare the invention for whichxf/we pray that a patent may be granted tojj^fus', and the method by which it is to be performed, to be particularly described in and by the following statement:- -1- Field of the Invention - 2 - 22 9 1 25 This invention relates to the constroction of a cloning and expression system for lactococcal strains which are used in dairy fermentations. This system also enables direct transfer and expression of foreign genes in these microorganisms resulting in production of their gene products. The Invention also relates in particular to a new shuttle vector and derivatives thereof. Bickground of Invention In the past few years, research directed towards Improving dairy foraentatton cultures by recombinant ONA technology has received considerable Interest. The dairy cultures are lactic acid bacteria which include « diverse group of strains (called ."starters") that are used to Initiate acid production for a variety of dairy and food fermentations. In addition to their use in the manufacture of dairy products, they are potentially useful bacterial hosts for the production of recombinant DNA products that can be used in food processing. The basic constituents of recombinant DNA technology which are used in strain improvement are well known to those skilled in the art and include: - the isolation (cloning) of a particular gene after restriction enzyme digestion, - the joining (ligation) of a gene or genes into a carrier (vector) to give a recombinant DNA molecule, - the transfer of the recombinant DNA molecule by transformation, transfection or electroporation into a host bacteriian, and - the expression of these genes in the host which is regulated by genetic control elements, e.g. selected promoters. t^'ji ir. ' L / i L.. J - 3 - Recombinant DNA technology is a powerful tool that can be used either to improve the genetic make-up of a microbial strain or to create new strains for the production of a particular gene product, e.g. enzymes, hormones, antigens. Such recombinant DNA products can be used in the food industry, the pharmaceutical industry, the chemical industry and the agricultural industry. Historically, research involving recombinant gentic manipulation of plasmids for the production of various substances has centred on Escherichia coli as a host organism. Escherichia coli is the best studied microorganism and the first discoveries and inventions in the field of recombinant DNA technology have been made using this organism as a host. However, E coli is not the most suitable organism for the commercial production of substances because it produces a number of toxic pyrogenic factors and inclusion bodies are also formed which renders the purification steps difficult. The genus Lactococcus (formerly group N Streptococcus) comprises strains of microorganisms which are used widely in dairy fermentations. Strains of this genus that are used as starters grow in milk efficiently. No strains of the genus are known to produce toxic substances. Interest in the molecular biology and genetics of this genus has been rapidly expanding. Extrachromosomal DNA in the form of large and small plasmids has been found in all studied strains of the genus Lactococcus (Davies, F. L., J. Dairy Sci., 48: 363-376 (1981)). Plasmids of these strains encode commercially important functions, e.g. lactose utilization, protein degradation, bacteriophage resistance, citrate utilization, antibiotic production (McKay, L. L., Antonie van Leewenhoek, 49: 2590274 (1983)). A number of the plasmid genes have been cloned and characterized (de Vos, W. M., FEMS Microbiol. Reviews, 46: 281-295 (1987)). - 4 - A variety of gene transfer processes have been discovered and analysed in the genus Lactococcus. Gene transfer by transduction, by conjugation, transformation and transfection have been reported (see Davies above). Recently, small cryptic (i.e. unknown function), high copy plasmids of these strains have attracted alot of interest. These small plasmids are potentially useful as vectors for gene cloning and expression. Vectors of this kind have been constructed and reported (see McKay above). PCT international application WO 85/03945 describes recombinant plasmids and lactic acid bacteria containing such plasmids. It also describes a process for preparing fermented food products, animal feedstuffs and ingredients thereof, and proteins using such bacteria. It is an object of this invention to go some way toward providing a vector system capable of direct transformation and expression of genetic information in lactococci, or at least to provide the public with a useful choice. Summary of Invention Accordingly, the invention may be said broadly to consist in a cloning vector of the family pFX which is replicable in a host selected from any one of the genera Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, Bacillus and Clostridium and in E. coli, said vector comprising: a marker sequence capable of expression in said host, a cloning site to enable the insertion of insert-DNA, and a replication sequence derived from plasmid pDI25 of L 1 act is subspecies 1 act is 5136 by which said vector can replicate in said 7 2 9125 - 5 - In another aspect, the invention may be said broadly to consist in a recombinant expression vector replicable in a host selected from any one of the genera Lactococcus, Streptocccus, Leuconostoc, Pediococcus, Lactobacillus, Bacillus, and Clostridium and E. coli, said vector comprising: DNA which when expressed gives said host an improved or new property; a control sequence compatible with said host operably linked to said DNA and capable of effecting expression thereof in said host; and a replication sequence derived from plasmid pD125 of L. lactis subsp. lactis 5136 to allow said vector to replicate in said host. In a further aspect the invention may be said to consist in a bacterium of the genera Streptococcus, Leuconostoc, Pediococcus, Lactobacillus, BaciJJjus or Clostridium or E. coli or any other suitable host provided with a cloning vector or expression vector as described above. In a further aspect the invention may be said broadly to consist in a method of giving an improved or enhanced property to a bacterium of the genera Streptococcus, Leuconostoc, Pediococcus, Lactobaci1lus, Baci1lus or Clostridium or E. col i which comprises transforming said bacterium wi th an expression vector as defined above and propagating the resultant transformant in a growth-sustaining medium. In a further aspect the present invention may be said to consist in a method for producing proteinase in proteinase-negative Lactococcus comprising electroporating lactococcus cells with a construction comprising: a) the vector pFXl, and b) a 6.5 kb Hindi 11 proteinase fragment of plasmid pD121 and propagating the resultant transformants in a growth sustaining medium. n 2 9 i 2 r> -6 - In a preferred embodiment the present invention may be said to consist in a method for producing proteinase in proteinase-negative Lactococcus comprising electroporating Lactococcus cells with the plasmid pFXIOl. The invention consists in the foregoing and also envisages constructions of which the following gives examples. Brief Description of the Drawin^s The invention may be more fully understood by having reference to the drawings accompanying the provisional specification: - Fig. 1 is an enzyme restriction map and schematic representation of the construction of pFXl. - Fig 2. is an enzyme restriction map and schematic representation of the construction of pFX2 and pFX3. - Fig. 3 is an enzyme restriction map and schematic representation of the construction of pFX4, pFX5 and pFX6. - Fig. 4 illustrates cloning of the pDI21 proteinase gene in the shuttle vector pFXl. A. The 6.5b Hindi 11 prt gene fragment cloned into pFXl. B. Autoradiogram after hybridization with 32p_iabe11ed recombinant phage XDNA (xNMl149 with 6.5 kb Hindlll prt gene fragment). Lane 1, pFXIOl Hindlll digest; lane 2, pFXl Hindi II digest, lane 3, DNA Hindlll M. W. standards; lane 4, recombinant phage \DNA as a positive control. - Fig. 5 is a series of electrophoretograms showing g-casein degradation by different strains of lactococci. Lane 1, 4125 (pFXIOl); lane 2, 4125 (pFXl); lane 3, 4125; lane 4, 4125 (pD121); lane 5, H2 (wild type); lane 6, e-casein control; lane 7, M. W. standards. - Fig. 6 represents plots of growth and acid production in RSMG by lactococci. H2 (wild type) [•], 4125 (pDI21) [a], 4125 (pFXIOl) [o], 4125 (pFXl) [o] and 4125 [a]. :^ni2r> - 7 - In the accompanying drawings: - Fig. 7 respresents the nucleotide sequence and inferred amino acid sequences (repA and repB) of the lactococcal portion (Hpall-Mbol) of pFX2. Nucleotides in lower case represent the region identical to staphylococcal plasmid pE194. A possible plus ori site {solid line) and direct repeat regions (double-dash) are indicated. Within the second direct repeat region are three iterons (stars). The inverted repeat region that could form an attenuator is indicated by facing arrows. Putative promoter region and ribosome binding sites are positioned with markers ~ and 0 respectively. RS/\ and important restriction sites are marked. Detailed Description of the Invention Lactic acid bacteria of the genus Lactococcus and in particular the species L. lactis subsp. lactis, L. lactis subsp. cremoris and L l.actis subsp. lactis variety diacetylactis, are biotechnologically important'and are used commercially, particularly for the manufacture of cheese and casein. It is well known that all wild-type lactococci carry a number of plasmids. Plasmids are self-replicating extrachromosomal ONA elements that are present in the bacteria. Plasmids encode a wide range of functions, for example, lactose utilization and protein degradation. Small, cryptic plasmids of high copy number, but unknown functions, are also found in these bacteria. A method of direct gene transformation to whole cells by electroporation has been widely used in a number of microorganisms, plant and animal cells (Newmann, E et al EMBO Journal, 7: 841-845 (1982)). The use of whole cells //* iflstpad of protoplasts has circumvented the difficulties inherent in the -v profoblast regeneration step and is a more efficient and rapid procedure. It ^ JANI9Q? "'/ a frirs Flow been found that this method is effective in lactococci for a wide ^2 9125 -8 - variety of plasmids (Powell, I B et al, Appl Environ Microbiol, 54: 655-660 (1988)). The linking of an effective tran format ion procedure with a vector system is an important combination for successful gene incorporation into lactococci. Vectors based on two cryptic plasmids of Lactococcus species have been reported (Chassy, B M FEMS Microbiol, Reviews 46: 297-312)). We have now made a new vector pFXl and its derivatives which are particularly suitable for transformation of lactococci and particularly L. lactis subsp. lactis and subsp. cremoris. The key features of this family of vectors which are based on a small cryptic, high copy plasmid of L. lactjhs subsp. lactis are: - 1. a high copy number (5.5 kb) cryptic plasmid pD125 of L. lactis 5136 was used in the primary construction; 2. pD125 is unrelated by DNA-DNA homology to previously described plasmids used as vectors in lactoccocci (Figure 1); 3. the size of the intially derived replicon pFXl is 5.5 KB; 4. the vector pFXl efficiently transforms both lactococci and E. coli; 5. the vector pFXl is stable without antibiotic selection in lactococci and E col i ; 6. a polylinker with two promoters can be inserted into a pFX derivative for easy cloning, transcript analyses and DNA sequencing. 7. modified versions of pFX with polylinker and three different reading frames for cloning lactococcal promoter containing fragments can be constructed for monitoring gene transcription and regulation. The lactococcal transformation method of Powell (1988) was used with some modifications. A gene pulser apparatus (Bio-rad Laboratories, Richmond, Ca, USA) was used for routine electroporation of lactococci. - 9 - Overnight lactoccal cultures were diluted in Ml7-g1u and grown at 30°C to OD 0.7 (600 nm). The cells were washed with ice cold EPB1 buffer (0.5 M sucrose, ImM MgCl, 7mM Na2HP04,pH7.4) and resuspended in 0.1 culture volume of BPB1 plus 250 U/ml lysozyme (Sigma, St Louis, Mo., USA), and incubated at 37°C for 20 min. The treated cells were further washed with EP82 buffer (0.5 M sucrose, ImM HEPES) and resuspended in 0.05 culture volume of EPB2. Volumes of 80 u 1 concentrated cells plus 1-5 ul of DNA were pulsed in 2 mm gene pulser cuvettes at 12 500 V/cm and 25 uF with a pulse controller at 200^ . Successful transformants were detected on a selective medium (e.g. M17) at 30°C containing chloramphenicol (Cm) at a final concentration of 5 ug/ml. Chloramphenicol resistant colonies appeared approximately 24 hours after transformation with pFX vectors. Comparative electrophoresis of DNAs from untransformed and transformed lactococci showed that only the transformed cells had hybridizing bands with electrophoretic mobilities corresponding to the vector used for transformation. The presence of the vector in transformed lactococci was further confirmed by transforming E. coli with plasmids prepared from these lactococcal transformants and reisolating the same plasmids from the E. coli transformants formed. The process of the present invention can be applied to host strains of Lactococcus lactis subsp. lactis (including variety diacetylactjs) and L. lactis subsp. cremoris. Both species are 'food grade' organisms and have GRAS (generally regarded as safe) status. £ jm. 'J'Jfj ] 'jr. -10- —ji.-.J No antibiotic selection was needed to maintain the vectors in the lactococcal hosts. After seven transfers in culture without Cm selection, >99% of L. lactis cultures contain the vector. Hybrid plasmids have been constructed using pFX vectors. Genes cloned into pFX vectors can remain as substantially as autonomously replicating plasmids after being introduced into lactococci or E. coli. The pFX vectors are capable of transformation (for example by electroporation) into Gram-positive hosts of the genera Streptcoccus, Leuconostoc, Pediococcus, Lactobacillus, Bacillus and Clostridium. The invention can be used for the cloning and expression of both prokaryotic and eukaryotic genes in Lactococcus as a host, preferably using a cloning vector of one of the types as described herein before. The invention can advantageously be used for the production of high levels of chemical substances, in particular polypeptides. The choice of a particular Lactococcus host strain and the optimal process conditions may vary with, inter alia, the gene and vector to be selected. The optimal slection and process conditions can be established by routine experimentation. These variations are all included within this invention. The invention is not limited to the use of lactococci as hosts, but can be used in any transformable Gram-positive bacteria. The invention is further exemplified by a detailed description of the cloning and expression of a proteinase gene from Lactococcus Lactis subsp. cremoris H2. , - N } rfqr Tto^fol lowing examples illustrate certain embodiments of the present invention. /'Ay 7 • =5^ _ r. 'w' \ 28 JAN 1992"' /> A e* ? 2 9125 -li- EXAMPLE 1 Construction of a Shuttle Vector for E coli and Lactococci The high copy cryptic 5.5 kb plasmid pD125 (Xu,F et al, Arch. Microbiol, 154 99- culture collection of the New Zealand Dairy Research Institute, Palmerston North, New Zealand) was double digested with Hpalll and Mbol to produce three fragments. They were ligated individually to the 1 kb Hpall-Mbol fragment of the B. subtil is plasmid pC194 (Old, R. W. et al., "Principles of Gene Manipulation", vol 3, p. 341 (1985)) which carries the promoterless chloramphenicol acetyl tranferase (cat) structural gene. The three ligation mixtures were separately transformed into E. coli HB101. Transformants were only obtained when the largest DNA fragment was ligated to the cat fragment. Recombinant plasmids from these transformants were able to replicate in both E. coli and lactococci. This plasmid vector was designated pFXl and a restriction map was determined (Fig. 1). The vector has been deposited in the culture collection of the Australian Government Analytical Laboratories, Pyinble, NSW, 2073, Australia on 15 May 1989 under no. 89/018350. pFXl could readily transform E. coli HB101, JM109, L. lactis subsp. lactis LM0230, MG1363 and 4125 strains at frequencies of 10^-10^ cfu/ug DNA, and L. lactis subsp. cremoris 4873 at 10^ cfu/ug DNA. The stability of pFXl in E. coli JM109 and HB101 after seven transfers in cultures without Cm selection was 95 and 97% respectively. In L. lactis subsp lactis MG1363 and 4125, it was > 99%. The smaller Hal region (1.9kb) was not essential for plasmid'pF^l replication, the Hindlll site in that region of pFXl could be used for cloning. The Clal digested derivative of pFXl was designated pFX2 (Fig 2). 104 (19SO)) of L. lactis subsp. lactis 5136 NZ DRI20026 of the - 12 - 229125 The new vectors obtained were designated pFX4, pFX5 and pFX6. These differ from each other by the presence of 3-,4- or 5-bp inserts between the polylinker and the lacZ gene. These differences permit the fusion of cloned genes to the lacZ in all three translational reading frames in lactococci. The nucleotide sequences of the polylinker regions contained within the coding region of the lacZ gene are given for each plasmid in Fig. 3. The three vectors herein described permit genes to be cloned in lactococci (fused to the £. coli lacZ gene) in all three translational reading frames. Such translational fusions are likely to greatly facilitate the investigation and characterization of regulatory systems in lactococci. mmLi Clonina and expression of Promoter Fragments in L lactis subsp. lactis A 2.0 or 0.4 kb EcoRl Lac fragment of the plasmid pDI-21 (Yu, P-L, sX. 3ll-» AppI. Microbiol. Biotech.. 2ft: 71-74 (1989)) was ligated into the polylinker vactors pFX4, pFX6 and pFX6. The ligated, mixtures were electroporated into Lactococcus lactis subsp. lactis strains and blue colonies were selected on 18 agar containing X-gal and IPTG. Further analysis of these cloned fragments by DHA sequencing showed the structures of typical lactococcal promoters. EXAHPLE 5 Clonlna and Expression of the Proteinase Gene 1n L. lactis subsp. lactis lactococcus lactis subsp. cremorlS H2 carries a lactose-proteinase plasmid pDI21. This plasmid had previously been transferred by conjugation to the plasmid-free Lactococcus JactlS, subsp. 1 act 1§ 4125. The 6.5 kb Hindlll proteinase gene fragment was isolated from strain 4125 (pDI21). Plasmid pFXl was digested with Hindlll and treated with calf intestine alkaline phosphatase (Promega Co., Wi., USA), and ligated to 6.5 Hindlll fragment. The ligation mixture was directly transformed in 1. lactis subsp. lactis 729125 0») -13- pFX2 was digested with Thai and blunt-end ligated to an EcoRI-Thal adaptor. The ligation mixture was transformed into L. lactis subsp. lactis 4125 by electroporation. A recombinant plasmid containing both EcoRI and Thai sites was thus obtained. Each of the three reading frames of the EcoRI and Oral 3.1 kb lacZ-polylinker DNA fragments was individually cloned into EcoRI-Thal digested plasmid pFX2. The ligation mixtures were separatelly electroporated into Lactococcus lactis subsp. lactis 4125. The new vectors obtained were designated pFX4, pFX5 and pFX6. These differ from each other by the presence of 3-,4- or 5-bp inserts between the polylinker and the lacZ gene. These differences permit the fusion of cloned genes to the lacZ gene in all three translational reading frames in lactococci. The nucleotide sequences of the polylinker regions contained within the coding region of the lacZ gene are given for each plasmid in Fig. 3. The three vectors herein described permit genes to be cloned in lactococci (fused to the E. coli lacZ gene) in all three translational reading frames. Such translational fusions are likely to greatly facilitate the investigation and characterization of regulatory systems in lactococci. EXAMPLE 4 Cloning and Expression of Lactose Metabolism Genes in L. lactis subsp. lactis A 2.0 kb EcoRI Lac fragment of the plasmid pDI-21 (Yu, P-L, et al., Appl. Microbiol ., 30: 71-74 (1989)) was ligated into the polylinker vectors pFX4, pFX5 and pFX6. The ligated mixtures were electroporated into Lactococcus lactis subsp. Lactis strains, selected on LB agar containing X-gal and IPTG and blue colonies were obtained with pFX5. Restriction analysis of the trtmsformants shewed the insert orientation and direction of transcription. I* EXAMPLE 5 -14- 229125 Cloning and Expression,of the Proteinase Gene in L. lactis subsp. lactis Lactococcus lactis subsp. creinoris H2 carries a plasmid pD 121 which encodes genes for both lactose metabolism (Lac) and the proteinase positive phenotype (prt). This plasmid had previously been transferred by conjugation to the plasmid-free Lactococcus lactis subsp. lactis 4125. The 6.5 kb Hindlll proteinase gene fragment was isolated from strain 4125 (pD121). Plasmid pFXl was digested with Hindlll and treated with calf intestine alkaline phosphatase (Promega Co., Wi., USA), and ligated to the 6.5 kb Hindlll fragment. The ligation mixture was directly transformed in L. lactis subsp. lactis 4125 by electroporation (10^ transformants/ug ligated DNA). Eight of 10 transformants tested were Prt+ by the milk-coagulation test. The insert orientation was determined following double digests with Hpall and Bglll. Recombinants with inserts in either orientation had the same milk-cogulating time, suggesting that the proteinase gene expressed from its own promoter. No re-arrangements in either the insert of the vector after transformation were detected (Fig. 4). The recombinant plasmid was designated pFXIOl. Plasmid pFXIOl showed the same stability in strain 4125 as did pFXl. Following seven transfers in Cm-free broth, 100 colonies all showed the presence of the proteinase gene by colony hybridization. Proteinase released from strain 4125 (pFXIOl) degraded 6-casein in a similar manner to that observed with the intact pD 121 in its original hosts (Fig. 5). L. ljJCtjis subsp. lactis 4125 (pFXIOl) grew more rapidly than 4125 (pFX 1) on citrate-milk agar plates. After 2 days incubation at 30°C, a white casein precipitate formed around the colonies due to a' ' results were observed with 4125 (pDI21). ? 0 1 O C ' s I 4. J fs. -15- Growth and acid production by L. lactis subsp. creinoris H2, L. lactis subsp. lactis 4125 (pDI21), 4125 (pFXIOl), 4125 (pFXl) and 4125 in RSMG at 30°C were compared. The Prt+ strain 4125 (pFXIOl) grew at a faster rate and produced more acid than 4125 and 4125 (pFXl) (Fig. 6). Growth and acid production by 4125 (pFXIOl) was, however, significantly slower than that of H2 or 4125 (pDI21). Plasmid pFXIOl was also electroporated in the piasmid-free lactococcal recipients L. lactis subsp. lactis LM0230 and MG 1363 (pFXIOl) coagulated milk in 18 h at 22°C, the same rate as the Prt+ strains H2 and 4125 (pDI21). As noted above, 4125 (pFXIOl) was significantly slower, requiring 30 h at 22°C to coagulate milk. The proteinase gene is thus expressed differently in different L. lactis subsp. lactis hosts. example 6 Sequencing the Recombinant Plasmid pFX2 pFX2 DNA was linearized with H pa11 and cloned into Smal sites of the sequencing vectors pGEM3Z and pGEM4Z (Promega). Overlapping templates were created by ExoIII digestion with Erase-a-Base™ system (Promega). The DNA sequence of each strand was determined using sequenase (USB) and the dideoxynucleotide chain termination method (Sanger et al. 1977) in the presence of ("*35$) dATP (Amersham). All enzymes were used according to the manufacturer's instructions. The software package developed by the Unversity of Wisconsin Genetics Computer Group, USA was used for analysis of DNA sequences and translated peptides. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> -16- WHAT WE CLAIM IS:

1. A cloning vector of the family pFX which is replicable in a host selected from any one of the genera Lactococcus, Leuconostoc. Pediococcus, Lactobaci 1 lus, BacilJIus and Clostridium and in E. col i, said vector comprising: a marker sequence capable of expression in said host, a cloning site to enable the insertion of insert-DNA, and a replication sequence derived from plasmid pD125 of L 1actis subspecies 1 actis 5136 by which said vector can replicate in said host.

2. A vector as defined in claim 1 wherein said replication sequence is included within a 4.5 kb Hpall - Mbol digestion fragment derived from plasmid pDI25.

3. A vector according to either of claims 1 or 2 wherein said marker sequence codes for chloramphenicol resistance (Cm^).

4. A vector according to any one of claims 1 to 3 wherein said cloning site comprises a polylinker.

5. A vector according to claim 4 wherein said polylinker is a sequence for the insertion of restriction fragments of Sac I, Bst XI, S_acII, E_agl, Not I, Xbal, Spel, BaniHI, Smal, PstI, EcoRI, EcoRV, Hindlll, C1 a I, San, (HincII), AccI, Xhol, Drall, Apal or KpnI.

6- A vector according to claim 4 or 5, further comprising the promoters T3 ^ ° \ "i^and T7 which are suitable for the generation of RNA transcripts and for I FFft/oo-, - ^NA sequencing. V n & .17. muim

7. A vector according to any one of claims 1 to 6 further comprising a Oral -EcoRI (lacZ) fragment and constructed to carry each of three reading frames to enable selection and characterisation of gene fragments carrying promoters. 8. The vector pFXl substantially as herein described with reference to figures 1 to 6 accompanying the provisional specification and the accompanying figure 7. 9. The vector pFX2 substantially as herein described with reference to figures 1 to 6 accompanying the provisional specification and the accompanying figure 7. 10. The vector pFX3, substantially as herein described with reference to figures 1 to 6 accompanying the provisional specification and the accompanying figure 7. 11. The vector pFX4, substantially as herein described with reference tofigures 1 to 6 accompanying the provisional specification and the accompanying figure 7. 12. The vector pFX5, substantially as herein described with reference to fiaures 1 to 6 accompanying the provisional specification and the accompanying figure 7. 13. The vector pFX6, substantially as herein described with reference to figures 1 to 6 accompanying the provisional specification and the accompanying "VJ: figure 7. 14. A recombinant expression vector replicable in a host selected from any one of the genera Lactococcus, Streptococcus, Leuconostoc, Pediococcus, Lactobacillus, Bacillus and Clostridium and in E. coli, said vector compri sing: DNA which when expressed gives said host an improved or new property; , ^ Y a control sequence compatible with said host operably linked to said j «2r- ' \ JANI992 and capable of effecting expression thereof in said host; and ' ' O -1

8- a replication sequence derived from plasmid pD125 of L. lactis subsp. lactis 5136 to allow said vector to replicate in said host. 15. A vector according to claim 14 wherein said replication sequence is included within a 4.5 kb Hpall - Mbol digestion fragment derived from plasmid pD125. 16. a vector according to either of claims 14 or 15 wherein said dna codes for chloramphenicol resistance (Cm^). 17. A vector according to any one of claims 14 to 16 wherein said DNA is heterologous to said host. 18. A vector according to any one of claims 14 to 17 wherein said DNA codes for a proteinase. 19. The vector pFXIOl, substantially as herein described with reference to figures 1 to 6 accompanying the provisional specification and.the accompanying figure 7. 20. A bacterium of the genera Streptococcus, Leuconostoc, Pediococcus, Lactobacillus, Bacillus or Clostridium or E. coli provided with one or more vectors according to any one of claims 1 to 19. 21. An E. coli or a lactic acid bacterium provided with one or more vectors according to any one of claims 1 to 19. „ 22. A Lactococcus bacterium provided with one or more vectors accordinq to any A*- -■;:■ one of claims 1 to 19.;■ V;\ 28 jM.mr;_ 'J Of) 1 vrr r; .19. -t-u-.i;23. A method of giving an improved or enhanced property to a gram-positive bacterium of the genera Streptococcus, Leuconostoc, Pediococcus, Lactobacillus^ Baci11 us or Clostridium which comprises transforming said bacterium with a vector according to any one of claims 14 to 19 and propagating the resultant transformant in a growth-sustaining medium.;24. A method of giving an improved or enhanced property to an E. coli or a lactic acid bacterium which comprises transforming said bacterium with a vector according to any one of claims 14 to 19 arid propagating the resultant transformant in a growth-sustaining medium.;25. A method of giving an improved or enhanced property to a Lactococcus bacterium which comprises transforming said bacterium with a vector according to any one of claims 14 to 19 and propagating the resultant transformant in a growth sustaining medium.;26. A method for producing proteinase in proteinase-negative Lactococcus comprising electroporating lactococcus cells with a construction comprising:;a) the vector pFXl, and b) a 6.5 kb Hindlll proteinase fragment of plasmid pD121 and;^ propogating the resultant transformants in a growth sustaining med i um.;27. A method for producing proteinase in proteinase-negative Lactococcus comprising electroporating Lactococcus cells with the plasmid pFXIOl.;£ n v 'o ■ O;, ;/ ^ ■;28.^ A method for preparing a protein, in which process a lactic acid bacterium \ 8 JAN 1992 ,'as claimed in either of claims 21 or 22 is used.;* C ■ '■ -20- 'J~2 in :ir> 29. A proteinase prepared by a method according to claim 26 or claim 27. 30. A food product, animal feedstuff or ingredient thereof, prepared by a method as claimed in claim 27. 31. A protein prepared by a method as claimed in claim 28. 32. A cloning vector of the family pFX substantially as hereinbefore described with reference tc figures 1 to 6 accompanying the provisional specification and tl'.e accompanying figure 7. 33. A recombinant expression vector substantially as hereinbefore described with reference to figures 1 to 6 accompanying the provisional specification and the accompanying figure 7. /■ fVlafJcy lUyr'SCf P, ijiiiw r Ajil-.or.sed AgoAte, A ! • I S •. S'Sk . i </div>